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2015v1.0
SIXTH EDITION

TEXTBOOK OF
DIAGNOSTIC
MICROBIOLOGY
Connie R. Mahon, M.S. Donald C. Lehman, Ed.D., MLS(ASCP)cm, SM(NRCM)
Director, Organization Development (Retired) Associate Professor
Health Resources and Services Administration Department of Medical Laboratory Sciences
Learning Institute University of Delaware
Rockville, Maryland Newark, Delaware
Adjunct Assistant Professor
Medical Laboratory Sciences
Integrative Health Sciences Department
School of Medicine and Health Sciences
The George Washington University
Washington, DC
Elsevier Saunders
3251 Riverport Lane
St. Louis, Missouri 63043

TEXTBOOK OF DIAGNOSTIC MICROBIOLOGY, ISBN: 978-0-323-48218-9


SIXTH EDITION
Copyright © 2019 Elsevier Inc. All Rights Reserved.
The contribution made by Kalavati Suvarna and Sumathi Nambiar is in public domain.
Previous editions copyrighted 2015, 2011, 2007, 2000

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any information storage and retrieval system, without
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This book and the individual contributions contained in it are protected under copyright by the Publisher
(other than as may be noted herein).

Notices

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and
using any information, methods, compounds or experiments described herein. Because of rapid advances
in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be
made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or
contributors for any injury and/or damage to persons or property as a matter of products liability,
negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas
contained in the material herein.

Library of Congress Cataloging-in-Publication Data

Names: Mahon, Connie R., editor. | Lehman, Donald C., editor.


Title: Textbook of diagnostic microbiology / [edited by] Connie R. Mahon, Donald C. Lehman.
Description: Sixth edition. | St. Louis, Missouri : Elsevier Saunders, [2019] | Includes bibliographical
references and index.
Identifiers: LCCN 2017050818 (print) | LCCN 2017051723 (ebook) | ISBN 9780323482127 (ebook) | ISBN
9780323482189
Subjects: | MESH: Microbiological Techniques | Communicable Diseases—diagnosis | Bacterial Infections—
diagnosis | Virus Diseases—diagnosis | Mycoses—diagnosis
Classification: LCC QR67 (ebook) | LCC QR67 (print) | NLM QW 25 | DDC 616.9/041—dc23
LC record available at https://lccn.loc.gov/2017050818

Content Strategist: Kellie White


Content Development Manager: Ellen Wurm-Cutter
Content Development Specialist: Alexandra York
Publishing Services Manager: Deepthi Unni
Project Manager: Kamatchi Madhavan
Marketing Manager: Emily Wall
Designer: Margaret Reid
To my husband Dan for his love and continued support
and understanding; my son Sean who inspires me; my daughter Kathleen,
for showing me courage; and my granddaughters Kelly Amelia, Natalie Page,
and Madeline Belle, who have given us so much pleasure.
CRM

To my wife Terri, for her constant support and


encouragement, and whose love makes me realize anything is possible,
and my grandchildren Shane, Athena, Jordan, and Vincent,
who keep me young at heart.
DCL

To George Manuselis, a dedicated microbiologist, educator,


and mentor, who inspired all.
This page intentionally left blank
Reviewers

Keri Brophy-Martinez, MHA/ED, MT(ASCP) Grace Leu-Burke MSCLS, MT(ASCP)


Department Chair/Professor Assistant Professor
Medical Laboratory Technology Medical Laboratory Science School of Allied Health
Austin Community College University of Alaska Anchorage
Austin, Texas Anchorage, Alaska

Delfina C. Domínguez, MT(ASCP), MS, PhD Nicholas M. Moore, MS, MLS(ASCP)cm


Professor Assistant Director, Division of Clinical Microbiology
Clinical Laboratory Science/Public Health Assistant Professor
The University of Texas at El Paso Departments of Pathology and Medical Laboratory Science
El Paso, Texas Rush University Medical Center
Chicago, Illinois
Frances Pouch Downes, BS, MT(ASCP), MPH, DrPH,
HCLD(ABB) Hamida Nusrat, PhD, PHM(CDPH)
Professor Faculty
Biomedical Laboratory Diagnostics Program Clinical Laboratory Science Internship Program
Michigan State University San Francisco State University
East Lansing, Michigan San Francisco, California
Public Health Microbiologist and Trainer
Joanna Ellis, MS, BS, MLS(ASCP) Napa-Solano-Yolo-Marin County Public Health Laboratory
Clinical Assistant Professor Fairfield, California
Clinical Coordinator
Clinical Laboratory Science Program Jennifer Sanderson, MS, MT(ASCP)
Texas State University Central Laboratory Automation Specialist
San Marcos, Texas Clinical Chemistry/Immunology
Siemens Healthineers
Denise Forwick-Whalley, MLT Deerfield, Illinois
Licensed Funeral Director & Embalmer
Microbiology Susan E. Saullo, RN, MS MT(ASCP)
Northern Alberta Institute of Technology Adjunct Instructor
Edmonton, Alberta, Canada Nursing
ITT Technical Institute
Shawn Froelich, MS, MLS(ASCP)cm Lake Mary, Florida
Assistant Professor
Medical Laboratory Science Michael Simpson, BA, MS, MT(ASCP)
Allen College – UnityPoint Health Professor of Clinical Laboratory Science (Full Time)
Waterloo, Iowa Laboratory Supervisor (Part Time)
Clinical Laboratory Science
Julie Gardner, MS, MBA, MLS(ASCP)cm College of Southern Nevada (Full Time)
Director of the Medical Laboratory Technician Program Diagnostic Center of Medicine (Part Time)
Assistant Professor of Biology Las Vegas, Nevada
University of Saint Francis
Crown Point, Indiana Richard B. States, DHSc, CNMT, RT(N)(ARRT)
Chair
Daniel J. Harrigan, MS, MB(ASCP)cm Diagnostic Services Department
Professor The University of Findlay
MLT Program, Department of Health Sciences Findlay, Ohio
Blackhawk Technical College
Monroe, Wisconsin

v
vi REVIEWERS

Jane M. Stevens, MS, MT(ASCP)SM Dorothy Yvonne Yaschuk, RT, ART, MEd
Manager School of Health Sciences
Department of Pathology College of New Caledonia
Clinical Microbiology Laboratory Prince George, British Columbia
Rush University Medical Center
Chicago, Illinois

Ronald L. Walker, MBA, CNMT, PET


Assistant Professor
Diagnostic Services Department
The University of Findlay
Findlay, Ohio
Contributors

Yousif Barzani, MD, MLS(ASCP)CM Connie F.C. Gibas, PhD


Assistant Professor Clinical Research Project Manager
Department of Integrated Health Sciences Department of Pathology and Laboratory Medicine
School of Medicine and Health Sciences University of Texas Health Science Center at San Antonio
The George Washington University San Antonio, Texas
Washington, DC
Amanda T. Harrington, PhD, D(ABMM)
Maximo O. Brito, MD, MPH Director, Clinical Microbiology Laboratory
Associate Professor of Medicine Associate Professor, Pathology
Division of Infectious Diseases Loyola University Medical Center
University of Illinois Maywood, Illinois
Chicago, Illinois
Chief of Infectious Diseases Michelle M. Jackson, PhD
Department of Medicine Microbiologist
Jesse Brown VA Medical Center Division of Nonprescription Drug Products
Chicago, Illinois Center for Drug Evaluation and Research
U.S. Food and Drug Administration
Nina Clark, MD Silver Spring, Maryland
Professor
Department of Medicine Deborah Josko, PhD, SM(ASCP)
Division of Infectious Diseases Associate Professor and Director—Medical Laboratory Science
Director, Transplant Infectious Diseases Program
Loyola University Medical Center Clinical Laboratory Sciences
Maywood, Illinois Rutgers, The State University of New Jersey—School of Health
Professions
James L. Cook, MD Newark, New Jersey
Clinical Professor of Medicine
Division of Infectious Diseases, Department of Medicine Arun Kumar, PhD
Loyola University Medical Center Assistant Professor
Maywood, Illinois Nanomedicine Research Laboratory
Staff Physician and Research Scientist Department of Medical Laboratory Sciences
Infectious Diseases Section Department of Biomedical Engineering
Edward Hines, Jr. VA Hospital UD Nanofabrication Facility
Hines, Illinois Center for Bioinformatics & Computational Biology (CBCB)
College of Health Sciences
Cliff Cymrot, MLS(ASCP), MT(AAB), MT(AMT), MHA University of Delaware
Assistant Professor Newark, Delaware
Medical Laboratory Science
George Washington University Steven D. Mahlen, PhD, D(ABMM)
Washington, DC Director, Microbiology
Adjunct Instructor Affiliated Laboratory, Inc.
CAHS School of Social Work Bangor, Maine
Cincinnati, Ohio
Frederic J Marsik, PhD
Robert C. Fader, PhD, D(ABMM) Microbiology Consultant
Section Chief, Microbiology New Freedom, Pennsylvania
Pathology Department
Baylor Scott & White Healthcare Kevin McNabb, PhD, MT(ASCP)
Scott & White Medical Center—Temple Director, Microbiology and Immunology
Temple, Texas New Hanover Regional Medical Center
Wilmington, North Carolina

vii
viii Contributors

Alfredo J. Mena Lora, MD Linda A. Smith, PhD, MLS(ASCP)CM, BBCM


Clinical Assistant Professor Professor
Associate Program Director, Infectious Diseases Fellowship Program University of Texas Distinguished Teaching Professor
Division of Infectious Diseases Department of Health Sciences
Department of Medicine The University of Texas Health Science Center at San Antonio
Chicago, Illinois San Antonio, Texas

Sarojini R. Misra, MS, SM(ASCP), SM(AAM) Kalavati Suvarna, PhD


Manager Senior Microbiologist
Microbiology Division of Anti-infective Products, Office of Antimicrobial Products,
Christianacare Health Services Center for Drug Evaluation and Research
Newark, Delaware U.S. Food and Drug Administration
Silver Spring, Maryland
Paula C. Mister, MS, MT(ASCP)SM
Educational Coordinator, Medical Microbiology Kimberly E. Walker, PhD, MT(ASCP)
The Johns Hopkins Hospital Manager, Public Affairs
Baltimore, Maryland American Society for Microbiology
Adjunct Faculty Washington, DC
School of Mathematics and Science
Community Colleges of Baltimore County A. Christian Whelen, PhD, D(ABMM)
Baltimore, Maryland State Laboratories Director
Hawaii Department of Health
Linda S. Monson, MS, MT(ASCP) Pearl City, Hawaii
Supervisory Microbiologist (Retired) Adjunct Professor and Graduate Faculty
San Antonio Military Medical Center Department of Microbiology and Office of Public Health Studies
Fort Sam Houston, Texas University of Hawaii
Honolulu, Hawaii
Sumati Nambiar MD, MPH
Director, Division of Anti-infective Products Nathan P. Wiederhold, PharmD
Center for Drug Evaluation Associate Professor & Director, Fungus Testing Laboratory
Food and Drug Administration Department of Pathology and Laboratory Medicine
Silver Spring, Maryland University of Texas Health Science Center at San Antonio
San Antonio, Texas
Lindsey E. Nielsen, PhD, ASCP(M, MB)
Deputy Chief, Microbiology Laboratories Christopher J. Woolverton, BS, MS, PhD
Department of Pathology and Area Laboratories Professor, Environmental Health Sciences
Brooke Army Medical Center Director, Center for Public Health Partnerships and Practice
San Antonio, Texas College of Public Health
Kent State University
Susan M. Pacheco, MD Kent, Ohio
Physician
Medicine PowerPoint Writer
Edward Hines, Jr. VA Hospital Elizabeth A. Gockel-Blessing, MLS(ASCP)CM
Hines, Illinois Associate Dean for Student and Academic Affairs
Assistant Professor Associate Professor, Department of Clinical Health Sciences
Department of Medicine Doisy College of Health Sciences
Loyola University Medical Center Saint Louis University
Maywood, Illinois St. Louis, Missouri

Gail E. Reid, MD, MSCTS Test Bank Writer


Assistant Professor Lorna Ruskin, EdD, MT(ASCP)
Department of Infectious Diseases Assistant Professor
Loyola University Medical Center Medical Laboratory Sciences
Maywood, Illinois Center for Allied Health Programs
University of Minnesota
Lauren Roberts, MS, MT(ASCP) Minneapolis, Minnesota
Microbiology Supervisor
St. Joseph’s Hospital & Medical Center
Phoenix, Arizona
Contributors ix

Laboratory Manual Writer Review Questions Writer


Jimmy L. Boyd, MS, MHS, MLS(ASCP) Joanna Ellis, MS, BS, MLS(ASCP)
Assistant Professor/Program Director Clinical Assistant Professor
Medical Laboratory Sciences Clinical Coordinator
Arkansas State University-Beebe Clinical Laboratory Science Program
Beebe, Arkansas Texas State University
San Marcos, Texas
Case Studies Writer
Nicholas M. Moore, MS, MLS(ASCP)CM
Assistant Director, Division of Clinical Microbiology
Assistant Professor
Departments of Pathology and Medical Laboratory Science
Rush University Medical Center
Chicago, Illinois
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Preface

T
his is the sixth edition of the Textbook of Diagnostic Although the identification of etiologic agents through culture
Microbiology. Since the inception and publication of the first remains the gold standard in microbiology for determining the
edition, the field of diagnostic microbiology has dramatically probable cause of an infectious disease, advances in molecular
changed and become more complex. Newly recognized pathogens diagnostic techniques and their application in clinical laboratories
continue to plague society in epidemic proportion. As examples, have increased our capabilities for microbial detection and
Ebola is a virus that produced severe outbreaks in West Africa in identification. Extensive biomedical research has focused on
2014–2015. Infection with it is often fatal, if untreated. The primar- nanomedicine—the potential applications of nanotechnology to
ily mosquito-borne Zika virus is linked to microcephaly, a birth medicine. We updated Chapter 11 by expanding the discussion
defect. Infection with it has been declared a global public health on the use of nanomedicine in diagnosing infectious diseases and
emergency by the World Health Organization. Highly pathogenic Chapter 12 by exploring the use and applications of nanotechnology
emerging coronaviruses that affect humans, including Middle in drug-delivery systems. In addition, a description of the applica-
East respiratory syndrome coronavirus (MERS CoV) and severe tion of matrix-assisted laser desorption–ionization time-of-flight
acute respiratory syndrome (SARS) virus, cause life-threatening (MALDI-TOF) mass spectrometry in microbial identification has
respiratory syndromes. This edition includes discussions on these been added to Chapter 11.
emerging public health issues.
As in previous editions, this edition maintains the characteristic
features of a well-designed and organized textbook. We maintain
Organization
the building-block approach to learning, critical thinking, and Part I remains the backbone of the textbook, providing important
problem solving, attributes that students of clinical laboratory background information; Part II focuses on laboratory identification
science and clinical laboratory technology, entry-level clinical of etiologic agents; and Part III on the organ system approach—the
laboratory scientists, and others have found valuable and effective. clinical and laboratory diagnoses of infectious diseases at various
The primary goal of the Textbook of Diagnostic Microbiology body sites.
is to provide a strong foundation for clinical laboratory science Part I presents basic principles and concepts of diagnostic
students, entry-level practitioners, and other health care profes- microbiology, including quality assurance, providing students with
sionals; therefore, discussions on organisms are limited to those a firm theoretic foundation. Chapters 7 (Microscopic Examination
that are medically important and commonly encountered, as well of Materials from Infected Sites) and 8 (Use of Colony Morphology
as new and re-emerging pathogens. The text provides students and for the Presumptive Identification of Microorganisms) still play
other readers with valuable learning tools, such as summary tables, a vital role in this text. These two chapters help students and
flowcharts, and descriptive illustrations, to help them comprehend practitioners who may have difficulty recognizing bacterial
the vast amount of information and reinforce learning. In response morphology on direct smear preparations and colony morphology
to our readers’ needs, we continued our efforts to enhance these on primary culture plates develop these skills with the use of
features that have made this textbook user-friendly. color photomicrographs of stained direct smears and cultures from
In this edition, we made considerable changes to show the vital clinical samples. These two chapters also illustrate how microscopic
nature and ever-evolving field of diagnostic microbiology. A more and colony morphology of organisms can aid in the initial identifica-
in-depth discussion on forensic microbiology has been included in tion of the bacterial isolate. Chapter 9 introduces the student/
Chapter 30, Agents of Bioterror and Forensic Microbiology. The reader to the principles behind various biochemical methods for
text has been updated to reflect pathogens newly recognized in the identification of gram-negative bacteria. This chapter contains
past decade and presents new applications of immunologic and/or several color photographs to help students understand the principles
molecular approaches to diagnose infections, identify infectious and visualize interpretations of these important tests.
agents, and determine antimicrobial resistance in microorganisms. Part II highlights methods for the identification of clinically
Despite the progress made and significant advances that have significant isolates. The chapters in Part II present medically
occurred in their control, prevention, and treatment, infectious important organisms through a taxonomic approach. Although
diseases remain a major threat to human health. The combined diseases caused by the organisms are discussed, the emphasis is
effects of rapid demographic, environmental, societal, technologic, on the characteristics and methods used to isolate and identify
and climatic changes, as well as changes in our way of life, have an each group of organisms. Numerous tables summarize the major
influence on the incidence of infectious disease. The sixth edition features of organisms and use schematic networks to show the
focuses on the continuing spread of infectious diseases and the relationships and differences among similar or closely related
emerging public health issues associated with them. species. Chapters devoted to anaerobic bacterial species, medically

xi
xii Preface

important fungi, parasites, and viruses affirm the significance of the context of the Case in Point at the beginning of the chapter
these agents. Chapter 29 includes a discussion on Zika virus and or case study at the beginning of a section within the chapter.
other viral pathogens, including SARS virus, the highly pathogenic The Case Check highlights a specific point in the text and intends
avian influenza virus, and MERS-CoV. Chapter 31 describes to help the learner connect the dots between the points under
biofilm—an increasingly complex entity. It has become evident discussion, as illustrated by the case study.
that microbial biofilms are involved in the pathogenesis of several To further reinforce learning, identification tables, flowcharts,
human diseases and may be a contributing factor for the failure and featured illustrations have been updated, and new ones have
of antimicrobial therapy. been added. Learning objectives and a list of key terms are also
The organ system approach in Part III has been the foundation provided at the beginning of each chapter. The list of key terms
of the Textbook of Diagnostic Microbiology and provides an includes abbreviations used in the text so that students can easily
opportunity for students and other readers to “pull things together.” find them in the text. At the end of each chapter, readers will find
The chapters in Part III begin with the anatomic considerations “Points to Remember” and “Learning Assessment Questions,”
of the organ system to be discussed and the role of the usual which help reinforce comprehension and understanding of important
microbiota found at a particular site in the pathogenesis of a concepts. Points to Remember includes a bulleted list of important
disease. It is important for students to be knowledgeable about concepts and highlights what the reader should have learned from
the usual inhabitants at a body site before they can appreciate the the chapter.
significance of the opportunistic infectious agents they are most The sixth edition of Textbook of Diagnostic Microbiology
likely to encounter. The case studies included in the chapters in is enriched by the expertise of contributors and elements to
Part III enhance problem-solving and critical-thinking skills and strengthen the learning strategy, such as full-color photographs
help students apply the knowledge they acquired from Parts I and and photomicrographs, an engaging and easy-to-follow design,
II. The case studies describe clinical and laboratory findings, learning assessment questions and answers, opening case scenarios,
providing students with opportunities to correlate these observations hands-on procedures, and lists of key terms to strengthen the
with possible etiologic agents. In most cases, the cause of the learning strategy.
illness is not disclosed in the case study; rather, it is presented
elsewhere in the chapter to give students the opportunity to figure Ancillaries for Instructors
out the explanations independently.
and Students
As in the case of previous editions, we continue to offer a variety
Pedagogic Features of instructor ancillaries specifically geared for this book. For
As in previous editions, the “Case in Point” feature introduces instructors, the Evolve website includes a test bank containing
the reader to an important pathogen, infectious disease, concept, more than 1200 questions. It also includes an electronic image
or principle that is discussed in the chapter text and is used to collection and PowerPoint slides. For students, the Evolve
lead the learner to the main context discussed in the chapter. The website will include a laboratory manual like it always has, but
Case in Point is followed by “Issues to Consider.” These points this edition will include new case studies and student review
are presented in a bulleted format, and learners are asked to think questions.
about them as they read the chapter.
“Case Checks,” a feature introduced in the previous edition, Connie R. Mahon
aims to reinforce understanding of the content or concept within Donald C. Lehman
Acknowledgments

We are grateful to all contributing authors, students, instructors, and many other individuals, who
have all made invaluable suggestions and comments on ways to improve this edition.

Connie R. Mahon
Donald C. Lehman

xiii
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PART I

Introduction to
Clinical Microbiology
CHAPTER

1  
Bacterial Cell Structure, Physiology,
Metabolism, and Genetics
Connie R. Mahon*, George Manuselis

CHAPTER OUTLINE
■ SIGNIFICANCE Common Stains Used for Microscopic Visualization
■ OVERVIEW OF THE MICROBIAL WORLD ■ MICROBIAL GROWTH AND NUTRITION
Bacteria Nutritional Requirements for Growth
Parasites Environmental Factors Influencing Growth
Fungi Bacterial Growth
Viruses ■ BACTERIAL BIOCHEMISTRY AND METABOLISM
■ CLASSIFICATION/TAXONOMY Metabolism
Nomenclature Fermentation and Respiration
Classification by Phenotypic and Genotypic Characteristics Biochemical Pathways from Glucose to Pyruvic Acid
Classification by Cellular Type: Prokaryotes, Eukaryotes, and Anaerobic Utilization of Pyruvic Acid (Fermentation)
Archaea Aerobic Utilization of Pyruvate (Oxidation)
■ COMPARISON OF PROKARYOTIC AND EUKARYOTIC Carbohydrate Utilization and Lactose Fermentation
CELL STRUCTURE ■ BACTERIAL GENETICS
Prokaryotic Cell Structure Anatomy of a DNA and RNA Molecule
Eukaryotic Cell Structure Terminology
Cytoplasmic Structures Genetic Elements and Alterations
■ BACTERIAL MORPHOLOGY Mechanisms of Gene Transfer
Microscopic Shapes
OBJECTIVES
After reading and studying this chapter, you should be able to:
1. Describe microbial classification (taxonomy), and accurately apply 11. Describe the stages in the growth of bacterial cells.
the rules of scientific nomenclature for bacterial names. 12. Explain the importance of understanding microbial metabolism in
2. List and define five methods used by epidemiologists to subdivide clinical microbiology.
bacterial species. 13. Differentiate between fermentation and oxidation (respiration).
3. Differentiate among archaeal, prokaryotic (bacterial), and eukaryotic 14. Name and compare three biochemical pathways that bacteria use to
cell types. convert glucose to pyruvate.
4. Compare and contrast prokaryotic and eukaryotic cytoplasmic and 15. Compare the two types of fermentation that explain positive results
cell wall structures and functions. with the methyl red or Voges-Proskauer tests.
5. Compare and contrast the cell walls of gram-positive and 16. Define the following genetic terms: genotype, phenotype,
gram-negative bacteria. constitutive, inducible, replication, transcription, translation, genome,
6. Compare the acid-fast cell wall with other gram-positive cell walls. chromosome, plasmids, insertion sequence element, transposon,
7. Apply the use of the following stains in the diagnostic microbiology point mutations, frameshift mutations, and recombination.
laboratory: Gram stain, acid-fast stains (Ziehl-Neelsen, Kinyoun, 17. Discuss the development and transfer of antimicrobial resistance in
auramine-rhodamine), acridine orange, methylene blue, calcofluor bacteria.
white, lactophenol cotton blue, and India ink. 18. Differentiate among the mechanisms of transformation, transduction,
8. List the nutritional and environmental requirements for bacterial and conjugation in the transfer of genetic material from one
growth and define the categories of media used for culturing bacterium to another.
bacteria in the laboratory. 19. Define the terms bacteriophage, lytic phage, lysogeny, and
9. Define the atmospheric requirements of obligate aerobes, temperate phage.
microaerophiles, facultative anaerobes, obligate anaerobes, and 20. Define the term restriction endonuclease enzyme, and explain the
capnophilic bacteria. use of such enzymes in the clinical microbiology laboratory.
10. Define aerotolerant anaerobe.

*My comments are my own and do not represent the view of Health Resources
and Services Administration of the Department of Heath and Human Services.

2
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 3

T
Case in Point his chapter provides a review of the structure, physiology,
metabolism, and genetics of prokaryotic and eukaryotic
A 4-year-old girl presents with symptoms of redness, burning, cells. It also gives examples of common stains used to
and light sensitivity in both eyes. She also complained of her visualize microorganisms microscopically. Each topic in this chapter
eyelids sticking together because of exudative discharge. A Gram emphasizes to clinical microbiologists the inherent importance
stain of the conjunctival exudates (product of acute inflammation of their efforts to culture, identify, and characterize the microbes
with white blood cells and fluid) showed gram-positive intracellular that cause disease in humans.
and extracellular, faint-staining, coccobacillary bacteria. The
organisms appeared to have small, clear, nonstaining “halos”
surrounding each cell. This clear area was noted to be between Significance
the stained organism and the amorphous (no definite form;
shapeless) background material. The Gram stain of the quality
Microbial inhabitants have evolved to survive in various ecologic
control organisms Staphylococcus aureus (gram-positive) and niches (place or location) and habitats (organism’s location and
Escherichia coli (gram-negative) revealed gram-positive reactions where its resources may be found). Some grow rapidly and
for both organisms. some grow slowly. Some can replicate with a minimal number
of nutrients present, whereas others require enriched nutrients
to survive. Variation exists in atmospheric growth conditions,
Issues to Consider temperature requirements, and cell structure. This diversity is
After reading the patient’s case history, consider: also found in the microorganisms that inhabit the human body
■ Role of microscopic morphology in presumptive identification as normal biota (flora), as opportunistic pathogens, or as true
■ Significance of observable cellular structures pathogens. Each microbe has its own physiology and meta-
■ Importance of quality control in assessing and interpreting bolic pathways that allow it to survive in its particular habitat.
direct smear results One of the main roles of a diagnostic or clinical microbiolo-
■ Unique characteristics of organisms, such as cellular gist is to isolate, identify, and analyze the bacteria that cause
structures, in initiating infection and disease in hosts disease in humans. Knowledge of microbial structure and
physiology is extremely important to clinical microbiologists in
three areas:
Key Terms • Culture of organisms from patient specimens
Acid-fast Microaerophilic • Characterization and identification of organisms after they have
Aerotolerant anaerobes Minimal medium been isolated
Anticodon Mycelia • Prediction and interpretation of antimicrobial susceptibility
Archaea Nomenclature patterns
Autotrophs Nutrient media Understanding the growth requirements of a particular bacte-
Bacteria Obligate aerobes rium enables the microbiologist to select the correct medium for
Bacteriophage Obligate anaerobes primary culture and optimize the chance of isolating the pathogen.
Capnophilic Pathogenic bacteria Determination of staining characteristics, based on differences
Capsule Phenotype in cell wall structure, is the first step in bacterial identification.
Codon Phyla Microscopic characterization is followed by observation of the
Competent Pili metabolic biochemical differences among organisms that form the
Conjugation Plasmids basis for most bacterial identification systems in use today. Recently
Differential media Pleomorphic advances in molecular biology methods, for example, nucleic
Dimorphic Prokaryotes acid amplification and matrix-assisted laser desorption/ionization,
Eukarya Protein expression have shifted identification away from biochemical testing. The
Eukaryotes Psychrophiles cell structure and biochemical pathways of an organism also
Facultative anaerobes Respiration (oxidation) determine its susceptibility to various antimicrobial agents. The
Family Restriction enzymes ability of microorganisms to change rapidly, acquire new genes,
Fermentation Selective media
and undergo mutations presents continual challenges to clinical
Fimbriae Species
microbiologists as they isolate and characterize the microorganisms
Flagella Spores
associated with humans.
Fusiform Strains
Genotype Taxa
Genus Taxonomy Overview of the Microbial World
Gram-negative Temperate
Gram-positive Thermophiles The study of microorganisms by the Dutch biologist and lens
Halophiles Transcription maker Anton van Leeuwenhoek has evolved immensely from its
Heterotrophs Transduction early historic beginnings. Because of Leeuwenhoek’s discovery
Hyphae Transformation of what he affectionately called wee beasties and animalcules
Krebs cycle Translation in a water droplet in his homemade microscope, the scientific
Lysogeny Transport medium community acknowledged him as the “father of protozoology and
Mesophiles Virion bacteriology.” Today, we know that there are enormous numbers
of microbes in, on, and around us in our environment. Many of
4 PART 1  Introduction to Clinical Microbiology

these microbes do not cause disease. The focus of this textbook through ingestion. Some are capable of locomotion (motile), whereas
is on microbes that are associated with human disease. others are nonmotile. They are categorized by their locomotive
structures: flagella (Latin: whiplike), pseudopodia (Greek: false
Bacteria feet), or cilia (Latin: eyelash). Many multicellular parasites (e.g.,
Bacteria are unicellular organisms that lack a nuclear membrane tapeworms) may be 7 to 10 meters long (see Chapter 28).
and true nucleus. They are classified as prokaryotes (Greek: before
kernel [nucleus]) and lack mitochondria, endoplasmic reticulum Fungi
(ER), or Golgi bodies. The absence of the preceding bacterial cell Fungi are heterotrophic eukaryotes that obtain nutrients through
structures differentiates them from eukaryotes (Greek eu: well or absorption. Yeasts are unicellular fungi that reproduce asexually.
good; Greek karyon: kernel). Table 1.1 compares prokaryotic and “True” yeasts do not form hyphae or mycelia. Most fungi are
eukaryotic cell organization; Fig. 1.1 shows both types of cells. multicellular, and many can reproduce sexually and asexually.
Multicellular fungi are composed of filaments called hyphae that
Parasites interweave to form mats called mycelia. Molds are filamentous
Certain eukaryotic parasites (organisms that live at the expense forms that can reproduce asexually and sexually. Certain fungi
of their hosts) exist as unicellular organisms of microscopic size, can assume both morphologies (yeast and hyphae/mycelial
whereas others are multicellular organisms. Protozoa are unicellular forms), growing as yeast at human temperature (37° C) and as
organisms within the kingdom Protista that obtain their nutrition the filamentous form at room temperature (22° C). These fungi

TABLE 1.1  Comparison of Prokaryotic and Eukaryotic Cell Organization

Characteristic Prokaryote Eukaryote

Typical size 0.4–2 µm in diameter 10–100 µm in diameter


0.5–5 µm in length >10 µm in length
Nucleus No nuclear membrane; nucleoid region of the Classic membrane-bound nucleus
cytosol
Genome
Location In the nucleoid, at the mesosome In the nucleus
Chromosomal DNA Circular; complexed with RNA Linear; complexed with basic histones and other
proteins
Genome: extrachromosomal Plasmids, small circular molecule of DNA In mitochondria and chloroplasts
circular DNA containing accessory information; most
commonly found in gram-negative
bacteria; each carries genes for its own
replication; can confer resistance to
antibiotics
Reproduction Asexual (binary fission) Sexual and asexual
Membrane-bound organelles Absent All
Golgi bodies Absent in all Present in some
Lysosomes Absent in all Present in some; contain hydrolytic enzymes
Endoplasmic reticulum Absent in all Present in all; lipid synthesis, transport
Mitochondria Absent in all Present in most
Nucleus Absent in all Present in all
Chloroplasts for photosynthesis Absent in all Present in algae and plants
Ribosomes: site of protein synthesis Present in all Present in all
(nonmembranous)
Size 70S consisting of 50S and 30S subunits 80S consisting of 60S and 40S subunits
Electron transport for energy In the cell membrane; no mitochondria In the inner membrane of mitochondria and
present chloroplasts
Sterols in cytoplasmic membrane Absent except in Mycoplasmataceae Present
Plasma membrane Lacks carbohydrates Also contains glycolipids and glycoproteins
Cell wall, if present Peptidoglycan in most bacteria Cellulose, phenolic polymers, lignin (plants),
chitin (fungi), other glycans (algae)
Glycocalyx Present in most as an organized capsule or Present; some animal cells
unorganized slime layer
Cilia Absent Present; see description of flagella
Flagella, if present Simple flagella; composed of polymers of Complex cilia or flagella; composed of MTs and
flagellin; movement by rotary action at the polymers of tubulin with dynein connecting
base; spirochetes have MTs MTs; movement by coordinated sliding
microtubules
Pili and fimbriae Present Absent

MT, Microtubule.
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 5

Division septum
Outer membrane
Peptidoglycan Mesosome (Pili) (Capsule)
(Capsule) layer Cytoplasmic
Inclusion membrane
body Inclusion
body
Peptidoglycan
layer

Porin
Cytoplasmic proteins
membrane Ribosome Periplasmic
Ribosome
(Flagellum) Surface proteins Chromosome space
(Flagellum)

A GRAM-POSITIVE GRAM-NEGATIVE

Centrosome Ribosomes
Centrioles
Smooth
endoplasmic Mitochondria
reticulum Smooth endoplasmic
reticulum
Cilia
Mitochondrion
Lysosome
Rough
endoplasmic Free
reticulum ribosomes
Peroxisome Golgi
apparatus

Vesicle
B Nuclear Nucleus Nucleolus
envelope
FIG. 1.1  Comparison of prokaryotic and eukaryotic cell organization and structures. A, Prokary-
otic gram-positive and gram-negative bacteria. B, Structure of the generalized eukaryotic cell.
(A, From Murray PR, Rosenthal KS, Pfaller MA: Medical microbiology, ed 6, Philadelphia, 2009,
Mosby; B, from Thibodeau GA, Patton KT: Anatomy and physiology, ed 6, St Louis, 2007, Mosby.)

are called dimorphic. Some systemic fungal diseases in human and metabolism. Because they lack enzymes, ribosomes, and
hosts are caused by dimorphic fungi (see Chapter 27). other metabolites, they “take over” host cell function using
the host cell machinery to reproduce. Growth (increase in size)
Viruses does not occur in viruses.
Viruses are the smallest infectious particles and cannot be seen Viruses are mostly host or host cell specific. For example,
under an ordinary light microscope. Often, we can see their effects human immunodeficiency virus infects T-helper lymphocytes,
on cell lines grown in the laboratory, such as inclusions, rounding not muscle cells, in humans, whereas other viruses, such as the
up of cells, and syncytium (fusion of host cells into multinucleated rabies virus, can infect dogs, skunks, bats, and humans. A virus
infected forms), where these characteristics become diagnostic that infects and possibly destroys bacterial cells is known as a
for many viral diseases. Viruses are neither prokaryotic nor bacteriophage (Greek phage: to eat). Viruses are classified and
eukaryotic They are distinguished from living cells by the following identified by their genome (DNA or RNA), host disease signs and
characteristics: symptoms, chemical makeup, and geographic distribution, the
• Viruses consist of deoxyribonucleic acid (DNA) or ribonucleic presence or absence of an envelope, their resistance to changes in
acid (RNA) but rarely both. Their genome may be double-stranded pH and temperature, their antigenicity (serologic methods), how the
DNA (dsDNA), single-stranded DNA (ssDNA), double-stranded virus replicates, and the virion (a complete virus outside a cell).
RNA (dsRNA), or single-stranded RNA (ssRNA).
• Viruses are acellular (not composed of cells), lack cytoplasmic
membranes, and are surrounded by a protein coat.
Classification/Taxonomy
• Viruses are obligate intracellular parasites that cannot self- Taxonomy (Greek taxes: arrangement; Greek nomos: law) is the
replicate. They require host cells for replication (increase in orderly classification and grouping of organisms into taxa (catego-
number does not involve mitosis, meiosis, or binary fission) ries). Taxonomy involves three structured, interrelated categories:
6 PART 1  Introduction to Clinical Microbiology

classification/taxonomy, nomenclature, and identification. It is based by use of the first letter (capitalized) of the genus followed by a
on similarities and differences in genotype (genetic makeup of period and the species epithet (e.g., S. aureus). The genus name
an organism, or combinations of forms of one or a few genes in followed by the word species (e.g., Staphylococcus species) may
an organism’s genome) and phenotype (observable physical and be used to refer to the genus as a whole. Species are abbreviated
functional features of an organism expressed by its genotype). “sp.” (singular) or “spp.” (plural) when the species is not specified.
Examples of genotypic characteristics include base sequencing When bacteria are referred to as a group, their names are neither
of DNA or RNA and DNA base composition ratio to measure the capitalized nor underlined (e.g., staphylococci).
degree of relatedness of two organisms (see later in this chapter
and Chapter 11). Examples of phenotypic characteristics include Classification by Phenotypic and
macroscopic (colony morphology on media) and microscopic (size, Genotypic Characteristics
shape, arrangement into groups or chains of cells) morphology, The traditional method of placing an organism into a particular
staining characteristics (gram-positive or gram-negative), nutritional genus and species is based on the similarity of all members in
requirements, physiologic and biochemical characteristics, antigenic numerous phenotypic characteristics. In the diagnostic microbiol-
markers, and susceptibility or resistance to antimicrobial agents ogy laboratory, this classification is accomplished by testing each
or chemicals. See Chapters 7, 8, 9, 12, and 13 for more detailed bacterial culture for various metabolic or molecular characteristics
information. and comparing the results with those listed in established tables
Taxa (plural of taxon), for example, the levels of classification, or databases. In many rapid identification systems, a numeric
are the categories or subsets in taxonomy. The formal levels of taxonomy is used in which phenotypic characteristics are assigned
bacterial classification in successively smaller taxa or subsets are a numeric value and the derived number indicates the genus and
domain, kingdom, division (or phylum in kingdom Animalia), species of the bacterium.
class, order, family, tribe, genus, species, and subspecies. Below Epidemiologists constantly seek means of further subdividing
the subspecies level, designations such as serotype or biotype may bacterial species to follow the spread of bacterial infections. Species
be given to organisms that share specific minor characteristics. may be subdivided into subspecies (abbreviated “subsp.”), on
Protists (protozoans) of clinical importance are named similarly to the basis of phenotypic differences; serovarieties (abbreviated
animals; instead of divisions, one uses phyla (plural of phylum), “serovar”), on the basis of serologic differences; or biovarieties
but the names of the others remain the same. Prokaryotes are (abbreviated “biovar”), on the basis of biochemical test result
placed in the domains Bacteria and Archaea (Greek: ancient, differences. Phage typing (based on susceptibility to specific
origin from the earliest cells), separate from the animals; plants bacterial phages) has also been used for this purpose. Current
and protists are placed in the domain Eukarya. The domains technology has allowed the analysis of genetic relatedness (DNA
Bacteria and Archaea include unicellular prokaryotic organisms. and RNA structure and homology) for taxonomic purposes. The
Clinical microbiologists traditionally emphasize placement analysis of ribosomal RNA (rRNA) gene sequencing has proved
and naming of bacterial species into three (occasionally four or particularly useful for this purpose. The information obtained
five) categories: the family (similar to a human “clan”), a genus from these studies resulted in the reclassification of some bacteria.
(equivalent to a human last name), and a species (equivalent to
a human first name). The plural of genus is genera. For example, Classification by Cellular Type:
there are many genera in the family Enterobacteriaceae. The Prokaryotes, Eukaryotes, and Archaea
proper word for the name of a species is an epithet. Although Another method of classifying organisms is by cell organization.
order and tribe may be useful for the classification of plants and Organisms fall into three distinct groups based on type of cell
animals, these taxa are not always used for the classification of organization and function: prokaryotes, eukaryotes, and archaea.
bacteria. For example, Staphylococcus (genus) aureus (species Taxonomists have placed all organisms into three domains that
epithet) belongs to the family Staphylococcaceae. In addition, have replaced some kingdoms: Bacteria, Archaea, and Eukarya.
there are usually different strains within a given species of the These three domains are the largest and most inclusive taxa. Each
same species. For example, there are many different strains of domain is divided into kingdoms on the basis of the similarities
S. aureus. If the S. aureus isolated from one patient is resistant of RNA, DNA, and protein sequences. The group prokaryotes
to penicillin and another S. aureus from a different patient is includes the domains Archaea and Bacteria (Eubacteria), whereas
susceptible to penicillin, the two isolates are considered to be fungi, algae, protozoa, animals, and plants are eukaryotic in nature
different strains of the same species. For an additional example, see and are placed in the domain Eukarya.
Corynebacterium diphtheriae in the section on transduction later in The domain Archaea (formerly Archaeobacteria) cell type
this chapter. appears to be more closely related to eukaryotic cells than to
prokaryotic cells and is found in microorganisms that grow under
Nomenclature extreme environmental conditions. Archaeal cell walls lack
Nomenclature provides naming assignments for each organism peptidoglycan, a major reason they are placed in a domain separate
in this textbook. The following standard rules for denoting bacterial from bacteria. These microbes share some common characteristics
names are used. The family name is capitalized and has an “-aceae” with bacteria; they too can stain gram-positive or gram-negative.
ending (e.g., Micrococcaceae). The genus name is capitalized and Gram-positive archaea have a thick wall and stain purple. Gram-
followed by the species epithet, which begins with a lowercase negative archaeal cells, in contrast to the typical gram-negative
letter; both the genus and the species should be italicized in print bacterial lipid membrane, have a layer of protein covering the
but underlined when written in script (e.g., Staphylococcus aureus cell wall and stain pink. See the Gram stain discussion later in
or Staphylococcus aureus). Often the genus name is abbreviated this chapter.
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 7

The structure of the cell envelope and enzymes of archaea makes them highly resistant to chemical agents, temperature
allows them to survive under stressful or extreme (extremophiles; change, starvation, dehydration, ultraviolet and gamma radia-
lovers of the extreme) conditions. Examples include halophiles tion, and desiccation. Under harsh conditions, each vegetative
(salt-loving cells) in Utah’s Great Salt Lake, thermophiles cell (active, capable of growing and dividing) produces inter-
(heat-loving cells) in hot springs and deep ocean vents, and the nally one endospore (inactive) that germinates under favorable
anaerobic methanogens that give off swamp gas and inhabit the environmental conditions into one vegetative cell. Endospores
intestinal tracts of animals. Because archaea are not encountered should not be confused with the reproductive spores of fungi (see
in clinical microbiology, they are not discussed further in this Chapter 27).
textbook. Spores appear as highly refractile bodies in the cell. Spores
In general, the interior organization of eukaryotic cells is more are visualized microscopically as unstained areas in a cell with
complex than that of prokaryotic cells (see Fig. 1.1). The eukaryotic the use of traditional bacterial stains (Gram) or with the use of
cell is usually larger and contains membrane-encased organelles specific spore stains. Schaeffer-Fulton stain is the most commonly
(“little organs”) or compartments that serve specific functions, used endospore stain. The size, shape, and interior location of the
whereas the prokaryotic cell is noncompartmentalized. Various spore, for example, at one end (terminal), subterminal, or central,
structures are unique to prokaryotic cells (see Fig. 1.1). Differences can be used as identifying characteristics. For instance, the terminal
also exist in the processes of DNA synthesis, protein synthesis, spore of Clostridium tetani, the etiologic (causative) agent of
and cell wall synthesis and structure. Table 1.1 compares the tetanus, gives the organism a characteristic tennis racquet–shaped
major characteristics of eukaryotic and prokaryotic cells. or lollipop-shaped appearance.
Pathogenic (disease-causing) bacteria are prokaryotic cells
that infect eukaryotic hosts. Targeting antimicrobial action against Cell Envelope Structures
unique prokaryotic structures and functions inhibits bacterial growth The cell envelope consists of the membrane and structures sur-
without harming eukaryotic host cells. This is one reason that rounding the cytoplasm. In bacteria, these are the plasma membrane
pharmaceutical companies have been successful in developing and the cell wall. Some species also produce capsules and slime
effective antimicrobial agents against bacterial pathogens, but layers.
they have been less successful in finding drugs effective against Plasma (Cell) Membrane. The plasma membrane is a
parasites and fungi, which are eukaryotic and similar to their phospholipid bilayer with embedded proteins that surrounds the
human hosts, and viruses, which use host cells for replication. cytoplasm. The prokaryotic plasma membrane (except for those
of members of the Mycoplasmataceae, which do contain sterols)
Comparison of Prokaryotic and is made of phospholipids and proteins and does not contain sterols.
Eukaryotic Cell Structure This is in contrast to eukaryotic plasma membranes, which do
contain sterols. The plasma membrane acts as an osmotic barrier
Prokaryotic Cell Structure (prokaryotes have a high osmotic pressure inside the cell) and is
Cytoplasmic Structures the location of the electron transport chain, where energy is
Bacteria do not contain a membrane-bound nucleus. Their genome generated. The general functions of the prokaryotic plasma
consists of a single circular chromosome. This appears as a diffuse membrane are identical to functions in eukaryotes (Fig. 1.2).
nucleoid or chromatin body (nuclear body) that is attached to a Cell Wall.  The cell wall of prokaryotes is a rigid structure that
mesosome, a saclike structure in the cell membrane. maintains the shape of the cell and prevents bursting of the cell
Bacterial ribosomes, consisting of RNA and protein, are found from the high osmotic pressure inside it. The different types of
free in the cytoplasm and attached to the cytoplasmic membrane. cell walls in bacteria have traditionally been categorized according
They are the site of protein synthesis. They are 70S in size and to their staining characteristics. The two major types of cell walls
dissociate into two subunits: 50S and 30S (see Table 1.1). The S are gram-positive and gram-negative (see Fig. 1.1A). Although
stands for Svedberg units, which refer to sedimentation rates (unit they stain poorly gram-positive, mycobacteria have a modified
of time) during high-speed centrifugation. The Svedberg unit is cell wall called an acid-fast cell wall, while mycoplasmas are
named for Theodor Svedberg, Nobel Prize winner and inventor bacteria that have no cell wall and therefore do not Gram stain.
of the ultracentrifuge. Larger particles have higher S values. The Gram-Positive Cell Wall. The gram-positive cell wall is
S value is not additive. When the previously mentioned two subunits composed of a very thick protective peptidoglycan (murein) layer.
50S and 30S bind together, there is a loss of surface area and the Because the peptidoglycan layer is the principal component of
two subunits produce a complex 70S in size. The same occurs in the gram-positive cell wall, many antimicrobial agents are effective
the eukaryotic cell, where the two subunits 60S and 40S combine against gram-positive organisms (e.g., penicillin) by preventing
to form an 80S complex. synthesis of peptidoglycan. Gram-negative bacteria, which have
Stained bacteria sometimes reveal the presence of granules in a thinner layer of peptidoglycan and a different cell wall structure,
the cytoplasm (cytoplasmic granules). These granules are storage are less affected by these agents.
deposits and may consist of polysaccharides such as glycogen, The peptidoglycan or murein layer consists of glycan (polysac-
lipids such as poly β-hydroxybutyrate, or polyphosphates. charide) chains of alternating N-acetyl-D-glucosamine (NAG) and
Certain genera, such as Bacillus and Clostridium, produce N-acetyl-D-muramic acid (NAM) (Fig. 1.3). Short peptides, each
endospores in response to harsh environmental conditions. consisting of four amino acid residues, are attached to a carboxyl
Endospores are small, dormant (inactive), asexual spores that group on each NAM residue. The chains are then cross-linked to
develop inside the bacterial cell as a means of survival. Endo- form a thick network via a peptide bridge (differing in number of
spores are not a means of reproduction. Their thick protein coat peptides) connected to the tetrapeptides on the NAM.
8 PART 1  Introduction to Clinical Microbiology

Carbohydrate chains

External
Glycolipid
membrane surface
Polar region
of phospholipid

Phospholipid
bilayer

Internal Cholesterol Protein Nonpolar region


membrane surface of phospholipid
Glycoprotein
Membrane
channel protein

FIG. 1.2  Structure of the plasma membrane. (From Thibodeau GA, Patton KT: Anatomy and
physiology, ed 6, St Louis, 2007, Mosby.)

CH2OH
(NAG) O

CH2OH OH
O O
(NAM) NH
CH2OH C O CH2OH
O CH3 (NAM) O O
(NAG) O
NH
OH C O CH2OH OH
O CH3 (NAG) O O
NH NH
HC CH3 OH C O
C O C O O CH3
CH3 L-Alanine NH HC CH3
D-Glutamate C O C O
Meso-diaminopimelate CH3 L-Alanine
D-Alanine
D-Glutamate
Meso-diaminopimelate
D-Alanine

FIG. 1.3  The structure of the peptidoglycan layer in the cell wall of Escherichia coli. The amino
acids in the cross-linking tetrapeptides may differ among species. NAG, N-acetyl-D-glucosamine;
NAM, N-acetyl-D-muramic acid. (From Neidhardt FC, Ingraham M, Schaechter M: Physiology of
bacterial cell: a molecular approach, Sunderland, MA, 1990, Sinauer Associates.)

Other components of the gram-positive cell wall that penetrate fever and shock conditions in patients infected with gram-negative
to the exterior of the cell are teichoic acid (anchored to the bacteria. The outer membrane functions in the following ways:
peptidoglycan) and lipoteichoic acid (anchored to the plasma • It acts as a barrier to hydrophobic compounds and harmful
membrane). These two components are unique to the gram-positive substances.
cell wall. Other antigenic polysaccharides may be present on the • It acts as a sieve, allowing water-soluble molecules to enter
surface of the peptidoglycan layer. through protein-lined channels called porins.
Gram-Negative Cell Wall.  The cell wall of gram-negative • It provides attachment sites that enhance attachment to host
bacteria comprises two layers: the inner peptidoglycan layer, which cells.
is much thinner than in gram-positive cell walls, and an additional Between the outer membrane and the inner membrane and
outer membrane unique to the gram-negative cell wall. The outer encompassing the thin peptidoglycan layer is an area referred to
membrane contains proteins, phospholipids, and lipopolysaccha- as the periplasmic space. Within the periplasmic space is a gel-like
ride (LPS). LPS contains three regions: an antigenic O–specific matrix containing nutrient-binding proteins and degradative and
polysaccharide, a core polysaccharide, and an inner lipid A (also detoxifying enzymes. The periplasmic space is absent in gram-
called endotoxin). The lipid A moiety is responsible for producing positive bacteria.
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 9

Slime layers or a glycocalyx are similar to capsules but are


Case Check 1.1 more diffuse layers surrounding the cell. They also are made of
The differential ability of the Gram stain makes it useful in classifying a polysaccharides and serve either to inhibit phagocytosis or, in
bacterium as gram-positive or gram-negative. As in the Case in Point at some cases, to aid in adherence to host tissue or synthetic implants.
the beginning of the chapter, correct interpretation and assessment of the
Glycocalyx production can be the first step in the formation of a
Gram-stained smear results are critical in the presumptive identification
of the organism present. See also Procedure 9 in Appendix C. The use of
biofilm; see Chapter 31.
quality control organisms with known Gram stain reactions ensures that
the Gram stain procedure is performed correctly. Bacteria with thick cell Case Check 1.2
walls containing teichoic acid retain the crystal violet–iodine dye complex
after decolorization and appear deep blue; they are gram-positive (e.g., The most common mechanism for evading phagocytosis used by many
S. aureus). Bacteria with thinner walls containing lipopolysaccharides do microorganisms is having a polysaccharide capsule on the surface.
not retain the dye complex; they are gram-negative (e.g., E. coli). The Microorganisms possessing a capsule are generally highly virulent (as in
alcohol-acetone decolorizer damages these thin lipid walls and allows the the Case in Point at the beginning of the chapter) until removal of the
stain complex to wash out. All unstained elements, such as Gram-negative capsule, at which point virulence becomes extremely low. Encapsulated
bacteria and products of inflammation, are subsequently counterstained strains of S. pneumoniae and H. influenzae are associated with highly
red by safranin dye. invasive infections and are known to be more virulent than nonencapsulated
strains. See also the section on ability to resist phagocytosis in Chapter
2. Antibodies produced against the capsule often lead to phagocytosis
and immunity against that bacterial strain.
Acid-Fast Cell Wall.  Certain genera, such as Mycobacterium
and Nocardia, have a gram-positive cell wall structure that also
contain a waxy layer of glycolipids and fatty acids (mycolic acid)
bound to the exterior of the cell wall. More than 60% of the cell Cell Appendages.  The flagellum is the organ of locomotion.
wall is lipid, and the major lipid component is mycolic acid, Flagella are exterior protein filaments that rotate and cause bacteria
which is a strong hydrophobic molecule that forms a lipid shell to be motile. Bacterial species differ in their possession of flagella
around the organism and affects its permeability. This makes from none (nonmotile) to many (Fig. 1.4). Flagella that extend
Mycobacterium spp. difficult to stain with the Gram stain. Myco- from one end of the bacterial cell are polar. Polar flagella may
bacteria are best stained with an acid-fast stain, in which the occur singly at one end (monotrichous) or both ends (amphitri-
bacteria are stained with carbolfuchsin, followed by acid-alcohol chous) or multiply in tufts at one end termed lophotrichous. Flagella
as a decolorizer. Other bacteria are decolorized by acid-alcohol, that occur all around the bacterium are peritrichous. The number
whereas mycobacteria and nocardiae retain the stain. Therefore and arrangement of flagella are sometimes used for identification
these bacteria have been designated acid-fast bacteria. The purposes. Flagella can be visualized microscopically with special
nocardiae are generally considered partially acid-fast and can flagellum stains.
more easily be decolorized with the acid-fast stain. In addition, Pili (plural of pilus) and fimbriae (plural of fimbria) are
the norcardiae will appear a darker blue in the Gram stain compared nonflagellar, proteinaceous, hairlike appendages that adhere some
with the faint blue for the mycobacteria. bacterial cells to one another and to host cells. Conjugation pili
Absence of Cell Wall.  Prokaryotes that belong to the genera are protein tubes that connect two bacterial cells and mediate
Acholeplasma, Mycoplasma and Ureaplasma are unique in that DNA exchange.
they lack a cell wall and contain sterols in their plasma membranes.
Because they lack the rigidity of the cell wall, they are seen in Eukaryotic Cell Structure
various shapes microscopically referred to as being pleomorphic. The following structures are associated with eukaryotic cells (see
Some gram-positive and gram-negative cells can lose their cell Table 1.1 and Fig. 1.1). In the diagnostic microbiology laboratory,
walls and grow as L-forms in media supplemented with serum the eukaryotic cell type occurs in medically important fungi and
or sugar to prevent osmotic rupture of the cell membrane. in parasites.

Surface Polymers
Various pathogenic bacteria produce a discrete organized covering
termed a capsule. Capsules are usually made of polysaccharide
polymers, although they may also be made of polypeptides. Lophotrichous
Capsules act as virulence factors in helping the pathogen evade
phagocytosis. During identification of certain bacteria by serologic
typing, capsules sometimes must be removed to detect the somatic Polar
(cell wall) antigens present underneath them. Capsule removal
is accomplished by boiling a suspension of the microorganism.
Salmonella Typhi must have its capsular (Vi) antigen removed for
the laboratory scientist to observe agglutination with Salmonella
somatic (O) antisera. The capsule does not ordinarily stain with Peritrichous
use of common laboratory stains, such as Gram stain or India
ink. Instead, it appears as a clear area (“halo-like”) between or
surrounding the stained organism and the stained amorphous FIG. 1.4  Three flagellar arrangements that occur in bacteria.
background material in a direct smear from a clinical specimen. Other variations can occur.
10 PART 1  Introduction to Clinical Microbiology

The basal body, or kinetosome, is a small structure located at the


Cytoplasmic Structures base of cilia or flagella, where microtubule proteins involved in
The nucleus of the eukaryotic cell contains the DNA of the cell movement originate.
in the form of discrete chromosomes (structures in the nucleus
that carry genetic information; the genes). They are covered with Bacterial Morphology
basic proteins called histones. The number of chromosomes in
the nucleus differs according to the particular organism. A rounded, Microscopic Shapes
refractile body called a nucleolus is also located within the nucleus. The largest bacterium known, Thiomargarita namibiensis, is found
The nucleolus is the site of rRNA synthesis. The nucleus is bounded in ocean sediment and generally has a diameter of 0.1 to 0.3 mm.
by a bilayered lipoprotein nuclear membrane. Most bacteria range in size from 0.4 to 2 µm. They occur in three
The ER is a system of membranes that occur throughout the basic shapes (Fig. 1.5):
cytoplasm. It is found in two forms. The “rough” ER is covered • Cocci (spherical)
with ribosomes, the site of protein synthesis. The ribosomes give • Bacilli (rod-shaped)
the ER the rough appearance. The smooth ER does not have • Spirochetes (spiral)
ribosomes on the outer surface of its membrane—hence the smooth Individual bacteria may form characteristic groupings. Cocci
appearance. Smooth ER does not synthesize proteins, but it does (plural of coccus) may occur singly, in pairs (diplococci), in chains
synthesize phospholipids. The major function of the Golgi apparatus (streptococci), or in clusters (staphylococci). Bacilli (plural of
or complex is to modify and package proteins sent to it by the bacillus) vary greatly in size and length from very short coccobacilli
rough ER, depending on the protein’s final destination. to long filamentous rods. The ends may be square or rounded.
Eukaryotic ribosomes, where protein synthesis occurs, are Bacilli with tapered, pointed ends are termed fusiform. Some
80S in size and dissociate into two subunits: 60S and 40S. They bacilli are curved. When a species differs in size and shape within
are attached to the rough ER. Eukaryotic cells contain several a pure culture, the bacterium is termed pleomorphic. Bacilli may
membrane-enclosed organelles. Mitochondria are the main site of occur as single rods or in chains or may align themselves side
energy production. They contain DNA and the electron transport
system that produces energy in the form of adenosine triphosphate
(ATP). Lysosomes contain hydrolytic enzymes for degradation of
macromolecules and microorganisms within the cell. Peroxisomes Microscopic Morphology of Bacteria
contain protective enzymes that break down hydrogen peroxide
and other peroxides generated within the cell. Chloroplasts, found COCCI
in plant cells, are the sites of photosynthesis. Chloroplasts are the
sites where light energy is converted into chemical energy (ATP). In clusters
Photosynthesis produces oxygen from carbon dioxide and water.
Fungi are not plants and have no chloroplasts.
In chains
Cell Envelope Structures
Plasma Membrane.  The plasma membrane (see Fig. 1.2)
In pairs
is a phospholipid bilayer with embedded proteins that envelops
the cytoplasm and regulates transport of macromolecules into and
out of the cell. A substantial amount of cholesterol is found in In tetrads
the plasma membrane of animals. Cholesterol has a stabilizing
effect and helps keep the membrane fluid. The polar heads of the
phospholipids are hydrophilic (water loving) and lie on both the BACILLI
intracellular and the extracellular fluids; their nonpolar tails are
hydrophobic (water hating) and avoid water by lining up in the
Coccobacilli
center of the plasma membrane “tail to tail.” This type of hydro-
phobic makeup of the interior of the plasma membrane makes it
potentially impermeable to water-soluble molecules. Proteins Bacilli of various sizes
perform several important functions of the membrane. They may
act as enzymes, hormone receptors, pore channels, and carriers.
Cell Wall.  The function of a cell wall is to provide rigidity Fusiform bacilli
and strength to the exterior of the cell. Most eukaryotic cells do
not have cell walls. However, fungi have cell walls principally
made of polysaccharides, such as chitin, mannan, and glucan. Palisading
Chitin is a distinct component of fungal cell walls.
Motility Organelles.  Cilia are short projections (3 to 10 µm),
usually numerous, that extend from the cell surface and are used SPIROCHETES
for locomotion. They are found in certain protozoa and in ciliated
epithelial cells of the respiratory tract. Flagella are longer projec-
tions (>150 µm) used for locomotion by cells such as spermatozoa. FIG. 1.5  The microscopic shapes and arrangements of bacteria.
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 11

by side (palisades). Spirochetes vary in length and in the number each step is important. The bacteria are initially stained purple
of helical turns (not all helical bacteria are called spirochetes). by the crystal violet that is bound to the cell wall with the aid of
iodine. When the decolorizer is applied to bacteria with a gram-
Common Stains Used for negative cell wall, the crystal violet washes out of the cells, which
Microscopic Visualization then take up the pink counterstain, safranin. For this reason,
Stains that impart color or fluorescence are needed to visualize gram-negative bacteria appear pink under the light microscope.
bacteria under the microscope. The microscopic staining charac- Bacteria with a gram-positive cell wall retain the primary crystal
teristics, shapes, and groupings are used in the classification of violet stain during the decolorizing treatment and appear purple.
microorganisms (Fig. 1.6). Cells in a direct smear from a patient specimen, such as epithelial
cells, white blood cells, red blood cells, and amorphous background
Gram Stain material, should appear pink (gram-negative) if the Gram stain
The Gram stain is the most commonly used stain in the clinical procedure was performed correctly.
microbiology laboratory. It places bacteria into one of two main
groups: gram-positive (blue to purple) or gram-negative (pink; Case Check 1.3
see Fig. 1.6A–B). Some organisms are gram-variable or do not Review of quality control slides is important in the detection of errors in
stain at all. As mentioned previously, the cell wall structure the performance of the Gram stain procedure and in interpretation of
determines the Gram-staining characteristics of a species. The results. As illustrated in the Case in Point at the beginning of the chapter,
Gram stain consists of gentle heat fixing (methyl alcohol may be the gram-positive control organism, S. aureus, stained gram-positive,
used instead for fixation) of the smear and the addition of four which is an acceptable result. However, the gram-negative control organism,
sequential components: crystal violet (the primary stain, 1 minute), E. coli, also appeared gram-positive, which is an unacceptable result and
indicative of an error in performing the Gram stain procedure. When
iodine (the mordant or fixative, 1 minute), alcohol or an alcohol-
such an error occurs, the results may not be reported until the discrepancy
acetone solution (the decolorizer, on and quick rinse), and safranin is resolved and the procedure is repeated with acceptable quality control
(the counterstain, 30 seconds). The time frames listed are not results.
exact and vary with the organism; rinsing with water between

A B

C D
FIG. 1.6  A, Gram stain of Lactobacillus species illustrating gram-positive bacilli, singly and in
chains. A few gram-negative–staining bacilli are also present. B, Gram stain of Escherichia coli
illustrating short gram-negative bacilli. C, Acid-fast stain, Carbol fuchsin-based. Sputum smear
demonstrating the presence of acid-fast Mycobacterium species (arrow) stained by the Kinyoun
or Ziehl-Neelsen carbol fuchsin method. D, Acid-fast stain, fluorochrome-based. Mycobacterium
species stained with the acid-fast fluorescent auramine-rhodamine stain. This stain is useful for
screening for the presence of acid-fast bacteria in clinical specimens. Continued
12 PART 1  Introduction to Clinical Microbiology

E F

G H
FIG. 1.6, cont’d  E, Acridine orange stain. Fluorescent stain demonstrating the presence of staphy-
lococci in a blood culture broth. This stain is useful for detecting bacteria in situations where
debris may mask the bacteria. F, Methylene blue stain. Methylene blue stain demonstrating the
typical morphology of Corynebacterium diphtheriae (arrows). G, Lactophenol cotton blue stain.
Lactophenol cotton blue–stained slide of macroconidia and hyphae of the fungal dermatophyte
Microsporum gypseum. H, India ink. An India ink wet mount of Cryptococcus neoformans dem-
onstrating the presence of a capsule (arrow). (A and B, Courtesy Dr. Andrew G. Smith, Baltimore,
MD; D, courtesy Clinical Microbiology Audiovisual Study Units, Health and Education Resources,
Inc., Bethesda, MD; E, courtesy Dr. John E. Peters, Baltimore, MD; and H, courtesy Dr. Andrew G.
Smith, Baltimore, MD.)

Acid-Fast Stains Acridine Orange


Acid-fast stains are used to stain bacteria that have a high lipid Acridine orange is a fluorochrome dye that stains both gram-positive
and wax content in their cell walls and do not stain well with and gram-negative bacteria, living or dead. It binds to the nucleic
traditional bacterial stains. Carbol fuchsin (a red dye) is used as acid of the cell and fluoresces a bright orange when a fluorescent
the primary stain (see Fig. 1.6C). The cell wall is treated to allow microscope is used. Acridine orange is used to locate bacteria in
penetration of the dye either by heat (Ziehl-Neelsen method) or blood cultures and other specimens where discerning bacteria
by a detergent (Kinyoun method). Acidified alcohol is used as a might otherwise be difficult (see Fig. 1.6E).
decolorizer, and methylene blue is the counterstain. Acid-fast
bacteria retain the primary stain and are red. Bacteria that are not Calcofluor White
acid-fast are blue. Calcofluor white is a fluorochrome that binds to chitin in fungal
Two other gram-positive genera, Nocardia and Rhodococcus, cell walls. It fluoresces a bright apple-green or blue-white,
may stain acid-fast by a modified method. Acid-fast staining is allowing visualization of fungal structures with a fluorescent
used to identify Saccharomyces, a yeast, and coccidian parasites, microscope. Calcofluor white was the original “bluing” used in
such as Cystoisospora belli (formerly known as Isospora belli), high-volume laundries to whiten yellow-appearing white cotton and
Cryptosporidium, and other coccidia-like bodies. A fluorochrome other fabrics.
(i.e., fluorescent) stain, auramine-rhodamine, also has been used
to screen specimens for acid-fast bacteria (see Fig. 1.6D). This Methylene Blue
stain is selective for the cell wall of acid-fast bacteria. Acid-fast Methylene blue traditionally has been used to stain C. diphtheriae
bacteria appear yellow or orange under a fluorescent microscope, for observation of metachromatic granules (see Fig. 1.6F). It is
making them easier to find. also used as a counterstain in acid-fast staining procedures. It is
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 13

sometimes used as a simple stain to detect white blood cells, such such as Haemophilus influenzae and the anaerobes, are fastidi-
as in stool samples. ous, requiring additional metabolites such as vitamins, purines,
pyrimidines, and hemoglobin supplied in the growth medium.
Lactophenol Cotton Blue Some pathogenic bacteria, such as Chlamydia spp., cannot be
Lactophenol cotton blue is used to stain the cell walls of medically cultured on laboratory media at all and must be grown in cell
important fungi grown in slide culture (see Fig. 1.6G). culture or detected by other means.

India Ink Types of Growth Media


India ink and nigrosin are negative stains used to visualize capsules A laboratory growth medium whose contents are simple and com-
surrounding certain yeasts, such as Cryptococcus spp. (see Fig. pletely defined is termed minimal medium. This type of medium is
1.6H). The fine ink particles are excluded from the capsule, leaving not usually used in the diagnostic microbiology laboratory. Media
a dark background and a clear capsule surrounding the yeast. that are more complex and made of extracts of meat or soybeans
are termed nutrient media (e.g., nutrient broth, trypticase soy
Endospore Stain broth). A growth medium that contains added growth factors, such
The Schaefer-Fulton spore stain is commonly used to stain bacterial as blood, vitamins, and yeast extract, is referred to as enriched
spores. To a heat-fixed smear, the primary stain, malachite green, (e.g., blood agar, chocolate agar). Media containing additives that
is applied (flooded) and heated to steaming for about 5 minutes. inhibit the growth of some bacteria but allow others to grow are
Then the preparation is washed for about 30 seconds to remove called selective media (e.g., MacConkey agar [MAC] selective
the primary stain. Next, the counterstain safranin is applied to for gram-negative bacteria by inhibiting gram-positive bacteria
the smear. The endospores appear green within pink-appearing and colistin–nalidixic acid selective for gram-positive bacteria
or red-appearing bacterial cells. by inhibiting gram-negative bacteria). Ingredients in media that
allow visualization of metabolic differences between groups or
species of bacteria are called differential media. MAC is also
Microbial Growth and Nutrition a differential medium because it distinguishes between lactose
All bacteria have three major nutritional needs for growth: fermenters (pink colonies) and nonlactose fermenters (clear
• A source of carbon (for making cellular constituents). Carbon colonies). A blood agar plate is differential because it distinguishes
represents 50% of the dry weight of a bacterium. between hemolytic and nonhemolytic organisms. When a delay
• A source of nitrogen (for making proteins and nucleic acids). between collection of the specimen and culturing is necessary,
Nitrogen makes up 14% of the dry weight. a transport medium is used. A transport medium is a holding
• A source of energy (ATP), for performing cellular functions. medium designed to preserve the viability of microorganisms
Smaller amounts of molecules, such as phosphate for nucleic in the specimen but not allow multiplication. Stuart broth and
acids and phospholipids of cell membranes and sulfur for protein Amies and Cary-Blair transport media are commonly used
synthesis, make up an additional 4% of the weight. Various metals examples.
and ions for enzymatic activity must also be present. Important
mineral ions, such as Na+, K+, Cl−, and Ca2+, are also required. Environmental Factors
Although the basic building blocks required for growth are the Influencing Growth
same for all cells, bacteria differ widely in their ability to use Three important environmental factors influence the growth rate
different sources of these molecules. of bacteria and must be considered when bacteria are cultured in
the laboratory:
Nutritional Requirements for Growth • pH
Bacteria are classified into two basic groups according to how • Temperature
they meet their nutritional needs. Members of one group, the • Gaseous composition of the atmosphere
autotrophs (lithotrophs), are able to grow simply, using carbon Most pathogenic bacteria grow best at a neutral pH. Diagnostic
dioxide as the sole source of carbon, with only water and inor- laboratory media for bacteria are usually adjusted to a final pH
ganic salts required in addition. Autotrophs obtain energy either between 7.0 and 7.5. Temperature influences the rate of growth
photosynthetically (phototrophs) or by oxidation of inorganic of a bacterial culture. Microorganisms are categorized according
compounds (chemolithotrophs). Autotrophs occur in environmental to their optimal temperature for growth. Bacteria that grow best
milieus. at cold temperatures are called psychrophiles (optimal growth
The second group of bacteria, the heterotrophs, require more at 10° to 20° C). Bacteria that grow optimally at moderate
complex substances for growth. These bacteria require an organic temperatures are called mesophiles (optimal growth at 20° to
source of carbon, such as glucose, and obtain energy by oxidiz- 40° C). Bacteria that grow best at high temperatures are called
ing or fermenting organic substances. Often, the same substance thermophiles (optimal growth at 50° to 60° C). Psychrophiles and
(e.g., glucose) is used as both the carbon source and the energy thermophiles are found environmentally in places such as the
source. Arctic seas and hot springs, respectively. Most bacteria that have
All bacteria that inhabit the human body fall into the heterotro- adapted to humans are mesophiles that grow best near human
phic group. However, nutritional needs differ greatly within this body temperature (37° C). Diagnostic laboratories routinely
group. Bacteria such as E. coli and Pseudomonas aeruginosa can incubate cultures for bacterial growth at 35° C. However, some
use a wide variety of organic compounds as carbon sources and pathogenic species prefer a lower temperature for growth; when
grow on most simple laboratory media. Other pathogenic bacteria, these organisms are suspected, the specimen plate is incubated
14 PART 1  Introduction to Clinical Microbiology

at a lower temperature. Fungal cultures are incubated at 30° C.


The ability to grow at room temperature (22° C) or at an elevated Stationary
temperature (42° C) is used as an identification characteristic for 9

log10 Number of bacteria


some bacteria.
Bacteria that grow on humans differ in their atmospheric 7 Death
requirements for growth. Obligate aerobes require oxygen for Exponential phase
growth. Aerotolerant anaerobes can survive in the presence of 5
oxygen but do not use oxygen in metabolism (e.g., Streptococcus
spp.). Obligate anaerobes cannot grow in the presence of oxygen. 3 Lag
Facultative anaerobes can grow either with or without oxygen.
If oxygen is present, the bacteria will utilize it via aerobic respira- 1
tion and grow faster than without oxygen. Capnophilic organisms
require an atmosphere enriched with extra carbon dioxide (5% 4 8 12 16 20
to 10%); an example is Neisseria gonorrhoeae. Because many FIG. 1.7  Typical growth curve of a bacterial culture.
bacteria grow better in the presence of increased carbon dioxide,
diagnostic microbiology laboratories often maintain their aerobic
incubators at a 5% to 10% carbon dioxide level.
Air contains approximately 21% oxygen and 1% carbon Determination of Cell Numbers
dioxide. When the carbon dioxide content of an aerobic incuba- In the diagnostic laboratory, the number of bacterial cells present
tor is increased to 10%, the oxygen content of the incubator is is determined in one of three ways:
decreased to approximately 18%. Obligate aerobes must have • Direct counting under the microscope: This method can be
oxygen to grow; incubation in air or an aerobic incubator with used to estimate the number of bacteria present in a specimen.
10% carbon dioxide present satisfies their oxygen requirement. It does not distinguish between live and dead cells.
Microaerophilic bacteria require a reduced level of oxygen to • Direct plate count: By growing dilutions of broth cultures on
grow. An example of a pathogenic microaerophile is Campylo- agar plates, one can determine the number of colony-forming
bacter jejuni, which requires 5% to 6% oxygen. This type of units per milliliter. This method provides a count of viable
atmosphere can be generated in culture jars or pouches with a cells only. It is used in determining the bacterial cell count in
commercially available microaerophilic atmosphere–generating urine cultures.
system. Obligate anaerobes must be grown in an atmosphere • Density measurement: The density (referred to as turbidity)
either devoid of oxygen or with significantly reduced oxygen of a bacterial broth culture in log phase can be correlated to
content. Facultative anaerobes are routinely cultured in an aerobic CFU/mL of the culture. This method is used to prepare a
atmosphere because aerobic culture is easier and less expensive than standard inoculum for antimicrobial susceptibility testing.
anaerobic culture, and the bacteria grow more rapidly. An example
is E. coli. Bacterial Biochemistry
Bacterial Growth
and Metabolism
Generation Time Metabolism
Bacteria replicate by binary fission, with one cell dividing into Microbial metabolism consists of the biochemical reactions bacteria
two cells. The time required for one cell to divide into two cells use to break down organic compounds and the reactions they use
is called the generation time or doubling time. The generation to synthesize new bacterial molecules from the resulting carbon
time of a bacterium in culture can be 20 minutes for a fast-growing skeletons. Energy for the new constructions is generated during
bacterium such as E. coli or 24 hours for a slow-growing bacterium the metabolic breakdown of a substrate. The occurrence of all
such as Mycobacterium tuberculosis. biochemical reactions in the cell depends on the presence and
activity of specific enzymes. Thus metabolism can be regulated
Growth Curve in the cell either by regulation of the production of an enzyme
If bacteria are in a growth state with enough nutrients and no itself (a genetic type of regulation, in which production of the
toxic products present, the increase in bacterial numbers is pro- enzyme can be induced or suppressed by molecules present in
portional to the increase in other bacterial properties, such as the cell) or by regulation of the activity of the enzyme (via feed-
mass, protein content, and nucleic acid content. Measurement of back inhibition, in which the products of the enzymatic reaction
any of these properties can be used as an indication of bacterial or a succeeding enzymatic reaction inhibit the activity of the
growth. When the growth of a bacterial culture is plotted during enzyme).
growth, the resulting curve shows four phases of growth: (1) a Bacteria differ widely in their ability to use various compounds
lag phase, during which bacteria are preparing to divide; (2) a as substrates and in the end products generated. Various biochemical
log phase, during which bacterial numbers increase logarithmically; pathways exist for substrate breakdown in the microbial world, and
(3) a stationary phase, in which nutrients are becoming limited the particular pathway used determines the end product and final
and the numbers of bacteria remain constant (although viability pH of the medium (Fig. 1.8). Microbiologists use these metabolic
may decrease); and (4) a death phase, when the number of nonviable differences as phenotypic markers in the identification of bacteria.
bacterial cells exceeds the number of viable cells. An example Diagnostic schemes analyze each unknown microorganism for (1)
of such a growth curve is shown in Fig. 1.7. utilization of various substrates as a carbon source, (2) production
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 15

Ethanol L– or D–Lactic acid


Propionic acid
+ 2H
Acetaldehyde H2 + CO2
Succinic acid + 2H
+ 4H
Oxaloacetic acid PYRUVIC ACID Formic acid

Acetolactic acid Acetyl CoA Acetoacetyl CoA


+ 4H + 4H
Ethanol Acetic acid Acetone Butyryl CoA
Acetoin
+ 2H + 4H
+ 2H
Isopropanol Butyric acid Butanol
2,3–Butanediol

FIG. 1.8  The fate of pyruvate in major fermentation pathways of microorganisms. (From Joklik
WK, et al: Zinsser microbiology, ed 20, Norwalk, CT, 1992, Appleton & Lange.)

of specific end products from various substrates, and (3) production


of an acid or alkaline pH in the test medium. Knowledge of D–Glucose
ATP
the biochemistry and metabolism of bacteria is important in the ADP
clinical laboratory. D–Glucose – 6 – PO4

Fermentation and Respiration D–Fructose – 6 – PO4


ATP
Bacteria use biochemical pathways to catabolize (break down) car-
bohydrates and produce energy by two mechanisms—fermentation D–Fructose – 1,6 – di PO4
and respiration (commonly referred to as oxidation). Fermentation
is an anaerobic process carried out by obligate, facultative, and Dihydroxyacetone–PO4 D–Glyceraldehyde–3–PO4
aerotolerant anaerobes. In fermentation, the electron acceptor is
an organic compound. Fermentation is less efficient in energy 1,3–Diphosphoglycerate
generation than respiration because the beginning substrate is not ADP
2 ATP
completely reduced; therefore all the energy in the substrate is not 2 NADH 3–Phosphoglycerate
released. Besides allowing growth in the absence of atmospheric
oxygen, fermentation is also important because it generates 2–Phosphoglycerate
nicotinamide adenine dinucleotide (NAD), a molecule necessary Ethanol
for maintaining the Krebs cycle. When fermentation occurs, a Lactate Phosphoenolpyruvate
mixture of end products (e.g., lactate, butyrate, ethanol, and acetoin) ADP
accumulates in the medium. Analysis of these end products is 2 ATP
Acetaldehyde Pyruvate
particularly useful for the identification of anaerobic bacteria.
End-product determination is also used in the Voges-Proskauer CO2
(VP) and methyl red tests, two important diagnostic tests used in
the identification of the Enterobacteriaceae. The term fermentation FIG. 1.9  Embden-Meyerhof-Parnas glycolytic pathway. (From
is often used loosely in the diagnostic microbiology laboratory to Joklik WK, et al: Zinsser microbiology, ed 20, Norwalk, CT, 1992,
Appleton & Lange.)
indicate any type of utilization—fermentative or oxidative—of a
carbohydrate—sugar—with the resulting production of an acid pH.
Aerobic respiration (oxidation) is an efficient energy-generating
process in which molecular oxygen (O2) is the final electron accep-
tor. Obligate aerobes and facultative anaerobes undergo aerobic first convert the sugar to glucose, which is processed by one of
respiration. Certain anaerobes can carry out anaerobic respiration, three pathways. These pathways are designed to generate pyruvic
in which molecules other than molecular oxygen, such as nitrate acid, a key three-carbon intermediate. The three major biochemical
and sulfate, act as the final electron acceptors. Anaerobic respiration pathways bacteria use to break down glucose to pyruvic acid
is less energy yielding than aerobic respiration. are (1) the Embden-Meyerhof-Parnas (EMP) glycolytic pathway
(Fig. 1.9), (2) the pentose phosphate pathway (Fig. 1.10), and
Biochemical Pathways from Glucose (3) the Entner-Doudoroff pathway (see Fig. 1.10). Pyruvate can
to Pyruvic Acid be further catabolized either fermentatively or oxidatively. The
The starting carbohydrate for bacterial fermentation or oxidation is three major metabolic pathways and their key characteristics are
glucose. When bacteria use other sugars as a carbon source, they described in Box 1.1.
16 PART 1  Introduction to Clinical Microbiology

Glucose
ATP
ADP
Glucose–6–PO4
NAD
NADH2
6–Phosphogluconic acid 2–Keto–3–deoxy–6–phosphogluconic acid
NAD
NADH2
Pentose PO4 + CO2 Pyruvic acid Glyceraldehyde–3–PO4
2 ADP
Glyceraldehyde–3–PO4 Acetyl PO4 Acetaldehyde + CO2 (Via
EMP 2 ATP
NADH2 NADH2 pathway) 2 NAD
2 ATP NAD NAD 2 NADH2

Lactate Acetaldehyde Ethanol Pyruvic acid

(Via EMP NADH2


pathway)
NAD

Ethanol Acetaldehyde + CO2

NADH2
NAD

Ethanol

FIG. 1.10  Alternative microbial pathways to the Embden-Meyerhof-Parnas (EMP) pathway for
glucose fermentation. The pentose phosphate pathway is on the left, and the Entner-Doudoroff
pathway is on the right. (From Joklik WK, et al: Zinsser microbiology, ed 20, Norwalk, CT, 1992,
Appleton & Lange.)

Anaerobic Utilization of Pyruvic formic. The strong acid produced is the basis for the positive
Acid (Fermentation) reaction on the methyl red test exhibited by these organisms.
Pyruvic acid is a key metabolic intermediate. Bacteria process • Butanediol fermentation: Members of the genera Klebsiella,
pyruvic acid further using various fermentation pathways. Each Enterobacter, and Serratia within the family Enterobacteriaceae
pathway yields different end products, which can be analyzed use this pathway for carbohydrate fermentation. The end
and used as phenotypic markers (see Fig. 1.8). Some fermentation products are acetoin (acetyl methyl carbinol) and 2,3-butanediol.
pathways used by the microbes that inhabit the human body are Detection of acetoin is the basis for the positive VP reaction
as follows: characteristic of these microorganisms. Little acid is produced
• Alcoholic fermentation: The major end product is ethanol. by this pathway. Thus organisms that have a positive VP reaction
This is the pathway used by yeasts when they ferment glucose usually have a negative reaction on the methyl red test, and
to produce ethanol. vice versa.
• Homolactic fermentation: The end product is almost exclusively • Butyric acid fermentation: Certain obligate anaerobes, including
lactic acid. Members of the genus Streptococcus and many many Clostridium spp., Fusobacterium, and Eubacterium,
members of the genus Lactobacillus ferment pyruvate using produce butyric acid as their primary end product along with
this pathway. acetic acid, carbon dioxide, and hydrogen.
• Heterolactic fermentation: Some lactobacilli use a mixed
fermentation pathway, of which, in addition to lactic acid, the Aerobic Utilization of
end products include carbon dioxide, alcohols, formic acid, Pyruvate (Oxidation)
and acetic acid. The most important pathway for the complete oxidation of a
• Propionic acid fermentation: Propionic acid is the major end substrate under aerobic conditions is the Krebs cycle or tricar-
product of fermentation carried out by Propionibacterium acnes boxylic acid cycle. In this cycle, pyruvate is oxidized, carbon
and some anaerobic non–spore-forming, gram-positive bacilli. skeletons for biosynthetic reactions are created, and the electrons
• Mixed acid fermentation: Members of the genera Escherichia, donated by pyruvate are passed through an electron transport
Salmonella, and Shigella within the family Enterobacteriaceae chain and used to generate energy in the form of ATP. This cycle
use this pathway for carbohydrate fermentation and produce a results in the production of acid and the evolution of carbon
number of acids as end products: lactic, acetic, succinic, and dioxide during aerobic respiration (Fig. 1.11).
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 17

Pyruvate
BOX 1.1  Three Major Metabolic Pathways
EMP Glycolytic Pathway (see Fig. 1.9) Acetyl–CoA
• Major pathway in conversion of glucose to pyruvate
• Generates reducing power in the form of NADH2
2H CO2
• Generates energy in the form of ATP
• Anaerobic; does not require oxygen
• Used by many bacteria, including all members of Enterobacteriaceae
Citrate
Pentose Phosphate (Phosphogluconate) Pathway Oxaloacetate
(see Fig. 1.10)
• Alternative to EMP pathway for carbohydrate metabolism cis–Aconitate
• Conversion of glucose to ribulose-5-phosphate, which is rearranged
into other 3-, 4-, 5-, 6-, and 7-carbon sugars Malate
• Provides pentoses for nucleotide synthesis
• Produces glyceraldehyde-3-phosphate, which can be converted to Isocitrate
pyruvate CO2
• Generates NADPH, which provides reducing power for biosynthetic Fumarate
reactions –Ketoglutarate
• May be used to generate ATP (yield is less than with EMP pathway) CO2
Succinate
• Used by heterolactic fermenting bacteria, such as lactobacilli, and
by Brucella abortus, which lacks some of the enzymes required in
the EMP pathway 2H 2H 2H 2H
Entner-Doudoroff Pathway (see Fig. 1.10)
• Converts glucose-6-phosphate (rather than glucose) to pyruvate and
glyceraldehyde phosphate, which can be funneled into other Electron transport
pathways and
• Generates one NADPH per molecule of glucose but uses one ATP oxidative phosphorylation
• Aerobic process used by Pseudomonas, Alcaligenes, Enterococcus
fecalis, and other bacteria lacking certain glycolytic enzymes
ATP
ATP, Adenosine triphosphate; EMP, Embden-Meyerhof-Parnas; NADH2,
nicotinamide adenine dinucleotide dehydrogenase; NADPH, nicotinamide FIG. 1.11  Krebs tricarboxylic acid cycle allowing complete
adenine dinucleotide phosphate.
oxidation of a substrate. (From Joklik WK, et al: Zinsser microbiol-
ogy, ed 20, Norwalk, CT, 1992, Appleton & Lange.)

Carbohydrate Utilization and by Frederick Miescher in 1869. In the 1920s, Phoebus A. T. Levine
Lactose Fermentation discovered that DNA contained phosphates, five-carbon sugars
The ability of microorganisms to use various sugars (carbohydrates) (cyclic pentose), and nitrogen-containing bases. Later, Rosalind
for growth is an integral part of many diagnostic identification Franklin discovered the helical structure by x-ray crystallography.
schemes. The fermentation of the sugar is usually detected by acid Most everyone is familiar with James Watson and Francis Crick,
production and a concomitant change of color resulting from a who described the three-dimensional structure of the DNA molecule
pH indicator present in the culture medium. Bacteria generally in the 1950s.
ferment glucose preferentially over other sugars, so glucose must
not be present if the ability to ferment another sugar is being tested. Anatomy of a DNA and RNA Molecule
The microorganism’s ability to ferment lactose is an important DNA is a double helical chain of deoxynucleotides. The helix is
step in classifying members of the family Enterobacteriaceae. a double strand twisted together, which many scientists refer to
These bacteria are classified as either lactose fermenters or lactose as a “spiral staircase” (resembling the handrail, sides, and steps
nonfermenters. Lactose is a disaccharide consisting of one molecule of a spiral staircase). A nucleotide is a complex combination of
of glucose and one molecule of galactose linked by a galactoside the following:
bond. The utilization of lactose by a bacterium requires two steps. • A phosphate group (PO4)
The first step requires an enzyme, β-galactoside permease, for • A cyclic five-carbon pentose (the carbons in the pentose
the transport of lactose across the cell wall into the bacterial are numbered 1′ through 5′) sugar (deoxyribose), which
cytoplasm. The second step occurs inside the cell and requires makes up the “handrails and sides”
the enzyme β-galactosidase to break the galactoside bond, releasing • A nitrogen-containing base, or the “steps,” either a purine
glucose, which then can be fermented. Thus all organisms that or a pyrimidine
can ferment lactose can also ferment glucose. A purine consists of a fused ring of nine carbon atoms and
nitrogen. There are two purines in the molecule: adenine (A) and
guanine (G). A pyrimidine consists of a single ring of six atoms
Bacterial Genetics of carbon and nitrogen. There are two pyrimidines in the molecule:
No discussion of bacterial genetics is complete without our first thymine (T) and cytosine (C). A nucleotide is formed when the
describing DNA and RNA. Historically, DNA was first discovered 5′ carbon of the sugar and one of the nitrogenous bases attaches
18 PART 1  Introduction to Clinical Microbiology

Pyrimidines Purines
NH2 O O NH2 O
H3C N N
N N N N N

N O N O N O N N NH2
N N
H H H H H
Cytosine Thymine Uracil
C T U Adenine Guanine
A G
DNA and DNA RNA
RNA only only DNA and RNA
FIG. 1.12  Molecular structure of nucleic acid bases. Pyrimidines: cytosine, thymine, and uracil.
Purines: adenine and guanine.

to the 1′ carbon of the pentose sugar. These are the basic building a detailed discussion of DNA and molecular diagnostics, see
blocks of DNA (Fig. 1.12). Chapter 11.
In the chain of deoxynucleotides, bonds form between the
phosphate group of one nucleotide and the 3′ sugar of the next Terminology
nucleotide. The base extends out from the sugar. Adenine of one The genotype of a cell is the genetic potential of the DNA of an
chain always pairs with thymine of the other chain, and cytosine organism. It includes all the characteristics that are coded for in
of one chain pairs with guanine of the other chain. The bases are the DNA of a bacterium and that have the potential to be expressed.
held together by hydrogen bonds. The information contained in Some genes are silent genes, expressed only under certain condi-
DNA is determined primarily by the sequence of letters along the tions. Genes that are always expressed are constitutive. Genes
“staircase.” The sequence ACGCT represents different information that are expressed only under certain conditions are inducible.
than the sequence AGTCC. This would be like taking the word The phenotype of a cell consists of observed characteristics
“stops” and using the same letters to form the word “spots” or expressed by the genome. The ultimate aim of a cell is to produce
“posts,” which have different meanings but all the same letters. the proteins that are responsible for cellular structure and function
The two complementary sugar phosphate strands run in opposite and to transmit the information for accomplishing this to the next
directions (antiparallel), 3′ to 5′ and 5′ to 3′, similar to one train generation of cells. Information for protein synthesis is encoded
with its engine going one way alongside a caboose of a train in the bacterial DNA and transmitted in the chromosome to each
going the opposite direction (Fig. 1.13). The direction is based generation. The general flow of information in a bacterial cell is
on what is found at the ends of the strands; for example, phosphate from DNA (which contains the genetic information) to messenger
attaches to the 5′ carbon of the sugar, and the OH group is attached RNA (mRNA; which acts as a blueprint for protein construction)
to the 3′ carbon of the sugar. to the actual protein itself.
DNA is also involved in the production of RNA. In RNA, the Replication is the duplication of chromosomal DNA for insertion
nitrogenous base thymine is replaced by uracil, another pyrimidine. into a daughter cell. Transcription is the synthesis of ssRNA, by
In contrast to DNA, RNA is single-stranded and short, not double- the enzyme RNA polymerase, using one strand of the DNA as a
stranded and long, and contains the sugar ribose, not deoxyribose. template. Translation is the actual synthesis of a specific protein
Human beings are 99.9% identical. In a human genome of 3 from the mRNA code. The term protein expression also refers to
billion “letters,” even one tenth of 1% translates into 3 million the synthesis of a protein. Proteins are polypeptides composed of
separate lettering differences, an important characteristic useful amino acids. The number and sequence of amino acids in a poly-
in forensic science but with related importance in diagnostic peptide and the character of that particular protein are determined
microbiology using the bacterial genome. Bacterial genetics is by the sequence of codons in the mRNA molecule. A codon is a
increasingly important in the diagnostic microbiology laboratory. group of three nucleotides in an mRNA molecule that signifies
Diagnostic tests have been developed that are based on identifying a specific amino acid. During translation, ribosomes containing
unique RNA or DNA sequences present in each bacterial species. rRNA sequentially add amino acids to the growing polypeptide
The polymerase chain reaction technique is a means of amplifying chain. These amino acids are brought to the ribosome by transfer
specific DNA sequences and detecting very small numbers of RNA (tRNA) molecules that “translate” the codons. The tRNA
bacteria present in a specimen. Genetic tests circumvent the need molecules temporarily attach to mRNA using their complementary
to culture bacteria, providing a more rapid method of identifying anticodon regions. An anticodon is the triplet of bases on the tRNA
pathogens. that bind the triplet of bases (codon) on the mRNA. It identifies
An understanding of bacterial genetics is also necessary to which amino acid will be in a specific location in the protein.
understand the development and transfer of antimicrobial resistance
by bacteria. The occurrence of mutations can result in a change Genetic Elements and Alterations
in the expected phenotypic characteristics of an organism and Bacterial Genome
provides an explanation for atypical results sometimes encountered The bacterial chromosome, also called the genome, consists of a
on diagnostic biochemical tests. This section briefly reviews some single, closed, circular piece of dsDNA that is supercoiled to fit
of the basic terminology and concepts of bacterial genetics. For inside the cell. It contains all the information needed for cell
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 19

3’ hydroxyl
Nucleotide
5’ phosphate
H HO
Base-pair Deoxyribose 3’
-O P=O sugar
Base H H H H
O
O A T H2C 3’
H2C5’ O
O
1’
H H -O P=O Phosphodiester
3’ H O bond
O H
5’
- H H
O P=O H H
O
O G C H2C
H2C O
O

H H H H -
O P=O A T

O H H O G C
-O P=O H H H A T
H
O C G
O H2C
H2C A T T A
O

H H H H -O P=O

H O
O H
-O P=O H H H H
O
O H2C
H2C C G
O
-
H H H H O P=O
H 3’ O
O H 3’
-O P=O H H H H
5’
O
O H2C5’ B
H2C T A
A O
-O P=O
H H
5’ phosphate
OH H
3’ hydroxyl

FIG. 1.13  A, Molecular structure of DNA showing nucleotide structure, phosphodiester bond
connecting nucleotides, and complementary pairing of bases (A, adenine; T, thymine; G, guanine;
C, cytosine) between antiparallel nucleic acid strands. B, 5′ and 3′ antiparallel polarity and “twisted
ladder” configuration of DNA.

growth and replication. Genes are specific DNA sequences that They are not essential for bacterial growth, so they can be gained
code for the amino acid sequence in one protein (e.g., one gene or lost. Genes that code for antimicrobial resistance (and sometimes
equals one polypeptide), but this may be sliced up or combined toxins or other virulence factors) are often located on plasmids.
with other polypeptides to form more than one protein. In front Antimicrobial therapy selects for bacterial strains containing
of each gene on the DNA strand is an untranscribed area containing plasmids encoding drug-resistance genes; this is one reason
a promoter region, which the RNA polymerase recognizes for antimicrobial agents should not be overprescribed. The number
transcription initiation. This area may also contain regulatory of plasmids present in a bacterial cell may range from one (low
regions to which molecules may attach and cause either a decrease copy number) to hundreds (high copy number). Plasmids are
or an increase in transcription. located in the cytoplasm of the cell and are self-replicating and
passed to daughter cells, similar to chromosomal DNA. They also
Extrachromosomal DNA Elements may sometimes be passed (nonsexually) from one bacterial species
In addition to the genetic information encoded in the bacterial to another through conjugation (horizontal transfer of genetic
chromosome, many bacteria contain extra information on small material by cell-to-cell contact). This is one way drug resistance
circular pieces of extrachromosomal, dsDNA called plasmids. is acquired.
20 PART 1  Introduction to Clinical Microbiology

Mobile Genetic Elements Plasmid DNA


Certain pieces of DNA are mobile and may jump from one place ready to be
Bacterial chromosome
taken into the
in the chromosome to another place. These are sometimes referred cell
to as jumping genes. The simplest mobile piece of DNA is an
insertion sequence (IS) element. It is approximately 1000 base
pairs long with inverted repeats on each end. Each IS element
codes for only one gene, a transposase enzyme that allows the IS A
element to pop into and out of DNA. Bacterial genomes contain
many IS elements. The main effect of IS elements in bacteria is Viral DNA
being injected
that when an IS element inserts itself into the middle of a gene, into the cell
it disrupts and inactivates the gene. This action can result in loss
of an observable characteristic, such as the ability to ferment a
particular sugar. Transposons are related mobile elements that
contain additional genes. Transposons often carry drug-resistance
Bacterial chromosome
genes and are usually located in plasmids. B
Mutations Plasmid DNA
Mutations are changes that occur in the DNA code and often containing the
result in a change in the coded protein or in the prevention of its Bacterial chromosome F factor entering
the F– cell
synthesis. Some mutations are silent, where a change in the DNA
sequence does not result in the substitution of a different amino F– cell
acid in the resulting protein. This is due to redundancy in protein
synthesis; more than one codon codes for the same amino acid.
A mutation may be the result of a change in one nucleotide base
F+ cell
(a point mutation) that leads to a change in a single amino acid
within a protein or may be the result of insertions or deletions in
the genome that lead to disruption of the gene or a frameshift C Bacterial chromosome
mutation, or both. A gene sequence must be read in the right FIG. 1.14  Methods of gene transfer into bacterial cells. A,
“frame” for the correct protein to be produced. This is because Bacterial transformation. Free or “naked” DNA is taken up by
every set of three bases in mRNA (a codon) specifies a particular a competent bacterial cell. After uptake, the DNA may take
amino acid, and when the reading frame is askew, the codons are one of three courses: (1) it is integrated into existing bacterial
interpreted incorrectly. Incomplete, inactive proteins are often the genetic material; (2) it is degraded; or (3) if it is a compatible
plasmid, it may replicate in the cytoplasm. B, Bacterial transduc-
result of frameshift mutations. Spontaneous mutations occur in
tion. A phage injects DNA into the bacterial cell. The phage
bacteria at a rate of about 1 in 109 cells. Mutations also occur as tail combines with a receptor of the bacterial cell wall and
the result of error during DNA replication at a rate of about 1 in injects the DNA into the bacterium. One of two courses may
107 cells. Exposure to certain chemical and physical agents can then be taken. In the lytic cycle, replication of the bacterial
greatly increase the mutation rate. chromosome is disrupted; phage components are formed and
assembled into phage particles. The bacterial cell is lysed,
Genetic Recombination releasing a mature phage. In the lysogenic cycle, the phage
DNA is incorporated into the bacterial genetic material, and
Genetic recombination is a method by which genes are transferred genes encoded by the phage DNA may be expressed from this
or exchanged between homologous (similar) regions on two DNA site. At a later time, the phage may be “induced,” and a lytic
molecules, forming new combinations of genes on a chromosome cycle then ensues. C, Bacterial conjugation. An F+ cell connects
This method provides a way for organisms to obtain new com- with an F− cell via sex pili. DNA is then transferred from the
binations of biochemical pathways and adapt to changes in their F+ cell to the F− cell.
environment.

Mechanisms of Gene Transfer take up naked DNA are referred to as being competent. Only a
Genetic material may be transferred from one bacterium to another few bacterial species, such as Streptococcus pneumoniae, Neisseria
in three basic ways: gonorrhoeae, and H. influenzae, do this naturally. Bacteria can be
• Transformation made competent in the laboratory, and transformation is the main
• Transduction method used to introduce genetically manipulated plasmids into
• Conjugation bacteria, such as E. coli, during cloning procedures.

Transformation Transduction
Transformation is the uptake and incorporation of free or naked Transduction is the transfer of bacterial genes by a bacteriophage
DNA into a bacterial cell (Fig. 1.14A). Once the DNA has been from one cell to another (see Fig. 1.14B). A bacteriophage consists of
taken up, it can be incorporated into the bacterial genome by a chromosome (DNA or RNA) surrounded by a protein coat. When
recombination. If the DNA is a circular plasmid and the recipient a phage infects a bacterial cell, it injects its genome into the bacterial
cell is compatible, the plasmid can replicate in the cytoplasm and cell, leaving the protein coat outside. The phage may then take a
be transferred to daughter cells during cell division. Cells that can lytic pathway, in which the bacteriophage DNA directs the bacterial
CHAPTER 1  Bacterial Cell Structure, Physiology, Metabolism, and Genetics 21

cell to synthesize phage DNA and phage protein and package it ■ Eukaryotes differ from prokaryotes in that they have membrane-
into new phage particles. The bacterial cell eventually lyses (lytic enclosed nuclei and organelles.
phase), releasing a new phage that can infect other bacterial cells. ■ Viruses cannot be seen under an ordinary light microscope, although
In some instances, the phage DNA instead becomes incorporated their cytopathic effects on cell lines are visible. They are obligate
into the bacterial genome, where it is replicated along with the parasites, and antibiotics are ineffective for treatment of viral
bacterial chromosomal DNA; this state is known as lysogeny, and infections. Viruses have DNA or RNA, but rarely both, in contrast
to prokaryotes and eukaryotes.
the phage is referred to as being temperate. During lysogeny,
■ A major way bacteria are classified in the diagnostic microbiology
genes present in the phage DNA may be expressed by the bacterial laboratory is by the Gram stain reaction. Whether an organism is
cell. An example of this in clinical microbiology is C. diphtheriae. gram-positive (blue or purple) or gram-negative (pink or red) is an
Strains of C. diphtheriae that are lysogenized with a temperate important first step in identifying bacteria and in determining
phage carrying the gene for diphtheria toxin cause disease. Strains appropriate antimicrobial therapy.
lacking the phage do not produce the toxin and do not cause disease. ■ Bacterial spores are formed as a result of harsh environments. They
Under certain conditions, a temperate phage can be induced, the are a means of survival, not reproduction.
■ The LPS contained in the outer membrane of gram-negative bacteria
phage DNA is excised from the bacterial genome, and a lytic
consists of three regions: an antigenic O–specific polysaccharide,
state occurs. During this process, adjacent bacterial genes may be a core polysaccharide, and an inner lipid A (also called endotoxin).
excised with the phage DNA and packaged into the new phage. The lipid A moiety is responsible for producing fever and shock
The bacterial genes may be transferred when the phage infects conditions in patients infected with gram-negative bacteria.
a new bacterium. In the field of biotechnology, phages are often ■ Bacteria utilize two biochemical pathways, fermentation and respira-
used to insert cloned genes into bacteria for analysis. tion, to catabolize carbohydrates to produce energy.

Conjugation
Conjugation is the transfer of genetic material from a donor Learning Assessment Questions
bacterial strain to a recipient strain (see Fig. 1.14C). Close contact 1. Explain the reason why the laboratory scientist in the Case in
is required between the two cells. In the E. coli system, the donor Point should repeat the Gram stain procedure on the exudate.
strain (F+) possesses a fertility factor (F factor) on a plasmid that 2. What might have occurred to make the Gram stain results invalid?
carries the genes for conjugative transfer. The donor strain produces 3. Differentiate the role of pili from the role of flagella.
a hollow surface appendage called a sex or conjugation pilus, 4. What is the role of the capsule in the pathogenesis of infectious
diseases?
which binds to the recipient F− cell and brings the two cells in
5. Why is lipopolysaccharide (LPS) a significant outer-membrane
close contact. Transfer of DNA then occurs. Both plasmids and structure in gram-negative bacteria?
chromosomal genes can be transferred by this method. When the 6. A bacterium that grows only on plates incubated in the absence
F factor is integrated into the bacterial chromosome rather than of oxygen would be categorized as a(n):
a plasmid, there is a higher frequency of transfer of adjacent a. Aerotolerant anaerobe.
bacterial chromosomal genes. These strains are known as high- b. Facultative anaerobe.
frequency recombination strains. c. Obligate anaerobe.
d. Obligate aerobe.
7. Fimbriae present on the outer surface of bacteria are used for:
Restriction Enzymes
a. Adherence to surfaces.
Bacteria have evolved a system to restrict the incorporation of b. Antimicrobial resistance.
foreign DNA into their genomes. Restriction enzymes are produced c. Sexual reproduction.
that cut incoming, foreign DNA at specific DNA sequences. The d. Bacterial motility.
bacteria methylate their own DNA at these same sequences so 8. All of the following are characteristic of fermentation except:
that the restriction enzymes do not cut the DNA in their own cell. a. It begins with the breakdown of pyruvic acid.
b. It follows glycolysis and produces reduced nicotinamide adenine
Many restriction enzymes with various recognition sequences dinucleotide (NADH).
have now been isolated from various microorganisms. The first c. It produces acids, alcohols, and gases.
three letters in the restriction endonuclease name indicate the d. It can occur in the presence of oxygen.
bacterial source of the enzyme. For instance, the enzyme EcoRI 9. Why are older bacterial cells more easily decolorized than cells
was isolated from E. coli, and the enzyme HindIII was isolated from younger colonies?
from H. influenzae type d. These enzymes are used in the field of 10. Why are spore-forming organisms more resistant than non–spore-
forming species?
biotechnology to create sites for insertion of new genes. In clinical
11. Explain the three ways in which genetic material can be transferred
microbiology, epidemiologists sometimes use restriction enzyme from one bacterium to another.
fragment analysis to determine whether strains of bacteria have 12. For the following DNA, write the complementary sequence. Include
identical restriction sites in their genomic DNA and therefore labeling the 3′ and 5′ end. 3′ TTACGGACAAC 5′: ________________.
likely came from the same source. 13. In RNA, thymine is replaced by ________________.
14. In bacteriophage, how does lysogeny differ from the lytic cycle?

Points to Remember BIBLIOGRAPHY


■ Many microorganisms inhabit our environment, most are
Baumann, R. W. (2017). Microbiology (5th ed.). San Francisco: Pearson.
nonpathogenic.
Murray, P. R., et al. (2016). Medical microbiology (8th ed.). St Louis:
■ Prokaryotes, such as bacteria, do not have membrane-enclosed
Mosby.
nuclei and organelles.
Pommerville, J. C. (2013). Fundamentals of microbiology: An introduction
(10th ed.). Burlington: Jones & Bartlett.
CHAPTER

2  
Host-Parasite Interaction
Steven Mahlen, Donald C. Lehman, Connie R. Mahon

CHAPTER OUTLINE
■ ORIGIN OF MICROBIAL BIOTA Ability to Resist Phagocytosis
Characteristics of Indigenous Microbial Biota Surface Structures That Promote Adhesion to Host Cells
Factors That Determine the Composition of the Usual and Tissues
Microbial Biota Ability to Survive Intracellularly and Proliferate
■ COMPOSITION OF MICROBIAL BIOTA AT DIFFERENT Ability to Produce Extracellular Toxins and Enzymes
BODY SITES ■ HOST RESISTANCE FACTORS
Normal Microbiota of the Skin Physical Barriers
Normal Microbiota of the Oral Cavity Cleansing Mechanisms
Normal Microbiota of the Respiratory Tract Antimicrobial Substances
Normal Microbiota of the Gastrointestinal Tract Indigenous Microbial Biota
Normal Microbiota of the Genitourinary Tract Phagocytosis
■ ROLE OF THE MICROBIAL BIOTA IN THE PATHOGENESIS Inflammation
OF INFECTIOUS DISEASE Immune Responses
■ ROLE OF THE MICROBIAL BIOTA IN THE HOST DEFENSE ■ MECHANISMS BY WHICH MICROBES MAY OVERCOME
AGAINST INFECTIOUS DISEASE HOST DEFENSES
■ MICROBIAL FACTORS CONTRIBUTING TO
PATHOGENESIS AND VIRULENCE
Pathogenesis
Virulence

OBJECTIVES
After reading and studying this chapter, you should be able to:
1. Define the following terms: parasitism, indigenous biota, commensal, 5. Evaluate the role of the indigenous biota in host defense against
symbiont, opportunist, resident biota, transient biota, carrier, true infectious diseases.
pathogen, opportunistic pathogen, and virulence. 6. Differentiate the mechanisms of infections caused by true pathogens
2. Explain how the following factors determine the composition of the from infections caused by opportunistic pathogens.
microbial biota at various body sites: 7. Discuss the conditions that must be present or events that must
• Amounts and types of nutrients available in the environment occur for a microorganism to cause disease.
• pH 8. Describe the characteristics of infectious agents that enable them to
• Oxidation-reduction potential cause disease in the host.
• Resistance to antibacterial substances 9. Describe the factors and mechanisms by which the human host is
3. List the predominant biota of various body sites in a healthy protected from microbial invasion.
individual. 10. Discuss the sequence of events in the phagocytosis and killing of an
4. Evaluate the role of the indigenous microbiota in the pathogenesis infectious agent.
of infectious disease. 11. Name the routes of transmission that microorganisms use to initiate
infection in a host, and give examples of each.

Case in Point department. He also said that he had developed right quadrant
abdominal pain over the last 24 hours. While in the emergency
A 71-year-old man was treated for right lower extremity cellulitis department the patient had five episodes of loose, watery diarrhea.
with a 10-day course of the antibiotic cephalexin. A few days A bacterial culture of the stool was negative for Salmonella, Shigella,
after completing the course of antibiotics, he started having loose, Campylobacter, Yersinia, and Vibrio species, but a polymerase chain
watery diarrhea. The patient described having many episodes of reaction assay for the Clostridium difficile toxin B gene was positive.
diarrhea per day; after 3 days of diarrhea he came to the emergency

22
CHAPTER 2  Host-Parasite Interaction 23

Issues to Consider the chapter describes the origin of the indigenous (resident)
microbial biota (microbiota) and the composition at different body
After reading the patient’s case history, consider:
■ Factors that predisposed this patient to his current
sites. It presents the role of the microbial biota at each body site
condition in the host immune defense and as a source of opportunistic
■ Clues that indicate the source of the infection infections. Factors that determine the composition of the microbiota
■ Significance of the microbiota in protecting the host against at different body sites are described. The second part of the chapter
pathogenic organisms discusses the virulence factors that contribute to the invasiveness
■ Role of the host innate and acquired immunity in protecting of organisms, protective mechanisms the host employs, and how
the host from infection microbes are able to evade the host’s defenses. Lastly, this chapter
■ Significance of microbial virulence factors in promoting describes factors that can make the host more susceptible to
infection infections and how microbes are transmitted.

Origin of Microbial Biota


Key Terms The fetus is in a sterile environment until birth. During delivery
Adaptive immunity Immunoglobulin M (IgM)
and the first few days of life, the newborn is introduced to the
Adhesin Immunoglobulins many and varied microorganisms present in the environment.
Anamnestic immune response Indigenous microbiota Each organism has the opportunity to find an area on or in the
Antigen Innate immunity infant into which it can adapt. Microorganisms that find their
Antibody Interferon niche colonize various anatomic sites and become the predominant
Bactericidal effect Lactoferrin organisms. Colonization is growth of microbiota in or on a body
Bacteriocin Leukocidin site without the production of damage or notable symptoms. Other
Carrier Lymphocyte microorganisms are transient or fail to establish themselves at all.
Carrier state Lymphokine As the infant grows, the microbial biota eventually becomes similar
Cell-mediated immune Lysozyme to the microbiota seen in older individuals.
response Opportunists Once established onto or into a particular body site of the host,
Chemotactic agent Opsonin microorganisms develop a particular relationship with that host.
Chemotaxis Opsonization The host-microbe relationship, depending on the circumstance, may
Colonization Panton-Valentine be one of symbiosis, commensalism, or parasitism. Symbiosis is
Commensalism Parasite defined as the association of two organisms living together. The
Complement Parasitism organisms are called symbionts. Symbiosis as a biological relation-
Diapedesis Pathogen ship between two or more organisms where both (host and organism)
Dissemination Pathogenicity benefit from one another may be described as mutualism. Lactobacilli
Endotoxin Pattern recognition receptor in the urogenital tract of women offer a mutual association: the
Exotoxin Phagocyte
lactobacilli provide the host protection by preventing colonization
Fimbriae Phagocytosis
of pathogenic species at that site while they derive nutrients from
Fomites Pili
the host. In the relationship where the organism benefits but there is
Glycolysis Receptor
Humoral immune response Resident microbial biota
no beneficial or harmful effect on the host, the association between
Iatrogenic infection Respiratory burst organisms is called commensalism. Proteus mirabilis is a commensal
Immune response Symbiosis species in the gastrointestinal tract of humans. In parasitism, one
Immune system Toll-like receptor species (microbe) benefits at the expense of the other (host). The
Immunogen Transient microbial biota parasite Entamoeba histolytica is a pathogenic intestinal ameba that
Immunoglobulin A (IgA) True pathogen derives nutrients from the host at the expense of the host, causing
Immunoglobulin E (IgE) Virulence intestinal ulcers and amebic dysentery.
Immunoglobulin G (IgG) Zoonoses
Characteristics of Indigenous
Microbial Biota
Microorganisms that are commonly found on or in body sites of

T
healthy persons are called normal or indigenous microbiota. The
he outcome from the interactions between host and pathogen different body sites may have the same or different microbiota,
is influenced by numerous factors. The status of the host’s depending on conditions. Local conditions select for organisms
immune system and ability of the host to defend itself that are suited for growth in a particular area. For example, the
from microbial invasion, combined with microbial factors inherent environment found on the dry skin surface is different from the
to the invading organism, often determine whether disease occurs. environment found on the moist surfaces in the oral cavity, and
To appreciate and understand the concepts involved in the patho- so the microbiota is different at the two sites.
genesis of infectious diseases, knowledge and understanding of Microorganisms that colonize an area for months or years
the host-pathogen relationship is important. represent resident microbiota, whereas microorganisms that
This chapter describes the interactions between the host and are present at a site temporarily represent transient microbiota.
infectious agents in the pathogenesis of disease. The first part of Transient biota comes to “visit” but does not usually stay. These
24 PART 1  Introduction to Clinical Microbiology

microorganisms are eliminated either by the host inherent immune found in breast-fed infants but have instead a colon microbiota
defenses or by competition with the resident biota. Some pathogenic similar to that seen in older children and adults (see Box 2.5). In
organisms may establish themselves in a host without manifesting areas of the body that have a low oxidation-reduction potential, the
symptoms. However, these hosts, called carriers, are capable of environment supports only organisms capable of fermentation, such
transmitting the infection. The condition of these hosts is called as is seen in the gingival crevices colonized with Bacteroides and
the carrier state. The carrier state may be acute (short-lived or Fusobacterium.
transient) or chronic (lasting for months, years, or permanently). The environmental conditions described here may change with
An example of a chronic carrier state is found in post–Salmonella age, nutritional status, disease states, and drug or antimicrobial
Typhi infection. This organism can establish itself in the bile duct therapy use. These changes can predispose an individual to infection
and can be excreted in the stool over years. In contrast, Neisseria by the indigenous biota, a type of infection referred to as an
meningitidis can be found in the nasopharynx of asymptomatic opportunistic infection. For example, two groups at increased risk
individuals during an outbreak of meningitis. After a few days for gram-negative bacillus pneumonia are diabetics and alcoholics.
or weeks at most, these individuals may no longer harbor the Antibiotics may reduce a particular population of bacteria, allowing
organism, in which case the carrier state would be termed acute. the proliferation of other organisms. An increase in age brings
The most transient of carrier states is the inoculation of a person’s with it a decrease in the effectiveness of the immune response.
hands or fingers with an organism, for example, Staphylococ- As a result, the incidence of infection caused by opportunistic
cus aureus that has colonized the person’s anterior nares and organisms increases.
is transmitted to the hands, that is carried only until the hands
are washed.
The organisms colonizing different body sites play a significant Case Check 2.1
role in providing host resistance to infections. The efficiency of The patient in the Case in Point at the beginning of this chapter developed
the microbial biota in providing protection to the human host is diarrhea following a course of antimicrobial therapy. As described in the
indicated by the relatively small number of infections caused by text, antibiotic use may alter the usual biota of nearly any body site. In this
these organisms in immunocompetent individuals. Nevertheless, case antibiotic use altered the normal bacterial biota of the gastrointestinal
these organisms may cause significant, often serious, infections or tract and allowed Clostridium difficile to cause an infection. In this case
may exacerbate existing infections in individuals lacking a fully C. difficile may have been a component of this patient’s bowel biota,
or, more likely, the patient acquired C. difficile from the environment.
responsive immune system. Knowing the benefit of the normal
microbiota, individuals can ingest probiotics, a suspension of
live bacteria that normally colonize the gastrointestinal tract, to
reestablish the microbiota. This is useful in some patients who Composition of Microbial Biota at
have taken oral broad-spectrum antimicrobial agents that have
reduced the number of colonizing bacteria in the intestines.
Different Body Sites
Human microbiome studies that use molecular sequencing strategies
to determine which organisms reside in or on the human body
Factors That Determine the have shown that the human host is colonized by a large number of
Composition of the Usual different species of microorganisms. For example, in the oral cavity
Microbial Biota alone, approximately 500 different species have been characterized.
Which microorganisms are present at a particular body site is The effectiveness of the various host defenses is evidenced by
influenced by nutritional and environmental factors, such as the the relatively low incidence of infection in immunocompetent
amount and types of nutrients available at the site. For example, individuals by members of the usual or indigenous microbiota.
more organisms inhabit moist areas than dry areas; these areas However, infections caused by members of the microbial biota
are dominated by diphtheroids, nonpathogenic corynebacteria. are frequently encountered among immunocompromised patients.
Although lipids and fatty acids are bactericidal to most bacteria, The clinical microbiologist must be able to recognize and identify
Propionibacterium spp. colonize the ducts of hair follicles the types of microorganisms found at the various body sites.
because these bacteria are able to break down the skin lipids
to fatty acids. The affinity of microorganisms for a specific site Normal Microbiota of the Skin
depends on the ability of the organisms to resist the antibacterial Normal skin has numerous mechanisms to prevent infection and
effects of substances such as fatty acids, bile, or lysozyme. The protect the underlying tissue from invasion by potential pathogens.
composition of the microbial biota is also affected by pH. For These mechanisms include physical separation of microorgan-
example, the female genital tract microbiota depends on the pH isms from the tissues, presence of fatty acids that inhibit many
of that environment, which in women of childbearing age is microorganisms, excretion of lysozyme by sweat glands, and
approximately 4.0 to 5.0. Many bacteria do not survive at this desquamation of the epithelium. The skin contains a wide variety
extreme pH range. Another example is the fecal biota found in of microorganisms, most of which are found on the most superficial
infants who are breast-fed, which differs from the fecal biota layers of cells and the upper parts of hair follicles. Scrubbing and
in infants fed with cow’s milk. Human milk has a high lactose washing may reduce the number of bacteria present on the skin
concentration and maintains a pH of 5.0 to 5.5, an environment by about 90% but do not completely eliminate the organisms
supportive of Bifidobacterium spp. Cow’s milk has a greater present, and their numbers return to normal within a few hours.
buffering capacity and is less acidic. Infants fed with cow’s milk The composition of the microbiota on the skin depends on the
do not have the high colonization rate by Bifidobacterium spp. activity of the sebaceous or sweat glands. Organisms concentrate
CHAPTER 2  Host-Parasite Interaction 25

BOX 2.1  Microorganisms Found on the Skin BOX 2.2  Microorganisms Found in the Mouth
Common Residents Common Residents
Candida spp. Staphylococcus epidermidis
Micrococcus spp. Streptococcus mitis
Staphylococcus spp. Streptococcus sanguinis
Propionibacterium spp. Streptococcus salivarius
Diphtheroids (Corynebacterium spp.) Streptococcus mutans
Peptostreptococcus spp.
Less Common or Transients
Veillonella spp.
Streptococcus spp.
Actinomyces israelii
Acinetobacter spp.
Bacteroides spp.
Gram-negative rods (fermenters and nonfermenters)
Prevotella/Porphyromonas
Moraxella spp.
Bacteroides oralis
Treponema denticola
Treponema refringens
the most in areas that are moist, such as the armpit, groin, and Less Common or Transients
perineum. The apocrine sweat glands in these areas secrete Staphylococcus aureus
substances metabolized by the skin bacteria, releasing odorous Enterococcus spp.
Eikenella corrodens
amines. Aerobic diphtheroids are usually found in moist areas
Fusobacterium nucleatum
such as the axillae and between the toes. Staphylococcus epider- Candida albicans
midis and Propionibacterium spp. reside in hair follicles and
colonize the sebaceous glands because they are resistant to skin
lipids and fatty acids as well as to superficial antiseptic agents
commonly used to cleanse the skin. The presence of skin bacteria BOX 2.3  Microorganisms Found in the Nose and
inhibits the growth of more pathogenic bacterial species, providing Nasopharynx
benefits to the host.
Common Residents
Microorganisms such as Propionibacterium acnes colonize Staphylococcus aureus
the deep sebaceous glands. Superficial antisepsis of the skin does Staphylococcus epidermidis
not eliminate this organism, which may be found as a contaminant Diphtheroids (Corynebacterium spp.)
in culture specimens obtained by invasive procedures (e.g., blood, Haemophilus parainfluenzae
cerebrospinal fluid), as a result of contamination of the needle. Streptococcus spp.
Box 2.1 lists the microorganisms most commonly found on the Less Common or Transients
skin. Other organisms have been isolated from the skin but are Streptococcus pneumoniae
found only occasionally or rarely and are not listed. Moraxella catarrhalis
Haemophilus influenzae
Normal Microbiota of the Oral Cavity Neisseria meningitidis
Moraxella spp.
The mouth contains large numbers of bacteria, with Streptococcus
being the predominant genus. Many organisms bind to the buccal
mucosa and tooth surface. Bacterial plaque that develops on teeth Streptococcus mutans, Streptococcus anginosus, and Streptococcus
may contain 1011 streptococci per gram. Plaque also results in a sanguinis. Moraxella catarrhalis, Neisseria spp., and diphtheroids
low oxidation-reduction potential at the tooth surface; this supports also colonize the upper respiratory tract. Obligate anaerobes reside
the growth of strict anaerobes, particularly in crevices and in the in the gingival crevices, where the anaerobic environment supports
areas between the teeth. Box 2.2 provides a partial list of micro- these organisms. The organisms found in the mouth, nasopharynx,
organisms found in the oral cavity. oropharynx, and nose, although similar, show some differences.
Box 2.3 lists common microorganisms encountered in the nose and
Normal Microbiota of the nasopharynx. Opportunistic pathogens such as S. aureus, found in
Respiratory Tract approximately 30% of healthy individuals, colonize the anterior
The respiratory tract, commonly divided into the upper and the nares. The population of the nasopharynx mirrors that of the nose,
lower respiratory tract, is responsible for the delivery of air from although the environment is different enough from the environment
the outside of the body to the pulmonary tissues responsible for of the nose to select for several additional organisms. Haemophilus
exchange of oxygen and carbon dioxide. The upper respiratory influenzae, Streptococcus pneumoniae, and N. meningitidis, all
tract is composed of the mouth, nasopharynx, oropharynx, and potential pathogens, are also found in the nasopharynx of healthy
larynx; the lower respiratory tract is composed of the trachea, individuals. Individuals who are hospitalized for several days may
bronchi, and pulmonary parenchyma. The trachea, bronchi, and become colonized in the upper respiratory tract by gram-negative
lungs are protected by the action of ciliary epithelial cells and by bacteria, particularly members of the Enterobacteriaceae.
the movement of mucus. The tissues of these structures are normally The oropharynx contains a mixture of streptococci. Many species
sterile as a result of this protective action. of the viridans group can be isolated, including Streptococcus mitis,
The mouth, nasopharynx, and oropharynx are colonized pre- S. mutans, Streptococcus milleri, S. sanguinis, and Streptococcus
dominantly with viridans streptococci, such as Streptococcus mitis, salivarius. In addition, diphtheroids and M. catarrhalis can be
26 PART 1  Introduction to Clinical Microbiology

BOX 2.4  Microorganisms Found in the Oropharynx BOX 2.5  Common Residents Found in the
Gastrointestinal Tract
Common Residents
α-Hemolytic and nonhemolytic streptococci Bacteroides spp.
Diphtheroids (Corynebacterium spp.) Clostridium spp.
Staphylococcus aureus Enterobacteriaceae
Staphylococcus epidermidis Enterococcus spp.
Streptococcus pneumoniae Eubacterium spp.
Streptococcus mutans Fusobacterium spp.
Streptococcus mitis Lactobacillus spp.
Streptococcus sanguinis Peptostreptococcus spp.
Streptococcus salivarius Peptococcus spp.
Moraxella catarrhalis Porphyromonas spp.
Haemophilus parainfluenzae Prevotella spp.
Bacteroides spp. Streptococcus spp.
Prevotella/Porphyromonas
Bacteroides oralis
Fusobacterium necrophorum
solid material. In fact, the colon contains over 70% of the all
Less Common or Transients microbes found in the body. Obligate anaerobes, such as Bacte-
Streptococcus pyogenes
roides, Clostridium, Prevotella, and Porphyromonas, far outnumber
Neisseria meningitidis
Haemophilus influenzae the facultative gram-negative bacilli, making up more than 90%
Gram-negative rods of the microbial biota of the large intestine. Gram-positive cocci
belonging to the genera Streptococcus and Enterococcus and yeasts
are also present in the large intestine.
readily isolated. Hospitalized patients often show colonization The gastrointestinal tract population may be altered by antibiot-
with gram-negative bacilli. The normal biota of the oropharynx ics. In some cases, certain populations or organisms are eradicated
is listed in Box 2.4. or suppressed, and other members of the indigenous biota are
able to proliferate. For example, Clostridium difficile or the yeast
Normal Microbiota of the Candida albicans can flourish in the intestinal tract of some people
Gastrointestinal Tract who are taking an oral broad-spectrum antimicrobial agent. This
The gastrointestinal tract comprises the esophagus, stomach, small alteration can be the cause of a severe necrotizing enterocolitis
intestine, and colon. The gastrointestinal tract is equipped with (C. difficile), diarrhea (C. albicans, S. aureus), or other superinfec-
numerous defenses and effective antimicrobial factors. Because tion. The bacteria constituting the usual intestinal biota also carry
intestinal pathogens are usually acquired by ingestion of organisms out various metabolic degradations and nutrient production that
contained in contaminated food or drink, host defenses against appear to play a role in the health of the host. The organisms
infections are present throughout the intestinal tract. Despite the found in the gastrointestinal tract are summarized in Box 2.5.
presence of antimicrobial factors, the intestinal tract is thought
to be colonized by over 35,000 bacterial species. The relation
Case Check 2.2
between the gut microbiota and human health is being increasingly
recognized. In the Case in Point, antimicrobial therapy led to an alteration of the
patient’s normal gastrointestinal tract biota. As shown in Box 2.5,
Microorganism population is lowest in the esophagus, about
Clostridium species, which may include C. difficile, are part of the normal
10 microbes per gram of content. Some microorganisms colonize gastrointestinal tract micrbiota in humans. In this case, clearing of some
the esophagus and others are present in ingested food as transient of the bacterial gut biota allowed this organism to grow unchecked and
biota. The stomach contains gastric juices, acids (pH 2), and cause an infection called C. difficile–antibiotic-associated diarrhea that
enzymes that help to protect the stomach from microbial attack. is often associated with prolonged antimicrobial therapy, as discussed in
Many microorganisms are susceptible to the acid pH of the stomach Chapter 22.
and are destroyed, except for the spore-forming bacterial species
in their spore phase and the cysts of parasites. Even with the
hostile environment of the stomach, some bacteria belonging to Normal Microbiota of the
the genera Streptococcus, Enterococcus, and Prevotella, and the Genitourinary Tract
opportunistic pathogen Helicobacter pylori, can inhabit the stomach. The kidneys, bladder, cervix, and fallopian tubes are normally
These organisms associate themselves with the stomach lining, sterile, although a few organisms originating from the perineum
protected by the layer of mucus that lines the stomach. Organisms can be found in the distal urethra, particularly in women. The
that are pH-susceptible and survive are generally protected by urethra is colonized in its outermost segment by organisms
being enmeshed in food, and they move to the small intestine. found on the skin. The composition of the vaginal microbiota is
The stomach acidity reduces the number of organisms that reach consistent with hormonal changes and age. Before puberty and
the small intestine. in postmenopausal women, vaginal biota primarily consists of
The small intestine contains fewer microorganisms compared yeasts, gram-negative bacilli, and gram-positive cocci. During
with those usually present in the colon. Microorganisms prevalent childbearing years, high estrogen levels promote the deposition
in the colon may produce a count of 1012 bacteria per gram of of glycogen in vaginal epithelial cells. Lactobacilli metabolize
CHAPTER 2  Host-Parasite Interaction 27

system is constantly primed by contact with microorganisms.


BOX 2.6  Microorganisms Found in the Animals born and raised in a germ-free environment have a poorly
Genitourinary Tract functioning immune system. Exposure to otherwise innocuous
Common Residents organisms can be fatal to such animals. Likewise, consider how
Lactobacillus spp. a sterile environment might affect a newborn. Antibody production
Bacteroides spp. would not be stimulated, and the mononuclear phagocyte system
Clostridium spp. would remain undeveloped. Serum immunoglobulin G (IgG) and
Peptostreptococcus spp. other antibodies effective against microorganisms would be sup-
Staphylococcus aureus pressed, which would make the individual more susceptible to
Staphylococcus epidermidis
pathogenic microorganisms. Without activation by microorganisms
Enterococcus spp.
Diphtheroids (Corynebacterium spp.) and the supporting action of antigen-presenting cells and cytokines,
cell-mediated immunity would not develop normally.
Less Common or Transients The microbial biota produces conditions at the microenviron-
Group B streptococci
Enterobacteriaceae
mental level that block colonization by extraneous pathogens.
Acinetobacter spp. When the composition of the indigenous biota is altered (e.g., by
Candida albicans antimicrobial therapy with broad-spectrum agents), other organ-
isms capable of causing disease may fill the void. For example,
gastroenteritis caused by Salmonella is generally not treated with
glycogen from vaginal epithelial cells to maintain a low pH, antimicrobial agents and is better eliminated by natural exclusion
creating an environment that is inhibitory to many organisms. by the colon biota. If the microbial biota is eliminated, such as
However, the low pH encourages colonization of the vagina with in patients receiving antimicrobial therapy, resistant or more
lactobacilli, anaerobic gram-negative bacilli, and gram-positive pathogenic species may be able to establish infection. The yeast
cocci. Microorganisms usually isolated from the genitourinary C. albicans may multiply and cause diarrhea or infections in the
tract are listed in Box 2.6. mouth or vagina when the normal biota has been eliminated, for
example.
Role of the Microbial Biota in the The microbial biota plays an important role in both health and
disease. Eradication of the usual biota may have profound negative
Pathogenesis of Infectious Disease effects, yet many common infections are caused by members of
Some organisms that make up the microbial biota live off the the usual biota. Knowledge of the role of these organisms in the
host’s nutrients, but in most cases, they provide some benefit to pathogenesis of an infectious disease is helpful in assessing their
the host, creating a symbiotic relationship with the host, as significance when they are isolated from clinical samples.
mentioned earlier. However, certain members of the normal
microbiota are opportunists; they cause disease when their habitat Microbial Factors Contributing to
is altered or when the host’s immune system is compromised. In Pathogenesis and Virulence
the case of trauma, either accidental or surgical, enough of the
normal microbial biota found in the traumatized area may reach Pathogenesis
sterile or other areas in the body where these organisms are not Pathogenicity is the ability of a microbe to produce disease in
part of the microbial biota. For example, patients who undergo an individual. An organism may be described as a true pathogen
surgery become susceptible to infections caused by organisms or as an opportunistic pathogen. True pathogens are organisms
that colonize the particular surgical site (e.g., abdominal cavity); recognized to cause disease in healthy immunocompetent individu-
leakage following perforation of the colon spills the contents of als a high percentage of the time. Bacterial species such as Yersinia
the colon into the peritoneal cavity, leading to an infection by the pestis and Bacillus anthracis are pathogenic in nearly all situations;
colon biota. when these species are recovered in clinical samples taken from
The host’s immune response may be reduced or altered because a body site, their clinical significance is well established.
of suppression by immunosuppressive drugs, chemotherapy, or Over the last several years, patient populations have changed.
radiation. Individuals with lymphoma, leukemia, or other blood Individuals with disease are living longer and are more likely to
disorders in which there is a functional defect in phagocytic activity undergo highly invasive medical procedures, organ transplanta-
or a decrease in the number of functioning cells or in which tion, and insertion of prosthetic devices, making them more
chemotactic activity is impaired also may have a reduced immune susceptible to infections. As a result, organisms that are found as
response. Members of the microbial biota also may initiate an normal biota are being seen with increasing frequency in clinical
infection or make an infection more serious in patients with chronic infections among immunosuppressed and immunocompromised
illnesses, including diabetes or severe hepatic disease such as individuals. H. influenzae colonizes the upper respiratory tract
cirrhosis. of healthy individuals without causing disease, but given the
opportunity, can rapidly produce a life-threatening infection.
Role of the Microbial Biota in the Host Organisms such as S. epidermidis under usual conditions do not
cause disease but can induce an infectious process in patients with
Defense Against Infectious Disease prosthetic devices. H. influenzae and S. epidermidis are called
The microbial biota provides beneficial effects, and the development opportunistic pathogens, and the infections they cause are called
of immunologic competence depends on this biota. The immune opportunistic infections. Table 2.1 lists common opportunistic
28 PART 1  Introduction to Clinical Microbiology

TABLE 2.1  Abbreviated List of Opportunistic TABLE 2.2  Common Routes of Transmissiona
Microorganisms
Route of Exit Route of Transmission Example
Conditions Compromising
Respiratory Aerosol droplet Influenza virus; tuberculosis
Host Defenses Organism(s)
inhalation
Foreign bodies (catheters, shunts, Staphylococcus epidermidis Nose or mouth → Common cold (rhinovirus)
prosthetic heart valves) Propionibacterium acnes hand or object →
Viridans streptococci nose
Serratia marcescens Salivary Direct salivary transfer Oral-labial herpes; infectious
Pseudomonas aeruginosa (e.g., kissing) mononucleosis
Aspergillus spp. Animal bite Rabies
Candida albicans Gastrointestinal Stool → hand → Enterovirus; hepatitis A
Alcoholism Streptococcus pneumoniae mouth and/or stool
Klebsiella pneumoniae → object → mouth
Burns Pseudomonas aeruginosa Stool → water or food Salmonellosis; shigellosis
Acinetobacter baumannii- → mouth
calcoaceticus complex Skin Skin discharge → air Varicella; poxvirus infection
Staphylococcus aureus → respiratory tract
Hematoproliferative disorders Cryptococcus neoformans Skin to skin Human papillomavirus
Varicella-zoster virus (warts); syphilis
Cystic fibrosis Pseudomonas aeruginosa Blood Transfusion or needle Hepatitis B; cytomegalovirus
Burkholderia cepacia prick infection; malaria; HIV
Immunosuppression (drugs, Candida albicans Insect bite Malaria; relapsing fever;
congenital disease) Pneumocystis jirovecii West Nile virus
Herpes simplex virus Genital Urethral or cervical Gonorrhea; herpes simplex;
Aspergillus spp. secretions secretions Chlamydia infection
Diphtheroids (Corynebacterium Semen Cytomegalovirus infection
spp.) Urine Urine → hand → Hospital-acquired urinary
Cytomegalovirus catheter tract infection
Staphylococcus spp. Urine → aerosol (rare) Tuberculosis
Pseudomonas spp. Eye Conjunctival Adenovirus
Zoonotic Animal bite Rabies
Contact with carcasses Tularemia
Arthropod Plague; Rocky Mountain
spotted fever; Lyme
microorganisms and the conditions with which they are most disease
commonly associated.
Because of these situations, our definition of a pathogen must HIV, Human immunodeficiency virus.
a
The examples cited are incomplete, and in some cases more than one
be expanded to apply to virtually any microorganism when condi- route of transmission exists.
tions for infection are met. In deciding whether a particular
organism that has been isolated is a pathogen, we also must consider
the human host from whom the organism was isolated and whether Routes of Transmission
that host has underlying disease that may affect susceptibility to The first step in initiating an infection is for the infectious agent
infection. For example, the potential pathogen list for a healthy to gain access to the host. The route by which a pathogen can be
20-year-old college student is much shorter than for a healthy transmitted to a susceptible host is an important factor in the
90-year-old person, a transplant recipient, or a 20-year-old college establishment of infection, which is often explained by the
student with acquired immunodeficiency syndrome (AIDS). Almost characteristics of the pathogen. The agent must be able to evade
any organism in the right place can cause an infection, and this host defenses and colonize the tissue at the point of entry. Although
needs to be considered when performing invasive procedures, some organisms may be naturally transmitted by more than one
because an inadvertent result of intervention can be the transfer route, most have a preferred route. These routes can be characterized
of an organism from where it is present as part of the indigenous as in the air (inhalation), via food and water (ingestion), through
biota to a place where it can replicate and cause infection. close contact (includes sexual transmission), through cuts and
An iatrogenic infection is an infection that occurs as the result bites, and via arthropods; animal diseases that can infect humans
of medical treatment or procedures. For example, many patients are transmitted through animal contact (zoonoses). The routes of
who have indwelling urinary catheters develop a urinary tract transmission are summarized in Table 2.2. Fig. 2.1 shows the
infection. Although placement of the catheter was a necessary routes of entry to and exit of microbes from the body.
procedure in the medical treatment of the individual, its use may
result in an infection. Patients who are given immunosuppressive Airborne Transmission
drugs because they have received a transplant are more susceptible Respiratory spread of infectious disease is common and is generally
to infection. Because any infection in such a patient would probably an efficient way to enter a host. Often, the respiratory secretions
be the result of the physician-ordered drug therapy, it would be are aerosolized by coughing, sneezing, and talking. Very small
considered an iatrogenic infection. particles, referred to as droplet nuclei, are the residue from the
CHAPTER 2  Host-Parasite Interaction 29

Infection
Influenza
Diphtheria
Shigella dysentery
Skin, throat, Ascending pyelonephritis
lung, intestine,
urinary tract

Lymph node Dengue


Malaria
Lymphatic Typhus

Bloodstream

Brain Poliomyelitis
Liver

Lungs Kidney Salivary gland


Skin

Hepatitis B
Yellow fever
Mumps
Rabies
Chickenpox
Yaws Measles Hematogenous
Rubella pyelonephritis
FIG. 2.1  Routes of entry and exit. (Reprinted from Mims CA, Nash A, Stephen J: Mims’ pathogenesis
of infectious disease, ed 5, San Diego, 2001, Elsevier Ltd, permission from Elsevier.)

evaporation of fluid from larger droplets and are light enough to especially true of the common cold–causing rhinovirus. The fingers
remain airborne for long periods. Pathogens that are spread through and hands are contaminated with infectious nasal secretions because
the air generally must be resistant to drying and inactivation by of hand-to-nose contact. The infectious viral particles are passed
ultraviolet light. Some infectious agents may be transmitted by from the infected individual to a susceptible recipient via hand-
dust particles that have become airborne. As discussed earlier in to-hand or hand-to-face contact. The recipient transmits the virus
this chapter, the body has many defenses against airborne infectious picked up from the infected individual by touching the face and
agents. The nasal turbinates, oropharynx, and larynx provide a nose. In this case the disease is transmitted via the respiratory route
twisting, mucus-lined passageway that makes direct access to the but not in the normal, classic manner of respiratory transmission.
lower respiratory tract mechanically difficult. In addition, the lower Transmission may also result from contact with inanimate
portions of the respiratory tract contain ciliary epithelium that objects contaminated with the infectious agent (fomites). For
sweeps organisms upward. For a microorganism to cause disease, example, a doorknob is contaminated by the hand and fingers of
it must circumvent these defenses, penetrate the mucous layer, an infected individual, and the virus is transmitted to a susceptible
and attach to the epithelium. The host also produces secretory person’s hand and fingers when that person opens the door. Control
IgA, lysozyme, alveolar macrophages, and other factors that act of such transmission is often as simple as frequent handwashing.
on the pathogen that manages to get beyond the physical defenses. Infections of the lower respiratory tract are less common but
Respiratory tract infections are the most common reason that more serious than infections of the upper respiratory tract. The
patients of all ages seek medical attention. Although most upper organisms causing these infections have managed to bypass host
respiratory tract infections are self-limiting and can be treated defenses, or the host defenses have been compromised (e.g., by
with over-the-counter medications, some are more serious. alcoholism, heavy smoking), allowing the pathogen access to the
Streptococcal sore throat, sinusitis, otitis media, acute epiglottitis, deeper portions of the respiratory tract.
and diphtheria can be serious and even life-threatening. Viral The most common microorganism causing lower respiratory
diseases causing the common cold and infectious mononucleosis tract infection of individuals older than 30 years of age is S.
are usually not life-threatening but can result in much discomfort pneumoniae. Although the pneumococcus is the most common
and absenteeism from work or school. cause of community-acquired pneumonia, it is also often seen
Although all the diseases mentioned can be spread via aerosols, in aspiration pneumonia, a common type of hospital-acquired
some may also be transmitted via the fingers and hands; this is pneumonia. Pneumococcal pneumonia begins suddenly and is a
30 PART 1  Introduction to Clinical Microbiology

serious, life-threatening disease, particularly in older patients. In aerosols, the susceptible host must be relatively close. However,
chronic lower respiratory tract infections, the survival of the for this discussion, close contact refers to passage of organisms by
infecting agent within phagocytes plays a role in the pathogenic salivary, skin, and genital contact. Two prominent infections passed
mechanism. As the agent of tuberculosis, a chronic debilitating by direct transfer of saliva (e.g., kissing) are herpes simplex virus
infection, M. tuberculosis is the classic example of an intracellular and Epstein-Barr virus. Skin-to-skin transfer of infectious disease
pathogen. This organism is highly virulent, is invasive, survives is not as common as for some of the other routes, but diseases
well, and multiplies within phagocytes. such as warts (human papillomavirus), syphilis, and impetigo
result when material from infectious lesions inoculates the skin
Transmission by Food and Water of a susceptible host. The list of sexually transmitted diseases is
Transmission of gastrointestinal infections is usually a result of a long one. In North America, the most commonly transmitted
ingestion of contaminated food or water. In some situations, venereal agents are human papillomavirus, Chlamydia trachomatis,
infection occurs via the fecal-oral route. The digestive tract is Neisseria gonorrhoeae, herpes simplex virus, Treponema pal-
colonized with vast numbers of different microorganisms. Under lidum subsp. pallidum (syphilis), Trichomonas spp., and human
usual conditions, the gut biota maintains a harmless relationship immunodeficiency virus (HIV).
with the host. Gastric enzymes and juices in the stomach prevent
survival of most organisms, but many survive and colonize the Cuts and Bites
small intestine and colon. The classic example of a bite-wound infection is rabies. However,
Gastrointestinal infections result from organisms that survive the human rabies is relatively rare. Of more concern with animal
harsh conditions of the stomach and competition with the microbial bites and especially human bites is infection by the mouth biota.
biota and then produce damage to the tissues of the gastrointestinal Dog-bite and cat-bite infections often yield Pasteurella multocida,
tract. This damage is a result of either a preformed toxin or disruption but the possibilities are extensive. Human bites are extremely
of the normal functioning of the intestinal cells by invasion of the dangerous because they are difficult to treat and because the human
pathogen or production of a toxin within the intestine. oral biota comprises many different organisms in extremely high
Organisms that can cause disease by means of a preformed toxin, numbers, including obligate anaerobic bacteria.
produced outside the body, include Clostridium botulinum, Bacillus
cereus, and S. aureus. The severity of disease ranges from mild Arthropods
diarrhea to rapidly fatal intoxication. Food poisoning by B. cereus Infectious agents can enlist the help of arthropods to be transmitted
and S. aureus is relatively common and is self-limiting. Botulism, among hosts. Infection following a mosquito, tick, flea, or mite
caused by C. botulinum, although rare, can be life-threatening. bite is a common occurrence in many parts of the world. Diseases
Other bacteria produce a toxin after infection of the intestinal spread by arthropods include malaria, relapsing fever, plague,
tract. Generally, to be effective as a pathogen, an organism must Rocky Mountain spotted fever, Lyme disease, West Nile fever,
survive, adhere to, and colonize the intestinal mucosa and either and untold numbers of regional hemorrhagic fevers. In most cases,
produce a toxin or invade deeper tissues. A commonly seen cause of the infectious agent multiplies in the arthropod, which then
diarrhea and intestinal infection is Escherichia coli. This organism transmits the agent while feeding on a human host.
is a member of the intestinal biota; however, some strains of E. coli
produce cytotoxins that cause alterations in the biochemical activity Zoonoses
of the intestinal epithelial cells, resulting in problems with fluid and The route of transmission known as zoonosis depends on contact
electrolyte control by the intestinal cells. These strains of E. coli, with animals or animal products. Certain organisms causing
referred to as enterotoxigenic, are a common cause of traveler’s disease in animals may also infect humans who have contact
diarrhea and other intestinal problems. Vibrio cholerae, the cause of with them. These diseases may be passed by animal bites (rabies),
cholera, produces an enterotoxin that causes the outpouring of fluid arthropod vectors (plague), contact with secretions (brucellosis),
from the cells into the lumen of the intestine. Massive amounts (20 L and contact with animal carcasses and products (tularemia, liste-
per day) of fluid can be lost. Other intestinal pathogens include C. riosis). The diseases are transmitted by routes already discussed.
difficile (see Case in Point), Shigella spp., Aeromonas hydrophila, The common factor is that, regardless of the route, the disease
Campylobacter jejuni, and Salmonella spp. The infective dose, is a disease of animals that is transmitted to humans. A partial
severity, and incidence of disease vary with the agent. list of zoonotic diseases and infecting organisms is provided
Numerous viruses also cause diarrheal disease. They multiply in Table 2.3.
within the cells of the intestinal mucosa and affect the normal
functioning of the cells. Viral agents in this category include Virulence
hepatitis A and E viruses, rotavirus, adenovirus, coxsackievirus, and Virulence is the relative ability of a microorganism to cause
Norovirus spp. The incidence of diarrhea caused by these agents is disease or the degree of pathogenicity. It is usually measured by
high, especially when people are in close contact (e.g., in daycare the numbers of microorganisms necessary to cause infection in
centers, nursing homes, military camps). Numerous parasites, such the host. Organisms that can establish infection with a relatively
as Cryptosporidium spp., Giardia lamblia, Entamoeba histolytica, low infective dose are considered more virulent than organisms
and Balantidium coli, also infect the gastrointestinal tract. that require high numbers for infection. For example, because
Shigella spp. cause disease with a relatively low infective dose
Close Contact (100 organisms), Shigella is considered to be a highly virulent
All of the routes of transmission of infectious diseases require organism. This generalization is misleading because the severity
close contact. For a respiratory pathogen to be transmitted via of disease caused by different organisms varies from one to another.
CHAPTER 2  Host-Parasite Interaction 31

susceptible to overwhelming infection. Therefore an extremely


TABLE 2.3  Zoonoses important event in the life of an invading pathogen that invades
the host is avoiding phagocytosis. There are many ways by which
Disease Organism microbial species evade phagocytosis, some are listed in Table 2.4.
Anthrax Bacillus anthracis The most common mechanism for evading phagocytosis that
Brucellosis Brucella spp. is used by many different microorganisms is that of having a
Erysipeloid Erysipelothrix rhusiopathiae polysaccharide capsule on the surface. Many of the microorganisms
Leptospirosis Leptospira interrogans possessing a capsule are highly virulent until removal of the capsule,
Tularemia Francisella tularensis at which point virulence becomes extremely low. Encapsulated
Ringworm Trichophyton spp.
strains of S. pneumoniae and H. influenzae are associated with
Microsporum spp.
Lyme disease Borrelia burgdorferi highly invasive infections and are known to be more virulent than
Plague Yersinia pestis nonencapsulated strains. The capsule is usually composed of
Rocky Mountain spotted fever Rickettsia rickettsii polysaccharides but can also be made of proteins or a combination
Yellow fever Flavivirus of protein and carbohydrate. The capsule inhibits phagocytosis
Encephalitis Alphavirus primarily by masking the cell surface structures that are recognized
Colorado tick fever Orbivirus by receptors on the surface of the phagocytic cell and in the same
Leishmaniasis Leishmania spp.
Rabies Rhabdovirus
manner inhibits the activation of complement by masking structures
Blastomycosis Blastomyces dermatitidis to which complement proteins bind.
Tuberculosis Mycobacterium bovis Another bacterial structure that protects organisms from
Q fever Coxiella burnetii phagocytosis is protein A. Protein A in the cell wall of S. aureus
Ornithosis Chlamydophila psittaci helps the organism avoid phagocytosis by interfering with the
Gastroenteritis Campylobacter spp. binding of the host’s antibodies to the surface of the organism.
Salmonella spp.
Antibodies bind to antigens via their Fab or antigen-binding portion.
Listeriosis Listeria monocytogenes
Giardiasis Giardia lamblia Protein A binds to the Fc portion of IgG (at the opposite end of
Toxoplasmosis Toxoplasma gondii the Fab sites), preventing opsonization and phagocytosis by turning
Tapeworms Taenia saginata the antibody around on the surface.
Taenia solium Some organisms evade phagocytic cell killing by releasing
Diphyllobothrium latum potent materials in tissues that kill phagocytes. Streptococci produce
Echinococcus spp. hemolysins that lyse red blood cells and induce toxic effects on
Trichinosis Trichinella spiralis
white blood cells and macrophages. Pathogenic staphylococci
release leukocidins that cause lysosomal discharge of white
blood cells into the cytoplasm. A staphylococcal leukocidin,
If a microorganism requires a relatively high infective dose but called Panton-Valentine leukocidin, is lethal to leukocytes and
the disease it causes is often fatal, we tend to think of the micro- contributes to the invasiveness of the organism. Other organ-
organism as highly virulent. A different organism may require a isms inhibit chemotaxis, which is the movement of white blood
low infective dose but produces a relatively mild disease. cells to sites of tissue damage, and the host is less able to direct
polymorphonuclear neutrophils (PMNs) and macrophages into
Microbial Virulence Factors the site of infection.
Infectious organisms have a wide variety of mechanisms or
virulence factors that allow them to persist in a host and cause Surface Structures That Promote
disease. Some virulence factors, such as capsules and toxins, Adhesion to Host Cells and Tissues
are used by many organisms. Other virulence factors tend to be Most infectious agents must attach to host cells before infection
specialized and specific to one particular organism, such as the occurs. In some diseases caused by exotoxins (e.g., botulism,
tissue tropism of N. gonorrhoeae. Virulence factors allow the staphylococcal food poisoning), adherence is not important.
pathogen to evade or overcome host defenses and cause disease and However, in virtually all other cases, the bacterium, virus, or
encompass functions such as inhibiting phagocytosis, facilitating fungus requires adherence to host cells before infection and disease
adhesion to host cells or tissues, enhancing intracellular survival can progress. The microbial surface structures that mediate attach-
after phagocytosis, and damaging tissue through the production ment are called adhesins. Host cells must possess the necessary
of toxins and extracellular enzymes. Many virulence factors are receptors for the adhesins. If the host or the infectious agent
well defined, such as the diphtheria and cholera toxins, the capsule undergoes a mutation that changes the structure of the adhesin
of S. pneumoniae, and the fimbriae of N. gonorrhoeae. Certain or the host receptor, adherence is not likely to take place, and the
microorganisms produce extracellular factors that appear to aid virulence of the infectious agent is affected. Fig. 2.2 illustrates
in infection, but the exact role of these factors is unknown. surface bacterial structures that are involved in the pathogenesis
of disease.
Ability to Resist Phagocytosis Virus infections depend on the target cell expressing an
Phagocytes, or phagocytic cells, such as macrophages and appropriate receptor for the virus adhesion molecule. Infection
polymorphonuclear cells, play a major role in defending the host of the cell occurs following attachment. The main adhesins in
from microbial invasion. These cells ingest bacteria and destroy bacteria are fimbriae (pili) and surface polysaccharides. Fimbriae
them. The lack of functioning phagocytic cells leaves the host enable bacteria to adhere to host cell surfaces, increasing the
32 PART 1  Introduction to Clinical Microbiology

secrete a toxin that causes the disease symptoms. Similarly, fimbriae


TABLE 2.4  Microbial Interference with Phagocytic are essential for N. gonorrhoeae to infect the epithelial cells of
Activities the genitourinary tract. Antibodies to the fimbriae of N. gonorrhoeae
are protective by preventing the organism from attaching to the
Mechanism (or epithelial cells. Similarly, antibodies to viral adhesions prevent
Microorganismsa Type of Interferenceb Responsible Factor)
infection.
Streptococci Kill phagocyte Streptolysin induces
lysosomal discharge Ability to Survive Intracellularly
into cell cytoplasm and Proliferate
Inhibit chemotaxis Streptolysin Some pathogens are able to survive within the phagocytic cell
Resist phagocytosis M substance after they have been engulfed. These organisms have developed
Resist digestion methods to prevent being killed intracellularly. Some organisms
Staphylococci Kill phagocyte Leukocidin induces prevent fusion of phagosomes and lysosomes, others have a
lysosomal discharge
resistance to the effects of the lysosomal contents, and still others
into cell cytoplasm
Inhibit opsonization Protein A blocks Fc escape from the phagosome into the cytoplasm.
portion of Ab To establish itself and cause disease, a pathogen must be able
Resist killing Cell wall mucopeptide to replicate after attachment to host cells. Numerous host factors
Bacillus anthracis Kill phagocyte Toxic complex work to prevent proliferation. Secretory antibody, lactoferrin,
Resist killing Capsular polyglutamic and lysozyme are produced by the host as a way to protect against
acid infection. To be successful in establishing infection, infectious
Haemophilus Resist phagocytosis Polysaccharide capsule
agents must be able to avoid or overcome these local factors.
influenzae (unless Ab present)
Streptococcus
Lactoferrin competes with bacteria for free iron, an essential trace
pneumoniae element; Neisseria meningitidis can use lactoferrin as a source
Klebsiella Resist digestion of iron and is therefore not inhibited by the presence of lactoferrin.
pneumoniae The nonpathogenic Neisseria spp. are usually unable to use the
Pseudomonas Resist phagocytosis “Surface slime” iron in lactoferrin and are inhibited by its presence.
aeruginosa (unless Ab present) (polysaccharide) Several pathogens (H. influenzae, N. gonorrhoeae, and N.
Resist digestion
meningitidis) produce an immunoglobulin A (IgA) protease that
Escherichia coli Resist phagocytosis O antigen (smooth
degrades the IgA found at mucosal surfaces. Other pathogens
(unless Ab present) strains)
K antigen (acid (influenza virus, Borrelia spp.) circumvent host antibodies by
polysaccharide) shifting key surface antigens. The host produces antibodies against
Resist killing K antigen the “old” antigens, which are no longer effective because the
Salmonella Typhi Resist phagocytosis Vi antigen organism now has “new” antigens that do not bind to antibodies
(unless Ab present) made against the old antigens.
Resist killing In most situations when an organism is engulfed by macro-
Cryptococcus Resist phagocytosis Polysaccharide capsule phages, lysosomal contents are released into the phagocytic
neoformans vacuoles and the organism is killed. However, if the engulfed
Treponema Resist phagocytosis Cell wall
organism is not exposed to intracellular killing and digestive
pallidum
Yersinia pestis Resist killing Protein-carbohydrate processes, it is able to survive and multiply inside the macrophage.
cell wall Bacterial species, such as Chlamydia, Mycobacterium, Brucella,
Mycobacteria Resist killing and Cell wall structure and Listeria species, are easily engulfed by macrophages and
digestion phagocytes. However, these species not only are able to survive
Inhibit lysosomal fusion ? inside the macrophages, but protected from the host’s other immune
Brucella abortus Resist killing Cell wall substance defenses, they are able to multiply intracellularly.
Toxoplasma gondii Inhibit attachment to ?
Pathogens exhibit an ability to penetrate and grow in tissues.
PMN
Inhibit lysosomal fusion ?
This process is called invasion. With some organisms, the invasion
is localized and involves only a few layers of cells. With others,
Adapted from Mims CA: The pathogenesis of infectious disease, ed 5, New it involves deep tissues; for example, the gonococcus organism
York, 2001, Academic Press. is invasive and may infect the fallopian tubes. With some organ-
Ab, Antibody; PMN, polymorphonuclear neutrophil.
a
isms, such as Salmonella spp., the organisms spread to distant
Often only the virulent strains show the type of interference listed.
b
Sometimes the type of interference listed has been described only in a
sites (organs and tissues). This is called dissemination. Other
particular type of phagocyte (polymorph or macrophage) from a particular organisms, such as Corynebacterium diphtheriae, do not spread
host, but it generally bears a relationship to pathogenicity in that host. beyond their initial site of infection, yet the disease they produce
is serious and often fatal because the exotoxins they produce are
organism’s colonizing ability, and providing resistance to phago- disseminated. Certain organisms that survive phagocytosis can
cytosis. Once bacteria are attached to nonphagocytic cells, be disseminated rapidly to many body sites, but the organisms
phagocytosis by white blood cells is less likely to occur. For themselves are not invasive. The phagocyte carries the organism to
example, the strains of E. coli that cause traveler’s diarrhea use other body sites, but the bacterium itself is incapable of penetrating
their fimbriae to adhere to cells of the small intestine, where they tissues.
CHAPTER 2  Host-Parasite Interaction 33

Adherence protein
Fibrilla
Pilus (e.g., M protein)
Protein F

Non-pili
adhesins Lipotechoic
Lipopolysaccharides acid
Capsule
Outer membrane Peptidoglycan
Peptidoglycan
Inner membrane Cell membrane

GRAM-NEGATIVE GRAM-POSITIVE
FIG. 2.2  Surface bacterial structures that are involved in the pathogenesis of disease. (From Kumar
V, Abbas AK, Fausto M: Robbins and Cotran pathologic basis of disease, ed 7, Philadelphia, 2005,
Saunders.)

exfoliatin, which causes rash and massive skin peeling or exfolia-


Ability to Produce Extracellular Toxins tion. Table 2.5 lists many bacterial exotoxins that are important
and Enzymes in disease.
Generally, disease from infection is noticeable only if tissue damage
occurs. This damage may be from toxins, either exotoxins or Endotoxins
endotoxins, or from inflammatory substances that cause host- Endotoxins are composed of the LPS portion of the outer membrane
driven, immunologically mediated damage. The ability of organisms on the cell wall of gram-negative bacteria. The cell wall of gram-
to produce exotoxins and extracellular enzymes is another major negative microorganisms is composed of two layers—the inner
factor that contributes to the virulence and invasiveness of organ- peptidoglycan layer and an outer membrane. The LPS is contained
isms. Toxins are poisonous substances produced by organisms in the outer membrane along with proteins and phospholipids (see
that interact with host cells, disrupting normal metabolism and Fig. 2.2). LPS contains three regions—an antigenic O–specific
causing harm. Exotoxins are produced by both gram-negative and oligosaccharide, a core polysaccharide, and an inner lipid A (also
gram-positive bacteria and are secreted by the organism into the called endotoxin). The lipid A portion of LPS is responsible for the
extracellular environment, or they are released on lysis of the toxic activity of endotoxin. LPS stimulates the release of proinflam-
organism. matory cytokines, e.g., tumor necrosis factor and interleukin 1,
Exotoxins can mediate direct spread of the microorganisms chemokines and other inflammatory mediators that aid in mounting
through the matrix of connective tissues and can cause cell and an innate immune response. These are the chemical mediators that
tissue damage. Some organisms produce soluble substances, such produce the effects of endotoxin that consist of dramatic changes
as proteases and hyaluronidases that liquefy the hyaluronic acid in blood pressure, clotting, body temperature, circulating blood
of the connective tissue matrix, helping bacteria to spread in cells, metabolism, humoral immunity, cellular immunity, and
tissues, promoting the dissemination of infection. Endotoxins resistance to infection. Endotoxin stimulates the fever centers in
are a constituent, the lipopolysaccharide (LPS), of the outer cell the hypothalamus, increasing body temperature within 1 hour after
membrane of gram-negative bacteria exclusively. Endotoxins, in exposure. Endotoxin exposure also causes hypotension, produc-
contrast to exotoxins, do not have enzyme activity, are secreted in ing severe hypotension within 30 minutes. Septic or endotoxic
only very small amounts, do not have specificity in their activity shock is a serious and potentially life-threatening problem. In
on host cells, are not very potent, and are not destroyed by heating. contrast to shock caused by fluid loss, such as shock seen in
Endotoxin is released in large amounts when the bacterial cell lyses. severe bleeding, septic shock is unaffected by fluid administra-
tion. The endotoxin also initiates coagulation, which can result in
Exotoxins intravascular coagulation. This process depletes clotting factors
Many bacterial exotoxins are highly characterized. Most are and activates fibrinolysis so that fibrin-split products accumulate
composed of two subunits: one is nontoxic and binds the toxin in the blood. These fragments are anticoagulants and can cause
to the host cells and the other is toxic. The toxin gene is com- serious bleeding. Another feature of patients with endotoxic shock is
monly encoded by phages, plasmids, or transposons. Only the severe neutropenia, which can occur within minutes after exposure.
organisms that carry the DNA coding for the toxin gene produce It results from sequestration of neutrophils in capillaries of the
toxin. Isolates of C. difficile, for example, have to be tested lung and other organs. Leukocytosis follows neutropenia because
for the presence of toxin genes, such as in the Case in Point. neutrophils are released from the bone marrow.
Diphtheria toxin inhibits protein synthesis and affects the heart, Endotoxin also produces a wide variety of effects on the immune
nerve tissue, and liver. Botulinum toxin is a neurotoxin that blocks system. It stimulates proliferation of B lymphocytes in some animal
nerve impulse transmission, causing flaccid paralysis, especially species, activates macrophages, activates complement, and has an
in infants. Streptococcus pyogenes and S. aureus both produce adjuvant effect with protein antigens. It also stimulates interferon
34 PART 1  Introduction to Clinical Microbiology

TABLE 2.5  Examples of Exotoxins of Pathogenic Bacteria

Bacterium Disease Caused in Humans Toxins

Bacillus anthracis Anthrax Lethal, edema-producing toxins


Bordetella pertussis Whooping cough Lethal, dermonecrotizing toxin
Clostridium botulinum Botulism 6 type-specific lethal neurotoxinsa
Clostridium difficile Antibiotic-associated diarrhea, pseudomembranous colitis Toxin A, enterotoxina
Toxin B, cytotoxina
Clostridium novyi Gas gangrene Alpha, lethal, dermonecrotizing
Beta, lethal, dermonecrotizing, hemolytic
Gamma, lethal, dermonecrotizing, hemolytic
Delta, hemolytic
Epsilon, lethal, hemolytic
Zeta, hemolytic
Clostridium perfringens Gas gangrene, food poisoning, enteritis necroticans Alpha, lethal, dermonecrotizing, hemolytica
Beta, lethal
Gamma, lethal
Delta, lethal
Epsilon, lethal, dermonecrotizing
Iota, lethal, dermonecrotizing
Theta, lethal, cardiotoxic, hemolytic
Kappa, lethal, proteolytic
Enterotoxina
Clostridium septicum Gas gangrene Alpha, lethal, hemolytic, necrotizing
Clostridium sordellii Gas gangrene Lethal toxina
Hemorrhagic toxina
Clostridium tetani Tetanus Tetanospasmin, lethal, neurotoxica
Neurotoxin, nonspasmogenic
Tetanolysin, lethal, cardiotoxic, hemolytic
Corynebacterium diphtheriae Diphtheria Diphtheria toxin, lethal, dermonecrotizinga
Escherichia coli Diarrhea Heat-labile enterotoxina
Heat-stable enterotoxin
Shiga toxin
Pseudomonas aeruginosa Pyogenic infections Exotoxin A
Staphylococcus aureus Pyogenic infections, enterotoxemia Alpha, lethal, dermonecrotizing, hemolytic
Beta, lethal, hemolytic
Gamma, lethal, hemolytic
Delta, hemolytic
Exfoliating toxina
Enterotoxina
Streptococcus pyogenes Pyogenic infections, scarlet fever, rheumatic fever Erythrogenic, nonlethal
Streptolysin O, lethal, hemolytic, cardiotoxic
Streptolysin S, lethal, hemolytic
Vibrio cholerae Cholera Cholera toxin, lethal, enterotoxica
Salmonella Typhimurium Enteritis Enterotoxin?a
Shigella spp. Dysentery Enterotoxina
Yersinia pestis Plague Murine toxin, cytotoxic pore-forming toxinsa

a
Toxins that produce harmful effects of infectious disease.

production and causes changes in carbohydrates, lipids, iron, and Host Resistance Factors
sensitivity to epinephrine. A severe infection with gram-negative
bacteria can lead to serious and often life-threatening situations. Physical Barriers
Bacterial exotoxins and endotoxins are compared in Table 2.6. Humans have evolved a complex system of defense mechanisms
to prevent infectious agents from gaining access to and replicating
in the body. Healthy skin is an effective barrier against infection.
Case Check 2.3 The stratified and cornified epithelium presents a physical barrier
In the Case in Point, diarrhea caused by C. difficile was diagnosed in a to penetration by most microorganisms. Organisms that can cause
patient who had been treated with an antimicrobial therapy for several infection by penetrating the mucous membrane epithelium usually
days. C. difficile produces potent virulence factors, including an enterotoxin
cannot penetrate unbroken skin. Only a few microorganisms are
called toxin A and a cytotoxin called toxin B. C. difficile is a significant
cause of diarrhea in patients who have received prolonged courses of capable of entering the body by way of intact skin. Some of these
antibiotic therapy in both outpatient and inpatient settings. microorganisms and others that normally enter when the skin barrier
is compromised are listed in Table 2.7. Most of the organisms
CHAPTER 2  Host-Parasite Interaction 35

TABLE 2.6  Differences Between Bacterial Exotoxins Cleansing Mechanisms


and Endotoxins Normally, the term cleansing brings to mind a liquid. However,
one of the most effective cleansing mechanisms humans have is
Characteristic Exotoxins Endotoxins the desquamation of the skin surface. The keratinized squamous
Organism type Gram-positive and Gram-negative
epithelium or outer layer of skin is being shed continuously. Many
gram-negative of the microorganisms colonizing the skin are disposed of with
Chemical nature Simple protein Protein-lipid- the sloughing of the epithelium.
polysaccharide More obvious is the cleansing action of the fluids of the eye
Stability to heating Labile Stable and the respiratory, digestive, urinary, and genital tracts. The eye
(100° C) is continually exposed to microorganisms, which means this organ
Detoxification by Detoxified Not detoxified
has some highly developed antimicrobial mechanisms. Tears bathing
formaldehyde
Neutralization by Complete Partial the cornea and sclera not only lubricate the eye but also wash
homologous antibody foreign matter and infectious agents away from the surface.
Biological activity Individual to toxin Same for all toxins Additionally, tears contain IgA and lysozyme.
The respiratory tract is also continuously exposed to micro-
organisms and is protected by nasal hairs, ciliary epithelium,
and mucous membranes. A continuous flow of mucus emanates
from the membranes lining the nasopharynx, which traps particles
TABLE 2.7  Microorganisms That Infect Skin or Enter
and microbes and sweeps them to the oropharynx, where they
the Body via the Skin are either expectorated or swallowed. The trachea is lined with
ciliary epithelium. These cells have hairlike extensions (cilia) that
Microorganisms Disease Comments
sweep particles and organisms upward toward the oropharynx.
Arthropod-borne Various fevers, 150 distinct viruses, This material is expectorated or swallowed. Heavy smokers have
viruses encephalitides transmitted by a significant reduction in ciliated epithelial cells and therefore are
infected arthropod more susceptible to respiratory infections. The purpose of these
bites mechanisms is to prevent infectious agents and other particles
Rabies virus Rabies Bites from infected
animals
from reaching the bronchioles and lungs. Under normal conditions,
Rickettsiaceae Typhus, spotted Infestation with they are very effective, and the air moving into and out of the
fevers infected arthropods lungs is sterile.
Leptospira Leptospirosis Contact with water Bacteria are swallowed into the gastrointestinal tract either as
containing infected part of the mouth biota and upper respiratory tract or in liquids
animal urine and food. Most bacteria are destroyed by the low pH found in
Staphylococci Boils, impetigo Most common skin
the stomach. However, some bacteria are able to survive and pass
invaders
Streptococci Impetigo, erysipelas
into the small intestine. The number of bacteria in the intestine
Bacillus anthracis Cutaneous anthrax Systemic disease increases as the distance from the stomach increases. The distal
following local portion of the colon contains the highest number of organisms.
lesion at inoculation The gastrointestinal tract is protected by mucous secretions and
site peristalsis that prevent the organisms from attaching to the intestinal
Treponema pallidum Syphilis, yaws Warm, moist skin is epithelium. Additionally, secretory antibody and phagocytic cells
and Treponema more susceptible
lining the mucosa defend the gastrointestinal tract against infection.
pertenue
Yersinia pestis Plague Bite from infected rat The genitourinary tract is cleansed by voiding urine. Conse-
flea quently, only the outermost portions of the urethra have a microbial
Plasmodium spp. Malaria Bite from infected population. The vagina contains a large population of organisms
mosquito as part of the indigenous biota. The acidity of the vagina, resulting
Dermatophytes Ringworm, athlete’s Infection restricted to from the breakdown of glycogen by the resident biota, tends to
foot skin, nails, and hair
inhibit transient organisms from colonizing.

Antimicrobial Substances
listed in Table 2.7 require help in breaking the skin barrier (e.g., Various substances produced in the human host have antimicrobial
animal or arthropod bite) or microscopic tears. Healthy, intact skin activity. Some are produced as part of the phagocytic defense and
is clearly the primary mechanical barrier to infection. The skin are discussed later. Others, such as fatty acids, hydrogen chloride
also has substantial numbers of microbial biota that are usually in the stomach, and secretory IgA have already been mentioned.
not pathogens, organisms that contribute to a low pH, compete for A substance that plays a major role in resistance to infection is
nutrients, and produce bactericidal substances. In addition, the low lysozyme, a low-molecular-weight (approximately 20,000) enzyme
pH resulting from long-chain fatty acids secreted by sebaceous that hydrolyzes the peptidoglycan layer of bacterial cell walls. In
glands ensures that relatively few organisms can survive and some bacteria, the peptidoglycan layer is directly accessible to
prosper in the acid environment of the skin. These conditions lysozyme. These bacteria are killed by the enzyme alone. In other
prevent colonization by transient, possibly pathogenic organisms. bacteria, the peptidoglycan layer is exposed after other agents
36 PART 1  Introduction to Clinical Microbiology

have damaged the cell wall (e.g., antibody and complement,


hydrogen peroxide). In these cases, lysozyme acts with the other TABLE 2.8  Tissue Distribution of Monocytes/
agents to cause death of the infecting bacteria. Lysozyme is found Macrophages
in serum, tissue fluids, tears, breast milk, saliva, and sweat.
Antibodies, especially secretory IgA, are found in mucous Cell Name Tissue Distribution
secretions of the respiratory, genital, and digestive tracts. They Monocyte Blood
may serve as opsonins, enhancing phagocytosis, or they may fix Kupffer cell Liver
complement and neutralize the infecting organism. Serum also Alveolar macrophage Lung
contains low-molecular-weight cationic proteins, termed β-lysins. Histiocyte Connective tissue
These proteins are lethal against gram-positive bacteria and are Microglial cell Central nervous system
released from platelets during coagulation. The site of action is Mesangial cell Kidney
Macrophage Spleen, lymph nodes
the cytoplasmic membrane. These antimicrobial substances and
systems work best together. A combination of antibody, comple-
ment, lysozyme, and β-lysin is significantly more effective in
killing bacteria than each alone or than any combination in which components that stimulate cell migration, the metabolic burst,
one or more are missing. and secretion of the lysosome contents into a phagosome. The
Proliferation of viruses is inhibited by interferon. The inter- PMN, an end-stage cell, has a circulating half-life of 2 to 7 hours
ferons are a group of cellular proteins induced in eukaryotic cells and makes up the majority of white blood cells in circulation of
in response to virus infection or other inducers. Uninfected cells healthy individuals. It can migrate to the tissues, where its half-life
that have been exposed to interferon are refractory to virus infection. is less than 1 week.
Numerous bacteria, viruses, and their products induce interferon Macrophages also originate in the bone marrow from stem
production. The interferon produced binds to surface receptors cells. They circulate as monocytes for 1 to 2 days and then migrate
on noninfected cells. This binding stimulates the cell to synthesize through the blood vessel walls into the tissues and reside in specific
enzymes that inhibit viral replication over several days. The antiviral tissues as part of the mononuclear phagocyte system. These cells
effect of interferon is only one action it exhibits. One type of are widely distributed in the body and play a central role in specific
interferon, interferon gamma, plays an especially important role immunity and nonspecific phagocytosis (Table 2.8).
in the immune response. It inhibits cell proliferation and tumor
growth and enhances phagocytosis by macrophages, the activity Chemotaxis
of natural killer cells, and the generation of cytotoxic T cells. Four activities must occur for phagocytosis to take place and be
effective in host defense: (1) migration of the phagocyte to the
Indigenous Microbial Biota area of infection (chemotaxis), (2) attachment of the particle to
Nonpathogenic microorganisms compete with pathogens for the phagocyte, (3) ingestion, and (4) killing. The PMNs circulate
nutrients and space. This competition lessens the chance that the through the body followed by movement into the tissues, an action
pathogen will colonize the host. Some normal microbiota species called diapedesis, which is the movement of the neutrophils between
produce bacteriocins, substances that inhibit the growth of closely the endothelial cells of the blood vessels into the tissues. The body
related bacteria. These proteins are produced by a variety of is under constant surveillance by these and other phagocytic cells.
gram-positive and gram-negative bacteria and appear to give the When an infection occurs, massive numbers of PMNs accumulate
secreting bacterium an advantage because they can eliminate other at the site. This accumulation is not a random event; rather, it is a
bacteria that would compete for nutrients and space. Some species directed migration of PMNs into the area needing their services.
of bacteria produce metabolic by-products that result in a micro- This migratory process is called chemotaxis. Several substances
environment hostile to potential pathogens. Vitamins and other serve as chemotactic agents, which attract phagocytic cells. These
essential nutrients are synthesized by certain bacteria in the intestine include certain complement components, a number of bacterial
and appear to contribute to the overall health of the host. products, products from damaged tissue cells, and products from
responding immune cells. The initial contact of the PMN with an
Phagocytosis invading organism may be random. As the organism causes the
Phagocytosis is an essential component in the resistance of the body’s defense mechanisms to respond via inflammation, directed
host to infectious agents. It is the primary mechanism in the host migration of phagocytes occurs (chemotaxis) (Fig. 2.3). The speed
defense against extracellular bacteria and numerous viruses and and magnitude of this response are easily visualized by recalling
fungi. The PMNs, macrophages, and monocytes are the body’s how quickly a splinter or similar injury becomes infected and
first line of defense. how much pus is produced.
The stem cells for neutrophils arise in the bone marrow, where
they differentiate to form mature neutrophils. During this matura- Attachment
tion, the cells synthesize myeloperoxidase, proteases, cathepsin, One of the most effective defenses bacteria have against phago-
lactoferrin, lysozyme, and elastase. These products are incorporated cytosis is the capsule. This structure prevents attachment of the
into membrane-bound vesicles called lysosomes. The lysosomes neutrophil’s membrane to the organism, which must occur before
appear as azurophilic granules on a Wright’s stain and contain ingestion can take place. Attachment is facilitated by the binding
enzymes, oxygen-reactive molecules, and other substances neces- of specific antibodies to the microorganism. The neutrophil
sary for the killing and digestion of engulfed particles. The PMN membrane has various receptors, including receptors for the Fc
also has receptors on the cell membrane for some complement portion of IgG1 and IgG3, and the C3b component of complement.
CHAPTER 2  Host-Parasite Interaction 37

Skin surface Diapedesis


Injury

Bacteria
Chemotaxins
PMNs Lumen of
blood vessel
FIG. 2.3  Phagocytosis: chemotaxis migration of phagocytes.

These three factors can bind to the invading microorganism,


resulting in the microorganisms being coated with one or more
of these factors. The receptor on the PMN for the particular factor
coating the bacterium binds to the factor and forms a bridge that
brings the bacterium into close physical contact with the leukocyte
membrane. The coating of the bacterium with antibody or comple-
ment components results in enhanced phagocytosis by the PMN.
This process or phenomenon is called opsonization.

Ingestion
The next step of phagocytosis is ingestion. This process occurs
rapidly after attachment. The cell membrane of the phagocytic FIG. 2.4  Transmission electron micrograph of engulfed bacterial
cell invaginates and surrounds the attached particle. The particle cells inside phagosomes (arrows).
is taken into the cytoplasm and enclosed within a vacuole called
a phagosome (Fig. 2.4). The phagosome fuses with lysosomes,
which are vacuoles containing enzymes and other antibacterial have frequent infections despite possessing high levels of serum
components. The combined structure is referred to as a phagolyso- antibody. Many of the organisms listed in Table 2.4 are common
some. The lysosomes release their contents into the phagosome. isolates, which is not surprising because they have developed a
The list of enzymes found within the lysosomes is long—more means to interfere with phagocytosis, increasing their pathogenicity.
than 20 enzymes, including proteases, lipases, RNase, DNase,
peroxidase, and acid phosphatase. Several of these are important Inflammation
in the killing and digestion of the engulfed bacterial cell. Inflammation is the body’s nonspecific response to injury or foreign
body. Fig. 2.5 illustrates the components involved in acute and
Killing chronic inflammatory responses. A hallmark of inflammation is
The phagocytosis of a particle triggers a significant increase in the accumulation of large numbers of phagocytic cells. These
the metabolic activity of the neutrophil or macrophage. This leukocytes release mediators or cause other cell types to release
increase is termed a metabolic or respiratory burst. The cell mediators. The mediators cause erythema as a result of greater
demonstrates increases in glycolysis, the hexose monophosphate blood flow, edema from an increase in vascular permeability, and
shunt pathway, oxygen use, and production of lactic acid and continued phagocyte accumulation, resulting in pus. The enzymes
hydrogen peroxide. The hydrogen peroxide produced at this time released by the phagocytes digest the foreign particles, injured
diffuses from the cytoplasm into the phagolysosome. It acts in cells, and cell debris. After the removal of the invader, the injured
conjunction with other compounds to exert a bactericidal effect. tissue is repaired.
In addition, other molecules from the lysosome have antimicrobial
action. They include lactoferrin, which chelates iron and prevents Immune Responses
bacterial growth; lysozyme; and several basic proteins. The usual The immune system response to infection is briefly discussed in
result is that a phagocytosed organism is quickly engulfed, killed, this chapter to provide the reader with an appreciation of its role
and digested. and complexity. The balance between health and infectious disease
Organisms that are “intracellular” (e.g., Mycobacterium tubercu- is complex and mediated by humoral and cellular factors. The
losis, Listeria monocytogenes, Brucella spp.) survive phagocytosis relative importance of each factor depends on the microbe, route
and can multiply within the phagocyte. Other defense mechanisms of infection, condition and genetic makeup of the host, and other
must play a major role in immunity to these intracellular organisms. factors yet to be clearly characterized. For example, a patient with
The importance of phagocytosis is seen in patients with defects AIDS becomes more susceptible to opportunistic organisms as
in the numbers or function of phagocytic cells. Such patients the immune system deteriorates.
38 PART 1  Introduction to Clinical Microbiology

Mast cell Fibroblast Macrophage

CONNECTIVE
TISSUE
CELLS

Smooth
muscle

Basophil
Platelets
VESSELS Clotting factors,
kininogens, and
complement
Polymorphonuclear Monocyte components Eosinophil
leukocyte Lymphocyte
Endothelium

Basement
membrane

CONNECTIVE
TISSUE
MATRIX
Elastic fibers Collagen fibers Proteoglycans
FIG. 2.5  Components involved in acute and chronic inflammatory responses. (From Kumar V,
Abbas AK, Fausto M: Robbins and Cotran pathologic basis of disease, ed 7, Philadelphia, 2005,
Saunders.)

Table 2.9 summarizes the defenses used by the human host


against infection. Classically, the term immunity has been defined
as a complex mechanism whereby the body is able to protect
Microbial biota,
itself from invasion by disease-causing organisms. This mechanism, cilia, mucus
known as the immune system, consists of numerous cells and Mucus and
protein molecules that are responsible for recognizing and removing Mucus, other secretions
ciliated cells
foreign substances. This general definition has been broadened
Saliva
over the years to mean a reaction to any foreign substance, including Skin, fatty acids,
proteins and polysaccharides as well as invading microorganisms. microbial biota
Research advances over the years have dramatically increased
our understanding of the basic immune response and given us
an appreciation of the cellular interrelationships. The immune
response can be divided into two broad categories: innate or natural
immunity with little or no specificity, and the adaptive or specific,
which is highly specialized.

Innate, or Natural, Immunity


Innate immunity, also referred to as natural or nonspecific immu-
nity, consists of several components. These include (1) physical
and chemical barriers such as the skin and mucous membranes,
(2) blood proteins that act as mediators of infection, and (3) a
cellular mechanism capable of phagocytosis such as neutrophils Gastro-
and macrophages and other leukocytes such as natural killer cells. Lower pH, intestinal biota,
Fig. 2.6 shows examples of the innate immune defenses located urogenital tract, gastric acid
at different body sites. This first line of defense has a limited microbial biota
capacity to distinguish one organism from another; however, Emptying of
previous exposure to a particular foreign substance is not required. the bladder
Physical barriers, mentioned earlier, may be as simple as the
keratinized outer layer of the skin. Also, the secretions along FIG. 2.6  Innate immune defenses located at different body
the mucous membranes and the ciliated epithelial cells of the sites.
CHAPTER 2  Host-Parasite Interaction 39

TABLE 2.9  Summary of Defenses of the Human or Animal Host to Infection and Evasion Mechanisms
Attributed to Various Microorganisms

Host Defense Mechanism of Evasion Microbial Example

Hydrodynamic flow Attachment Fimbriae, surface proteins, lipoteichoic acid,


pseudomembrane of diphtheria
Mucous barrier Attachment Mannose-sensitive fimbriae
Penetration Mucinase
Deprivation of essential nutrients Systems of high-affinity uptake Iron metabolism, siderophores
Lysozyme in secretions Resistance to lysis Substitution of peptidoglycan
Surface Igs Absent or low immunogenicity Hyaluronic acid, capsules
Antigenic heterogeneity Fimbriae, capsules, LPS, M protein
Masking of antigens Capsules, IgA-binding proteins
Destruction of immunoglobulins IgA protease
Unbroken surface (epithelial cell Penetration Neisseria gonorrhoeae, Shigella spp.
surface)
Serum defenses
Recognition by antibody Antigenic heterogeneity Fimbriae, capsules, LPS, M protein
Masking of antigen Capsules, Ig-binding proteins
Destruction of antibody Borrelia
Antigenic variation
Complement system Failure to activate alternative pathway Sialic acid capsules
Inactivation of complement components Cleavage of C3b
Resistance to bacteriolysis CoIV plasmid
Formation of abscess Bacteroides fragilis capsule
Localization
Fibrin tapping Fibrinolysis Streptococcus spp.
Abscess formation Collagenase, elastase Pseudomonas, Clostridium
Secondary immune response Nonspecific B-cell activation LPS, lipoprotein
Inhibition of delayed hypersensitivity Anergy of miliary tuberculosis
Rapidly fatal (toxin) Anthrax, plague, Clostridium
Phagocytosis Inhibition of chemotaxis Brucella, Salmonella, Neisseria, Staphylococcus, Pseudomonas
Inhibition of attachment and ingestion Capsules, M protein, Ig-binding proteins, gonococcal pili
Inhibition of metabolic burst Salmonella Typhi
Inhibition of degranulation Mycobacteria
Resistance to permeability-inducing Gram-positive cell wall, smooth LPS, polyanionic capsules
cationic protein
Resistance to oxidative attack Catalase, superoxide dismutase, carotenoid pigments
Escape from phagosome Mycobacterium bovis, Legionella pneumophila
Destruction of phagocyte Streptococcus pneumoniae, Streptococcus pyogenes,
Staphylococcus aureus, Pseudomonas aeruginosa

Ig, Immunoglobulin; IgA, immunoglobulin A; LPS, lipopolysaccharide.

respiratory tract promote trapping and removal of microorganisms. PRRs allows innate immune system cells to initiate the innate
In addition, many secretions provide a chemical barrier, such immune response. Phagocytic cells ingest and kill microorganisms,
as the acidic pH of the stomach and vagina. Saliva and tears whereas activated complement components contribute to a wide
contain enzymes such as lysozyme, and the sebaceous glands of variety of immunologic events, including promotion of attachment
the skin contain oils and fatty acids capable of inhibiting inva- and engulfment of bacteria by neutrophils (opsonization) and
sion by pathogenic organisms. The normal biota of these sites attraction of neutrophils to sites of infection (chemotaxis). Col-
adds another dimension to the host’s ability to resist invading lectively, these immunologic defense mechanisms, along with
pathogens. host tissue damage caused by the invading organisms, combine
Once the physical and chemical barriers to infection have been to produce an acute inflammatory response in the host. The
penetrated, nonspecific mechanisms of innate immunity become importance of natural immune mechanisms rests in their rapid
involved. Innate immune system cells have receptors, called pattern response to invading organisms. However, these mechanisms are
recognition receptors (PRRs), that recognize conserved sequences effective primarily against extracellular bacterial pathogens, playing
on the surfaces of microorganisms. The first type of PRR discovered only a minor role by themselves in immunity to intracellular
was the toll-like receptors (TLRs). Eleven different TLRs bacterial pathogens, viruses, and fungi.
(TLR1 to TLR11) have been identified in humans, and each has
specificity for components of different microorganisms. For Adaptive, or Specific, Immunity
example, TLR4 recognizes LPS from many species of gram- Adaptive immunity enhances the protective capability of innate
negative bacteria. Recognition of these microbial components by immunity. This arm of the immune response is specific for distinct
40 PART 1  Introduction to Clinical Microbiology

molecules, responding in particular ways to different types of multiple divisions and differentiates into plasma cells that actively
foreign substances and developing memory, which allows for a secrete proteins known as immunoglobulins, or antibodies. All
more vigorous response on repeated exposures to the same foreign antibody molecules derived from a single clone of B cells are of
invader. Lymphocytes and their products, such as antibodies, are a single specificity (recognize a unique antigen), identical to the
the major constituents of the adaptive or specific immune response. antibody receptor molecule on the original activated B cell. These
Antibodies are produced in response to immunogens, substances antibody molecules circulate in the bloodstream and lymphatics,
that are capable of inducing the adaptive immune response. An bathe body tissues, and bind to infectious agents or substances
antigen is a molecule that can bind specifically to an antibody to aid the host in eliminating them from the body. The detection
or T-cell receptor. and quantification of these antibody molecules, obtained from a
An interrelationship exists between the mechanisms of the patient’s serum, constitute the primary goal of diagnosing infectious
innate and the adaptive responses. For instance, inflammation, diseases through serologic methods.
a nonspecific response of the innate system, provides a signal
that triggers an adaptive immune response. The adaptive immune Classification and Characteristics of Antibodies
system enhances the protective mechanisms of the innate system. Antibody molecules, found in serum and other body fluids and
For instance, the activation of complement (a component of innate secretions, may be classified into one of five distinct immuno-
immunity) by invading bacteria is enhanced by the presence of globulin groups or classes. The classes differ from one another
specific antibodies (components of adaptive immunity). This in several ways, including chemical structure, serum concentration,
activation leads to phagocytic clearance and elimination of the half-life, and functional activity.
bacteria. The adaptive immune response adds a high degree of The immunoglobulin G (IgG) antibody class constitute about
specialization to the passive mechanisms of the innate response. 70% to 75% of the total serum immunoglobulin pool. Their half-life
The nature of the adaptive immune response varies according to in serum is about 3 to 4 weeks. IgG can cross the maternal placenta
the type of organism and is designed to eliminate it efficiently. to the fetus, conferring some protection in both the prenatal and
For example, antibodies are produced by B lymphocytes and the postnatal periods. Structurally, IgG is a protein with a molecular
plasma cells in response to bloodborne organisms and aid in their weight of about 150,000 consisting of four polypeptides (two
elimination. However, the response to phagocytosed immunogen is identical light chains and two identical heavy chains) bridged by
primarily by T lymphocytes that produce chemicals that enhance several disulfide bonds (Fig. 2.7). Although the amino acid sequence
the activities of the phagocytic cells. The nature and origin of of some regions of the polypeptides is nearly identical among all
these cells are described next. Most importantly, the specific IgG molecules (conserved regions), one of the ends of each
response remembers each time it encounters a particular foreign polypeptide is highly variable. These variable regions create two
immunogen. This is called immunologic memory. Subsequent active sites, fragment of antigen binding (Fab) on each IgG
exposure to that immunogen stimulates an increased and specific molecule (see Fig. 2.7). Thus IgG antibodies are said to be
defense. Ultimately, the adaptive or specific immune response is bivalent—capable of binding two antigen molecules.
the second line of defense and improves the first significantly. Antibodies of the immunoglobulin M (IgM) class account
Lymphocytes originate in the bone marrow from stem and for 10% to 15% of serum immunoglobulins. Their half-life in
progenitor cells. Lymphocytes mature and take up residence in serum is about 5 days, and IgM cannot cross the placenta. A
various body tissues and organs, including the thymus, lymph developing fetus in the second or third trimester as well as a
nodes, and spleen. They are a diverse group of cells that can be newborn may respond to an infectious agent with an IgM antibody
classified into two major types on the basis of cell surface response of its own. The IgM molecules are large; the molecule
markers—T (thymus-derived) cells and B (bone marrow–derived) has a molecular weight of about 900,000 and consists of a pentamer
cells. The uniqueness of these cells lies in the presence of specific or five basic subunits—each composed of two heavy chains and
cell surface receptor molecules that recognize and bind a unique
immunogen, activating the cell to divide, differentiate, and secrete
numerous effector substances. The millions of lymphocytes found Fab fragment
in the body have been preengineered during embryogenesis and
Light chain
throughout life in the primary lymphoid tissues to recognize a
vast array of substances as foreign while learning which substances
constitute self. Thus the result of an encounter with the antigen Disulfide bonds s
s
is an expanded clone or clones of activated lymphocytes.
Antigen
Nature of the Immune Response to Heavy
s s
s s

binding
Infectious Agents chains
regions
Although both humoral immunity and cell-mediated immunity Fc fragment
are important in protecting humans from a wide variety of infectious
s
s

agents, each contributes differently, in terms of overall importance


according to the type of pathogen and virulence mechanisms.
Although B lymphocytes play the predominant role in the humoral Light chain
immune response, T lymphocytes mediate cellular immunity. Fab fragment
Each B lymphocyte has surface receptors that recognize only one
type of antigen. After antigen binding, the B lymphocyte undergoes FIG. 2.7  Immunoglobulin G.
CHAPTER 2  Host-Parasite Interaction 41

Disulfide bonds Primary Secondary

IgG
IgG-like

Antibody response
subunit

IgM
J chain

Time
First Second
exposure exposure
to antigen to antigen
Antigen-binding sites
FIG. 2.9  Primary versus secondary response.
FIG. 2.8  Immunoglobulin M.

IgE-specific serologic tests for the diagnosis of a few parasitic


TABLE 2.10  IgG Versus IgM agents have been developed. The role of serum IgD during infection
is unknown, except that it functions as a receptor on B lymphocytes
Immunoglobulin Class
for antigen.
Property IgG IgM
Primary and Secondary Antibody Responses
Molecular weight 150,000 900,000
Number of 4-polypeptide subunits 1 5
After exposure to an infectious agent, the host’s acquired humoral
Number of antigen-binding sites 2 10 immunity may respond through the production of various classes
Serum concentration (mg/dL) 800–1600 50–200 of antibody directed to one or more antigens associated with the
Percentage of total immunoglobulin 75 10 agent. If the host has not been previously exposed to the antigen,
Ability to cross placenta + − a primary immune response, characterized by the relatively rapid
Half-life (days) 23–25 5–8 appearance of IgM antibodies, occurs. IgM levels usually peak
in 1 to 2 weeks followed by a gradual decline to undetectable
levels over the next few months. At the time when IgM levels
two light chains (similar to an IgG molecule) and linked to another have nearly peaked, IgG (and in some cases IgA) antibodies become
polypeptide chain (J chain) by disulfide bonds (Fig. 2.8). The detectable and their levels continue to increase for about 1 month,
IgM molecule has up to 10 antigen-binding sites available. Both surpassing peak IgM levels. IgG levels remain elevated for months
IgG and IgM antibodies are commonly assayed in a variety of and then decline slowly, often persisting at low but detectable
serologic tests. The differences in size and configuration of IgG levels for years (Fig. 2.9).
and IgM molecules result in differences in functional activity of A subsequent exposure to the same antigen elicits a secondary
the molecules in serologic tests (Table 2.10). or anamnestic immune response, characterized by a rapid increase
Immunoglobulin A (IgA) antibodies represent 15% to 20% in IgG levels, a prolonged elevation, and a more gradual decline
of the total serum immunoglobulin pool. IgA constitutes the (see Fig. 2.9). IgM synthesis plays a minor role in a secondary
predominant immunoglobulin class in certain body secretions, immune response. Serologic tests that are designed to detect
such as saliva, tears, and intestinal mucosa. Because of this separately IgG and IgM antibodies take advantage of the differences
association of IgA with mucosal surfaces, it provides protection in IgM production between a primary and a secondary immune
against microorganisms invading at those sites. Serum IgA occurs response. Thus a positive test result for IgM is considered indicative
primarily as a dimer composed of two subunits (each similar to of a current or very recent infection, whereas the presence of IgG
an IgG molecule) linked together by a J chain. However, when alone suggests a previous infection or exposure. Similarly, the
found in secretions, the molecule also contains a secretory presence of significant levels of IgM (with or without IgG) in a
component that stabilizes the molecule. Although significant newborn suggests in utero infection (IgM can be synthesized by
increases in serum IgA levels may occur in association with certain the fetus and cannot cross the placenta), whereas IgG only in the
infections, the function of serum IgA is unclear, and few serologic newborn is indicative of passive maternal transfer of IgG across
tests for the diagnosis of infectious disease are designed specifically the placenta, not in utero infection.
to detect IgA antibody.
The remaining two immunoglobulin classes, immunoglobulin Cell-Mediated Immune Response
D (IgD) and immunoglobulin E (IgE), are found in very low In contrast to humoral immunity, the primary effector cell in the
concentrations in serum (<1%). Immunoglobulin E (IgE) levels cell-mediated immune response is the T lymphocyte. The T cell
increase during infection by numerous parasites and may play a does not secrete antibody molecules; however, the result of antigen
role in eliminating these infectious agents from the host. Total binding, activation, cell division, and differentiation of the T cell
serum IgE levels may increase during parasitic infection, and is the production of a number of low-molecular-weight proteins
42 PART 1  Introduction to Clinical Microbiology

known as lymphokines. Lymphocytes affect their immunologic the infection occurs during fetal life or in a neonate. For example,
function through direct cell-to-cell contact or through the activity although the fetus, when infected by the rubella virus, responds
of the lymphokines on other cells, such as macrophages. The and makes its own antibodies, it is believed that the antibodies
cell-mediated immune response can target host cells harboring are often weak and unable to contain the infection. Because the
intracellular pathogens for destruction. Killing the host cell aborts T-cell response is also poor, the virus is able to persist in the fetus
the replication of the pathogen. The measurement and diagnostic and during the neonatal period. Hence microorganisms can persist
significance of cell-mediated immunity are beyond the scope of if they are able to survive in the host during prenatal infections
this book, and cellular immune function tests generally are not without producing an overt form of disease.
performed in microbiology or serology laboratories. Certain microorganisms do cause immunosuppression in the
In essence, the wide variety and complexity of infectious agents infected individual. The decreased immune response is often
necessitate flexibility in the immune mechanisms of the host. more far-reaching than simply to the immunogen of the involved
Immunity to extracellular bacterial pathogens, such as S. aureus microorganism. Viruses, certain bacteria, and protozoans are
and S. pyogenes, is mediated primarily by antibody functioning examples of infectious agents likely to cause immunosuppression
either alone (neutralization of toxins and blocking the binding in the infected host. These agents multiply in macrophages or
of bacteria to host cells) or with complement and neutrophils in lymphoid tissues. The exact mechanisms of immunosuppres-
(chemotaxis and phagocytosis of bacterial cells). Immunity to sion have not been defined for all infecting agents. However,
intracellular bacterial pathogens, such as M. tuberculosis, is primar- individuals infected with viruses such as Epstein-Barr virus and
ily cell mediated, through the activities of T lymphocytes, lym- cytomegalovirus show depressed T-cell or antibody responses
phokines, and macrophages. If antibody is produced, it plays little to other unassociated immunogens. Reduced immunoreactivity
role in eliminating this pathogen because the pathogen is sequestered caused by an infectious agent is exemplified by that caused by
(hidden) intracellularly where antibody cannot reach. the HIV, which targets CD4+ T cells. Because HIV destroys the
Meanwhile, viral infections often elicit both humoral and cell- major cells that defend the host against viral, fungal, and protozoan
mediated immune responses. Antibody may bind directly to and infections, the infected person becomes susceptible to opportunistic
neutralize viral particles (render the virus noninfectious—unable infections caused by these organisms.
to infect cells) when they are found free in the bloodstream or Certain organisms are able to change their surface antigens
other body fluids. For example, central nervous system infection systematically during the course of a single infection, even while
by arboviruses (which cause encephalitis) or by certain enterovi- inside the host, evading the host immune defenses. This occurs
ruses (which cause meningitis) can be prevented if neutralizing in relapsing or recurring fever infections with Borrelia recurrentis.
antibody against these organisms is present when the virus reaches After an initial incubation period of 2 to 15 days following
the bloodstream and before it enters the central nervous system. transmission of the spirochetes from a tick or louse, large numbers
However, some viruses cause infections that spread cell-to-cell (e.g., of the organism are found in the blood. The infected individual
herpes simplex virus); they would not be subject to the neutralizing experiences high temperature, rigors, severe headache, muscle
effect of antibodies. In these cases, cell-mediated immunity plays a pains, and weakness. The febrile period lasts for about 3 to 7
predominant role in eliminating the agent. Immunity to both fungal days but ends quickly with the induction of an immune response.
and parasitic infections is also primarily cell mediated; antibody However, a similar but less severe course of symptoms recurs
plays little or no role in prevention of or recovery from infection several days to weeks later. The relapses are caused by antigen
resulting from these agents. Although antibodies may not have variation by the borreliae. Spirochetemia worsens during febrile
protective value for certain infectious agents, they nonetheless may periods and diminishes between recurrences.
have diagnostic and prognostic value in serologic tests. Methods for Intracellular parasites such as Brucella spp., Listeria spp.,
detecting the presence and significance of antibodies for diagnostic and mycobacteria avoid the host’s immune response by surviv-
purposes are discussed in Chapter 10. ing inside infected cells. This is another evasion strategy used
by microorganisms—making themselves unavailable as targets
Mechanisms by Which Microbes May to the host’s immune system. Macrophages that engulfed these
microbial species protect them from antibacterial substances and
Overcome Host Defenses support their growth inside the macrophage. For example, during
Infectious agents are able to establish disease despite the host’s the exoerythrogenic cycle in liver cells, the parasite Plasmodium
defenses. The strategies that microbes employ to counter the host’s spp. avoids being a target for the immune response. Malarial
defenses consist of inducing tolerance or immunosuppression, parasites also infect red blood cells and cause disease while being
change in the appropriate target for the immune response, and protected from the host’s defense mechanisms.
antigenic variation. Sometimes the host immune system fails to Hosts produce antibodies against specific immunogenic stimuli
respond to specific immunogens of the infecting microorganisms. as part of an immune defense. However, if the antibodies produced
This failure to respond is not necessarily due to immunosuppression. against an infecting organism are of low avidity or have a weak
The inability to induce an immune response to a microbial antigen, antimicrobial effect on the infecting organisms, the ability of the
referred to as tolerance, may be due to a “feeble antigen”—that infected host to control the infection is decreased. Therefore in
is, an immunogen or immunogenic component of an organism certain microbial infections, antibodies, although produced, provide
that is incapable of stimulating an immune response from the little or no protection to the host.
host. The host fails to initiate a response or is sometimes slow in Similarly, interferons play a significant role in the host defense
responding. This lack of response suggests tolerance to this against foreign invaders. The main function of these cytokines is
immunogen. Tolerance to an organism may also develop when to stimulate the expression of major histocompatibility complex
CHAPTER 2  Host-Parasite Interaction 43

(MHC) proteins by T cells. Interferons are also antiviral;


11. What is the difference between true pathogens and opportunistic
interferon alpha and interferon beta work against double-stranded pathogens?
RNA viruses, whereas interferon gamma is produced after the 12. How does inflammation play a role as a host immune defense?
activation of T cells. There are instances when viruses escape 13. What is the difference between exotoxins and endotoxins?
the effects of interferons, either because they are resistant to the 14. What cells and soluble mediators are involved in the innate immune
antiviral effects or because the induction of interferon in the host response versus the adaptive immune response? What cells and
soluble mediators are involved in the humoral response versus
does not take place. For example, vaccinia virus is able to resist
the cellular response?
the effects of interferons by inactivating interferon gamma. Other 15. How can microorganisms evade the immune response?
viruses can produce persistent infections because these viruses 16. How are organisms transmitted?
do not induce interferon production. 17. What are the steps involved in phagocytosis, and how can
microorganisms evade each step?
18. What are the differences between and functions of IgG and IgM?
19. What are zoonoses, and what organisms are considered zoonotic
Points to Remember agents?
■ Humans do not exist in a sterile environment. Colonization of the
body by microorganisms begins at birth.
■ The usual microbial biota present at each site in the body is dictated
by nutritional and environmental factors. BIBLIOGRAPHY
■ Some species of the usual microbial biota may be opportunists, Andreasen, A. S., et al. (2008). Human endotoxemia as a model of systemic
capable of causing disease in an immunocompromised host. inflammation. Current Medicinal Chemistry, 15, 1697.
■ The usual microbial biota benefits the normal host by priming the Chapel, H., et al. (2006). Essentials of clinical immunology (5th ed.).
immune system, outcompeting potential pathogens for nutrients, Malden, MA: Wiley-Blackwell.
and creating a hostile environment for other microbes. Hentschel, U., Steinert, M., & Hacker, J. (2000). Common molecular
■ True pathogens cause disease in all individuals. mechanisms of symbiosis and pathogenesis. Trends in Microbiology,
■ The host defense system against microorganisms includes physical, 8, 226.
mechanical, and chemical barriers; components of the innate immune Hiemstra, P. S., & Bals, R. (2004). Series introduction: innate host defense
system, such as phagocytes, complement, cytokines, and the products of the respiratory epithelium. Journal of Leukocyte Biology, 75, 3.
of inflammation; and the components of the adaptive immune Jandhyala, S. M., et al. (2015). Role of the normal gut microbiota. World
response. Journal of Gastroenterology, 21, 8787.
■ Microbes have mechanisms to evade the host’s defenses, including Kamradt, T., & Mitchison, N. A. (2001). Tolerance and autoimmunity.
ability to evade phagocytosis, production of enzymes and exotoxins, The New England Journal of Medicine, 344, 655.
ability to induce tolerance in the adaptive immune system or to Kumar, V., Abbas, A., & Aster, J. (Eds.), (2010). Robbins and Cotran
suppress the adaptive immune system, and ability to avoid recognition pathologic basis of disease (8th ed.). Philadelphia: Saunders.
by the adaptive immune system by varying the antigens present Lanzavecchia, A., & Sallusto, F. (2000). Dynamics of T lymphocyte
on the surface of the microorganism. responses: intermediates, effectors, and memory cells. Science, 290, 92.
Mandell, G., Bennett, J., & Dolin, R. (2010). Mandell, Douglas, and
Bennett’s principles and practice of infectious diseases (7th ed.). New
York: Churchill Livingstone.
McKenzie, S. B. (2010). Clinical laboratory hematology (2nd ed.). Upper
Learning Assessment Questions Saddle River, NJ: Prentice Hall.
1. How did the patient in the Case in Point at the beginning of this Medzhitov, R., & Janeway, C., Jr. (2000). Innate immunity. The New
chapter develop diarrhea caused by Clostridium difficile? England Journal of Medicine, 343, 338.
2. What is the difference between resident and transient biota? Mims, C., Nash, A., & Stephen, J. (2001). Mims’ pathogenesis of infectious
3. What organisms would be expected to be potential contaminants disease (5th ed.). San Diego: Academic Press.
of improperly collected blood cultures? Moran, N. A., & Baumann, P. (2000). Bacterial endosymbionts in animals.
4. What is a carrier? Current Opinion in Microbiology, 3, 270.
5. What is the significance of the carrier in the pathogenesis of Nelson, K. E., & Masters Williams, C. F. (2006). Infectious disease
disease? epidemiology: theory and practice (2nd ed.). Burlington: Jones &
6. What determines the composition of the indigenous biota at Bartlett.
different body sites? Nemazee, D. (2000). Receptor selection in B and T lymphocytes. Annual
7. Of the following, which would most likely cause an opportunistic Review of Immunology, 18, 19.
infection in the genitourinary tract of a woman of childbearing Owen, J. A., Punt, J., & Stranford, S. A. (2013). Innate immunity. In
age? Kuby immunology (7th ed.). New York: W.H. Freeman and Company.
a. Chlamydia trachomatis Sompayac, L. M. (2008). How the immune system works (3rd ed.). Malden,
b. Neisseria gonorrhoeae MA: Wiley-Blackwell.
c. Candida albicans Stevens, D. L., et al. (2015). Clostridium. In J. H. Jorgensen, et al. (Eds.),
d. Trichomonas vaginalis Manual of clinical microbiology (11th ed., p. 940). Washington, DC:
8. A long-term resident species of bacteria in the gastrointestinal ASM Press.
tract produces vitamin K, which is required for blood clotting in Versalovic, J., et al. (2015). The human microbiome. In J. H. Jorgensen,
mammals. What type of host-parasite relationship does this et al. (Eds.), Manual of clinical microbiology (11th ed., p. 226).
represent? Washington, DC: ASM Press.
9. How does the resident microbial biota help protect the host from Wilson, J. W., et al. (2002). Mechanism of bacterial pathogenicity.
bacterial infection? Postgraduate Medical Journal, 78, 216.
10. In the Case in Point, what virulence factor(s) did the infecting Winn, W., et al. (Eds.), (2006). Koneman’s color atlas and textbook of
organism use? diagnostic microbiology (6th ed.). Philadelphia: Lippincott Williams
& Wilkins.
CHAPTER

3  
The Laboratory Role in
Infection Control
Sarojini R. Misra

CHAPTER OUTLINE
■ GENERAL CONCEPTS IN INFECTION PREVENTION AND Environmental Culturing
CONTROL PRACTICE Reporting
Infection Prevention and Control in Health Care Settings ■ EDUCATION
Infection Control Surveillance Laboratory Scientists and Infection Prevention and Control
Frequently Identified Microbes Practitioners
■ OUTBREAK INVESTIGATION Safety
Local Outbreaks ■ EMERGING AND REEMERGING PATHOGENS
Widespread Outbreaks Emerging Pathogens
Steps of an Outbreak Investigation Reemerging Pathogens
Investigation Support from the Laboratory Response Plans

OBJECTIVES
After reading and studying this chapter, you should be able to:
1. Delineate the various roles the laboratory and laboratory scientist 7. List microorganisms commonly encountered in health care–
may play in an infection prevention and control program. associated infections in hospitals.
2. List the facilities and settings in which an infection prevention 8. Describe agencies and entities to which an infection prevention and
control program is important. control program would provide reports.
3. Define surveillance and the ways in which surveillance is conducted. 9. Discuss educational activities that encompass an infection
4. Describe outbreak investigation and the steps followed in an prevention and control program.
outbreak investigation. 10. Correlate the activities of the laboratory with the safety and
5. List the ways in which a microbiology laboratory can support an prevention activities of an infection prevention and control program.
outbreak investigation. 11. Describe the roles of the microbiology laboratory and the
6. Define when and how environmental culturing is appropriate in an epidemiology program in preparing for potential bioterrorism
infection prevention and control program. activities.

isolates of the resistant Acinetobacter. The infection prevention


Case in Point and control practitioner, microbiology laboratory scientist, ICU
An 82-year-old man was admitted to an intensive care unit (ICU) nurse manager, and physician in charge of the ICU met to review
from an extended care facility (ECF); he was confused and the situation. Contact precautions were implemented for the
short of breath. His chest radiograph revealed consolidation in patients. Education was initiated that highlighted hand hygiene
the left lower lobe. A bronchoalveolar lavage was performed, practices, and environmental cultures were taken. The occurrence
and the specimen was sent to the laboratory for culture and of the multidrug-resistant strain of Acinetobacter decreased and
antimicrobial susceptibility testing. An endotracheal tube was eventually no new cases were observed.
placed, and the patient was attached to a ventilator for respiratory
support. The cultures grew a multidrug-resistant strain of Aci-
netobacter baumannii. Within 3 days, two more patients in the Issues to Consider
ICU had respiratory cultures positive for A. baumannii. Over the After reading the patient’s case history, consider:
next 4 days, three additional ventilated patients had tracheal ■ The role of the microbiology laboratory in an infection
aspirate cultures that grew the same microbe. prevention and control program
The microbiology laboratory scientist notified the infection ■ The surveillance of health care–associated infections (HAIs)
prevention and control team of the unusual occurrence of several and the required laboratory support

44
CHAPTER 3  The Laboratory Role in Infection Control 45

■ The information needed in an outbreak investigation which are characteristics seen in clinical laboratory scientists and
■ The role of the laboratory scientists as an educator in technologists.
infection prevention and control
■ Bioterrorism and emerging pathogens Infection Prevention and Control in
Health Care Settings
Infection prevention and control practices are important in many
Key Terms health care settings. Table 3.1 lists different health care environ-
ments in which infection control practices play a vital role. In
Antibiogram Index case all the settings, the goal is to prevent the spread of infectious
Antimicrobial pressure Infection control risk agents and reduce dissemination of infections by assisting with
Baseline data assessment assessment, planning, implementation, and evaluation of national
Case definition Infection prevention and
infection control practices. In public health or community settings,
Catheter-associated urinary control practitioner (IPCP)
the transmission of microorganisms occurs through many events
tract infection (CAUTI) Infection rate
in daily living. Microbes are spread at home, in daycare centers,
Central line–associated Intravascular device
in schools, in crowds, and by individuals. There are sexually
bloodstream infection Molecular epidemiology
(CLABSI) Outbreak transmitted diseases (STDs), diseases spread by respiratory routes,
Communal living Outbreak investigation diseases spread by contact, foodborne diseases, and waterborne
Community-acquired infection Prevalence diseases, all of which occur in the public arena.
Data mining Public health In acute care hospitals, infections occur as a surgical site
Emergency response plans Pulsed-field gel infection (SSI), central line–associated bloodstream infection
Emerging pathogens electrophoresis (PFGE) (CLABSI), and catheter-associated urinary tract infection
Endotracheal Reemerging pathogens (CAUTI), in ventilator-associated pneumonia (VAP). They are
Environmental cultures Standard precautions found in adults, teenagers, children, neonates, healthy individuals,
Epidemiologic curve Surgical site infection (SSI) and immunocompromised patients. Infections occur as community-
Extended care facility (ECF) Surveillance acquired infection, health care–associated infection (HAI), and
Hand hygiene Targeted surveillance iatrogenic infection (infections due to the activities of a health
Health care–associated Total surveillance care provider, e.g., physician). HAIs and iatrogenic infections
infection (HAI) Ventilator-associated occur because of instrumentation, increased use of antimicrobial
Iatrogenic pneumonia (VAP) agents, breaks in aseptic techniques, and lack of hand hygiene.
Ambulatory care settings include outpatient surgery, chronic
dialysis, and infusion centers, as well as physician offices and

E
emergency care facilities. Infection control must be practiced in
very year, an estimated 722,000 health care–associated these settings, even though the patients may not be seen again.
infections (HAIs) occur that result in 75,000 deaths. HAIs In ECFs, patients are frequently immunosuppressed by disease,
are infections that originate in health care facilities. These age, or therapy. ECFs include skilled nursing facilities, nursing
infections cost the health care system countless dollars. An effective homes, assisted living centers, rehabilitation centers, and hospice
infection control program must be established in a health care care settings. Because of their suppressed defenses, these patients
setting to recognize and prevent HAIs. This chapter covers the are prime candidates for acquiring infections. Patients who are at
settings, activities, and analytic practices involved in implementing home and receive home care from family or a professional home
an effective infection control program, specifically from the labora- care provider sometimes acquire infections. Most often, home care
tory scientists’ perspective. involves intravascular-related or device-related care. As in ECFs,
the immune defenses of these patients are often suppressed by
General Concepts in Infection their disease or therapy.
Prevention and Control Practice
The clinical laboratory and the laboratory scientist play a vital
role in the functioning of an effective infection prevention and TABLE 3.1  Health Care Settings Involving Infection
control program. In some settings, the laboratory scientists Prevention and Control
provide supportive data; in other settings, they may function
as a part-time infection prevention and control practitioner Setting Description
(IPCP) as well as a part-time laboratorian. Not infrequently, the Public health Community settings (e.g., daycare centers, schools)
laboratory scientist may leave the laboratory and become the Acute care Hospitals
IPCP or work as a member of an infection prevention and control Ambulatory care Outpatient surgery, chronic dialysis and infusion
department. centers, physicians’ offices, emergency care
As the term implies, infection prevention and control involves facilities
Extended care Skilled nursing facilities, nursing homes, assisted
activities aimed at preventing and reducing the dissemination of
living centers
infections in persons in broad and varied settings. The activities Home care Skilled nursing provided in the home
range from surveillance activities, to outbreak investigation, to Communal living Prisons and jails, behavioral health facilities
education. They require an inquisitive and analytic mind, both of
46 PART 1  Introduction to Clinical Microbiology

Communal living programs can also be settings in which Infections are generally defined by site, risk factors, and pro-
infections occur. These community living programs might include cedures, as shown in Table 3.2; primary infections are infections
prisons and behavioral health facilities. In these facilities, infections that occur at one site—for example, a primary bacteremia. A
might be found that are spread by contact (illicit tattooing), by primary bacteremia would not have another site as the source
intimate contact with blood and body fluids, or simply due to of the infection, as would be seen in urosepsis, in which case
overcrowding. the primary site would most likely be the urinary tract. Using
The microbiology laboratory may receive specimens from any primary bacteremia as an example, the laboratorian recognizes a
of these settings, all of which represent opportunities for infections bloodstream infection (BSI) by the recovery of clinically significant
to spread and opportunities for infection control to be practiced. organisms from blood cultures, whereas the IPCP determines
Microbiology laboratory scientists must know who their clients whether the infection was health care–associated and primary. If
are and be poised to insert themselves into infection control the infection control program was monitoring CLABSIs, the IPCP
activities for the varied customer base. would determine whether the BSI was related to an intravascular
device. For this reason, the site of the blood culture draw (e.g.,
Infection Control Surveillance peripheral internal jugular catheter, femoral catheter) is important
Infection control programs include many activities aimed at prevent- to include with the specimen description on the microbiology
ing the spread of infections. To determine where to direct these laboratory report.
preventive activities most efficiently, an effective infection control The incidence of SSIs is frequently reviewed by the IPCP. To
program must collect data on existing infections. By comparing the laboratorian, the isolation of pathogens from the surgical site
baseline figures with periodic numbers, the IPCP can recognize an is indicative of postsurgical wound infection; the IPCP determines
outbreak (sudden increase in the occurrence of a disease), upward whether the infection was superficial or deep, or an organ space
trends, and positive effects of interventions. Ongoing, systematic infection. Therefore the site of the wound culture is an important
collection of these data and the analysis and interpretation of the component of the specimen description on the laboratory report.
details surrounding a disease or event is termed surveillance. The The IPCP relates the infection to other risk factors, such as the
laboratory contributes to these records on a daily basis. length of surgery, degree of contamination of the surgical site
(gunshot wound to the abdomen versus a hernia repair), and whether
Surveillance Definitions any breaks in surgical technique occurred.
An important part of infection prevention and control is surveil- Because urinary tract infections (UTIs) are determined by
lance; data can be compared internally, locally, and nationally if microbial growth from a urine specimen, the method of urine
standard definitions are used. Table 3.2 lists the common terms specimen collection should be described and differentiated between
used in surveillance and their definitions. The Centers for Disease a voided clean-catch urine specimen and a specimen collected by
Control and Prevention (CDC) has recommended definitions catheterization. The definition of a health care–associated UTI
that are generally used in the infection control profession. Most includes the presence or absence of a urinary catheter. Health
IPCPs are concerned about infections that are acquired within the care–associated pneumonias are difficult to assess from the perspec-
health-care facility. These infections occur after the patient arrives tives of the laboratory and IPCP. From the IPCP perspective, the
(generally not within the first 48 hours) and were not incubating in criteria to define a hospital-acquired pneumonia include the
the community before the patient arrived. HAIs can be followed presence of an endotracheal tube or some other respiratory device,
within a setting to determine when infection numbers increase and whether the pneumonia was incubating or present when the
above baseline, thereby requiring investigation and intervention. patient arrived. In the laboratory, the microbiologist should include
in the specimen description on the laboratory report the type and
source of the respiratory specimens (e.g., bronchoalveolar lavage,
tracheal aspirate, expectorated sputum). With sputum specimens,
TABLE 3.2  Surveillance Definitions the quality of the specimen as evaluated by the presence of white
blood cells and single bacterial morphotypes, and the absence of
Term Definition
squamous epithelial cells, is an important adjunct. The use of
Primary infection Infection related to a single, specific these definitions is important for guiding the IPCP and allowing
site, not from multiple sites the comparison of data.
Bloodstream infection Infection found in the bloodstream General or Targeted Surveillance.  Infection prevention
Central line–associated Bloodstream infection related to the and control programs may have all the HAIs within the setting
bloodstream infection presence of an intravascular device
under surveillance or, because of budget or personnel constraints,
such as a central venous catheter
Surgical site infection (SSI) Infection at a site where a surgical
they may be observing for only specific infections. In total surveil-
procedure was performed; usually lance programs, all infections are recorded and analyzed to
risk is stratified by length of surgery, determine whether the infections are health care–associated. Risk
site of infection, and degree of assessments determine whether the situation is a high or low risk
anticipated contamination and whether the infections are occurring in an unanticipated number.
Urinary tract infection Infection of the urinary tract, frequently The infection rate, the speed of spread or frequency of an infectious
associated with a urinary catheter
disease within a population, is determined and analyzed to establish
Ventilator-associated Pneumonia in a patient associated with
pneumonia a ventilator device such as an whether the number of infections has increased or decreased.
endotracheal tube or a tracheotomy These rates can be compared with previous rates in the setting or
with rates in similar local or national health care facilities.
CHAPTER 3  The Laboratory Role in Infection Control 47

There are several ways of calculating infection rates. For laboratory-generated results, generally from a laboratory informa-
example, the SSI rate is defined as the number of infections per tion system (LIS). For example, the IPCP or microbiologist reviews
number of procedures expressed as a percentage. Infection rates positive cultures and categorizes them into groups based on sites,
of CLABSIs and VAPs are calculated as the number of infections units, organisms, or procedures. From this categorization, the
per 1000 device days. If an unexpected change in rates is seen, IPCP may recognize a trend, such as an upward trend in the
the IPCP may conduct further investigations to determine, if number of Pseudomonas aeruginosa isolates from a skilled nursing
possible, the cause of the change and to propose a course of action facility or an increased number of positive cultures in laminectomy
to reverse the situation. From the results of the investigation, the patients. Even more important is when the IPCP determines that
IPCP would recommend the appropriate intervention, such as a there are more S. aureus isolates than usual in the home health
change in procedure, education, or increased emphasis on hand care setting. In this case an initiation of detailed investigations
hygiene. The IPCP would continue to monitor the infection to may be indicated. These fact-gathering activities are more formal
determine whether the infection rate decreases. and specific than the informal monitoring done by the laboratory
Unlike in total surveillance, targeted surveillance involves a scientist on the basis of experience.
close watch of only specific, high-risk, high-volume procedures.
For example, the program may follow only CLABSIs in the ICU
and SSI in gynecologic surgical procedures, whereas another Case Check 3.1
program may conduct surveillance on VAP in the neonatal ICU In the Case in Point at the beginning of the chapter, a multidrug-resistant
and adult ICU. Which infections to target would largely be based strain of Acinetobacter baumannii was recovered from the bronchoalveolar
on review of previously ascertained data on infection rates (baseline lavage of the index case. Within days, respiratory cultures from a number
data), on recognition of high-volume and high-risk procedures of patients grew A. baumannii. This upward trend was reported to the
within the setting, and sometimes on requests by others (e.g., IPCP and intervention measures were administered.
insurance companies, physicians). This process is often termed a
risk assessment. The targeted surveillance may change from year
to year, depending on recognized outbreaks or changes in the Statistical correlation by interfacing with the LIS offers a more
number of procedures performed. All the surveillance data must sophisticated data review system for the purpose of data mining.
be carefully collected, use high-quality laboratory data, and be In this type of data analysis, a multitude of events from a database
protected from legal discovery, as determined by the risk manage- are searched, analyzed, and then reported to the IPCP. Data mining
ment team for the setting. removes much of the daily details of the culture result review. It
Baseline Data.  To recognize when infections constitute an also adds other health care parameters to the analysis that are
outbreak or when an upward trend is occurring, the infection frequently not available to the IPCP without detailed examination.
prevention and control program must have established baseline This type of analysis may require the LIS and other hospital
data; that is, the historical occurrence of infections over time. information systems to interface with the data mining system.
Data at the national level derived from the National Healthcare No matter how the culture results are screened and analyzed,
Safety Network (NHSN) may also serve as the baseline data. a timely review is an integral component of an infection control
However, baseline data from within the specific health care setting program. Not only are the patients and their health care providers
more accurately reflect events within that setting. The baseline dependent on high-quality microbiology results, but the effective-
data are collected and analyzed by numbers, by percentages, or ness of an infection control program is also dependent on these
by rates per 1000 device days. Baseline data are an important results.
component of the decision making process regarding which types Cases.  The laboratory must be attuned to what is happening
of infections to target. in the community and in various community settings in regard
to infection control issues. Infection control in the public health
Data Gathering arena affects the local microbiology laboratory. For example,
Culture Review.  Many infection control programs rely heavily there may be an increase in the number of cases of whooping
on a review of culture results, which is initiated informally by the cough in surrounding counties. Even though the laboratory in the
laboratory scientist, who may then inform the IPCP of what appears immediate county may not have seen any Bordetella pertussis
to be an increased number of specific isolates. For example, the isolates, it needs to be aware of these other cases. Similarly, the
laboratory scientist may notice the isolation of an increased number anticipated appearance of influenza cases is a seasonal occurrence
of Serratia marcescens or Salmonella spp. isolates, which may that also influences the activities of a microbiology laboratory. The
be infrequently seen in this particular health care facility. Or the laboratory can be proactive in educating health care providers on
laboratory scientist might think that there is an unusual number of specimen collection and transport if those are unique to a specific
methicillin-resistant Staphylococcus aureus (MRSA) isolates from public health concern. Awareness of infection control activities
skin sites in a prison setting. The appearance of an increase in the within the public health setting allows the laboratory to acquire
number of surgical site specimens with various microorganisms the necessary media or reagents to meet emerging needs.
may trigger a call to the IPCP. These are informal data-gathering Potential infection control issues that might become apparent
activities that are significant to the infection control program. To within a setting can drive activities in the microbiology labora-
report data, laboratorians use their experience and sense of what tory. For example, a product used in the health care setting is
is usual regarding types of specimens and microbes encountered. recalled because of suspected bacterial contamination. Effective
A more formal data analysis involves the daily review of culture communication between the infection control program and micro-
results reported by the laboratory or IPCP. Such a review requires biology laboratory is needed to establish screening techniques
48 PART 1  Introduction to Clinical Microbiology

in anticipation of cases involving contaminated products that adjunct to an infection control program by providing a multiplicity
might arise in the health care setting. Laboratory scientists need of types of data.
to increase their knowledge of the specific product, types of
specimens to anticipate, and expected isolates, which may be Frequently Identified Microbes
unique or previously unrecognized by the laboratory. Collaboration Although the types of microorganisms seen in HAIs vary from
among the microbiology laboratory, infection prevention and control setting to setting, common pathogens are encountered. This discus-
program, and health care customer becomes paramount. Reporting sion of the laboratory’s role in infection control addresses some
of community-acquired cases to the laboratory and by the laboratory of the common microbes to focus the attention of the laboratory
to the infection control team is an important tool in surveillance scientist on in the health care setting and typical body sites they
activities. infect. Table 3.3 lists some of the most common organisms identi-
Laboratory Support and Data Gathering. Besides fied. The most frequent HAIs are pneumonia, SSIs, gastrointestinal
providing culture results, the microbiology laboratory also pro- illness, UTIs, and primary BSIs.
vides the IPCP with other details. These may include specimen
contamination rates, numbers of isolates per site, and/or number Public Health and Community Setting
of isolates per unit within the health care facility. Knowledge of The laboratory serving a public health or community setting is
specimen contamination rates helps the IPCP with the interpretation likely to identify infectious diseases of infection control importance
of culture results and provides guidance in developing educational that are less frequently seen in other health care situations. Some
activities to improve the collection of quality specimens. For of the microbes frequently associated in waterborne or foodborne
example, if respiratory specimens are frequently contaminated community outbreaks include Giardia lamblia, Cryptosporidium,
with upper respiratory tract microbiota, interpretation of the results Salmonella, Shigella, and Campylobacter. STDs such as syphilis,
for the health care provider and IPCP becomes more difficult. gonorrhea, and chlamydia are community-acquired infectious
Contaminated specimens also prove costly for the patient and diseases identified in public health laboratories and in laboratories
health insurance payer because they do not provide useful or serving acute care facilities. Some organisms that are important
appropriate information and cultures might need to be repeated. in public health settings may be more likely to be recovered in
Attempts to determine VAP become futile if the specimens are an acute care hospital but are reported to a public health jurisdiction
contaminated with upper respiratory tract microbiota. A similar for the latter to follow up on as a potential outbreak. Organisms
situation is encountered in blood culture contamination, which such as Neisseria meningitidis, encephalitis viruses, coronaviruses
lessens the likelihood of high-level interpretive abilities. Blood (severe acute respiratory syndrome [SARS]), and West Nile virus
culture contamination rates above 3% are generally considered high are examples.
and may indicate educational opportunities for the microbiology
laboratory and infection control program. However, it is generally Acute Care Setting
difficult to lower the contamination rate below 2%. Although a great variety of infectious agents can cause concern as
The types of pathogens isolated from given specimens represent HAIs in acute care settings, some are more frequently seen than
important information that can be generated by the laboratory to others. These are listed in Table 3.3. S. aureus, especially MRSA,
support the infection control program. For example, if S. aureus is an important health care–associated pathogen, causing BSIs,
is frequently isolated from skin infections in a jail setting, the SSIs, VAPs, and other infections. Although community-acquired
health care provider can anticipate the success of antistaphylococcal
antimicrobials in routinely treating those infections. If MRSA,
however, is more frequently isolated, the health care provider
would change the empiric therapy. TABLE 3.3  Health Care Settings and Common
The prevalence of a particular pathogen is another piece of Microbes of Infection Control Significance
information that the microbiology laboratory can provide to the
IPCP. Prevalence is the number of cases of disease that occur in Health Care Setting Microorganisms and Infectious Diseases
a given moment in time or specific period in a given population. Public health Neisseria meningitidis, Salmonella spp., Shigella
Therefore not only knowing what pathogens are isolated from a spp., Campylobacter spp., Giardia lamblia,
given body site but also being familiar with what pathogens are Cryptosporidium spp., Chlamydia spp.,
frequently isolated from a given location in a health care facility is syphilis, gonorrhea, HIV, hepatitis B virus,
important to the IPCP. For example, a skilled nursing facility may hepatitis C virus
Acute care Clostridium difficile, Staphylococcus aureus,
be reassured that the lack of negative-air-flow rooms is acceptable
MRSA, Enterococcus spp., Escherichia coli,
if Mycobacterium tuberculosis has not been isolated from any of Klebsiella pneumoniae, Pseudomonas
its patients in the past 7 years. aeruginosa, coagulase-negative staphylococci
Being able to recognize which pathogens are isolated from Ambulatory care Hepatitis B virus, hepatitis C virus, HIV, S.
patients in a medical ICU may provide the opportunity for the aureus, MRSA, P. aeruginosa, Enterococcus
IPCP to inform health care providers about the effects of anti- spp.
microbial pressure. For example, if extended-spectrum β-lactamase Extended care C. difficile, P. aeruginosa, Candida albicans, S.
facilities, home aureus, MRSA, Acinetobacter, Enterococcus
(ESBL)–producing Klebsiella pneumoniae isolates were seen in
care spp., coagulase-negative staphylococci
that medical ICU, the physicians might be advised to limit the Communal living S. aureus, MRSA, hepatitis C, lice
use of antimicrobial agents that tend to induce the formation of
ESBLs. The microbiology laboratory then serves as an important HIV, Human immunodeficiency virus; MRSA, methicillin-resistant S. aureus.
CHAPTER 3  The Laboratory Role in Infection Control 49

MRSA has become more prevalent, health care–associated MRSA is Catheter-Related Bloodstream Infections
worrisome because of its resistance to multiple antimicrobial agents Medical Intensive Care Unit
and the opportunity to infect a compromised population of patients. 9
Similarly, enterococci are pathogens of concern in HAIs because

Rate per 1000 catheter days


8
of their possible resistance to vancomycin and their potential
7
ability to pass that resistance on to other bacteria. Escherichia
coli and other members of the family Enterobacteriaceae, such 6
NHSN
as Klebsiella pneumoniae, are common fecal organisms seen in 5
a variety of HAIs in various sites, including BSIs, SSIs, VAPs, 4
and UTIs. In patients who are immunosuppressed by disease or
3
by therapy, P. aeruginosa is seen as a cause of HAIs. Clostridium Infection
difficile is an organism whose toxins cause diarrhea associated 2
with health care infections. Recovery of the organism is not the 1
significant finding, but demonstration of the toxin is significant. 0
JAN FEB MAR APR MAY JUN JUL AUG SEP
Ambulatory Care Setting Month
Ambulatory care settings include a variety of different locations. FIG. 3.1  Plot of the rate of central line–associated bloodstream
However, the commonly encountered microorganisms of infection infections in a medical intensive care unit. Shown are the
control importance do not vary. Usually, the patients have a chronic National Healthcare Safety Network (NHSN) benchmark and
calculated monthly infection rate. The increase in the rate above
illness, are immunosuppressed, and have infectious diseases caused the baseline and above the NHSN benchmark in September
by opportunistic pathogens. Other patients are likely to acquire would trigger an investigation of a possible outbreak.
community-acquired infections, such as hepatitis B virus, hepatitis
C virus, and human immunodeficiency virus (HIV) infection. Other
bacterial isolates include pathogens seen in other settings, such infection rates that might trigger an outbreak investigation. In
as S. aureus, MRSA, vancomycin-resistant enterococci (VRE), this figure, the baseline infection rate is seen for January through
and P. aeruginosa. August as 3.3 per 1000 catheter days. In addition, the NHSN
benchmark of 5.0 per 1000 catheter days is also plotted. In
Extended Care Facility and Home Care Settings September, the infection rate rose above the baseline and the
Patients in ECF and home care settings are frequently immunosup- NHSN benchmark. This might indicate that an outbreak has
pressed by disease or therapy and often need intravascular or taken place or that the change in the rate of infections may have
other device-related care. The microbes identified in these patients other causes. A more detailed investigation would reveal further
are often opportunistic pathogens. Infectious causative agents of information.
infection control significance identified in these patients include
P. aeruginosa, Candida, S. aureus, MRSA, VRE, Acinetobacter
spp., and C. difficile. Case Check 3.2
As represented by the Case in Point, an outbreak may also involve the
Communal Living unexpected isolation of a microorganism. During the investigation and
People who are housed together in some form of communal living introduction of infection control interventions, an epidemiologic curve is
setting, such as a prison or behavioral health facility, often have created. Fig. 3.2 shows the epidemiologic curve and recovery of the
common pathogens similar to those in the other settings described Acinetobacter sp. The case first described on the fourth of the month is
earlier. The infectious diseases are more likely related to the termed the index case, and it was to be determined whether the other
infections that followed were related to that case. The investigation used
activities of the persons in the facility. For example, S. aureus
the laboratory results provided by microbiologists.
and MRSA are recovered from prisoners who practice illicit
tattooing with nonsterile shared equipment, whereas lice and
hepatitis C virus are more frequently seen in behavioral health
settings because of the community source of the clients and their Widespread Outbreaks
intimate contact with blood and body fluids. Widespread outbreaks occur outside the confines of a given health
care setting. These might be found in a statewide or worldwide
outbreak. An example of a statewide outbreak is the occurrence of
Outbreak Investigation diarrheal disease in persons attending a state fair. Fig. 3.3 depicts
When numbers of isolates or infection rates increase above the an epidemiologic curve from such an outbreak. This involved
baseline, or when an isolate of a rare or potential bioterrorism reported cases in 565 fair attendees. A total of 47 cultures were
agent is recovered, an outbreak may have occurred. The microbiol- obtained, and 26 of those cultures were positive for Salmonella
ogy laboratory may be the first to recognize the event and will spp. Contaminated well water at the fairgrounds was suspected as
likely participate in the outbreak investigation. the source, and increased hand hygiene and use of bottled water
were instituted 5 days after the first case was reported. After the
Local Outbreaks interventions, the numbers of suspected cases decreased. Cases
In a given setting, an outbreak may be suspected when an involved individuals from seven states and Canada. Some were food
unanticipated increase in infections occurs. Fig. 3.1 shows handlers at the fair in addition to fair attendees. Many suspected
50 PART 1  Introduction to Clinical Microbiology

Recovery of Acinetobacter Imagine the complexity of an investigation of this case. This


Intensive Care Unit scenario was recognized as a potential outbreak and not an actual
Number of patients with Acinetobacter

5 outbreak when the investigation was undertaken. The recognition


of measles by the microbiology laboratory set the investigation
4 into motion, not the occurrence of any additional cases. Several
issues must be examined.
3 • Did the student have measles?
Because of routine use of a vaccine most, but not all, people
2 in the United States are immune. The people on the flight from
Index case Interventions Russia to London were the first people exposed, followed by
1
those on the flight from New York to Chicago.
• Were they immune?
• Where did they go after their flight landed?
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 • How does the health department follow up with them?
Day cases were identified The incubation period for measles is approximately 10 days
FIG. 3.2  Epidemiologic curve of the occurrence of Acinetobacter (8 to 13 days for the fever, 14 days for the rash). People are
spp. in respiratory specimens in an intensive care unit. Data contagious before the development of the fever and rash. Those
were gathered as the result of a suspected outbreak. The graph who are not immune are susceptible.
plots the number of patients with Acinetobacter spp. infection All three of these examples (Acinetobacter spp., Salmonella
by the day on which the cases were identified. Both the index
spp., measles) represent scenarios of potential infectious disease
case and the point at which infection control interventions
were implemented are indicated on the graph. outbreaks. The investigation of these outbreaks depends heavily
on the support of the microbiology laboratory to assist in ruling
in or ruling out the infection and in identifying cases and sources.
Outbreak of Diarrhea Laboratory support is described in detail later.
State Fair
180 Steps of an Outbreak Investigation
Intervention
160 When an outbreak is suspected, steps are taken to investigate the
140 event. The laboratory is integral in several of the steps. Table 3.4
lists the steps that are followed in an outbreak investigation.
120 Culture confirmed The first step is to establish a case definition. This step ensures
Number

100
Suspected that the rest of the investigation is based on a single definition.
80 This may involve the microbiology laboratory in the search for
60 a specific pathogen or the recovery of several pathogens.
40 Fair ended
The second step is to confirm that an outbreak exists. One
Fair started needs to be certain that all the suspected cases match the case
20
0
definition and that there is more than an expected number of
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 cases. At this point, the investigator seeks as much consultative
Day cases were identified assistance as possible. The laboratory is frequently asked for
FIG. 3.3  The epidemiologic curve of an outbreak investigation additional input.
of diarrhea over a 15-day period at a state fair. The number The third step is to find additional cases that might be added
of cases includes suspected cases (black bars) and culture-proven to the initial number of cases. Additional suspected cases may be
cases (colored bars). Infection control interventions were discovered by more detailed investigation or by the new occurrence
implemented on day 8 of the outbreak, and the number of
cases decreased.
of cases. The laboratory might be asked to review microbiology
data from a previous period to determine whether unrecognized
cases have occurred.
cases (>500) were reported but not cultured. The Salmonella spp. The fourth step is to gather as much information as possible
isolates recovered were all the same serotype. Well water, food, about the cases with respect to person, place, and time. Persons
and food preparation sites were cultured. suspected of being part of the outbreak should be interviewed to
Another example involves the investigation of a potential find what the victims have in common. An epidemiologic curve
measles outbreak involving an international flight from Russia to may be constructed to assist in the visualization of the outbreak
the United States. The patient involved in the suspected index numbers over time.
case flew from Russia to London, where she changed planes for The fifth and sixth steps are to form a hypothesis about the
a flight to New York. She then changed planes in New York for event and then to test that hypothesis. In the fifth step, a tentative
a flight to Chicago, where she lived. The patient was a college hypothesis is established as a best guess about the likely reservoir,
student who had visited her parents in Russia for the summer. source, and means of transmission. In testing that hypothesis, a
While in Russia, she had contact with several young children control group is established; then the event is compared in the
who had measles. On her return flight to the United States, she incident and control groups. Again, the microbiology laboratory
complained of a fever but did not develop a rash until she had may provide insight into the hypothesis and its relationship to
been home in Chicago for a day. the control group.
CHAPTER 3  The Laboratory Role in Infection Control 51

at the state fair discussed earlier, consider the number of fecal


TABLE 3.4  Steps of an Outbreak Investigation specimens that the health department laboratory processed, although
patient cultures are only one component of the investigative activity.
Steps Description Cultures from other sites may provide additional significant
1. Verify diagnosis of Establish a case definition. information. Well water and food may have been cultured in
suspected cases. addition to specimens from the patients with diarrhea and food
2. Confirm that an Be certain that all suspected cases meet handlers. In the outbreak of infections caused by Acinetobacter
outbreak exists. the definition. spp., the laboratory may have cultured respiratory therapy equip-
3. Find additional cases. Investigate to determine whether ment and water samples.
additional cases exist.
One of the major difficulties in a large outbreak (e.g., the
4. Characterize cases. Collect as much information as possible
about the cases, including people, state fair outbreak) is the reduced ability to collect and transport
place, and time elements. Develop an specimens from persons from out of state. Individuals may have
epidemiologic curve. cultures processed in their home state, but information about those
5. Form a hypothesis. Establish a “best guess” hypothesis results may be difficult to retrieve. In the state fair example,
about the outbreak. only a few of the affected individuals had cultures processed
6. Test the hypothesis. Test the hypothesis with control groups and results included in the investigation. In the example of 565
and data collected.
7. Institute control Implement intervention activities to
suspected cases, only 47 cultures were processed. Reasons for this
measures. control the outbreak. low number of cultures include the following: (1) the diarrhea
8. Evaluate effectiveness Determine whether the implemented lasts 24 to 48 hours and people may not seek medical help; (2)
of control measures. activities have an impact on the people not from the immediate area may not know of the potential
outbreak. Does the number of cases outbreak and the need to provide culture material; (3) people
diminish or disappear? do not want to be bothered with the expense and time spent to
9. Communicate the Document the investigation and
collect cultures; and (4) the specimens may be collected too late
findings. communicate with all involved parties.
in the infection or not transported properly, so no organisms are
recovered.
In addition to the actual culture results, the laboratory may be
The seventh step in the investigation may actually occur at asked to determine the serologic relationship of the isolates.
any point along the investigation time line. The establishment of • Were all the Salmonella spp. of the same serotype?
interventions to stop the outbreak probably occurs from the initial • What was the epidemiologic profile of the serotype?
recognition and heightened awareness of a problem. The formal Both the isolate identification and serologic relatedness may
steps of the intervention process might not be developed until be important determinants in an outbreak investigation.
after the hypothesis is developed and tested. Undoubtedly, interven-
tions of some type (e.g., increased hand hygiene) are introduced Antibiograms
early in the investigation. Antibiograms, patterns of sensitivity and resistance to antimicrobial
The eighth step, which comes after the development of inter- agents in bacteria, can often be used in the investigation of an
ventional strategies, is to evaluate the effectiveness of the interven- outbreak. As discussed in Chapter 13, there are times when
tions. Did the outbreak cease or at least decrease in its intensity? antibiograms must be viewed with suspicion. Although these
The ninth step, the final step and one sometimes overlooked, is laboratory figures are not always as precise as other results, they
to communicate the findings of the investigation. This must include may provide guidance about the microbial relatedness of the
a written report that is kept on file and provided to all responsible isolates. Table 3.5 demonstrates comparisons of two antibiograms
individuals. of isolates with the antibiogram of the index case. If the isolates
It is not unusual for an outbreak to end before all data have all had identical antibiograms (e.g., isolate 1) or if some of the
been collected and analyzed. This is probably because of an early isolates were different in their susceptibility patterns (isolate 2),
intervention. However, an early end to the outbreak does not the inclusion or exclusion from the case definitions might have
ameliorate the need for communication and a written report. been affected. For example, relatively susceptible S. aureus can
be distinguished from MRSA in an outbreak of skin infections
Investigation Support from in prisoners.
the Laboratory
The microbiology laboratory plays a crucial role in providing Molecular Epidemiology
investigative support in an outbreak investigation and in the creation Molecular epidemiology is the analysis of molecules, such as
of routine surveillance information. The availability of culture proteins and nucleic acids, for the detection, identification, and
reviews, which may result in the initiation of an outbreak investiga- characterization of microorganisms to generate isolate-specific
tion, was discussed earlier. Other types of laboratory support are markers to assess epidemiologic relatedness. This field of study
often important as well. began in the 1970s with plasmid profiling. In this assay, plasmids
from bacterial isolates are compared. Because of the low typeability
Cultures and Serology of some bacteria, this technique is used infrequently today.
In an outbreak investigation and collection of routine surveillance Pulsed-field gel electrophoresis (PGFE) is a strain typing
data, the collection, processing, reporting, and reviewing of technique that can be an important adjunct to epidemiologic
pertinent cultures becomes critical. In the Salmonella spp. outbreak investigations. In this method, enzyme-digested chromosomal
52 PART 1  Introduction to Clinical Microbiology

TABLE 3.5  Comparison of Antibiograms from TABLE 3.6  Pathogens Related to


Microbial Isolates Waterborne Infections
Isolate Viruses Bacteria Parasites

Antimicrobial Agent Index Case 1 2 Noroviruses Salmonella spp. Entamoeba histolytica


Rotavirus Campylobacter spp. Giardia lamblia
Ampicillin-sulbactam R R S Hepatitis A Yersinia enterocolitica Cryptosporidium spp.
Piperacillin R R S Hepatitis E Escherichia coli (O157:H7) Naegleria spp.
Cefepime R R S Legionella spp. Acanthamoeba spp.
Imipenem S S S Pseudomonas spp.
Ciprofloxacin R R S Mycobacterium spp.
Gentamicin R R S Aeromonas spp.
Tobramycin R R R
Amikacin R R I

I, Intermediate; R, resistant; S, sensitive.


fungi). High-volume air samplers are the preferred method for
collection, although settle plates may also be used. The results of
fragments of bacteria are separated electrophoretically. The patterns these microbiological cultures must be reported to the infection
of the fragments are compared among strains of microbes recovered control team and evaluated.
in a possible outbreak. Strains with dissimilar patterns would be
determined to be unrelated. However, if the patterns are similar, Water
the strains can be identified as possibly related. This potential Water is incriminated in outbreaks in many of the settings for
relatedness is an additional epidemiologic tool that can be incor- which microbiology laboratories provide service. Outbreaks can
porated into the investigation of an outbreak. PFGE has been occur in various environmental situations, such as those associated
used since the 1980s, and because of its high discriminatory power, with contaminated drinking water (e.g., hospitals, ECFs, prisons)
reproducibility, and nearly 100% typeability it has remained the or recreational water (e.g., swimming pools, whirlpools, lakes,
reference method. A number of other techniques involving primarily streams). They may take place in homes, aboard a plane or ship, in a
amplification of genomic sequences by polymerase chain reaction city or state, or in a foreign country. In the United States an average
and gene sequencing are also used. of 46 waterborne outbreaks were reported each year between 1971
and 2013, resulting in 642,782 outbreak-associated illnesses from
Environmental Culturing 50 states and six territories. Waterborne pathogens often cause
diarrheal illness. Other waterborne diseases include respiratory
Case Check 3.3 illnesses (e.g., legionellosis), hepatitis (hepatitis A or hepatitis
As part of an effective infection control program, the microbiology labora- E), skin infections (from Pseudomonas spp. or mycobacteria),
tory may be called on to perform cultures of various environmental sites. and central nervous system infections (Naegleria spp.). Because
Recommendations for environmental infection control have been extensively
of these infection control implications, the laboratory must be
discussed in a CDC document, “Guidelines for Environmental Infection
Control in Health-Care Facilities.” The environment is rarely implicated prepared to offer diagnostic services or recommend laboratories
in disease transmission, except with immunosuppressed patients. Although that do offer those services for waterborne pathogens.
environmental cultures are generally to be avoided, as in the Case in Table 3.6 lists examples of waterborne pathogens. Some
Point, there are times when they become an important and often required waterborne agents can be recovered by routine microbiology
element of an infection control program that affects the microbiology procedures such as cultures, but others may require specialized
laboratory. techniques. When asked to perform environmental cultures
of water, the microbiology laboratory must determine which
specific pathogens are sought, if that information is known. If
Air legionellosis is suspected, for example, the laboratory needs to
Usually, infections traced to air quality occur during construction have standard operating procedures for recovering this microbe. If
activities in a health care setting. Because microbes in the air can the outbreak involves diarrheal diseases of an unknown cause or
be incriminated in HAIs, cultures of the air can be a component causes, the recovery techniques must be broader and may require
of air quality investigations. Initially, an infection control risk the use of a specialized laboratory. The IPCPs involved in the
assessment should be conducted to determine whether the air outbreak investigation must be consulted before routine culturing
is the likely source of infectious particles. Such an assessment of environmental water is undertaken.
is necessary in construction activities and must be done before In some settings, routine water cultures must be performed
any decision to culture the air can be made. The CDC makes no because of specific guidelines. For example, for chronic dialysis
recommendation regarding routine microbiological air sampling centers, the CDC recommendations include performing bacterio-
before, during, or after construction. If a fungal infection such logic assays of water and dialysis fluids at least once a month
as aspergillosis occurs during or immediately after construction, with standard methods. The infection control program or managers
an outbreak investigation may be initiated and control measures of the specific area of concern must be familiar with regulations
implemented. Such an investigation may involve collecting and guidelines addressing water cultures. The laboratory must be
environmental samples (e.g., searching for sources of airborne aware of the standard methods to ensure that proper procedures
CHAPTER 3  The Laboratory Role in Infection Control 53

and proper media are used. The laboratory scientist, IPCP, and as they are suspected or identified. Some of these requirements
manager should maintain a close working relationship to ensure are federally mandated, whereas some may be designated by the
compliance with culturing requirements. state. It is imperative that the laboratory scientist knows which
infectious diseases are reportable to what agency and in what
Surfaces time frame they are to be reported.
The culturing of environmental surfaces should be performed
only under the combined direction of the laboratory and infection Reporting to Committees and Programs
control program. In an outbreak investigation, surface culturing Depending on the setting served by the microbiology laboratory,
would be needed; however, such cultures should not be routinely there may be expectations of reports of infectious diseases to
obtained. The laboratory must be consulted before environmental committees in that setting. For example, in an acute care hospital,
surface cultures are undertaken to ensure that proper procedures infection prevention and control committees may expect reports
and media are used. The laboratory scientist should be instrumental with various types of microbiological information. Annual anti-
in the interpretation of the results. biograms, lists of reportable diseases, pathogens recovered in
certain hospital units, isolates recovered from certain sites, and
Reporting blood culture contamination rates are examples of reports that
The role of the microbiology laboratory does not stop with provid- might be requested.
ing culture results. Depending on statutory requirements, the In other settings, physicians may expect periodic updates,
laboratory might also be responsible for the reporting of certain including antibiograms and pathogen prevalence. They may expect
infectious diseases to public health jurisdictions. Other groups these to be delineated by office practice, physician, site, or patient
may expect reports as well, such as committees, persons managing type. Those making these requests may be in home health care,
specific programs, and the news media. extended care, or communal living settings.
Schools and businesses might request updated reports especially
Reporting to Public Health related to outbreaks that affect their operation. These reports must
There is a requirement to report the identification or suspicion of be tempered by public health needs and individual confidentiality
certain infectious diseases to the local, state, or federal public health restrictions. Recently insurance companies and community
entities. As shown in Table 3.7, diseases designated as class A1 are advocacy groups have expressed interest in knowing about infection
considered public health concerns and must be reported as soon control rates. Some states require periodic reporting of these rates.
The microbiology laboratory may be involved in providing data
for these reports.
TABLE 3.7  Examples of Reportable Diseasesa Reporting to the Media
Class Description Examples
Among the activities of a microbiology laboratory, discussing
microbiology and infection control activities with the media (e.g.,
A1 Diseases of major public Anthrax, botulism television, radio, newspapers) may become necessary. Media
health concern—reported (foodborne), diphtheria, relations for the laboratory should be discussed with the risk
immediately on plague, rabies, smallpox, management area associated with the laboratory. Media relations
recognition of a case, cholera, meningococcal
represent an educational opportunity that might be investigated
suspected case, or disease, measles, tularemia,
positive laboratory results yellow fever before the laboratory scientist speaks to media personnel. One
A2 Diseases of public health Encephalitis (viral), foodborne must balance the public’s need for knowledge as perceived by
concern needing timely disease outbreaks, hepatitis the news media with privacy restraints for the patient, laboratory,
response—reported by A, Legionnaires’ disease, and setting.
the end of the next pertussis, syphilis,
business day after
recognition of a case,
tuberculosis, typhoid fever,
vancomycin-resistant
Education
suspected case, or Staphylococcus aureus, Laboratory Scientists and Infection
positive laboratory results vancomycin-intermediate S.
aureus, tetanus Prevention and Control Practitioners
A3 Diseases of significant Brucellosis, giardiasis, hepatitis The role of the microbiology laboratory in education is a further
public health concern— B, hepatitis C, Lyme extension of its activities related to infection control. Laboratory
reported by the end of disease, Rocky Mountain scientists must not only keep themselves educated in their contribu-
the work week spotted fever, trichinosis
tion to the infection control team, but also keep the infection
B Diseases reported only by Chickenpox, influenza
number of cases—
control personnel educated regarding the laboratory’s contribution
reported by the end of to the team. Seminars, scientific articles and books, computer-based
the work week learning, and discussion with the infection control personnel are
C Report of outbreak, unusual Blastomycosis, histoplasmosis, ways in which laboratory scientists can maintain their knowledge
incidence, or epidemic— scabies, staphylococcal skin of infection control and laboratory techniques that can aid the
reported by the end of infections, toxoplasmosis infection control program. The laboratorian should continuously
next working day
educate the IPCP regarding the abilities of the laboratory in
a
These are reporting regulations from the Ohio Administrative Code and contributing meaningful information to the infection control
may vary from state to state. program. As new techniques become available or old techniques
54 PART 1  Introduction to Clinical Microbiology

are replaced, the laboratory scientist needs to relay that information


to the IPCP. BOX 3.1  Examples of Emerging and
Similar information needs to be provided for others associated Reemerging Pathogens
with the health care setting and infection control program. Ancillary Emerging Pathogens
personnel, such as housekeeping and maintenance personnel, • Avian influenza virus
benefit from knowing the laboratory perspective of an infection • Coronavirus (SARS)
control program: • Viral hemorrhagic fever viruses
• What cleaning and disinfecting agents work against viruses? • West Nile virus
• How long does the agent need to be in contact with the environ- • Zika virus
ment to inactivate the virus? Reemerging Pathogens
• When do the maintenance personnel need to worry about fungi? • Anthrax (Bacillus anthracis)
• What does mold growing on wet drywall look like? • Botulism (Clostridium botulinum)
• Plague (Yersinia pestis)
These and other questions arise among ancillary health care
• Rocky Mountain spotted fever (Rickettsia rickettsii)
personnel and the laboratory scientist needs to be prepared to • Tularemia (Francisella tularensis)
respond.
Consultation with the microbiology laboratory often is sought
when construction is anticipated. Sometimes that education needs
to be provided, even when it is not sought. control program. Whether dealing with emerging diseases, such
• What microbes may be harbored in standing water? as Zika virus in 2016, or reemerging diseases, such as Rocky
• When should high-efficiency particulate air (HEPA) filters be Mountain spotted fever, the laboratory must stay closely aligned
used? with the infection control activities in the setting that the laboratory
• What might spread through a facility if proper barriers are not serves. Box 3.1 lists examples of emerging pathogens and
used to control demolition dust and debris? reemerging pathogens.
These are some questions that might be addressed by the
microbiology laboratory scientist while acting as a consultant to Emerging Pathogens
the infection control program. Sometimes infectious agents that have not been previously rec-
ognized appear. Agents such as the West Nile virus, the coronavirus
associated with SARS, and the avian influenza virus are three
Case Check 3.4 examples of emerging pathogens. The laboratory must quickly
Whereas the investigation could not conclusively determine why the learn not only how to identify these agents in human infections
outbreak in the Case in Point occurred, the use of contact precautions but also how to collaborate with the infection control program in
and improved hand hygiene stopped new cases. It is likely that the
dealing with these agents in the human population.
causative agent, Acinetobacter baumannii, was in the hospital environment
and was spread among the patients by improper hygiene. The presence • How are these microbes spread?
of a ventilator in the affected patients bypassed host defenses in the • What reservoirs may harbor them?
compromised patients. • What is their incubation period?
• What antimicrobial agents can successfully treat them?
• How can the environment be disinfected?
Safety These are questions that the infection control program must
The infection control program affects the microbiology laboratory by ask. The laboratory scientist, IPCP, and infectious disease physicians
emphasizing the need for laboratory safety. As discussed in Chapter all must combine their knowledge to address these issues. The
4, safety in the microbiology laboratory encompasses the infection educational role of the laboratory once again becomes evident in
control program. Hand hygiene is a critical part of laboratory safety. teaching specimen collection, specimen transport, disinfection,
Hand hygiene involves handwashing when hands are soiled or the and safety methods.
use of alcohol hand rubs when hands are not soiled. The practice
of standard precautions further extends infection control to the Reemerging Pathogens
microbiology laboratory. Gloves are always to be worn when blood The reemergence or potential reemergence of pathogens once
or body fluids are being handled. Proper disposal of microbiological thought to be eliminated demands collaboration with the infection
waste, according to state or local regulations and national guidelines, control program. The pathogens that cause Rocky Mountain spotted
is another critical component of the infection control program fever, anthrax, and plague are agents that clinical microbiologists
in the microbiology laboratory. Receiving available vaccines and have learned about but are not often identified by microbiology
testing for diseases (e.g., for hepatitis B, chickenpox, influenza, laboratories. The laboratory scientist must relearn information once
meningococcus, tuberculin skin test [TST]) are steps to protect thought to be of low importance. Media selection, identification
laboratory personnel. All these functions connect the microbiology techniques, and safety precautions must all be reexamined and
laboratory safety program and the infection control program. implemented. Interaction with the infection control program
strengthens the establishment of prevention and control strategies.
Emerging and Reemerging Pathogens Response Plans
With the advent of terrorist activities worldwide, the microbiology With the potential use of biological agents in terrorism, the
laboratory has become an integral part of that area of the infection development of emergency response plans is paramount. The
CHAPTER 3  The Laboratory Role in Infection Control 55

microbiology laboratory is an integral part of that infection control


c. Outbreaks of public health concern (e.g., scabies)
activity. Safety, specimen collection, agent identification, and agent d. All of the above
control are components of the response plan to which the laboratory 6. Microbial pathogens of potential bioterrorism activity include:
can contribute its input. a. Bacillus anthracis, Staphylococcus aureus, West Nile virus
Whether in everyday activities or activities in response to an b. Yersinia pestis, Staphylococcus aureus, hepatitis C virus
outbreak or emerging diseases, the microbiology laboratory and c. Bacillus anthracis, Yersinia pestis, Francisella tularensis
d. Bacillus anthracis, Escherichia coli, coronaviruses
laboratory scientists constitute a critical component of an efficient
7. Environmental cultures are usually to be avoided, except in:
and successful infection control program. The lessons of the chapter a. An outbreak investigation
must be carefully learned and implemented. b. The occurrence of infections following construction
c. Compliance with specific regulatory requirements
d. All of the above
Points to Remember 8. The formal steps in an outbreak investigation include:
a. Establishing a case definition and culturing air and water
■ The microbiology laboratory interacts with the infection control b. Establishing a case definition, forming and testing a hypothesis,
program in many different health care settings. and communicating findings
■ Surveillance is important to establish baseline data and recognize c. Forming and testing a hypothesis, performing PFGE, and
the need to investigate potential outbreaks. calculating an infection rate
■ The microbiology laboratory supports outbreak investigations by d. Confirming an outbreak exists, calculating an infection rate,
providing consultative services, including epidemiologic correlation and performing serology and culture tests
of isolates. 9. Infection control programs rely on microbiology laboratory support
■ Although infrequently performed, environmental cultures may play in:
a role in outbreak investigation. a. Public health settings
■ Microbiology laboratory scientists must recognize their role in b. Acute care facilities
providing reports to health departments, committees, the infection c. Home care settings
control program, and on occasion, the public. d. All of the above
■ The microbiology laboratory must maintain competencies in new 10. The microbiology laboratory interacts with the infection control
techniques and the identification of reemerging and emerging program by providing:
infectious diseases. a. Culture results
■ In anticipation of potential bioterrorism events, the microbiology b. Antibiograms and pathogen prevalence reports
laboratory must be equipped to participate in emergency prepared- c. Environmental cultures when appropriate
ness programs. d. All of the above

Learning Assessment Questions BIBLIOGRAPHY


1. Surveillance is defined as: Association for the Advancement of Medical Instrumentation. (2001).
a. The systematic collection and analysis of data
Water treatment equipment for hemodialysis applications, ANSI/AAMI
b. The review of health care–associated infections in laboratory
RD62-2001. Arlington, VA: American National Standards Institute.
personnel Baron, E. J., et al. (2005). Blood cultures IV. Cumitech 1C. Washington,
c. The recognition of emerging pathogens
DC: American Society for Microbiology.
d. The development of an infection control risk assessment
Boyce, J. M., & Pittet, D. (2002). Guideline for Hand Hygiene in Health-
2. Microbes commonly encountered in health care–associated infec-
Care Settings. Recommendations of the Healthcare Infection Control
tions in hospitals include:
Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA
a. Salmonella spp., Shigella spp., hepatitis C virus, Neisseria
Hand Hygiene Task Force. Society for Healthcare Epidemiology of
meningitidis
America/Association for Professionals in Infection Control/Infectious
b. Staphylococcus aureus, Pseudomonas aeruginosa, MRSA,
Diseases Society of America. MMWR. Recommendations and Reports:
Escherichia coli
Morbidity and Mortality Weekly Report. Recommendations and Reports,
c. Pseudomonas aeruginosa, Salmonella spp., hepatitis C virus,
51(RR-16), 1.
Giardia spp.
Centers for Disease Control and Prevention. (2016). Foodborne (1973–2013)
d. All of the above
and waterborne (1971–2013) disease outbreaks—United States. MMWR.
3. Pulsed-field gel electrophoresis (PFGE) might be performed to:
Recommendations and Reports: Morbidity and Mortality Weekly Report.
a. Identify staphylococcal species
Recommendations and Reports, 63, 79.
b. Assist in an outbreak investigation
Centers for Disease Control and Prevention. Healthcare-associated infec-
c. Develop a new isolation precaution
tions and statistics. Available at: https://www.cdc.gov/hai/surveillance/
d. All of the above
index.html. (Accessed 23 February 2017).
4. The occurrence of surgical site infections is generally calculated Carrico, R., et al. (2009). APIC text of infection control and epidemiology
as:
(3rd ed.). Washington, DC: Association for Professionals in Infection
a. The rate of infections in 1000 device-related events
Control and Epidemiology.
b. The percentage of infections in 100 device-related events Edwards, J. R., et al. (2007). National Healthcare Safety Network (NHSN)
c. The percentage of infections in surgical sites or procedures Report, data summary for 2006. American Journal of Infection Control,
d. All of the above
35, 290.
5. Health departments frequently require the reporting by the labora- Magill, S. S., et al. (2014). Multistate point-prevalence survey of health
tory of: care–associated infections. The New England Journal of Medicine,
a. Diseases of major health concerns (e.g., smallpox)
370, 13.
b. Diseases needing timely response (e.g., foodborne outbreaks) Sehulster, L., et al. (2003). Guidelines for environmental infection control
in health-care facilities. Recommendations of CDC and the Healthcare
56 PART 1  Introduction to Clinical Microbiology

Infection Control Practices Advisory Committee (HICPAC). MMWR. https://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf. (Accessed


Recommendations and Reports: Morbidity and Mortality Weekly Report. 25 February 2017).
Recommendations and Reports, 52(RR-10), 1. Trees, E., et al. (2015). Molecular epidemiology. In J. H. Jorgensen, et al.
Siegel, J. D., et al. 2007 guideline for isolation precautions: preventing (Eds.), Manual of clinical microbiology (11th ed., p. 131). Washington,
transmission of infectious agents in healthcare settings. Available at: DC: ASM Press.
CHAPTER

4  
Control of Microorganisms:
Disinfection, Sterilization, and
Microbiology Safety
Michelle M. Jackson,* Clifford Cymrot

CHAPTER OUTLINE
■ DISINFECTION AND STERILIZATION ■ ENVIRONMENTAL PROTECTION AGENCY REGULATIONS
■ STERILIZATION VERSUS DISINFECTION ON CHEMICAL SURFACE DISINFECTANTS
■ FACTORS THAT INFLUENCE THE DEGREE OF KILLING ■ FOOD AND DRUG ADMINISTRATION REGULATIONS ON
Types of Organisms CHEMICAL SKIN ANTISEPTICS
Number of Organisms Hygienic Handwashing and Waterless Handrubs
Concentration of Disinfecting Agent Surgical Hand Scrub and Waterless Surgical Handrubs
Presence of Organic Material Presurgical Skin Disinfection
Nature of Surface to Be Disinfected ■ MICROBIOLOGY SAFETY
Contact Time ■ GENERAL LABORATORY SAFETY
Temperature Safety Program for the Clinical Laboratory
pH Hazardous Waste
Biofilms Chemical Safety
Compatibility of Disinfectants Fire Safety
■ METHODS OF DISINFECTION AND STERILIZATION Storage of Compressed Gases
Physical Methods Electrical Safety
Chemical Methods Miscellaneous Safety Considerations
■ DISINFECTANTS VERSUS ANTISEPTICS Safety Training
Alcohols ■ BIOTERRORISM AND THE CLINICAL MICROBIOLOGY
Aldehydes LABORATORY
Halogens Laboratory Response Network
Chlorine and Chlorine Compounds Safety During a Possible Bioterrorism Event
Detergents: Quaternary Ammonium Compounds Packaging and Shipping of Infectious Substances
Phenolics
Heavy Metals
Gases

OBJECTIVES
After reading and studying this chapter, you should be able to:
1. Define the following terms: sterilization, disinfection, and antiseptic. 7. Describe EPA regulations on chemical surface disinfectants and FDA
2. Differentiate the functions and purposes of a disinfectant and an regulations on chemical skin antiseptics.
antiseptic. 8. Discuss the appropriate use of the following skin antiseptics in
3. Describe the general modes of antimicrobial action. health care settings by health care personnel: handwash or handrub,
4. Describe the way each physical agent controls the growth of surgical hand scrub or surgical handrub, and patient preoperative
microorganisms. skin preparation.
5. Give the mechanism of action for each type of chemical agent 9. Describe the hazards that can be encountered in a microbiology
commonly used in antiseptics and disinfectants. laboratory.
6. Describe the different heat methods and their respective 10. List the elements included in an exposure control plan.
applications. 11. Discuss the practice of standard precautions.
12. Differentiate standard precautions and transmission-based
*This section was prepared by the author in her private capacity. No official support precautions.
or endorsement by the Food and Drug Administration is intended or implied.

57
58 PART 1  Introduction to Clinical Microbiology

13. Define and give examples of engineering controls, work practice 18. Explain the information that must be included in safety data sheets.
controls, and personal protective equipment. 19. Describe the components of basic fire safety and electrical safety
14. Discuss the World Health Organization classification of infectious within the microbiology laboratory.
microorganisms by risk group. 20. Discuss the special safety considerations that must be addressed in
15. Compare and contrast the three types of biosafety cabinets. the clinical microbiology laboratory during a possible bioterrorism
16. Compare and contrast the four categories of biosafety levels. event.
17. Given an infectious agent and test procedure, determine the
appropriate safety precautions to use to ensure an exposure event
does not occur.

Sentinel laboratories Transmission-based


Case in Point Sporicidal precautions
Dr. Adams is a pediatrician at a busy metropolitan medical clinic. Standard precautions Transient biota
This morning, she arrived late to work because she had to take Sterilization Work practice controls
her sick son to her mother’s house and drop off her dog at the Surgical hand scrub
vet. When Dr. Adams arrived at the clinic, many children were
waiting for her, so she immediately began seeing her patients.
At one point, she thought about washing her hands, but she
felt guilty about coming to work late and did not want to keep
her patients waiting any longer. Besides, her hands did not look
Disinfection and Sterilization
dirty. Safety in the laboratory cannot be overemphasized. Quantification
of the risk of working with an infectious agent is difficult. Risk
to an individual increases with the frequency and type of organism
Issues to Consider and level of contact with the agent, as demonstrated by the Case
After reading the patient’s case history, consider: in Point at the beginning of this chapter. Each laboratory must
■ Potential risks that the physician is taking in spreading
develop and institute a plan that effectively minimizes exposure
germs to her patients to infectious agents. This chapter provides information on standard
■ Importance of handwashing and use of an appropriate
disinfection and sterilization techniques and laboratory safety
antiseptic
guidelines for the clinical laboratory. This section provides a
■ Quality control plan to minimize the risks to patients
practical overview of the following topics:
• Sterilization and disinfection
• Chemical and physical methods of disinfection and sterilization
• Principles and application of each method
• Common disinfectants and antiseptics used in health care
Key Terms settings
Antisepsis Health care antiseptic drug • Principles and applications of disinfectants and antiseptics
Antiseptic products • Regulatory process of disinfectants and antiseptics
Antiseptic drug Health care personnel
Biofilms handwash
Biosafety cabinet Laboratory Response Network Sterilization Versus Disinfection
Biosafety level (BSL) Microbial load The scientific use of disinfection and sterilization methods
Bloodborne pathogens Moist heat originated more than 100 years ago when Joseph Lister introduced
Destruction National Fire Protection the concept of aseptic surgery using carbolic acid, now called
Disinfectants Association (NFPA) phenol. Since then, the implementation of effective sterilization
Disinfection New drug application (NDA)
and disinfection methods has remained crucial in the control
Employee right-to-know Over-the-counter (OTC)
of infections in the laboratory and health care facilities (health
Engineering controls Pasteurization
care–associated infections).
Environmental Protection Patient preoperative skin
Agency (EPA) preparation To understand fully the principles of disinfection and steriliza-
Exposure control plan Personnel protective tion, we need to have accurate definitions of certain terms. Steriliza-
Fast-acting antiseptic equipment (PPE) tion refers to the destruction of all forms of life, including bacterial
Filtration Persistent spores. By definition, there are no degrees of sterilization—it is
Food and Drug Prions an all-or-nothing process. Chemical or physical methods may be
Administration (FDA) Recognized as safe and used to accomplish this form of microbial destruction. The word
Generally recognized as safe effective sterile is a term that is relevant to the method used. For example,
and effective (GRASE) Resident biota (flora) a solution that has been filtered through a certain pore-size filter
Germ theory Risk groups (<0.22 µm) is often referred to as sterile. Even though the filtered
Hazard-rating diamond Safety data sheets (SDSs) solution may be free of large microorganisms such as bacteria
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 59

and fungi and their spores, in actuality, any infectious agents (e.g., viruses containing lipid-rich envelopes are more susceptible to the
viruses) that are smaller than the pore size of the filter were not effects of detergents and wetting agents. Microorganisms living
removed, and therefore the filtered solution is not truly sterile. together in communities, referred to as biofilms, also provide
Disinfection refers to a process that eliminates a defined scope protection to the microorganisms against chemical and physical
of microorganisms, including some spores. Physical or chemical means of destruction.
methods may be used, but most disinfectants are chemical agents The organisms known today to be the most resistant to the
applied to inanimate objects. A substance applied to the skin for actions of heat, chemicals, and radiation are prions. Prions are
the purpose of eliminating or reducing the number of bacteria naked pieces of protein. Prions are thought to be the agents that
present is referred to as an antiseptic. Antiseptics do not kill cause a number of degenerative diseases of the nervous system
spores. (transmissible spongiform encephalopathy—mad cow disease,
Creutzfeldt-Jakob disease). These agents are transmitted to
Factors That Influence the humans through contaminated medicinal products, therapeutic
devices, body fluids, and food products. These infectious agents
Degree of Killing are extremely resistant to chemical and physical methods of
Before discussing methods used to kill microorganisms, a review destruction. Prions can withstand temperatures exceeding 121° C
of the factors that influence the degree of killing of organisms for several hours while immersed in acid or basic solutions. It is
is important. The following factors play a significant role in best to handle all body secretions as potentially being contami-
the selection and implementation of the appropriate method of nated with this agent. When an object or material is thought to
disinfection: be contaminated with a prion, special methods need to be taken
• Types of organisms to destroy the agent. Simple disinfection or sterilization may not
• Number of organisms be sufficient.
• Concentration of disinfecting agent
• Presence of organic material (e.g., serum, blood) Number of Organisms
• Nature of surface to be disinfected Another factor to consider is the total number of organisms
• Contact time present, referred to as the microbial load (bioburden). If the
• Temperature number of organisms is plotted against the time they are exposed
• pH to the killing agent (exposure time) logarithmically, the result is a
• Biofilms straight line (Fig. 4.2). The death curve is logarithmic. Because the
• Compatibility of disinfectants and sterilants microbial load is most likely composed of organisms with differing
degrees of susceptibility to killing agents, not all the organisms
Types of Organisms die at the same time. The microbial load determines the exposure
Organisms differ greatly in their ability to withstand chemical and time that is necessary for 99.9% elimination of the microorgan-
physical treatment (Fig. 4.1). This variability is due to the bio- isms. In general, higher numbers of organisms require longer
chemical composition of microorganisms and various mechanisms exposure times.
that they can use to protect themselves. For example, bacterial
endospores have coats rich in proteins, lipids, and carbohydrates Concentration of Disinfecting Agent
as well as cores rich in dipicolinic acid and calcium, all of The concentration of a disinfecting agent is also important.
which protect the spores. Cell walls of mycobacteria are rich The amount of disinfectant needed to destroy microorganisms
in lipids, which may account for their resistance to chemical varies with the different agents. Manufacturers’ instructions on
and environmental stresses, particularly desiccation. In contrast, preparation, dilution, and use must be followed very carefully.

Most resistant

Prions

Bacterial spores

Mycobacteria

Nonlipid viruses

Fungi

Bacteria

Lipid viruses Least resistant

FIG. 4.1  Different types of organisms and their resistance to killing agents.
60 PART 1  Introduction to Clinical Microbiology

disinfectants or sterilants for a much longer time than their vegeta-


108
tive counterpart before they are killed.
Log of number of organisms

106 Temperature
Disinfectants are generally used at room temperature (20° to 22° C).
104 Their activity is generally increased to some degree by an increase
in temperature and decreased by a decrease in temperature. A
102 disinfectant that is used on the workbench generally works faster
than if the disinfectant is used on a cold surface such as the walls
0 of a refrigerator. Disinfection of blood spills in a refrigerator can
5 10 15 20 take longer than disinfection of blood spills on a room temperature
Exposure time (minutes) countertop. Disinfectants and sterilants can be rendered inactive
FIG. 4.2  Effect of exposure time versus number of organisms. by too high or too low a temperature.

pH
Concentrated disinfectants, such as povidone-iodine, may actually The pH of the material to be disinfected or sterilized can affect
allow microorganisms to survive because there is not enough free the activity of the disinfecting or sterilizing agent. It is critical to
iodine to kill microorganisms. Proper concentrations of disinfecting make sure at what pH the agent is active and what the pH of the
agents ensure the inactivation of target organisms and promote material to be exposed to the agent is at the time the process will
safe and cost-effective practices. be done.

Presence of Organic Material Biofilms


Organic material, such as blood, mucus, and pus, affects killing Biofilms are considered a community of bacteria or other micro-
activity by inactivating the disinfecting agent. In addition, by organisms. These communities are generally layers of microorgan-
coating the surface to be treated, organic material prevents full isms that often have a protective material over them that shields
contact between object and agent (see the section discussing them from outside environmental factors. These communities of
glutaraldehyde later in this chapter). Bleach (sodium hypochlorite) microorganisms can be on the surface of either inanimate or animate
is easily inactivated by organic material. For optimal killing activity, objects. A critical place where biofilms are seen in the hospital
instruments and surfaces should be cleansed of excess organic is on catheters. Other places may be inside pipes that carry water
material before disinfection. and on deionizing columns used to make processed water. When
dealing with the disinfection of objects that may have a biofilm,
Nature of Surface to Be Disinfected it is critical to realize that the presence of the biofilm makes
Certain medical instruments are manufactured of biomaterials disinfection more difficult. Microorganisms in a biofilm can be
that exclude the use of certain disinfection or sterilization methods characteristically different than when they are planktonic, or
because of possible damage to the instruments. For example, singular and free floating. This means disinfectants that the microbes
endoscopic instruments are readily damaged by the heat generated were susceptible to singularly can be resistant within a biofilm
in an autoclave. Alternative methods must be used for this class (sessile). To disinfect materials that may have a biofilm present,
of instruments. the concentration of the disinfectant may need to be increased,
the contact time may need to be increased, or both.
Contact Time
The amount of time a disinfectant or sterilant is in contact with Compatibility of Disinfectants
the object is critical. Too little contact time does not allow the A common mistake is to believe that two disinfectants are better
agent to work properly. The contact time is a function not only than one. This is not necessarily incorrect, but when more than one
of the agent itself but also of the bioburden on the object, the disinfectant is used, the compatibility of the disinfectants must be
type of microorganism that is to be killed, and the presence of taken into consideration. Some disinfectants may inactivate other
organic material and the temperature at which the agent is being disinfectants. For example, the use of bleach and a quaternary
used. When disinfecting or sterilizing a contaminated object, it ammonium compound together may negate the activity of both
is critical to know what organisms may be present and the contact disinfectants.
time to use, which is based on the microorganism that is most
resistant. The amount of time that an agent is in contact with an Methods of Disinfection
object can also determine whether it is disinfecting or sterilizing
the object. For example, glutaraldehyde can be used as a disinfectant
and Sterilization
or a sterilant, with the difference being the amount of time the Having discussed the way factors that affect the survival of
glutaraldehyde is in contact with the contaminated object. When microorganisms influence disinfection and sterilization, we now
glutaraldehyde is used as a sterilant, the contact time is much look at the ways in which methods are selected. E. H. Spaulding
longer than when it is used as a disinfectant. Alcohol and iodine categorized medical materials into three device classifications:
preparations (e.g., Betadine) must be in contact with an object 1. Critical materials
for at least 1 to 2 minutes for them to kill microorganisms. The 2. Semicritical materials
spores of both bacteria and fungi must be in the presence of 3. Noncritical materials
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 61

TABLE 4.1  Device Classification and Methods of Effective Disinfection

Killing Action Against

Device Classification Disinfection Method Spores Mycobacteria Nonlipid Viruses Fungi Bacteria

Critical Sterilization
 Steam + + + + +
  Dry heat + + + + +
 Gas + + + + +
 Chemical + + + + +
  Ionizing radiation + + + + +
Semicritical High-level disinfection
  2% glutaraldehyde ± + + + +
  Chlorine dioxide ± + + + +
  Wet pasteurization − + + + +
Low-level disinfection
  Sodium hypochlorite − + ± + +
  Quaternary ammonium compounds − − ± + +
  Ethyl alcohol, isopropyl alcohol (70%–90%) − − + + +
 Phenolics − ± + + +
 Iodophors − − + + +

+, Positive kill; −, no kill; ±, variable.

Critical materials are materials that invade sterile tissues or than moist heat. This method may be used for heat-stable substances
enter the vascular system. These materials are most likely to that are not penetrated by moist heat, such as oils. Dry heat is
produce infection if contaminated, and they require sterilization. commonly used to sterilize glassware.
Before semicritical materials come into contact with mucous Boiling and pasteurization are methods that achieve disinfection
membranes, they require high-level disinfection agents. Noncritical but not sterilization; these methods do not eliminate spores. Boiling
materials require intermediate-level to low-level disinfection before (100° C) kills most microorganisms in approximately 10 minutes.
contact with intact skin. High-level disinfectants have activity Pasteurization, used mostly in the food industry, reduces food-borne
against bacterial endospores, whereas intermediate-level disin- pathogens and organisms responsible for food spoilage. It is
fectants have tuberculocidal activity but not sporicidal activity. generally performed at 72° C (161° F) for 15 seconds. The main
Finally, low-level disinfectants have a wide range of activity against advantage of pasteurization is that treatment at this temperature
microorganisms but do not demonstrate sporicidal or tuberculocidal reduces spoilage of food without affecting its taste. Table 4.2
activity. Table 4.1 presents a summary of these principles. summarizes the applications of heat.

Physical Methods Filtration


As mentioned earlier, sterilization and disinfection can be performed Filtration methods may be used with both liquid and air. Filtration
by both physical and chemical methods. Although several physical of liquids is accomplished through the use of thin membrane
methods are available, this discussion is restricted to the methods filters composed of plastic polymers or cellulose esters containing
most commonly used in a laboratory or hospital setting. pores of a certain size. The liquid is pulled (vacuum) or pushed
(pressure) through the filter matrix. Organisms larger than the
Heat size of the pores are retained. Filters with various pore sizes are
Because of its reliable effects, ease of use, and economy, heat is available. Most bacteria, yeasts, and molds are retained by pore
the most common method used for the elimination of microorgan- sizes of 0.45 and 0.80 µm; however, this pore size may allow
isms. Heat can be used in several ways. Moist heat, or heat under passage of Pseudomonas-like organisms, and therefore a 0.22-µm
steam pressure, is the agent used in autoclaves. Putting steam size is available for critical sterilizing (e.g., parenteral solutions).
under 1 atm of pressure, or 15 psi, achieves a temperature of Membranes with pore sizes of 0.01 µm are capable of retaining
121° C. At this temperature, all microorganisms (except for prions) small viruses. The most common application of filtration is in the
and their endospores are destroyed within approximately 15 minutes sterilization of heat-sensitive solutions, such as parenteral solutions,
of exposure. The time varies according to the density of the vaccines, and antibiotic solutions. Filtration of air is accomplished
material; important factors are that the moisturized heat comes with the use of high-efficiency particulate air (HEPA) filters. HEPA
in contact with the material and the contact time is sufficient. An filters are able to remove microorganisms larger than 0.3 µm and
added advantage of moist heat is the shorter time required for are used in laboratory hoods and in rooms of immunocompromised
sterilization than for dry heat sterilization. Heat in water is patients.
transferred more readily to a cool body than heat in air. Moist
heat is the sterilization method of choice for heat-stable objects. Radiation
Dry heat may also be used as a sterilizing agent, although it Radiation may be used in two forms—ionizing and nonionizing.
requires much longer exposure times and higher temperatures Ionizing radiation, in the form of gamma rays or electron beams, is
62 PART 1  Introduction to Clinical Microbiology

of short wavelength and high energy. This method of sterilization chemical agents may be used for sterilization. These are known
is used by the medical field for the sterilization of disposable as chemosterilizers. All disinfectants are regulated by the U.S.
supplies such as syringes, catheters, and gloves. Nonionizing Environmental Protection Agency (EPA). Agents that are clas-
radiation in the form of ultraviolet rays is of long wavelength and sified as sterilants are regulated by the U.S. Food and Drug
low energy. It damages deoxyribonucleic acid (DNA) by forming Administration (FDA) when they are to be used to sterilize devices
thymine and cytosine dimers. Because of its poor penetrabil- that will come in contact with patients. Chemical agents exert
ity, usefulness is limited; it can be used to disinfect surfaces, their killing effect by the following mechanisms:
although the parameters (distance to surface, potential microorgan- • Reaction with components of the cytoplasmic membrane
isms to be destroyed) under which it is to be used need to be • Denaturation of cellular proteins
determined. • Reaction with the thiol (–SH) groups of enzymes
• Damage of RNA and DNA
Chemical Methods The fact that agents can exert one or a combination of actions
Just as physical methods are used mainly to achieve sterilization, on microorganisms is important to remember. Damage to the
chemical agents are used mainly as disinfectants. However, some integrity of the cytoplasmic membrane causes the cytoplasm and
its contents to leak out, resulting in cell death. Denaturation of
proteins effectively disrupts the metabolism of the cells. Some
TABLE 4.2  Control of Microorganisms Using Heat agents specifically react with the thiol (–SH) groups of enzymes,
Methods inactivating them. Thiol groups occur usually in the amino acid
cysteine. Finally, damage to RNA and DNA inhibits the replication
Temperature of the organism. For ease of discussion, the chemical agents are
Method (° C) Time Required Applications grouped on the basis of chemical composition. Table 4.3 sum-
marizes the applications of chemicals commonly used as disin-
Boiling water 100 15 min Kills microbial
fectants and antiseptics.
(steam) vegetative forms;
endospores survive
Autoclave 121.6 15 min at Sterilizes and kills Disinfectants Versus Antiseptics
(steam under 15 psi endospores
pressure) The germ theory of disease was one of the most important
Pasteurization contributions by microbiologists to the general welfare of the
  Batch method 63 30 min Disinfects and kills worldwide population. The medical community gradually grew
milk-borne
aware of the problem of nosocomial (hospital-acquired) infections
pathogens and
vegetable forms; and the need to practice asepsis to prevent the contamination of
endospores survive wounds, dressings, and surgical instruments. The germ theory of
  Flash method 72 15 s Same, but shorter disease also contributed to the development of antimicrobial
time at higher chemotherapeutics.
temperature Ignaz Semmelweis (1816–1865) and Joseph Lister (1827–1912)
Over (dry heat) 160–180 1.5–3 h Sterilizes; keeps are considered to be important pioneers for the promotion of
materials dry
asepsis. More than 100 years ago, Semmelweis demonstrated
Adapted from VanDemark PJ, Batzing BL: The microbes: an introduction to that routine handwashing can prevent the spread of disease. Sem-
their nature and importance, Redwood City, CA, 1987, Benjamin-Cummings. melweis worked in a hospital in Vienna where maternity patients

TABLE 4.3  Chemical Agents Commonly Used as Disinfectants and Antiseptics

Type Agent(s) Action(s) Applications and Precautions

Alcohols (50%–70%) Ethanol, isopropanol, benzyl alcohol Denature proteins; make lipids soluble Skin antiseptics
Aldehydes (in solution) Formaldehyde (8%), glutaraldehyde React with NH2, –SH, and –COOH Disinfectants; kill endospores; toxic to
(2%) groups humans
Halogens Tincture of iodine (2% in 70% Inactivates proteins Skin disinfectants
alcohol)
Chlorine and chlorine compounds React with water to form hypochlorous Used to disinfect drinking water;
acid (HClO); oxidizing agents surface disinfectants
Heavy metals Silver nitrate (AgNO3) Precipitates proteins Eye drop (1% solution)
Mercuric chloride (HgCl2) Reacts with –SH groups; lyses cell Disinfectant: toxic at high
membrane concentrations
Detergents Quaternary ammonium compounds Disrupt cell membranes Skin antiseptics; disinfectants
Phenolics Phenol, carbolic acid, Lysol, Denature proteins; disrupt cell Disinfectants at high concentrations;
hexachlorophene membranes used in soaps at low concentrations
Gases Ethylene oxide Alkylating agent Sterilization of heat-sensitive objects

Adapted from VanDemark PJ, Batzing BL: The microbes: an introduction to their nature and importance, Redwood City, CA, 1987, Benjamin-Cummings.
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 63

were dying at an alarming rate. Most of the maternity patients cotton balls contaminated with spores that were used to prepare
who died had been treated by medical students who worked on the skin for blood collection. Alcohols are inactivated by the
cadavers during an anatomy class before beginning their rounds presence of organic material. Because their action is greatly
in the maternity ward. Because the students did not wash their reduced in concentrations less than 50%, alcohols should be
hands between touching the dead and the living (handwashing used in concentrations between 60% and 90%. For alcohols to
was an unrecognized hygienic practice at the time), pathogenic be effective, they must be allowed to evaporate from the surface
bacteria from the cadavers were transmitted by the hands of the to which they were applied. Alcohols inactivate microorganisms
students to the mothers. The result was a death rate five times by denaturing proteins, dehydrating cells, and dissolving lipids.
higher for mothers who delivered in the hospital in contrast to Alcohols are used principally as antiseptics and disinfectants.
the mothers who delivered at home. Of women who delivered Commonly, alcohols are used to disinfect laboratory surfaces and
their babies in hospitals, 25% died of childbed fever (puerperal gloved hands.
sepsis), later found to be caused by infection with Streptococcus The 2015 proposed rule for health care antiseptic drug
pyogenes. In an experiment considered quaint at best by his col- products has reclassified ethanol (60% to 95%) as category I, safe
leagues, Semmelweis insisted that his students wash their hands and effective for health care personnel handwash, surgical hand
before treating the mothers; deaths on the maternity ward were scrub, and patient preoperative skin preparation (for preparation
reduced fivefold. Despite these remarkable results, Semmelweis’s of the skin before injection) to category III, need more data and
colleagues greeted his findings with hostility, in part because testing. After reviewing currently available scientific evidence,
of the results implying that physicians were unclean. Because the FDA tentatively determined there are important scientific
of this hostility, he eventually resigned his position. Later in data gaps for health care antiseptic products marketed under the
another maternity clinic, he had similar dramatic results when previous 1994 tentative final monograph (TFM) proposed rule
handwashing was implemented. Ironically, Semmelweis died in to support the products’ effectiveness at reducing infections, and
1865 of infection with S. pyogenes, with his views still largely meet safety requirements. The 2015 proposed rule states that more
ridiculed. extensive data than previously proposed in the 1994 TFM are
After the death of Semmelweis, Lister, an academic surgeon, necessary to demonstrate that over-the-counter (OTC) monograph
benefited by reading Pasteur’s works about bacteria as causes of topical antiseptics used in the health care setting are generally
infection before he ventured into studies of antisepsis. In 1867 recognized as safe and effective (GRASE). At the present time,
Lister introduced handwashing and the use of phenol as an the FDA is evaluating additional safety and efficacy of alcohols
antimicrobial agent for surgical wound dressings to British surgery. for use as health care antiseptics. Use of ethanol as an antiseptic
His principles were gradually, although reluctantly, adopted in is generally for preparing skin sites from which blood is to be
Britain, and the mortality rate for amputation decreased from 45% collected or where an inoculation is to be placed. Alcohols are
to 15%. The Listerian technique was approved in the United States flammable, so they should not be used in the presence of ignition
at the first official meeting of the American Surgical Association sources such as open flames or incinerators. They may be used
in 1883, 20 years after Semmelweis’s initial publications. This as a housekeeping disinfectant for damp-dusting furniture and
was the beginning of infection control. lights or wiping electrical cords without leaving a residue on
Health care workers’ hands are frequently contaminated by treated surfaces. They are nonstaining and can disinfect semicritical
direct contact while caring for a patient or by indirect contact instruments.
while touching a contaminated surface or device. Several factors
should be included in the evaluation of a disinfectant and an Aldehydes
antiseptic. A prerequisite for a disinfectant or antiseptic is its Formaldehyde
effectiveness against the expected spectrum of pathogens. The Formaldehyde is generally used as formalin, a 37% aqueous
implementation of effective disinfection and antiseptic chemicals solution, or formaldehyde gas. Formaldehyde gas is often used
remains crucial in the control of nosocomial infections. to disinfectant biosafety hoods. This type of procedure should
be left to professionals. Although formalin can be used as a
Alcohols chemosterilizer in high concentrations, its usefulness is limited
The two most effective alcohols used in hospitals for disinfection by its irritability factor and its potential carcinogenicity. Form-
purposes are ethyl alcohol and isopropyl alcohol. Alcohols have aldehyde is a carcinogen, and the U.S. Occupational Safety and
excellent in vitro bactericidal activity against most gram-positive Health Administration (OSHA) has set worker exposure limits.
and gram-negative bacteria. They also kill Mycobacterium tuber- It is not recommended that formaldehyde in any form be used
culosis various fungi, and inactivate certain enveloped viruses; as a disinfectant or sterilant on a routine basis. tuberculosis
however, they are not sporicidal and have poor activity against has been known to survive for many years in tissue fixed in
nonenveloped viruses. Because alcohols are not sporicidal solutions, formaldehyde.
alcohol may actually be contaminated with spores. Any solution
of an alcohol that is used as an antiseptic or disinfectant should Glutaraldehyde
be filtered through a 0.22-µm filter to remove any spores that may Glutaraldehyde is a saturated five-carbon dialdehyde that has broad-
be present. In addition, because alcohols are not sporicidal, when spectrum activity and rapid killing action and remains active in the
alcohol is used to saturate cotton balls to be used to prepare the presence of organic matter. Glutaraldehyde is extremely susceptible
skin for blood collection or inoculation, the cotton balls should to pH changes and is active only in an alkaline environment.
be sterile. There have been many reports of false-positive blood When used as a 2% solution, it is germicidal in approximately 10
cultures that have been traced back to the use of alcohol-soaked minutes and sporicidal in 3 to 10 hours. Its killing activity is due
64 PART 1  Introduction to Clinical Microbiology

to inactivation of DNA and RNA through alkylation of sulfhydryl


and amino groups. Even though glutaraldehyde is not inactivated Chlorine and Chlorine Compounds
by organic material, it does not penetrate organic material well. Chlorine and chlorine compounds are some of the oldest and most
Therefore objects coated with organic material should be cleaned commonly used disinfectants. They are usually used in the form of
before glutaraldehyde is used. Glutaraldehyde solutions may be hypochlorite, such as the liquid sodium hypochlorite (household
reused; however, a decrease in the activity of the glutaraldehyde bleach) and solid calcium hypochlorite. Their killing activity is
may be seen owing to accumulation of organic material, dilution, based on the oxidative effects of hypochlorous acid, formed when
and change in the pH of the solution. Because it does not corrode chloride ions are dissolved in water. Hypochlorites are inexpensive
lenses, metal, or rubber, it is the sterilizer of choice for medical and have a broad spectrum of activity; however, they are not used as
equipment that is not heat-stable and cannot be autoclaved as sterilants because of the long exposure time required for sporicidal
well as for material that cannot be sterilized with gas. It is a safe, action and their inactivation by organic matter. Hypochlorites are
high-level disinfectant for most plastic and rubber items, such corrosive; therefore concentrated bleach solutions should not be
as items used for administering anesthetic agents or respiratory used for disinfection. The activity of hypochlorite solutions is
therapy. greatly influenced by the pH of the surrounding medium. These
Glutaraldehyde is bactericidal, pseudomonacidal, fungicidal, solutions are commonly used as surface disinfectants (e.g., for
and virucidal (against human immunodeficiency virus [HIV] and tabletops). A solution containing 0.5% to 1% sodium hypochlorite is
hepatitis B virus [HBV]) with a minimum of 10 minutes’ exposure generally used for disinfection. Such solutions are generally stable
at a temperature between 20° and 30° C. It is also tuberculocidal. for no longer than 30 days, with 50% of the original concentration
Variations in formulations of products available affect the exposure of chlorine dissipating by 30 days. However, current practice
time and temperature of the solution, especially after reuse. A 2% recommends replacing the solution and making a fresh dilution
solution at 25° to 30° C may be 100% tuberculocidal. Users should daily. As with the use of any disinfectant, proper contact time
follow the label instructions of the manufacturer. Most of the is critical for bleach solutions. Solutions should be allowed a
products labeled as cold sterilants are sporicidal in a minimum contact time of a least 3 minutes, and longer if organic material
of 10 hours’ exposure at room temperature. It remains active in is present. A 1 : 10 dilution of a 5.25% concentration of sodium
the presence of organic matter and does not coagulate protein hypochlorite is recommended by the Centers for Disease Control
material. and Prevention (CDC) for cleaning up blood spills. The most
common use of chlorine is disinfection of water.
Halogens
Iodophors Detergents: Quaternary
Iodine can be used as a disinfectant in one of two forms: tincture or Ammonium Compounds
iodophor. Tinctures are alcohol and iodine solutions, used mainly Quaternary ammonium compounds are derived by substitution
as antiseptics. An iodophor is a combination of iodine and a neutral of the four-valence ammonium ion with alkyl halides. They are
polymer carrier that increases the solubility of the agent. This cationic, surface-active agents, or surfactants, that work by reducing
combination allows the slow release of iodine. Iodophors must the surface tension of molecules in a liquid. Their effectiveness
be diluted properly for them to be effective. Improperly diluted is reduced by hard water and soap, and they are inactivated by
iodophors may not kill microorganisms because of the lack of excess organic matter. Their action is mediated through disruption
free iodine in solution. Iodophors have the added advantage of of the cellular membrane, resulting in leakage of cell contents.
being less irritating, nonstaining, and more stable than iodine in its Certain bacteria, particularly gram-negative bacteria such as
pure form. Iodophors may be used as antiseptics or disinfectants, Pseudomonas aeruginosa, are intrinsically resistant to quaternary
depending on the concentration of free iodine. The best known ammonium compounds. Pseudomonas spp. growing in ammonium
iodophor is povidone-iodine (Betadine), which is mainly used acetate–containing quaternary ammonium compounds and disease
as an antiseptic. Povidone-iodine provides slow and continuous outbreaks associated with contaminated quaternary ammonium
release of free iodine. Free iodine degrades microbial cell walls compounds have been reported. Because they are not sporicidal
and cytoplasm, denatures enzymes, and coagulates chromosomal or tuberculocidal, the use of quaternary ammonium compounds
material. Iodophors are commonly used as skin preparation agents is limited to disinfection of noncritical surfaces such as benchtops
for sites where blood is to be drawn for blood cultures. When and floors.
iodophors are used for this purpose, it is critical that there is the
proper amount of contact time, which is generally more than 30 Phenolics
seconds. All iodine tinctures and iodophors must be completely Phenolics are molecules of phenol (carbolic acid) that have been
removed from the skin to avoid irritation. Iodophors are used only chemically substituted, typically by halogens or alkyl, phenyl, or
to disinfect because they are not sporicidal. Their bactericidal benzyl groups. These groups reduce the toxicity of phenol and
action is due to the oxidative effects of molecular iodine and increase its effectiveness. The most common phenolics are ortho-
hypoiodic acid, both of which are found in solution. The FDA phenylphenol and ortho-benzyl-para-chlorophenol. Phenolics have
has reclassified povidone-iodine 5% to 10% as category I for a fairly broad spectrum of activity but are not sporicidal. The
use as a topical antiseptic in health care personnel handwash, addition of detergents to the phenol formulation makes products
surgical hand scrub, and patient preoperative skin preparation that clean and disinfect in one step. They are stable, biodegradable,
to category III. At the present time, the FDA is evaluating and relatively active in the presence of organic material. Their
safety and efficacy data of iodophors for use as health care mechanism of inactivation is disruption of cell walls, resulting
antiseptics. in precipitation of proteins. At lower concentrations, phenolics
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 65

are able to disrupt enzyme systems. Their main use is in the control procedures have been unsuccessful. Hexachlorophene
disinfection of hospital, institutional, and household environments. should be used only as long as necessary for infection control.
They are also commonly found in germicidal soaps.
Chloroxylenol
Chlorhexidine Gluconate Chloroxylenol (parachlorometaxylenol [PCMX]) is a halogen-
Chlorhexidine gluconate (CHG) has been used for more than 30 substituted phenolic compound that has been used in the United
years in the hospital setting. In 1976 the FDA granted approval States since the 1940s. PCMX at concentrations of 0.5% to 4%
of CHG for use as a topical antiseptic on the basis of its high acts by microbial cell wall disruption and enzyme inactivation.
level of antimicrobial activity, low toxicity, and strong affinity for PCMX has good activity against gram-positive bacteria, but it is
binding to the skin and mucous membranes. CHG was not an OTC less active against gram-negative bacteria, M. tuberculosis, fungi,
drug monograph active ingredient at that time. CHG disrupts the and viruses. The antimicrobial activity of PCMX is unaffected
microbial cell membrane and precipitates the cell contents. CHG by organic materials such as blood or sputum, but it is neutralized
(0.5% to 4%) is more effective against gram-positive than gram- by nonionic surfactants and polyethylene glycol. It is considered
negative bacteria and has less activity against fungi and tubercle intermediate-acting to slow-acting and has minimal persistent
bacilli. CHG is inactive against bacteria spores except at elevated effect of more than a few hours. PCMX has low antimicrobial
temperatures. Lipid-enveloped viruses (e.g., herpesvirus, HIV, efficacy compared with iodines, iodophors, and CHG in reducing
respiratory viruses, influenza virus, cytomegalovirus) are rapidly skin biota. The FDA has reclassified PCMX (0.24% to 3.75%)
inactivated. Nonenveloped viruses (e.g., rotavirus, adenovirus, from category I for safety and category III for effectiveness for
enteroviruses) are not inactivated by exposure to CHG. short-term use such as patient preoperative skin preparation to
Numerous studies indicate that CHG is safe and nontoxic. It category III for both. The FDA is currently evaluating additional
is not absorbed through the skin and has a low skin-irritancy safety and efficacy of PCMX for use as a health care antiseptic
potential. However, severe skin reactions may occur in infants under the OTC drug review.
younger than 2 months. The potential for allergic contact sensitiza-
tion and photosensitization is reported to be minimal. However, Triclosan
CHG should not come into contact with the eyes, the middle ear, Triclosan is a diphenyl ether that disrupts the cell wall. The reaction
or meninges. Although CHG is not as rapidly effective as the time is intermediate, and the persistence is excellent. It has good
alcohols, a major attribute of CHG is its persistence, in that it activity against gram-positive bacteria, gram-negative bacteria, and
binds to the skin and remains active for at least 6 hours. Although viruses. It has fair activity against M. tuberculosis and poor activity
it is not significantly affected by organic matter such as blood, it against fungi. Triclosan is not significantly affected by organic
is pH-dependent—hence the formulation significantly affects matter such as blood but is affected by pH and the presence of
activity. The optimum pH range of 5.5 to 7.0 corresponds to the surfactants and emollients, and formulation significantly affects activ-
pH of body surfaces and tissues. ity. Triclosan can be absorbed through intact skin. Some short-term
CHG is used extensively for disinfection of the hands of animal studies have shown that exposure to high doses of triclosan
surgical personnel and provides whole-body disinfection of is associated with a decrease in the levels of some thyroid hormones.
patients undergoing surgery. Low-concentration (0.5% to 1%) The FDA currently does not know the significance of those findings
CHG is added to alcohol-based preparations to provide greater to human health. Other studies have raised the possibility that
residual activity than alcohol alone. The immediate bactericidal exposure to triclosan contributes to making bacteria resistant to
action of CHG surpasses that of antiseptic preparations containing antibiotics. The FDA currently does not have enough information
povidone-iodine, triclosan, hexachlorophene, or chloroxylenol. available to assess the level of risk that triclosan poses for the
Its persistence, which prevents regrowth of microorganisms on development of antibiotic resistance. The FDA published a final
the skin, is comparable to that of hexachlorophene or triclosan. rule for consumer antiseptic hand and body washes containing the
CHG has a broader spectrum of activity than the others, especially majority of the antibacterial active ingredients, including triclosan
against gram-negative bacteria. and triclocarban, that they will no longer be able to be marketed.
Manufacturers have not proven that triclosan is safe for daily
Hexachlorophene use over a long period. Additionally, manufacturers have not shown
Hexachlorophene is primarily effective against gram-positive bac- that this ingredient is any more effective than plain soap and
teria. It is a chlorinated bisphenol that interrupts bacterial electron water in preventing illness and the spread of certain infections.
transport, inhibits membrane-bound enzymes at low concentra- At the present time, the FDA is reevaluating safety and efficacy
tions, and ruptures bacterial membranes at high concentrations. of triclosan for use as a health care antiseptic for health care
Gram-positive bacteria are killed by 3% hexachlorophene within personnel handwashes and surgical hand scrubs.
15 to 30 seconds, but a longer time is needed for gram-negative Triclosan has been added to many consumer products, including
bacteria. Hexachlorophene has residual activity for several hours clothing, kitchenware, furniture, toothpaste, and toys to prevent
after application and has a cumulative effect after multiple uses. bacterial contamination. The EPA regulates the use of triclosan
Hexachlorophene has been associated with severe toxic effects, in these products and is presently reevaluating its assessment of
including deaths. It can be absorbed through damaged skin of the effects of triclosan when used in these products.
adults and the skin of premature infants. The FDA classified 3%
hexachlorophene to be available only by prescription and designated Heavy Metals
it as unsafe for OTC distribution. Hexachlorophene is indicated to Disinfectants containing heavy metals are rarely used in clinical
control outbreaks of gram-positive infections when other infection applications; they have been replaced by safer and more effective
66 PART 1  Introduction to Clinical Microbiology

compounds. Heavy metal disinfectants are slowly bactericidal; for certain inanimate, hard nonporous surfaces or incorporation of
their action is primarily bacteriostatic. Because of the toxic effects antimicrobial pesticide products into substances under the pesticide
of mercuric chloride and other mercury compounds, their use as law—the Federal Insecticide, Fungicide, and Rodenticide Act. An
disinfectants has declined, and they are used mainly as preservatives EPA registration number is granted only when the requirements
for paint. Silver nitrate (1% eye drop solution) had been used as of laboratory test data, toxicity data, product formula, and label
a prophylactic treatment to prevent gonococcal (Neisseria gonor- copy are approved. The label of a disinfectant that is effective
rhoeae) conjunctivitis in newborns. It has been largely replaced against a specific major group of microorganisms only (e.g.,
by erythromycin drops. Copper and copper alloys (e.g., brass, gram-positive or gram-negative) must specify the major group
bronze) are the first solid surface materials ever to get approval against which it is effective. Label claims of effectiveness as a
by the EPA to be considered antimicrobial for health care use. “general disinfectant” or representations that the product is effective
They are being evaluated for use lining rails and door handles in against a broad spectrum of microorganisms are acceptable if the
health care facilities to mitigate the spread of microorganisms. product is effective against both gram-positive and gram-negative
bacteria. Label claims for use of disinfectants in hospital or medical
Gases environments are acceptable only for products that are effective
Ethylene Oxide as general or broad-spectrum disinfectants as well as disinfectants
Ethylene oxide is the gas most commonly used for sterilization. against the nosocomial bacterial pathogen P. aeruginosa. The
Because it is explosive in its pure form, it is mixed with nitrogen disinfectant label should indicate several highlighted points
or carbon dioxide before use. Factors such as temperature, time, important in selecting the appropriate agents for the designated
and relative humidity are extremely important in determining the use (Box 4.1).
effectiveness of gas sterilization. The recommended concentration
is 450 to 700 mg of ethylene oxide per liter of chamber space at
55° C to 60° C for 2 hours. A relative humidity of 30% is optimal Food and Drug Administration
for the destruction of spores. The killing mechanism of ethylene Regulations on Chemical Skin
oxide is the alkylation of nucleic acids in the spore and vegetative
cell. Gas sterilization is widely used in hospitals for materials
Antiseptics
that cannot withstand steam sterilization. This method is also used When developing an antiseptic drug product, a manufacturer can
extensively by the manufacturing industry for the sterilization of pursue two options: the new drug application (NDA) process or
low-cost thermoplastic products. the OTC drug review known as the monograph system. NDAs are
defined by law as being recognized as safe and effective. A new
Hydrogen Peroxide chemical entity never before marketed in the United States would
Vaporized hydrogen peroxide (H2O2) is primarily used as a sterilant be classified as a new drug and, in most cases, initially approved for
in the pharmaceutical and medical device manufacturing industries. prescription use only. The approved NDA is manufacturer-specific
It is active against all vegetative microorganisms, bacterial and allows only that particular sponsor to market the product. Other
endospores, and fungal spores.

Peracetic Acid
Peracetic acid is used in a gaseous form as a sterilant primarily BOX 4.1  Type of Information to Review on a
in the pharmaceutical and medical device manufacturing industries. Disinfectant Label
It is active against all vegetative microorganisms and bacterial
and fungal spores. Front Panel
• Product name, brand, or trademark
Hydrogen Peroxide and Peracetic Acid • Ingredient statement (concentration or strength)
The combination of H2O2 and peracetic acid vapors is used in • “Keep Out of Reach of Children”
• EPA registration number and establishment number
the pharmaceutical and medical device manufacturing industries.
Similar to each of its individual components, the combination of Back Panel
H2O2 and peracetic acid is active against all vegetative forms of • Precautionary statements
microorganisms and bacterial and fungal spores. The major advantage • Hazards to humans and domestic animals
• First aid
to the use of the combination of H2O2 and peracetic acid over each • Environmental hazard
of its individual components is a shorter contact time. The activity • Physical or chemical hazard
against prions of the combination of H2O2 and peracetic acid as • Directions for use
well as each of the individual components is not fully known. • How to use the product
• Application sites and rates
• Worker protection issues
Environmental Protection Agency •

Aftercare
Equipment
Regulations on Chemical Surface • Treated surfaces
Disinfectants •

Cleaning supplies
Storage and disposal
The Antimicrobial Division of the EPA regulates the registration on
EPA, Environmental Protection Agency.
the use, sale, and distribution of antimicrobial pesticide products
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 67

manufacturers wanting to market a similar product would also


need to seek FDA approval through an NDA. The FDA considers Hygienic Handwashing and
a drug safe enough to approve when the benefits outweigh the Waterless Handrubs
risks. This risk-to-benefit assessment is critical in the drug approval The main goal of handwashing is to eliminate transient biota.
process. Transient biota is contracted from the environment or from other
OTC drugs are defined as generally recognized as safe and people. In most cases, these organisms are not part of the established
effective (GRASE) for their intended use as long as they are normal biota.
neither misbranded nor marketed using false or misleading state-
ments. The FDA classifies OTC drug products into three categories:
(1) category I, GRASE for the claimed therapeutic indication; Case Check 4.1
(2) category II, not GRASE or having unacceptable indications; The Case in Point at the beginning of this chapter exemplifies the sig-
and (3) category III, insufficient data available to permit final nificance of handwashing in the prevention of disease and pathogen
classification. transmission. Routine handwashing in health care settings is performed
A manufacturer desiring to market a monographed (therapeutic in the following situations:
classes of ingredients that are GRASE) drug need not seek clearance • Removing physical dirt (including blood, excretions, secretions, or
from the FDA before marketing the drug. In this case marketing discharge from lesions)
• Before and after routine patient contact
is not exclusive, and all data and information supporting GRASE
• After contact with infected or colonized patients or their immediate
status are publicly available. Monographs mainly address active surroundings
ingredients in the product, and final formulations are not subject • In high-risk units such as intensive care and burn units
to monograph specifications in most cases. Manufacturers are free • On entering protective isolation units and leaving source isolation units
to include any inactive ingredients that serve a pharmaceutical • Before antiseptic procedures (e.g., dressing techniques, minor invasive
purpose as long as those ingredients are considered safe and do procedures)
not interfere with product effectiveness or required final product
testing. In some instances, although the product may contain
GRASE ingredients, the final formulation may need to meet a For example, the health care worker in the Case in Point at
monograph testing procedure. An example would be the antiseptic the beginning of this chapter may acquire microbes, including
drug products that are for health care personnel handwash, methicillin-resistant Staphylococcus aureus (MRSA), during direct
surgical hand scrub, and patient preoperative skin preparation. contact with animals, patients, or contaminated surfaces. Hands
Table 4.4 lists terms and definitions frequently used for topical should always be washed immediately after arriving at work.
antiseptics in health care settings. These products are required to Although the health care worker’s hands appeared to be clean,
meet in vivo and in vitro efficacy testing requirements to ensure her hands harbored many bacteria and infectious microorganisms
that the formulated products are effective as an antiseptic. Inactive after having contact with her sick child and her dog. She should
ingredients and emollients, when included in the products, may have stopped seeing patients and washed her hands as soon as
inhibit the antiseptic action; therefore testing must be performed she remembered, and she should wash her hands before and after
to show effectiveness. contact with each patient. Although transient organisms are easily

TABLE 4.4  Food and Drug Administration Product Categories of Topical Antiseptics

Category Definition

Antiseptic drug Representative of a drug, in its labeling, as an antiseptic shall be considered to be representation that it is
a germicide, except in the case of a drug purporting to be, or represented as, an antiseptic for inhibitory
use as a wet dressing, ointment, dusting powder, or such other use as involves prolonged contact with
the body
Broad-spectrum activity Properly formulated drug product, containing an ingredient included in the monograph, that possesses in
vitro activity against the microorganisms listed in §333.470(a)(1)(ii), as demonstrated by in vitro
minimum inhibitory concentration determinations conducted according to methodology established in
§333.470(a)(1)(ii)
Health care antiseptic drug product Antiseptic-containing drug product applied topically to the skin to help prevent infection or help prevent
cross contamination
Antiseptic handwash or health care Antiseptic-containing preparation designed for frequent use; it reduces the number of transient
personnel handwash drug product microorganisms on intact skin to an initial baseline level after adequate washing, rinsing, and drying; it
is broad-spectrum, fast-acting, and, if possible, persistent
Surgical hand scrub drug product Antiseptic-containing preparation that significantly reduces the number of microorganisms on intact skin; it
is broad-spectrum, fast-acting, and persistent
Patient preoperative skin preparation Fast-acting, broad-spectrum, and persistent antiseptic-containing preparation that significantly reduces the
drug product number of microorganisms on intact skin

Federal Register on the tentative final monograph for health-care topical antiseptic drug products (June 17, 1994, 59 FR 31402).
68 PART 1  Introduction to Clinical Microbiology

removed from the upper layer of the skin along with dirt particles over the hands and forearms, which must be kept wet with the
and oil, they may become part of the resident biota of individuals. handrub solution for the scheduled period of 3 to 5 minutes by
Interventions against the bacterial load of the hands should balance adding additional portions as necessary and continuing to rub.
two goals: protecting the skin with its resident biota and killing Before the application of an alcohol, the hands must be dry, and the
the transient biota. Intact skin on health care workers’ hands helps alcohol must have completely evaporated before donning gloves.
to protect both patients and health care workers from getting or
transmitting nosocomial infections. Presurgical Skin Disinfection
The FDA requires that all antiseptic handwashing and handrub- To be effective, preoperative skin preparation formulations must
bing products used in the hospital setting reduce the number of degerm an intended surgical site rapidly as well as provide a high
sampled test bacteria by 2.5 log10 on each hand within 5 minutes level of bacterial inactivation and persistent antimicrobial activity,
after the first wash/rub application. The technique involves treating up to 6 hours after preparing the skin. A patient preoperative skin
the hands with the antiseptic product according to the manufac- preparation drug product is defined as a fast-acting, broad-spectrum,
turer’s instructions for the specified time. The lower third of the and persistent antiseptic-containing preparation that significantly
forearm is also washed. After completion of the wash, hands and reduces the number of microorganisms on intact skin. Similar to
forearms are rinsed under tap water (40° C ± 2° C) and dried the surgeon’s hands, the patient’s operation site requires surgical
thoroughly with disposable or sterilized towels. disinfection, directed against resident as well as transient biota,
Waterless handrubs (alcohol handrubs)—either liquid or gel—are and it often requires maximum disinfection in a single treatment,
used for hygienic hand antiseptics. They also can be used as an without the benefit from progressive effects of repeated application.
alternative to routine handwashing when there is no visible soiling The FDA requires that the product reduce the number of bacteria
and for patient contacts. They are often more convenient than by 2 log10 per square centimeter on an abdomen test site and 3
handwashing and can be particularly useful if sinks are not readily log10 per square centimeter on a groin test site within 30 seconds
available. Dispensers for alcohol handrubs can be fitted next to after product use and that the bacterial count for each test site
sinks and placed beside each bed or carried around by each health does not exceed baseline 6 hours after product use. For preinjection
care worker. The technique involves rubbing small portions (3 to sites, the product must reduce the number of bacteria by 2 log10
5 mL) of a fast-acting antiseptic, usually an alcoholic preparation, per square centimeter on a dry skin test site within 30 seconds
into the hands and rubbing until dry or for a preset duration recom- of product use. It has long been debated whether or not a preopera-
mended by the manufacturer. All areas of the hands must be tive skin preparation is adequate in degerming the skin before
covered completely with the antiseptic, including the subungual making a surgical incision. Many surgeons recognize this problem,
spaces of the fingers. and to promote greater degerming of the surgical site area, patients
are requested to wash the area daily, or more often, before surgery.
Surgical Hand Scrub and Waterless The intent is to reduce the microbial population at the surgical
Surgical Handrubs site area so that the region is prepared before surgery; the organisms,
The objective of the surgical hand scrub and waterless surgical then being few in number, would be virtually totally removed
handrubs is to eliminate the transient biota and most of the resident from the skin.
biota. Resident biota can be persistently isolated from the hands
of most people. These organisms include coagulase-negative
staphylococci, Corynebacterium spp. (diphtheroids or coryneforms),
Microbiology Safety
Propionibacterium spp., and Acinetobacter spp.
The rationale is to limit bacterial exposure of the surgeon’s
Case in Point
hands in case the surgical glove is punctured or torn. Tiny holes A 35-year-old infectious disease specialist complained of experienc-
are observed in 30% or more of surgeons’ gloves after an operation, ing fever, chills, myalgia, and severe headache for the past several
even when high-quality gloves are used. A surgical hand scrub days. The patient had been otherwise healthy until 2 weeks ago,
drug product is defined as an antiseptic containing a preparation when he began to experience fatigue and felt slightly swollen
that significantly reduces the number of microorganisms on intact lymph nodes. The patient recalled that he had demonstrated to
skin; it is broad-spectrum, fast-acting, and persistent. The FDA his medical students and residents a culture of Brucella melitensis
requires that the product reduce on the first wash/rub the number isolated from a patient’s blood sample. While handling the Petri
of bacteria by 2 log10 on each hand within 1 minute after application dish without gloves, he picked up the telephone to answer a
page. He touched his mustache while talking on the telephone,
and that the count on each hand does not subsequently exceed
as he habitually does when he speaks.
the baseline within 6 hours. Surgical hand scrub procedures are
performed according to the manufacturer’s instruction but for no
longer than 5 minutes. They offer the advantage of cleaning and Issues to Consider
disinfecting the hands at the same time. After reading the patient’s case history, consider:
Surgical handrubs, alcohol solutions (60% to 70% ethyl alcohol ■ Potential risks to which laboratory scientists are exposed
or propyl alcohol) with an emollient and with or without an added while working in the microbiology laboratory
antiseptic as recommended for hygienic waterless handrubs, are ■ Exposure control plan to minimize the risks
used, but larger volumes and longer exposure times are needed ■ Laboratory safety guidelines to protect laboratory personnel
than for hygienic waterless handrubs. Surgical handrub applica-
tion technique is accomplished by pouring a specified amount of The concept of laboratory safety has changed drastically since
antiseptic into the cupped dry hands and rubbing vigorously all the early 1980s. Before 1980, safety practices in most clinical
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 69

laboratories were lax. Mouth pipetting was a widely used technique, • Describe the safe handling, storage, and disposal of chemicals
and eating, drinking, and smoking in the laboratory, although and radioactive substances.
discouraged, were common. Beginning in the early 1980s, this • Clearly outline the laboratory or hospital policies for correct
relaxed attitude toward safety among personnel changed dramati- procedures in the event of fire, natural disasters, and bomb
cally. The impetus behind the change was the arrival in the United threats.
States of a previously unheard of disease with an apparent 100% • Perform initial safety training for all employees in all aspects
mortality rate. This disease became known as acquired immuno- of laboratory safety, and update training annually.
deficiency syndrome (AIDS). In addition to being a global calamity, • Teach correct techniques for lifting and moving heavy objects
AIDS initiated a major rethinking of employee risk for laboratory- and patients.
acquired infections (LAIs) in hospitals around the United States. The safety program needs to be ongoing and consistent with
Beginning with an emphasis on reducing the risks of biological current federal and state regulations. Most important, it must be
hazards (biohazards), safety became a priority for laboratory presented in a way that encourages employees to incorporate the
personnel. The attitude of “What you don’t know can’t hurt you,” safety practices into their daily routines and take responsibility
common among laboratory employees, rapidly went out of date. for keeping the work environment safe.
Safety in the clinical laboratory is a major concern, and work
practices need to be continually reevaluated. Studies have shown Occupational Safety and Health Administration
laboratorians are at increased risk of infections compared with Laboratorians must always remember that they work in a hazardous
the general population. Laboratory exposures do occur, and often environment. Hazards can be classified as biological, chemical,
they cannot be traced back to a specific event. In 2012 the CDC radiologic, or physical. Training programs are instituted in all of
published “Guidelines for Safe Work Practices in Human and these areas for employees who are exposed to any of these hazards.
Animal Medical Diagnostic Laboratories” in the Morbidity and It is imperative for the individual to follow the rules that are set
Mortality Weekly Report, Supplement. This report was developed forth in the safety procedure manuals.
to improve safety in the laboratory by increasing safe practices, The mission of the OSHA is to protect workers within the
to encourage laboratorians to consider safety issues, and to foster United States. The clinical laboratory falls under these regulations.
a culture of safety. Safety in the clinical laboratory encompasses In 1991 the OSHA created and released the Bloodborne Pathogens
biological, chemical, electrical, radioactive, and fire hazard Final Standard to protect health care workers. This standard was
protection. revised in 2001 in conformance with Public Law 106-430, the
Needlestick Safety and Prevention Act.
General Laboratory Safety Exposure Control Plan
Safety in the clinical laboratory is the responsibility of the institu- The OSHA Bloodborne Pathogens Standard clearly states the
tion, laboratory directors, laboratory managers and laboratory safety requirements that the employer must have in place to protect
employees. Laboratory employees must be provided with a safe the employee from bloodborne pathogens. Employers are required
work environment. Laboratory directors, managers, and employees to have an exposure control plan, which must be reviewed and
must know the current safety regulations; safety procedure manuals updated annually. This plan must be available to all employees
must be provided; and training in safe laboratory practices must and should include the following:
occur on an annual basis through in-service education and should • A determination of tasks and procedures that may result in an
be the duty of an assigned safety officer. Although the provision occupational hazard
of a safe work environment is ultimately the employer’s responsibil- • A plan to investigate all exposure incidents and a plan to prevent
ity, it cannot be achieved without the commitment of all persons these from reoccurring
in that environment to practice safe techniques for their own and • Methods of compliance for standard precautions
their coworkers’ protection. • Engineering and work practice controls
• Personal protective equipment (PPE)
Case Check 4.2 • Guidelines for ensuring that the work site is maintained in a
clean and sanitary manner
The Case in Point at the beginning of this section illustrates the importance
• Guidelines for the handling and disposal of regulated
of a safety program for the clinical laboratory. Safety in the laboratory
is the responsibility of all laboratory personnel. All people who come waste
through the laboratory must also observe the safety guidelines to ensure • A training program for all employees
proper protection and reduce risk of exposure to potential hazardous
biological agents. Standard Precautions
In 1985 the CDC instituted safety guidelines for the handling of
blood and body fluids. These guidelines, called universal precau-
Safety Program for the tions, were intended to protect hospital personnel from bloodborne
Clinical Laboratory infections. In 1996 these guidelines were updated and renamed.
The comprehensive safety program for the clinical laboratory These new guidelines, standard precautions, are still in effect.
needs to fulfill the following: These guidelines require that blood and body fluids from all patients
• Address biological hazards by performing biological risk be considered infectious and capable of transmitting disease. Blood
assessments and developing safety procedures for working and all body fluids, including secretions and excretions except
with these hazards. sweat, regardless of whether visible blood is present, are considered
70 PART 1  Introduction to Clinical Microbiology

infectious. Standard precautions also include nonintact skin and laboratory equipment, the use of safety needles and single-use
mucous membranes. holders, eyewash stations, emergency showers, and plastic shield
To ensure that the guidelines required in standard precautions barriers. Ideally, laboratories should have negative air pressure,
are followed within the laboratory, engineering controls and work access to the laboratory should be limited, and there should be a
practice controls are instituted, and employers must provide PPE. plan to prevent insect infestation.
Standard precautions address the following:
• Handwashing must be done after touching blood, body fluids, Work Practice Controls
secretions, excretions, and any items considered contaminated. Altering the manner in which a task is performed to reduce the
Hands must be washed after removal of gloves and between likelihood of exposure to infectious agents is defined by the OSHA
patients. as work practice controls. Examples of work practice controls
• Gloves should be worn when handling blood, body fluids, include the following:
secretions, excretions, and any items considered contaminated. • No mouth pipetting
Clean gloves must be put on before touching mucous membranes • No eating, drinking, smoking, or applying cosmetics in the
and nonintact skin. Hands must be washed after removal of laboratory
gloves. • Disinfection of workstations at the end of each shift and after
• Mask, eye protection, or face shield must be worn anytime any spill of infectious material
there is a potential for splashes or sprays of blood, body fluids, • No recapping or breaking of contaminated needles
secretions, and excretions. • Disposal of needles in an appropriate puncture-resistant
• Laboratory coats must be worn to protect skin and clothing container
when contact with blood, body fluids, secretions, and excretions • Procedures performed in a manner that minimizes splashing
could occur. and the generation of air droplets
• Appropriate sharps disposal must be implemented with care • Specimens transported by way of well-constructed containers
to prevent injuries with sharps, needles, and scalpels. These with secure lids to prevent leakage of infectious materials
devices must be placed in appropriate puncture-resistant contain- • Frequent handwashing
ers after use.
• Environmental control must include procedures for routine Personal Protective Equipment
care, cleaning, and disinfection of environmental surfaces. Specialized clothing or equipment that is worn by an employee
for protection is defined by the OSHA as personnel protective
Transmission-Based Precautions equipment (PPE). PPE must be provided and maintained by the
The second set of precautions for the health care setting are called employer; examples include gloves, laboratory coats, masks,
transmission-based precautions. Standard precautions are still respirators, face shields, and safety glasses. For PPE to be protective
followed, and transmission-based precautions are added precau- and considered appropriate, blood and body fluids must not be
tions that are used when the patient is known or suspected to be able to penetrate the PPE material. The equipment must be acces-
infected or colonized with an infectious agent that requires extra sible to the employee and must be worn whenever there is the
measures to prevent spread or transmission of the agent. The potential for exposure to infectious material; it must be removed
categories of these precautions are contact precautions, droplet before leaving the work area and must be placed in an area
precautions, and airborne precautions. Contact precautions are used designated for PPE. Gloves should be removed whenever they
to stop the spread of infectious agents that may be transmitted become contaminated, and disposable gloves should never be
through direct or indirect contact with the patient or with the washed and reused. Hands must be washed after the removal of
patient’s environment. Examples of these types of infectious agents gloves.
are multidrug-resistant organisms such as vancomycin-resistant PPE must fit properly to be the most effective. Respirators
enterococci, MRSA, and Clostridium difficile. that are used for protection against airborne transmission of
Droplet precautions are used to stop the spread of infectious infectious agents must be fit-tested to ensure the protection of the
agents that can be transmitted by close respiratory contact or by worker. Fig. 4.3 illustrates goggles, masks, and laboratory garments
exposure of mucous membranes to respiratory secretions. Examples appropriate for use in laboratory workstations.
of infectious agents that can be transmitted by this route include
Neisseria meningitidis, Bordetella pertussis, and influenza virus. Biological Risk Assessment
The final category is airborne precautions. These precautions are For an infection to occur, including an LAI, there must be a
used for infectious agents, such as M. tuberculosis, varicella virus, susceptible host, the infectious agent must have a route of transmis-
and rubeola virus, that can remain airborne and infectious over sion to the susceptible host, and the concentration of the agent
long distances. For further information on what safety procedures must be high enough to cause disease. The biological hazards
should be used for each category of precautions, refer to the 2007 in the microbiology laboratory come from two major sources:
Guideline for Isolation Precautions: Preventing Transmission of (1) processing of the patient specimens and (2) handling of the
Infectious Agents in Healthcare Settings published by the CDC. actively growing cultures of microorganisms. Either activity puts
the employee at risk of potential contact with infectious agents.
Engineering Controls The major routes of LAIs in the clinical laboratory are parenteral
Engineering controls are defined by the OSHA as controls that inoculations (e.g., needlesticks or contaminated sharps), spills and
isolate or remove the hazard from the workplace. Examples of splashes onto skin or mucous membranes, ingestions (e.g., putting
engineering controls include the use of closed tube sampling by pens or fingers into the mouth, mouth pipetting), and inhalation
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 71

A B

C
FIG. 4.3  A, Eye protection used when chemical splashing could occur. B, Integrated eye protection
and mask. C, Laboratory coats. The material of the white laboratory coat slows the penetration
of liquids that splash or soak it. The coat on the right is disposable.

of infectious aerosols. Families of microbiology personnel and deaths of a laboratory worker’s wife and infant who were infected
persons who work in adjacent laboratories may also be at risk. through handling his contaminated laboratory coat. Emerging
Many infectious agents pose a high risk to laboratory employees. pathogens, such as severe acute respiratory syndrome coronavirus,
M. tuberculosis has long been known to cause tuberculosis in pose a major risk because there is often a lack of knowledge
laboratory workers exposed to aerosols created in processing and experience among those working with these emerging
sputum samples. A laboratory accident involving a spill of active infectious agents.
M. tuberculosis, which could easily aerosolize through the ventila- Bloodborne pathogens also pose a high risk. HBV can be
tion system, is every microbiologist’s nightmare. Brucella spp. transmitted to laboratory workers through needlestick injuries or
and Francisella tularensis are other infectious agents that can cuts from other sharp instruments that have been contaminated with
be transmitted through inhalation of an aerosol created during an infected patient’s blood or body fluids or through contact with
the processing or handling of specimens (e.g., blood that may mucous membranes. The CDC stated that before the institution
harbor these organisms) or cultures of the organism. Coccidioides of the hepatitis B vaccine in 1982, more than 10,000 health care
immitis, the most infectious fungus, can infect several people in workers were accidentally infected with this bloodborne pathogen
a room if culture plates on which the organism is growing are annually. By 2001 the number had decreased to less than 400. HIV
not sealed with tape or are open in the absence of a biosafety and hepatitis C virus are other bloodborne pathogens that may be
hood. In the 1970s, Bacillus anthracis was responsible for the transmitted to laboratory personnel from contaminated specimens
72 PART 1  Introduction to Clinical Microbiology

through a needlestick injury or another percutaneous route. All


these infectious agents must be handled with extreme care. BOX 4.2  Classification of Infective Microorganisms
Because microbiology laboratory personnel frequently deal by Risk Group
with various infectious agents—viral, fungal, bacterial, parasitic, Risk Group 1 (No or Low Individual and
and mycobacterial—LAIs are an obvious hazard and should be Community Risk)
a concern for all laboratorians. All laboratorians must understand A microorganism that is unlikely to cause human or animal disease.
that they are at risk of an LAI because of the environment in
Risk Group 2 (Moderate Individual Risk, Low
which they work, and it should be the goal of laboratory manage- Community Risk)
ment and laboratory workers to minimize this risk and follow all A pathogen that can cause human or animal disease but is unlikely to
of the safety procedures employed by the laboratory for their be a serious hazard to laboratory workers, the community, livestock,
protection. A more recent example of the risk to laboratorians or the environment. Laboratory exposures may cause serious infection,
and their family members of LAIs was an outbreak of Salmonella but effective treatment and preventive measures are available, and the
risk of spread of infection is limited.
typhimurium infections. Between August 2010 and June 2011,
109 individuals were infected with this organism in over 38 states. Risk Group 3 (High Individual Risk, Low
An epidemiologic study was conducted, and one possible link Community Risk)
identified was exposure to microbiology laboratories, including A pathogen that usually causes serious human or animal disease but
does not ordinarily spread from one infected individual to another.
teaching laboratories and clinical laboratories. The CDC, along
Effective treatment and preventive measures are available.
with other organizations, identified areas where biosafety and
laboratory safety improvements should be made. Some of the Risk Group 4 (High Individual and Community Risk)
A pathogen that usually causes serious human or animal disease and
recommendations for students, laboratorians, laboratory directors
that can be readily transmitted from one individual to another, directly
and managers, and faculty included the following: or indirectly. Effective treatment and preventive measures are not usually
1. Laboratorians and students should know that the bacteria handled available.
in the laboratory can make people sick. These organisms can
From World Health Organization: Laboratory biosafety manual, ed 3, Geneva,
also make family members sick. Individuals must not take Switzerland, 2004, World Health Organization.
items into the laboratories that will be taken home, such as
laboratory coats, pens, books, laboratory report forms, cell
phones, and keys. biosafety levels (BSLs) discussed later in this chapter, but are
2. Students should have dedicated writing utensils and supplies at not always equal (Box 4.2). Other factors that must be considered
their work stations, and these should not leave the laboratory. when one is determining the correct BSL needed are the mode
3. Laboratorians and students must be aware of the organisms of transmission, the microbiological procedures that will be
with which they are working and what the signs and symptoms performed, and the experience of the staff members. A biological
are if they get infected with one of these organisms. risk assessment is a comprehensive attempt to determine what
4. Laboratorians and students must be trained and proficient in controls should be used to protect the worker and environment
biosafety practices and techniques. from exposure. It is a subjective process, and different strategies
5. Laboratory coats should always be worn over personal clothing can be used to perform the assessment. Five steps have been
(appropriate PPE should be worn). Individuals should not leave outlined in the CDC’s “Guidelines for Safe Work Practices in
the laboratory with PPE on; PPE always should be disposed Human and Animal Medical Diagnostic Laboratories” for perform-
of properly. ing a risk assessment. These are as follows:
6. Handwashing sinks and supplies must be provided. Laborato- 1. Identify the hazards associated with an infectious agent or
rians and students should always wash their hands before leaving material.
the laboratory. 2. Identify the activities that might cause exposures to the agent
Biological risk assessment is an important part of every or material.
microbiology laboratory safety program. Biological risk assessment 3. Consider the competencies and experience of laboratory
is a process used to recognize the hazardous characteristics of personnel.
infectious agents that may be encountered in the clinical microbiol- 4. Evaluate and prioritize risks (evaluate the likelihood that an
ogy laboratory. Also included in the risk assessment process are exposure would cause an LAI and the severity of consequences
the laboratory practices that could result in an infectious exposure, if such an infection occurs).
the likelihood that an LAI will occur, and the consequences of 5. Develop, implement, and evaluate controls to minimize the
that infection. Through this process, appropriate safety practices risk for exposure.*
can be identified to protect laboratorians.
The hazardous risk characteristics of an agent are determined Processing of Patient Specimens
by the agent’s ability to infect and cause disease in humans or Labeling only specimens from patients with known hepatitis or
animals; its virulence; the availability of treatment for the disease; AIDS and requiring “extra precautions” for dealing with these
and whether there are any preventive measures, such as a vaccine,
that can be used to prevent disease by the microorganism. The
World Health Organization (WHO) defined four risk groups for *From Department of Health and Human Services, Centers for Disease Control
and Prevention: Guidelines for safe work practices in human and animal medical
infectious agents based on the hazardous characteristics listed
diagnostic laboratories. MMWR Suppl 61(01) January 6, 2012, p.1, Available at:
previously. The four risk groups, defined in the WHO classification https://www.cdc.gov/mmwr/preview/mmwrhtml/su6101a1.htm?s_cid=su6101a1_w.
of infectious microorganisms by risk group, correlate with the Accessed Oct. 2, 2017.
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 73

specimens does not provide adequate protection for laboratory 8. Any cultures suspected of growing other potentially aerosolized
workers. Many patients come into emergency departments or are infectious agents, such as M. tuberculosis or Brucella spp.,
admitted to the hospital with no diagnosis. These patients may should be worked on only in a biosafety cabinet.
be in the early stages of either disease and may be asymptomatic In an effort to educate individuals at risk of LAI, the CDC
but still contagious. Because the incidence of both hepatitis publishes a manual entitled Biosafety in Microbiological and
and AIDS is high, the likelihood of exposure to one or both of Biomedical Laboratories every few years, which contains important
these bloodborne pathogens is also elevated. For this reason, information on the degree of risk of biological agents for laboratory
standard precautions should be used when handling all patient workers and the safety procedures that should be used when
samples. working with infectious agents.
As stated earlier, when samples are received in the microbiology
laboratory, often not enough information is available to assess the Case Check 4.3
risk involved with processing the sample. Microbiologists do not
Shigella sonnei was isolated from the stool culture of the medical laboratory
know what infectious agents are present in the sample and will
scientist from the Case in Point. The laboratory supervisor investigated
not know until the agent or agents have been identified. For this the incident. It was discovered that the medical laboratory scientist had
reason, it must be assumed that the specimen contains agents her cell phone with her in the laboratory and used the phone during her
that correlate with at least a BSL-2, unless there is additional work at the laboratory bench. She stated that at the end of her shift she
information suggesting that there may be an agent present from wiped the phone with alcohol. It appears that the laboratory-acquired
a higher-risk group. A common practice is to perform all speci- infection occurred because of the medical laboratory scientist not following
safety protocols in the microbiology laboratory.
men processing in a biosafety cabinet because of the uncertainty
regarding the infectious agents that might be present in the
sample. Biological Safety Cabinets
Biological safety cabinets (BSCs) are a form of engineering control
that is used throughout the microbiology laboratory. These hoods
Case in Point are a type of containment barrier that protects the worker from
A medical laboratory scientist was reading cultures at the bench. the aerosolized transmission of organisms. Any procedure that
During her work, she isolated and identified Shigella sonnei. She has the ability to create aerosols should be performed in a BSC.
started having watery diarrhea 48 hours later, which progressed Microbiology samples should also be handled within a BSC. There
to abdominal cramps and bloody stools. Because of her signs are three types of BSC: class I, class II, and class III (Fig. 4.4).
and symptoms, her primary care practitioner ordered a stool A BSC must be used properly, and it is imperative that the
culture. laboratory worker understand the functions and limitations of the
unit. The most common type of BSC used in a microbiology
laboratory is the class II. Box 4.3 provides a few guidelines to
Issues to Consider
follow to ensure the proper use of a BSC.
After reading the patient’s case history, consider:
■ The identity of the organism causing the infections
Biosafety Levels
■ The potential for a laboratory-acquired infection
LAIs may occur through error, accident, or carelessness. However,
■ The importance of laboratory safety procedures
the mode of transmission is unknown in many LAIs. For this reason,
strict guidelines have been instituted to protect the worker during
Working with Actively Growing Cultures all laboratory tasks. Earlier in this chapter, the classification of
Many of the guidelines in place for the protection of microbiology infectious microorganisms into risk groups was discussed. When
personnel against exposure to bloodborne pathogens also apply considering which BSL to use, the laboratorian should know the risk
to working with cultures of microorganisms at the bench. Some group for the agents being handled, the mode of transmission for
of the safety precautions that should be used in the microbiology the agents, and the procedures that will be performed (e.g., whether
laboratory are the following: aerosols will be created). The Biosafety in Microbiological and
1. Hands must be washed frequently and kept away from the Biomedical Laboratories manual from the CDC has recommended
nose, mouth, and eyes. Hands should be washed after removal the BSLs that should be used when working with particular agents
of gloves. and performing certain procedures. Four BSLs with guidelines
2. Appropriate PPE should be worn. were established for each level for protection.
3. Adhesive bandages or small finger cots should be worn directly Biosafety Level 1.  Infectious agents that would be classified
over cuts or hangnails. as requiring BSL-1 containment are agents that are well classified
4. Plates should not be waved in front of the face to determine and are not known to cause disease consistently in healthy adults.
the odor of the organism. These agents pose a minimal threat to laboratory personnel and
5. Splash guards are recommended for any procedure that may the environment. Safety guidelines for handling these agents include
produce splashes, such as work at the blood culture bench. the following:
Gram-negative coccobacilli from a blood culture should be 1. Access to the laboratory is limited or restricted, and there must
handled in a biosafety cabinet. be a biohazard sign posted at the entrance of the laboratory.
6. Do not use open flames in the laboratory. 2. Employees must wash their hands after they have removed
7. Any plates growing a fungus should immediately be sealed, their gloves, after they have handled live organisms, and before
and the culture should be worked on in a biosafety cabinet. they leave the laboratory.
74 PART 1  Introduction to Clinical Microbiology

Exhaust Exhaust HEPA filters

HEPA filter

Air

Air

A B

Exhaust

Filter

Gloves

C
FIG. 4.4  A, The class I biological safety cabinet (hood) uses an exhaust fan to move air inward
through the open front. The air is circulated within the safety hood, passing through a high-
efficiency particulate air (HEPA) filter before reaching the environment outside the hood. B, The
class II biological safety hood is the most common in microbiology laboratories. Air is pulled
inward and downward by a blower and passed up through the air flow plenum, where it passes
through a HEPA filter before reaching the work surface. A percentage of the remaining air is
HEPA filtered before reaching the environment. C, The class III biological safety hood is a self-
contained ventilated system for highly infectious microorganisms or materials and provides the
highest level of personal protection. The closed front contains attached gloves for manipulation
on the work surface. Note: Chemical fume hoods cannot be used as biological safety cabinets,
and biological safety cabinets cannot be used as chemical fume hoods.

3. Employees must follow basic work practice controls. Laboratory work can be conducted on open bench tops in a
4. Employees must follow OSHA guidelines for handling needles BSL-1 laboratory. Employees should be trained in laboratory
and sharps. procedures and supervised by a scientist with training in microbiol-
5. Work surfaces must be decontaminated after completion of ogy or a related science. Standard microbiology practices should
work and anytime there has been a spill of potentially infectious be followed at all times. Examples of BSL-1 organisms include
material. Bacillus subtilis and Enterobacter aerogenes.
6. Laboratory coats and gloves should be worn and other PPE, Biosafety Level 2. Infectious agents that require BSL-2
such as face shields and eye protection, may be needed when containment and practices are agents that pose a moderate potential
there is a potential for splashes or sprays of infectious or other hazard for the employees and the environment. The guidelines
hazardous materials. are the same as for BSL-1 with added precautions for BSL-2
7. Cultures and stock material must be decontaminated before agents as follows:
disposal. 1. Employees in the laboratory must have specific training in the
8. An insect and rodent control plan must be in effect. handling of pathogenic agents and should receive annual updates
9. The laboratory facility should be designed so that it can be or additional training as needed.
easily cleaned. Carpets and rugs should not be used. The labora- 2. Access to the laboratory is limited when work is being con-
tory must be equipped with handwashing sinks, eyewashes, ducted. The laboratory director is ultimately responsible for
and adequate illumination. determining who may enter or work in the laboratory.
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 75

of these pathogenic and potentially lethal organisms. A few of


BOX 4.3  Proper Use of a Biological Safety Cabinet the special BSL-3 guidelines are as follows:
1. Be aware of the functioning and limitations of the unit. You and 1. The handling of infectious materials, samples, and cultures
your specimen are being protected solely by an air curtain barrier, must occur within a BSC or other physical containment device.
and anything that disrupts this curtain threatens your safety. Ensure 2. Personnel must wear appropriate PPE. Gloves must be worn
that the cabinet has been inspected within the last year and that the to protect hands from exposure to hazardous materials.
air pressure readings across the HEPA filter are within specifications. 3. The BSL-3 laboratory should be separated from the other parts
2. Plan your work, anticipating the order of events. of the building and should be accessed through two self-closing
3. Turn on the incandescent lights and the blower fan. Be sure the
doors. An anteroom may be used for access.
UV light is off. Wait 15 to 30 minutes to ensure satisfactory
establishment of the air curtain. 4. The BSL-3 laboratory requires a ducted air ventilation system
4. Wash your hands, then gown and glove. Wear a mask or other that must provide sustained directional air flow. This direc-
personal protective equipment as appropriate. tional air flow pulls air from “clean” areas toward “potentially
5. Decontaminate the interior work surfaces by cleansing them contaminated” areas (negative air pressure).
thoroughly with 70% ethanol. 5. The ceilings and floors must be solid, and any seams must be
6. Organize all necessary work materials and place them in the cabinet.
sealed.
Do not place any extra items in the cabinet. Ensure that both
the front intake grill and the rear-wall or floor exhaust grills are
6. All parts of the laboratory must be constructed for easy cleaning
unobstructed. and decontamination.
7. Segregate clean and contaminated items and place them to minimize Examples of BSL-3 organisms include M. tuberculosis, St.
subsequent movement with the BSC. The discard bucket/pan should Louis encephalitis virus, and Coxiella burnetii.
be near the rear of the cabinet but not obstructing the exhaust Biosafety Level 4. BSL-4 containment and practices are
grill. required when working with agents that are dangerous and exotic.
8. Do not use open flames in the unit. Instead, use disposable supplies
These agents have a high risk of causing life-threatening infections,
or a microburner/incinerator.
9. All arm movements into and within the cabinet should be slow can be transmitted by aerosols, or have an unknown risk of
and deliberate so as to minimize disruption of the air curtain. Allow transmission. As in all microbiology practices, specific training
adequate time at the conclusion of movement for the air curtain is required. Laboratory personnel must receive thorough training
to reestablish itself. in the handling of these dangerous agents, and they must be trained
10. When work is completed, remove all nonpermanent items from in how to use the containment barriers that are in place for protec-
the BSC and allow the cabinet fans to continue running for at least tion. The BSL-4 facility either is located in a separate building
30 minutes to ensure thorough filtering of the inside air (assuming
or is in an isolated zone within a building. This facility is isolated
the fans are not left on permanently). Turn on the UV lights to
disinfect the interior of the cabinet. from all other areas, and access is strictly controlled. The guidelines
listed for BSL-3 must be followed along with additional guidelines
BSC, Biological safety cabinet; HEPA, high-efficiency particulate air; UV,
for handling BSL-4 agents. There are two types of BSL-4 labo-
ultraviolet.
Data from Kruse RH, Puckett WH, Richardson JH: Biological safety cabinetry, ratories, cabinet and suit. In the cabinet laboratory, all work is
Clin Microbiol Rev 2:207, 1991. performed within a class III BSC. In a suit laboratory, personnel
wear a positive pressure protective suit to perform all work. Some
of the additional guidelines are:
3. Laboratorians should receive immunizations or tests for agents 1. Access is strictly controlled, and the supervisor has the
handled or for agents that could potentially be in the laboratory ultimate responsibility for who has access. A logbook is
environment (e.g., hepatitis B vaccination and the tuberculosis maintained to document all personnel who enter and exit the
skin test.) laboratory.
4. A biosafety manual must be developed and updated. 2. As required by all levels, a biohazard sign must be posted
5. A BSC must be used whenever there is a potential for creating outside the door with the potential hazards; the laboratory
infectious aerosols or splashes or when high concentrations of director’s name; and any special requirements, such as immu-
infectious agents are used. The recommended BSC is class II. nizations, for entering the area.
6. All PPE must be worn. Gloves must be worn to protect hands 3. All personnel must demonstrate high proficiency in standard
from exposure to hazardous materials. and special microbiology practices.
7. Extreme precautions are taken with contaminated sharp items. 4. Policies and procedure are established on the collection and
The use of needles and syringes is restricted within the labora- storage of serum samples from at-risk personnel.
tory to only times when there is no alternative equipment that 5. Personnel enter and exit the laboratory through the clothing
can be used. change room. When personnel leave the laboratory, they must
Examples of BSL-2 organisms include HBV, HIV, Salmonella completely change clothes and shower. The clothes are then
spp., and Toxoplasma gondii. decontaminated before being laundered.
Biosafety Level 3. BSL-3 containment and practices are 6. The BSL-4 laboratory has a dedicated nonrecirculating ventila-
required for infectious agents that are either indigenous or exotic. tion system, which is filtered through a HEPA filter before
These agents have the potential for aerosol transmission, and being exhausted.
diseases with these agents may have serious lethal consequences. 7. All material is decontaminated before leaving the BSL-4
The guidelines for BSL-2 laboratories must be followed along laboratory.
with the more stringent guidelines needed to handle BSL-3 agents Examples of BSL-4 agents include Marburg and Congo-Crimean
safely. Laboratory personnel must have specific training in handling hemorrhagic fever viruses.
76 PART 1  Introduction to Clinical Microbiology

Hazardous Waste
The clinical laboratory is responsible for the proper handling
and disposal of all of the waste it generates. The scope of
hazardous waste comprises more than just infectious waste. The
Clinical Laboratory Waste Management Approved Guideline,
2nd edition, by the Clinical Laboratory and Standards Institute,
provides information for laboratory managers on the regulations
governing hazardous wastes and addresses the following types of
waste: chemical, infectious, radioactive, sharps, multihazardous,
and nonhazardous. The guideline also emphasizes methods to
reduce waste generation and methods to reduce the volume of
and toxicity of unavoidable wastes.

Disposal of Infectious Waste


In addition to laboratory employees who work with potentially
infectious material, the general public must be protected from
exposure to these same materials after the laboratory or hospital
has disposed of them. The surfacing of contaminated needles and
other sharps along lake and ocean beaches led to a public outcry
to make hospitals accountable for their infectious waste disposal.
In an effort to deal with this problem, the U.S. Congress passed
the Medical Wastes Tracking Act in 1988 to regulate the states
of New York, New Jersey, Connecticut, and Rhode Island as well
as Puerto Rico. These regulations went into effect on June 24,
1989, and expired on June 21, 1999. This act allowed the EPA
to gather information and focus attention on the problem of medical
waste and provided a basis for other states and federal agencies
to develop policies on the disposal of medical waste.
The microbiology laboratory’s safety program must follow
state and local regulations for the safe disposal of its infectious
FIG. 4.5  Biohazard bag used to dispose of culture plates and
wastes, usually by either autoclaving or incineration. Warning
other nonsharp contaminated materials. The bag is sealed and
signs containing the warning symbol for biohazardous materials incinerated. In the center of the bag is the biohazard symbol.
must be placed on all biohazard wastes and material disposal
containers. Figs. 4.5 and 4.6 show disposal containers appropriate
for contaminated and biohazardous materials. laboratory personnel should have a thorough working knowledge of
the hazards of the chemicals with which they come into contact, or
Hazardous Waste Reduction employee right-to-know, and must receive chemical safety train-
The EPA has also made recommendations for hazardous waste ing annually. The National Research Council publication Prudent
reduction through the following methods: Practices in the Laboratory: Handling and Disposal of Chemicals
1. Substitute less hazardous chemicals when possible. can be used by laboratory managers when developing a chemical
2. Develop procedures that use less of a hazardous chemical. hygiene plan for a laboratory. All chemicals in the workplace must
3. Recycle chemicals when possible. be identified and clearly labeled with the National Fire Protection
4. Segregate infectious wastes from uncontaminated trash. Association (NFPA) 704 hazard-rating diamond, stating risk for
5. Substitute micromethodology in antimicrobial susceptibility flammability, reactivity, and health (Fig. 4.7).
testing and identification of organisms to reduce the volume
of chemical reagents as well as infectious waste. Safety Data Sheets
Safety data sheets (SDSs) are provided by the manufacturer or
Chemical Safety distributor for hazardous chemicals. Although there is no accepted
Chemicals used in the clinical laboratory can be hazardous to the format regarding what is contained in an SDS, some of the
laboratorian. The toxic risks of a chemical are related to the extent information that should be provided is as follows:
of exposure and to the inherent toxicity of the chemical. The pos- • Name, address, and telephone number of the manufacturer
sible routes of exposure for chemicals are through the skin, eyes, • Chemical and synonym names and a list of the hazardous
mucosa, gastrointestinal tract, and respiratory tract. The OSHA ingredients
addresses employee safety with hazardous chemicals in 29 Code of • Chemical characteristics, such as vapor pressure and flash point,
Federal register (CFR) 1910.1200, Hazard Communication Standard and the physical characteristics (potential for fire, explosion,
(HCS). The HCS focuses on the proper classification of chemicals and reactivity)
by manufacturers and importers as well as the safety practices for • Health hazards of the hazardous chemical (signs and symptoms
employers and employees. It requires a laboratory chemical hygiene of exposure and any medical conditions that can be caused by
plan and a hazard communication program, and states that all clinical exposure)
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 77

A B
FIG. 4.6  A and B, Examples of biohazard containers for disposable needles, glass slides, and other
sharp materials.

HAZARDOUS MATERIALS CLASSIFICATION


CLASIFICACION DE MATERIALES PELIGROSOS BOX 4.4  Hazardous Chemicals Commonly Used in
HEALTH HAZARD FIRE HAZARD the Microbiology Laboratory
4 – Deadly Flash points
4 – Below 73°F
3 – Extreme danger
2 – Hazardous 3 – Below 100°F Flammables
1 – Slightly hazardous 2 – Below 200°F
0 – Normal material 1 – Above 200°F • Methanol
0 – Will not burn
PELIGRO PARA LA SALUD • Acetone
PELIGRO DE
4 – Mortal
3 – Gravemente peligroso
INFLAMABILIDAD • Ethanol
2 – Peligroso Puntos de inflamabilidad
1 – Ligeramente peligroso 4 – Por debajo de 73°F
Potential or Proven Carcinogens

2
0 – Material normal 3 – Por debajo de 100°F
2 – Por debajo de 200°F
1 – Por encina de 200°F • Formaldehyde
0 – No arde
• Aniline (crystal violet) stain

3 1 • Auramine-rhodamine (Truant) stain


Irritants and Corrosives
SPECIFIC HAZARD
W REACTIVITY
4 – May detonate
• Hydrogen peroxide
• Acids: HCl, H2SO4, acetic acid
3 – Shock and heat may
Oxidizer
Acid
OX
ACID detonate • NaOH
Alkali ALK 2 – Violent chemical change
Corrosive COR 1 – Unstable if heated
Use NO WATER W 0 – Stable
Radiation hazard
An example of an SDS is shown in Fig. 4.8. These documents
REACTIVIDAD
PELIGRO ESPECIFICO 4 – Puede explotar
Oxidante
Acido
OX
ACID
3 – Puede explotar en caso
de choque o de should be kept on file and made available to every employee.
Other examples of common hazardous chemicals found in the
Alcalino ALK calentamiento
Corrosive COR 2 – Cambio químico violento
1 – Inestable en caso de
NO USAR AGUA
Peligro de radiación
W
calentamiento
0 – Estable
microbiology laboratory are listed in Box 4.4.

FIG. 4.7  Hazardous material classification symbol. (Courtesy Chemicals Inventory


Laboratory Safety Supply, Inc., Janesville, WI.) The laboratory must maintain a current inventory of chemicals,
which must be updated annually, along with the SDSs for those
• The primary routes of entry and the target organs particular chemicals. The following four sources should be
• Precautions for safe handling and use, including appropriate consulted in preparing an inventory:
hygienic practices 1. 29 CFR Part 1910, Subpart Z, Toxic and Hazardous Substances,
• Any generally applicable control measures, such as appropriate OSHA
engineering controls, work practices, or PPE 2. National Toxicology Program Annual Report on Carcinogens
• Emergency and first aid procedures 3. International Agency for Cancer Research Monographs
• Spill cleanup procedure 4. Manufacturers’ SDSs
• Disposal recommendations Text continued on p. 84
78 PART 1  Introduction to Clinical Microbiology

Page 1/7
Safety data sheet
according to 1907/2006/EC, Article 31
Date Prepared: 24.03.2011 Revision: 11.02.2011

1 Identification of the substance/mixture and of the company/undertaking


· Product identifier
· Trade name: Streptococus pneumoniae Antibody-Coated Latex
(Pneumoslide Test Kit)
· Article number: 240840
· Relevant identified uses of the substance or mixture and uses advised against
· Sector of Use
SU3 Industrial uses: Uses of substances as such or in preparations at industrial sites
SU22 Professional uses: Public domain (administration, education, entertainment, services,
craftsmen)
SU20 Health services
SU24 Scientific research and development
· Application of the substance / the preparation Preparation
· Details of the supplier of the safety data sheet
· Manufacturer/Supplier:
BD Diagnostic Systems Europe
Regulatory Compliance Department
Becton Dickinson France
11, rue Aristide Bergès
38800 Le Pont de Claix
France
Telephone: 33 (0) 476 68 36 36
Email: msds_europe@bd.com
· Further information obtainable from: Technical Service Representative
· Emergency telephone number: Tel: 33 (0) 476 68 36 36

2 Hazards identification
· Classification of the substance or mixture
· Classification according to Regulation (EC) No 1272/2008
The product is not classified according to the CLP regulation.
· Classification according to Directive 67/548/EEC or Directive 1999/45/EC
This product contains no hazardous constituents, or the concentration of all chemical
constituents are below the regulatory threshold limits described by the Occupational Safety
and Health Administration Hazard Communication Standard or European Directive 67/548/
EEC and 1999/45/EC.
Void
· Information concerning particular hazards for human and environment:
The product does not have to be labelled due to the calculation procedure of the "General
Classification guideline for preparations of the EU" in the latest valid version.
· Classification system:
The classification is according to the latest editions of the EU-lists, and extended by company
and literature data.
· Label elements
· Labelling according to EU guidelines:
Observe the general safety regulations when handling chemicals
(Contd. on page 2)
EU

FIG. 4.8  Safety data sheet. (Courtesy and copyright Becton, Dickinson and Company, Franklin
Lakes, NJ.)
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 79

Page 2/7
Safety data sheet
according to 1907/2006/EC, Article 31
Date Prepared: 24.03.2011 Revision: 11.02.2011

Trade name: Streptococus pneumoniae Antibody-Coated Latex


(Pneumoslide Test Kit)

(Contd. of page 1)
The product is not subject to identification regulations under EU Directives and the Ordinance
on Hazardous Materials (German GefStoffV).
· Other hazards
· Results of PBT and vPvB assessment
· PBT: Not applicable.
· vPvB: Not applicable.

3 Composition/information on ingredients
· Chemical characterization: Mixtures
· Description: Mixture consisting of the following components.
· Dangerous components:
Natural Rubber Latex 99,88%
CAS: 2682-20-4 2-methyl-4-isothiazolin-3-one 0,1%
EINECS: 220-239-6 T R24/25; C R34; Xi R43
Acute Tox. 3, H301; Acute Tox. 3, H311; Skin Corr. 1B, H314;
Skin Sens. 1, H317
· Additional information For the wording of the listed risk phrases refer to section 15.

4 First aid measures


· Description of first aid measures
· General information No special measures required.
· After inhalation Seek medical treatment in case of complaints.
· After skin contact Immediately wash with water and soap and rinse thoroughly.
· After eye contact
Rinse opened eye for several minutes under running water. If symptoms persist, consult a
doctor.
· After swallowing If symptoms persist consult doctor.

5 Firefighting measures
· Extinguishing media
· Suitable extinguishing agents
CO2, powder or water spray. Fight larger fires with water spray or alcohol resistant foam.
· Advice for firefighters
· Protective equipment: No special measures required.

6 Accidental release measures


· Personal precautions, protective equipment and emergency procedures Not required.
· Environmental precautions: Damp down dust with water spray.
(Contd. on page 3)
EU

FIG. 4.8, cont’d  Continued


80 PART 1  Introduction to Clinical Microbiology

Page 3/7
Safety data sheet
according to 1907/2006/EC, Article 31
Date Prepared: 24.03.2011 Revision: 11.02.2011

Trade name: Streptococus pneumoniae Antibody-Coated Latex


(Pneumoslide Test Kit)

(Contd. of page 2)
· Methods and material for containment and cleaning up: No special measures required.
· Reference to other sections No dangerous substances are released.

7 Handling and storage


· Handling
· Precautions for safe handling No special measures required.
· Information about fire - and explosion protection: No special measures required.
· Conditions for safe storage, including any incompatibilities
· Storage
· Requirements to be met by storerooms and receptacles: 2 - 8 ºC
· Information about storage in one common storage facility:
Store away from oxidizing agents.
· Further information about storage conditions:
Store in cool, dry conditions in well sealed receptacles.

8 Exposure controls/personal protection


· Additional information about design of technical facilities: No further data; see item 7.
· Control parameters
· Ingredients with limit values that require monitoring at the workplace:
The product does not contain any relevant quantities of materials with critical values that
have to be monitored at the workplace.
· Additional information: The lists valid during the making were used as basis.
· Exposure controls
· Personal protective equipment
· General protective and hygienic measures
The usual precautionary measures are to be adhered to when handling chemicals.
· Respiratory protection:
In case of brief exposure or low pollution use respiratory filter device. In case of intensive or
longer exposure use self-contained respiratory protective device.
· Protection of hands:

Protective gloves.

· Eye protection: Safety glasses


· Body protection: Protective work clothing. (Contd. on page 4)
EU

FIG. 4.8, cont’d 


CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 81

Page 4/7
Safety data sheet
according to 1907/2006/EC, Article 31
Date Prepared: 24.03.2011 Revision: 11.02.2011

Trade name: Streptococus pneumoniae Antibody-Coated Latex


(Pneumoslide Test Kit)

(Contd. of page 3)

9 Physical and chemical properties


· Information on basic physical and chemical properties
· General Information
· Appearance:
Form: Solid.
Colour: According to product specification
· Odour: Characteristic
· Change in condition
Melting point/Melting range: Not determined
Boiling point/Boiling range: Not determined
· Flash point: Not applicable
· Flammability (solid, gaseous) Product is not flammable.
· Danger of explosion: Product does not present an explosion hazard.
· Density: Not determined
· Solubility in / Miscibility with
Water: Insoluble
· Solvent content:
Solids content: 100,0 %

10 Stability and reactivity


· Reactivity
· Chemical stability
· Thermal decomposition / conditions to be avoided:
No decomposition if used according to specifications.
· Possibility of hazardous reactions No dangerous reactions known
· Hazardous decomposition products: No dangerous decomposition products known

11 Toxicological information
· Information on toxicological effects
· Acute toxicity:
· Primary irritant effect:
· on the skin: No irritant effect.
· on the eye: No irritating effect.
· Sensitization: No sensitizing effects known.
(Contd. on page 5)
EU

FIG. 4.8, cont’d  Continued


82 PART 1  Introduction to Clinical Microbiology

Page 5/7
Safety data sheet
according to 1907/2006/EC, Article 31
Date Prepared: 24.03.2011 Revision: 11.02.2011

Trade name: Streptococus pneumoniae Antibody-Coated Latex


(Pneumoslide Test Kit)

(Contd. of page 4)
· Additional toxicological information:
The product is not subject to classification according to the calculation method of the General
EU Classification Guidelines for Preparations as issued in the latest version.
When used and handled according to specifications, the product does not have any harmful
effects to our experience and the information provided to us.

12 Ecological information
· Toxicity
· Acquatic toxicity: No further relevant information available.
· Ecotoxical effects:
· Other information:
The ecological effects have not ben thoroughly investigated, but currently none have been
identified.
· Additional ecological information:
· General notes: Generally not hazardous for water.
· Results of PBT and vPvB assessment
· PBT: Not applicable.
· vPvB: Not applicable.

13 Disposal considerations
· Waste treatment methods
· Recommendation
Smaller quantities can be disposed of with household waste.
Must be specially treated adhering to official regulations.
· Uncleaned packaging:
· Recommendation: Disposal must be made according to official regulations.
· Recommended cleansing agents: Water, if necessary together with cleansing agents.

14 Transport information
· Land transport ADR/RID (cross-border)
· ADR/RID class: -
· Maritime transport IMDG:
· IMDG Class: -
· Marine pollutant: No
· Air transport ICAO-TI and IATA-DGR:
· ICAO/IATA Class: -
· UN "Model Regulation": -
(Contd. on page 6)
EU
FIG. 4.8, cont’d 
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 83

Page 6/7
Safety data sheet
according to 1907/2006/EC, Article 31
Date Prepared: 24.03.2011 Revision: 11.02.2011

Trade name: Streptococus pneumoniae Antibody-Coated Latex


(Pneumoslide Test Kit)

(Contd. of page 5)
· Special precautions for user Not applicable.

15 Regulatory information
· Safety, health and environmental regulations/legislation specific for the substance or
mixture
· IARC (International Agency for Research on Cancer)
None of the ingredients is listed.
· Labelling according to EU guidelines:
Observe the general safety regulations when handling chemicals
The product is not subject to identification regulations under EU Directives and the Ordinance
on Hazardous Materials (German GefStoffV).
· Chemical safety assessment: A Chemical Safety Assessment has not been carried out.

16 Other information
Disclaimer:
To the best of our knowledge, the information contained herein is accurate. However, neither
BD or any of its subsidiaries assums any liabilities whatsoever for the accuracy or
completeness of the information contained herein. Final determination of suitability of any
material is the sole responsibility of the user. All materials may present unknown hazards and
should be used with caution. Although certain hazards described herein, we cannot
guarantee that these are the only hazards that exist.
· Relevant phrases
H301 Toxic if swallowed.
H311 Toxic in contact with skin.
H314 Causes severe skin burns and eye damage.
H317 May cause an allergic skin reaction.
R24/25 Toxic in contact with skin and if swallowed.
R34 Causes burns.
R43 May cause sensitisation by skin contact.
· Department issuing MSDS:
Environmental, Health & Safety
Created by Michael J. Spinazzola
· Contact: Technical Service Representative
· Abbreviations and acronyms:
ADR: Accord européen sur le transport des marchandises dangereuses par Route (European Agreement
concerning the International Carriage of Dangerous Goods by Road)
RID: Règlement international concernant le transport des marchandises dangereuses par chemin de fer
(Regulations Concerning the International Transport of Dangerous Goods by Rail)
IMDG: International Maritime Code for Dangerous Goods
IATA: International Air Transport Association
IATA-DGR: Dangerous Goods Regulations by the "International Air Transport Association" (IATA)
ICAO: International Civil Aviation Organization
(Contd. on page 7)
EU

FIG. 4.8, cont’d  Continued


84 PART 1  Introduction to Clinical Microbiology

Page 7/7
Safety data sheet
according to 1907/2006/EC, Article 31
Date Prepared: 24.03.2011 Revision: 11.02.2011

Trade name: Streptococus pneumoniae Antibody-Coated Latex


(Pneumoslide Test Kit)

(Contd. of page 6)
ICAO-TI: Technical Instructions by the "International Civil Aviation Organization" (ICAO)
GHS: Globally Harmonized System of Classification and Labelling of Chemicals
EU

FIG. 4.8, cont’d 

Chemical Storage Chemical Spills


Chemicals should never be stored alphabetically. Chemicals must Spillage in the laboratory is unavoidable and unpredictable.
be stored according to established rules of compatibility to prevent Many chemical manufacturers develop charts describing the
contact between reactive substances. Do not store alkali metals procedure for managing spills. Acid and base spill kits and
(e.g., sodium, potassium) with carbon dioxide, chlorinated flammable spill kits should be kept in areas where such sub-
hydrocarbons, or water. Acids and bases should never be stored stances are used. Equipment such as protective clothing, scoops
together. Also, acids such as acetic acid and sulfuric acid should and dust pans, forceps for picking up glass, and buckets should
never be stored with oxidizing agents. Halogens are incompatible be kept in a designated area. In the event of a large spill, the
with ammonia, acetylene, and hydrocarbons. Flammable chemicals Environmental Health and Safety Department should be called for
should be stored in a flammable storage cabinet. Within the labora- assistance.
tory, chemicals should be stored in amounts reflective of the daily
requirements. Bulk stocks should be stored in specially designated Signage
areas and not in the laboratory. It is good practice to store corrosives Signage is important safety equipment. Warning signs and symbols,
in trays as secondary containment because of the potential for as shown in Fig. 4.9, must be placed in appropriate locations.
shelves to become damaged. Employees must be able to recognize each of these symbols and
must be knowledgeable about the danger each indicates and the
Hazardous Chemical Classification proper precautions that must be observed.
Chemical manufacturers and importers classify chemicals as
required by OSHA Standard 1910.1200(d). Chemical manufacturers Fire Safety
and importers determine the hazard classes and the category of Bunsen burners and other open-flame burners in most cases have
each chemical by considering the comprehensive range of scientific been replaced with other methods or techniques, such as fixing
literature related to a chemical. There is no requirement for slides with methanol or slide warmers and using disposable loops.
manufacturers or importers to perform tests to determine a chemical Bunsen burners and open-flame burners are among the biggest
classification. Chemical manufacturers and importers determine sources of fire hazards in the clinical microbiology laboratory.
whether a chemical is carcinogenic by referring to the National Employees must strive not to be careless or negligent in the use
Toxicology Program and the International Agency for Research of this equipment. Flammable materials should never be opened
on Cancer annual reports. End-users of chemicals are not required near a Bunsen burner in use. Open flames should not be left
to classify chemicals; however, they may conduct their own unattended, and the laboratorian should always be sure to turn
classification studies if they deem such procedures appropriate off the gas when finished. As was previously noted, never place
for their facility. an open flame inside of a BSC.
Another hazard associated with Bunsen burners and other
Laboratory Safety for Hazardous Chemicals open-flame burners is the fuel. Bunsen burners use gas. A leaking
Laboratory fume hoods are one of the most important pieces of gas line may be a source of explosion or may cause illness in
equipment for protecting workers from exposure to hazardous employees who work near the leak. The gas hose should be
chemicals. Fume hoods must be provided to prevent inhalation inspected on a regular basis for cracks, holes, pinched points or
of fumes and should be evaluated at least annually for adequate other defects, and the hose should be replaced if any defects are
face velocity (average velocity of the air drawn through the face found. If a leak is suspected or the appliance is newly connected,
of the hood) and proper operation. Fume hoods should be vented. leaks can be checked for by lightly spraying the tubing with soapy
PPE, including appropriate gloves, laboratory coats, and eyewear, water. If bubbles appear when the gas is on, there is a leak at that
must be used in the case of a small spill to protect the workers point. Other burners use a flammable liquid as the fuel, which is
from exposure. A dust mask or respirator may be appropriate in itself a hazard. Other sources of ignition include heating ele-
depending on the material spilled. ments, hot plates, and spark gaps in motors and light switches.
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 85

WARNING SIGNS POTENTIAL DANGER


AND SYMBOLS TABLE 4.5  Classes of Fire Extinguishers

Symbol Class Use

A Fires in ordinary combustible materials, such as


A
Flammable wood, cloth, paper, rubber, and many plastics;
contains water or dry chemical to cool
B B Fires in flammable liquids, gases, and greases;
contains CO2 or dry chemical to smother
C C Fires involving electrical equipment, for which the
electrical nonconductivity of the extinguishing
media is important; contains CO2 or dry chemical
to smother without damaging the equipment
Toxic hazard
Poisonous

fire emergency. The drills should include both exit and nonexit
procedures. Exit drills familiarize personnel with the escape routes
and location of fire doors and stairwells. Nonexit procedures alert
Carcinogenic personnel to the potential for evacuation if the fire is located
Cancer-causing agent elsewhere in the building. The laboratory should be kept free of
clutter (e.g., do not leave boxes, cords, reagents, etc., on the floor),
and exits should always remain clear of obstructions.

Thermal Injuries
Corrosive Personnel should be warned of any hot surface or situation in
Harmful to mucous membranes, which the potential for burns is present. Use of long thermal
skin, eyes, or tissues gloves that extend to the shoulder is recommended when reaching
into autoclaves or hot-air ovens. Signs should be posted warning
employees about hot instruments or flasks that have just been
sterilized. Burns may also come from extremely low temperatures
found with use of liquid nitrogen or freezers that maintain tem-
peratures less than −70° C.
Radiation
Radioactive material present Storage of Compressed Gases
Flammable and nonflammable gas cylinders in use in the labora-
tory must always be properly restrained and stored in bulk and
FIG. 4.9  Miscellaneous warning signs and symbols.
secured in vented areas some distance from the laboratory. In
the laboratory, cylinders must be located well away from open
flames and other heat sources. Because a leaking pressurized
The OSHA requires employers to have an emergency action gas cylinder is a potential “missile,” care must be taken to avoid
plan, which includes what to do in the event of a fire. The emer- accidental breakage or removal of the pressure valve on top. The
gency action plan must be written, and it must be kept in the metal cap that protects this valve on top of the cylinder must be
workplace and available to all employees. All personnel must be kept in place during transportation and when the cylinder is not
thoroughly trained in the procedure for responding to a fire in use.
emergency. Most institutions use the acronym RACE:
Rescue: Remove anyone who is in danger. The safety of Electrical Safety
handicapped personnel (e.g., deaf or physically disabled) Today’s microbiology laboratories contain many instruments. All
should be a priority. laboratory electrical equipment must conform to national electrical
Alarm: Know where the nearest fire pull box or alarm station safety standards and codes. Each instrument must undergo regular
is located and the number to call to report the fire. preventive maintenance to ensure that it is functioning properly
Contain: Close doors to contain fire and smoke. and in the best repair. Electrical cords should be checked for
Extinguish: Use the properly rated fire extinguisher on small fraying. All cords should have grounded (three-pronged) plugs.
fires (Table 4.5). The College of American Pathologists, an organization that provides
If the situation is out of control, the best course of action is to accreditation to laboratories, requires electrical grounding and
evacuate. The fire evacuation plan must be posted, and employ- leakage checks on instruments before they are put in use, after
ees should be familiar with fire exit locations and evacuation repairs or modifications, and when there is a suspected problem.
procedures. Periodic fire drills should be conducted to ensure Electrical equipment should never be placed near safety showers
that all personnel react quickly and efficiently in case of a real because of the risk of electrocution.
86 PART 1  Introduction to Clinical Microbiology

Miscellaneous Safety Considerations Laboratory Response Network


Back Safety The CDC developed the Laboratory Response Network (LRN)
The best way to care for the back is to prevent back injuries. in 1999. This program developed a network of laboratories that
Carrying heavy trays of culture plates, lifting heavy loads into could respond quickly and effectively to biological and chemical
and out of autoclaves, and sitting or standing improperly all can terrorism. The LRN developed three levels of laboratories: (1)
contribute to back stress or injury. The following are some ways sentinel laboratories, (2) reference laboratories, and (3) national
to prevent back injuries: laboratories. The roles and responsibilities of each of these levels
• Use the legs to lift, not the back. are defined to provide rapid and safe identification of biological
• Keep loads close to the body when transporting them. agents during a bioterrorism event.
• Ask for assistance or use a cart when a load is too heavy.
• Use good posture. Sentinel Laboratories
• Stay physically fit. Sentinel laboratories constitute most hospital-based microbiology
laboratories and are divided into two levels. Advanced sentinel
First Aid Training clinical laboratories function at the frontlines and have the most
All personnel should be trained in cardiopulmonary resuscitation capability. The role of these sentinel laboratories is to recognize
and other lifesaving first aid so that they will be able to act quickly possible bioterrorism agents and to perform basic testing to rule
in an emergency involving either fellow workers or patients. out these agents or to refer suspicious specimens or isolates to
the LRN reference laboratory. Basic sentinel clinical laborato-
Immunizations ries have fewer analytical capabilities but may handle suspect
The OSHA requires that the hepatitis B vaccine be offered samples and would refer these specimens to an LRN reference
free of charge to all personnel who are at risk of exposure to laboratory. The key to the success of this level of the LRN is the
bloodborne pathogens. Microbiology personnel working with rapid recognition of a bioterrorism event. The American Society
sputum specimens or mycobacterial cultures should be screened for Microbiology has published laboratory guidelines for the
for exposure to M. tuberculosis with an annual tuberculosis infectious agents that could be used in a bioterrorism event.
skin test. Laboratorians must be aware of what organisms are on this list
and be familiar with the guidelines established when dealing with
Safety Training these agents.
All clinical laboratories must offer their employees safety training, The potential infectious agents of bioterrorism are divided into
and this training must be documented. The training must include the following categories:
the following safety issues: Category A agents pose the greatest public health threat because
• Fire: Knowledge of how and when to report fires, the location they are easily transmitted and are highly infectious.
of the nearest alarm box and fire extinguishers, how to use Examples include agents that cause smallpox, anthrax, and
fire extinguishers in small fires, and blocking of fire doors tularemia.
• Hazardous materials management: How to use SDSs Category B agents have moderate morbidity and low mortal-
• Proper storage of gases ity and are not as easily transmitted as category A agents.
• Bloodborne pathogens program: Appropriate practice of Examples include agents that cause Q fever, melioidosis,
infection control, how to handle sharps, compliance with and typhus fever.
exposure control plan, and handling of biological spills Category C agents are classified as emerging pathogens.
Safety training programs for microbiology personnel should Examples include Nipah virus and hantavirus.
be conducted annually to keep safe techniques fresh in everyone’s
mind. New personnel should be thoroughly trained in safety Reference Laboratories and
procedures. It is a good idea to focus on one safety topic at a National Laboratories
time and to make the training sessions enjoyable. Safety is each The role of reference laboratories and national laboratories in the
employee’s responsibility. LRN is to perform confirmatory testing. At the present time, more
than 100 laboratories are members of the reference laboratory
Bioterrorism and the Clinical category. The national laboratories in the LRN are the CDC, the
U.S. Army Medical Research Institute of Infectious Diseases, and
Microbiology Laboratory the Naval Medical Research Center.
Clinical microbiology laboratorians are critical players in the early
detection of a bioterrorism event. In the September 2001 anthrax Safety During a Possible
incident, laboratorians performed the testing that identified the Bioterrorism Event
infectious agent. The CDC has developed a program that identifies The agents in categories A and B have been associated with LAIs.
how health care workers and other public health officials should The biggest threat with handling these agents is not recognizing
respond to an event. This program (Emergency Preparedness and the risk associated with these infectious agents and not following
Response Program) addresses biological agents and diseases, the safety procedures instituted in the laboratory for protection.
laboratory information, training, preparedness and planning, and Specimen processing of samples from a possible bioterrorism
surveillance. Refer to Chapter 30 for more information on agents event should occur within a class II BSC. However, it is likely
of bioterror. that the first patients seen in an event will not be identified as
CHAPTER 4  Control of Microorganisms: Disinfection, Sterilization, and Microbiology Safety 87

victims of bioterrorism. Therefore the cultures from these patients


Learning Assessment Questions
could cause the greatest risk to laboratorians.
The agents that pose the greatest risk are agents that are 1. What is the difference between sterilization and disinfection?
transmitted by aerosols, and all laboratorians must remember 2. Explain situations in which you would use a disinfectant and an
antiseptic.
that many of the procedures that are performed in the laboratory
3. Describe the difference between physical and chemical methods
create aerosols, such as pipetting, flaming loops, streaking plates, of disinfection and sterilization.
and centrifugation. When a bioterrorism agent is suspected, all 4. What method is required to kill endospores effectively?
manipulations of the culture should be performed using BSL-3 5. List and describe factors that influence the degree of killing during
guidelines. Sentinel laboratories should not accept environmental disinfection and sterilization.
samples for testing because of the unknown nature of the sample. 6. Explain EPA regulations on chemical surface disinfectants and FDA
As soon as an agent has been identified as a possible select agent, regulations on chemical skin antiseptics.
7. Transient biota of the skin is defined as:
the sample should be referred to the appropriate LRN reference a. Organisms that are contracted from the environment
laboratory. For more information on the responsibilities of a b. Organisms that are contracted from other persons
sentinel laboratory, visit the American Society for Microbiology c. Organisms that are not part of the established normal biota
website. d. All of the above
8. Which of the following characteristics should be considered when
selecting an antimicrobial agent?
Packaging and Shipping of a. Spectrum of activity
Infectious Substances b. Rate of action
c. Mechanism of action
Laboratory personnel who are involved in the packaging and d. All of the above
shipping of infectious materials must be trained and certified before 9. Give the mechanism of action for each type of chemical agent
shipping infectious material. Personnel must be retrained on a commonly used in antiseptics and disinfectants.
regular basis. The International Air Transport Association, the 10. Explain the use of health care personnel handwash, surgical hand
International Civil Aviation Organization, and the U.S. Department scrub, and patient preoperative skin preparation.
11. OSHA requires employers to offer the hepatitis B virus vaccine
of Transportation regulations must be followed when packaging
free of charge to employees who are at risk for exposure to
and shipping infectious agents. Laboratory personnel must be hepatitis B virus. True of False?
aware of what agencies will transport these materials to the 12. SDSs are important to employees because they contain information
identified LRN reference or national laboratory. relating to which of the following?
a. Bloodborne pathogens
b. Fume hoods
c. Chemical safety
Points to Remember d. Fire extinguishers
■ Physical and chemical methods may be used in the process of 13. Which of the following would be a correct definition of standard
sterilization to remove all forms of life. precautions?
■ Disinfection involves removal of pathogenic organisms but may not a. Wearing only gloves to handle blood and body fluids
include removal of bacterial or other spores; most disinfectants are b. Viewing all specimens as potentially infectious and using the
chemical agents. appropriate protective equipment
■ Factors that influence the degree of killing include types of organisms c. Delaying testing of blood and body fluids pending results of
and number of organisms present, concentration of disinfecting HIV and hepatitis B antigen testing
agent, amount of soil present, and nature of the surface to be d. Flagging only specimens that come from patients who are
disinfected. known HIV carriers for “extra precautions”
■ Antiseptics are designed to reduce the bacterial load of living tissues. 14. What type of filter does a class II BSC use to filter out infectious
■ Disinfectants are designed to be used on inanimate objects to kill agents?
or destroy disease-producing microorganisms. a. Millipore filters
■ There are two options a manufacturer can pursue in seeking approval b. HEPA filters
for a disinfectant product: submission of an NDA or OTC drug c. Dust filters
review known as the monograph system. d. Charcoal filters
■ Antimicrobial agents for health care personnel use must meet certain 15. Infectious agents can enter the body through which of the following
standards that demonstrate the product’s safety and efficacy. routes?
■ Major sources of biological hazards come from patient samples a. Inhalation
during processing and handling of actively growing culture b. Ingestion
materials. c. Inoculation
■ Protective equipment should be used appropriately. d. All of the above
■ Safety policies and procedures should always be followed. 16. Employees can remember the steps to take in case of a fire by
■ The microbiology safety program includes proper and safe disposal remembering which of the following acronyms?
of infectious waste material. a. RUSH
■ OSHA regulations for bloodborne pathogen protection should be b. REST
followed. c. RACE
■ Chemical and fire safety hazards must be identified, and measures d. RISK
to prevent chemical spills should be employed.
■ Continuing education programs to train laboratory personnel in all
aspects of laboratory safety and exposure control should be in
place.
88 PART 1  Introduction to Clinical Microbiology

Clinical Laboratory Standards Institute. (2011). Clinical laboratory waste


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Olson, L. K., et al. (2012). Prospective, randomized in vivo comparison (2012). Occupational exposure to hazardous chemicals in laboratories,
of a dual-active waterless antiseptic versus two alcohol-only waterless 29 CFR 1910.1450, FR 17887. Available at: http://www.osha.gov.
antiseptic for surgical hand antisepsis. American Journal of Infection (Accessed 23 March 2017).
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Paulson, D. S. (2003). Nosocomial infection. In D. S. Paulson (Ed.), 1988. Available at: http://www.epa.gov. (Accessed 23 March 2017).
Handbook of topical antimicrobials. New York: Marcel Dekker. U.S. Environmental Protection Agency. (2000). Environmental manage-
Phillips, N. F. (Ed.), (2007). Berry and Kohn’s operating room technique ment guide for small laboratories. Available at: http://www.epa.gov.
(11th ed.). St Louis: Mosby. (Accessed 23 March 2017).
Rotter, M. L. (2004). Special problems in hospital antisepsis. In A. P. Voss, A., & Nulens, E. (2003). Prevention and control of laboratory acquired
Fraise, P. A. Lambert, & J. Y. Maillard (Eds.), Russell, Hugo and infections. In P. R. Murray (Ed.), Manual of clinical microbiology (8th
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sterilization. Malden, MA: Blackwell. World Health Organization. (1999). Infection control guidelines for trans-
Rutala, W. A., & Weber, D. J. (2008). Guideline for disinfection and missible spongiform encephalopathies. Report of a WHO Consultation,
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skin preparation. Journal of Perioperative Practice, 22, 95. in bacteria: an overview. Microbial Drug Resistance (Larchmont,
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agents of biocrime or bioterrorism. Journal of Clinical Microbiology,
41, 2801.
CHAPTER

5  
Performance Improvement in the
Microbiology Laboratory
Sarojini R. Misra

CHAPTER OUTLINE
■ GENERAL GUIDELINES FOR ESTABLISHING The Customer Concept
QUALITY CONTROL Fixing the Process
Temperature Benchmarking
Thermometer Calibration Commercially Purchased Monitors
Media Quality Control ■ EVALUATING AND INTERPRETING DIAGNOSTIC
Reagent Quality Control LABORATORY TESTS
Antimicrobial Susceptibility Quality Control ■ ANALYTIC ANALYSIS OF TESTS
Personnel Competency Analytic (Technical) Sensitivity and Specificity
Use of Stock Cultures Accuracy
Quality Control Manual ■ CLINICAL ANALYSIS OF TESTS
■ PERFORMANCE IMPROVEMENT Clinical (Diagnostic) Sensitivity
Vision and Mission Statements ■ OPERATIONAL ANALYSIS OF TESTS
Individualized Quality Control Plan Incidence of Disease
Proficiency Testing Prevalence of Disease
Indicators of Performance Improvement: Process Versus Predictive Values of Tests
Outcome Efficiency of Tests
Establishing Performance Monitors ■ CHOOSING A LABORATORY METHOD
Problem-Action Form ■ TEST VALIDATION

OBJECTIVES
After reading and studying this chapter, you should be able to:
1. Define quality control (QC) as it applies in the clinical microbiology 5. Discuss the 10-step plan for establishing quality monitors.
laboratory. 6. Describe the customer concept.
2. Discuss the general guidelines for establishing a QC program, 7. Define benchmarking.
including how to monitor equipment maintenance and performance, 8. Define and differentiate analytic sensitivity and specificity and
culture media and reagent performance, personnel competency, use clinical sensitivity and specificity.
of stock cultures, and the development and updating of procedure 9. Compare prevalence and incidence of disease.
manuals. 10. Discuss the importance of prevalence in computing predictive values
3. Describe proper documentation and institution of appropriate of tests.
corrective action. 11. Discuss predictive values of tests and show how they are computed.
4. Define performance improvement and, if it differs from QC, describe 12. Apply the concepts of predictive values of tests to several clinical
how it differs. examples.

was negative for group A streptococci. A follow-up culture was


Case in Point positive for group A streptococci 24 hours later.
A 5-year-old girl was complaining of a sore throat. A low-grade
fever (99° F [37° C]) was noted when the patient was seen in
the physician’s office. Her pharynx was red, exudate was present, Issues to Consider
and her tonsils were swollen. The physician requested that a After reading the patient’s case history, consider:
group A streptococcal direct antigen test be performed; the test ■ All the intricate processes involved in quality assurance

90
CHAPTER 5  Performance Improvement in the Microbiology Laboratory 91

■ How the sensitivity and specificity of the laboratory test is


used in the clinical diagnosis of a disease TABLE 5.1  Three Stages of Activities That Affect
■ How the prevalence of a disease in a population might Laboratory Testing Activity Outcomes
affect the predictive value of tests
■ Why it is important to validate constantly methods and Stage Activities
procedures used in the laboratory
Preanalytic Test ordering
Order transcription
Key Terms Specimen collection
Specimen identification
Accuracy Focused monitors Specimen transport
Analytic activity Incidence Analytic Sample testing
Analytic sensitivity Individualized quality control Postanalytic Result delivery
Analytic specificity plan (IQCP) Result review
Bayes’s theorem Negative predictive value Action taken on basis of result
Benchmarking (NPV)
Bias ORYX
Clinical (diagnostic) sensitivity Outcome monitors
Clinical (diagnostic) specificity Performance improvement (PI) Quality assurance involves taking measures to ensure high-
Clinical Laboratory Postanalytic activity quality patient care. This process involves monitoring all the
Improvement Act of 1988 Positive predictive value (PPV) components of a system or procedure (preanalytic, analytic, and
(CLIA ‘88) Preanalytic activity postanalytic) and implementing changes when suboptimal per-
Clinical and Laboratory Precision formance is identified. Quality assurance is measured by patient
Standards Institute (CLSI) Predictive value outcome; hence patient care might be adversely affected before
College of American Prevalence problems are identified.
Pathologists (CAP) Preventive maintenance In 1995, the Joint Commission on Accreditation of Healthcare
Competency Process monitors Organizations, now called The Joint Commission (TJC),
Continuous quality Proficiency testing underwent a change in philosophy. Individual disciplines and
improvement (CQI) Q-probes departments were replaced with functions critical to patient care.
Continuing education (CE) Quality control (QC) New regulations placed accreditation emphasis on the organization’s
Cross-functional teams Test validation performance of these functions, thereby making them everyone’s
Customer concept The Joint Commission (TJC) responsibility. This new process, called performance improvement
Detection limit Total quality management (PI), replaced quality assurance as a more proactive process of
Facilitator (TQM) ensuring the quality of health care.
The current TJC accreditation process called “Shared Visions—

P
New Pathway” became effective in January 2004 and focuses on
roviding accurate results in laboratory testing is of the upmost performance measurement of organizational systems critical to
importance, and the issue of quality in the laboratory is patient safety, quality of care, treatment, and services. This new
complex. The emphasis and terminology have changed initiative involves the use of ORYX, a new TJC requirement for
tremendously in recent years. Laboratories have always taken the submission and evaluation of performance measurement data.
measures to control the testing performed on patient specimens. Unlike past accreditation processes, which provided snapshot
This effort has been termed quality control (QC); it is defined views of an organization’s performance, this new requirement
as the measures designed to ensure the medical reliability of provides an ongoing view of laboratory performance, thus providing
laboratory data. Examples are checking media and reagents with continuous opportunities for quality improvements. ORYX helps
specific organisms to determine whether expected results are organizations in their quality improvement efforts.
obtained and documenting that instrumentation meets all operating In the ever-changing health care arena, the pursuit of quality
parameters before it is used on patient samples. continues with concepts that integrate all aspects of health care.
Laboratory professionals now realize that QC is only a small These concepts, called total quality management (TQM) and
part of the issue of quality. Even when the laboratory has effectively continuous quality improvement (CQI), are continuous, incre-
controlled media, reagents, and instruments, the quality of the mental improvement processes that reflect the organization-wide
test result is poor if the specimen had degraded before arriving philosophy that quality is everybody’s responsibility. QC is a
in the laboratory but was still tested. Suppose a specimen contained small but important part of TQM and CQI, and as such, it con-
the wrong patient name; again, the media can be top quality, the tributes to the organizational goal of providing quality patient
incubator temperature accurate, and the laboratory scientist very care. This section presents two major quality issues, applications
competent, but if the results are reported for the wrong patient, of QC and PI.
quality patient care management did not exist for the intended
patient. Actual laboratory testing is called an analytic activity. General Guidelines for Establishing
It is important to realize that preanalytic, analytic, and postanalytic
activities all affect quality. An outcome can be interrupted or
Quality Control
destroyed at any point in the process. Table 5.1 attempts to clarify All QC activities that take place must be recorded to prove their
these three stages by giving examples of each type of activity. existence. All record sheets must list tolerance limits, when
92 PART 1  Introduction to Clinical Microbiology

applicable, so that the person recording the results will always


know whether the value being recorded is acceptable. Corrective TABLE 5.2  Frequency of Equipment Testing
action must also be recorded when any measurement falls outside
a tolerance limit. The responsibility for QC may rest mainly with Equipment Test Type Frequency
one person, but in reality everyone must participate if a program is Incubator Temperature, CO2, Daily
to be successful. A QC program for a microbiology laboratory must GasPak jar Anaerobiosis, Each use
include procedures for control of the following items: temperature, catalyst-heated
equipment, media, reagents, susceptibility testing, and personnel. Anaerobe chamber Anaerobiosis, humidity, Daily
temperature
Temperature Biohazard hood Air flow (done by Annually or any time
specialist) hoods are moved
Daily temperature checks are required on all temperature-dependent Centrifuge Check of rpm Every 6 months
equipment: Microscope Cleaned and adjusted Four times per year
• Incubators or as needed
• Heating blocks Autoclave Temperature Each load
• Water baths Spore testing Weekly
Balance Accuracy of weights Annually
• Refrigerators
• Freezers rpm, Revolutions per minute.
Incubator and refrigerator thermometers are easier to read if
they are permanently immersed in glycerol. This helps prevent
temperature fluctuations when the door is opened to read the gives examples of some laboratory equipment, the type of testing
thermometer. Before use, each thermometer must be checked done, and the frequency of testing.
against a reference thermometer from the National Institute of A preventive maintenance program must be established as
Standards and Technology (NIST), formerly the National Bureau an additional control measure. Preventive maintenance performed
of Standards (NBS). The most efficient method is to check a large on equipment generally involves tasks such as oiling and cleaning,
batch of thermometers at the same time and at the temperature replacing filters, and recalibrating instruments. Keeping an instru-
ranges likely to be used. A common practice in clinical microbiol- ment in top shape and functioning at the proper level will increase
ogy is to test all thermometers at −20° C, 2° to 8° C, 37° C, and its lifetime and help control the quality of the results. Fig. 5.1
56° C. The NIST thermometer comes with certification papers shows an example of a preventive maintenance log sheet.
that list correction factors to be used in various temperature ranges.
These correction factors are applied to all values obtained with Media Quality Control
individual laboratory thermometers. Laboratories arbitrarily Each batch of prepared media must be quality controlled to docu-
determine the acceptable temperature variance. For most routine ment sterility and performance. Documentation must show that
work, thermometers that differ by 1° C or more from the reference the media support the growth of appropriate microorganisms,
thermometer are discarded. and if appropriate, inhibit growth of specific microorganisms
or produce the correct biochemical response. Records must be
Thermometer Calibration maintained for 2 years. The criteria are established by the Clinical
Thermometers are calibrated by batch on arrival in the laboratory. and Laboratory Standards Institute (CLSI) and are listed in
Procedure 15 in Appendix C outlines the calibration procedure. document M22-A2. Commercial media are always tested by the
Once the thermometer has passed calibration and is placed in use, manufacturer. The laboratory must obtain a statement of QC from
repeated calibration of the thermometer should not be necessary. the manufacturer for all media that the laboratory will not retest.
Alternatively, thermometers already checked against an NIST This certificate must be retained for as long as the laboratory
thermometer can be purchased. Certificates of calibration should be uses the specified media. Only certain types of media must be
kept for the life of the thermometer or until the expiration date on retested by the user, usually because of complexity or history of
the certificate; after that date, the thermometer can be recalibrated failure rate. Examples of media that require retesting are chocolate
or discarded. For environmental and safety reasons, nonmercury agar, selective media for pathogenic Neisseria, and Campylobacter
thermometers are recommended. Mineral spirits with nontoxic, media. Fig. 5.2 lists specialty commercial media that have been
red-dyed alcohol are used in place of the mercury. Thermometers retested by the laboratory. A list of all media requiring retesting
should be checked daily to ensure that gas bubbles have not been can be found in the CLSI document M22-A2.
introduced into the liquid, making reading the temperature difficult. Media not quality controlled by the laboratory must still undergo
Gas bubbles can be eliminated by centrifugation or by placing observation for moisture, sterility, breakage, and appearance with
the thermometer at a high or low temperature. every lot or shipment received:
• Moisture: Plates should be free of moisture before use but
Equipment Quality Control should never show signs of drying around the edges.
Equipment used in the clinical microbiology laboratory must be • Sterility: Plates should be free of contaminants.
tested for proper performance at intervals appropriate for each • Breakage: Petri dishes should not be cracked or broken.
item. This process may involve checking the percentage of CO2 • Appearance: Blood-based plates should not show signs of
in an incubator daily or measuring the revolutions per minute of hemolysis, and any other plate that deviates from the normal
a centrifuge twice a year. Sometimes frequency of testing is dictated color should not be used. Any deterioration should be reported
by a regulatory agency, and other times it is arbitrary. Table 5.2 to the manufacturer.
CHAPTER 5  Performance Improvement in the Microbiology Laboratory 93

MEDIA, REAGENTS, AND SMALL EQUIPMENT JANUARY FEBRUARY MARCH

MEDIA, REAGENTS:

1. Check refrigerators and freezers for outdated 1/21/09 AF 2/24/09 LH 3/28/09 DS


material.
2. Check for "received" and "opened" dating. 1/21/09 AF 2/24/09 LH 3/28/09 DS

THERMOMETERS: Calibrate by batch on arrival.

PIPETTORS: Calibrate. 3/10/09 MS

pH METER: (Rooms 337 and 354)


1. Clean the exterior. 1/21/09 AF 2/24/09 LH 3/28/09 DS
2. Replace water in the electrode holder. 1/21/09 AF 2/24/09 LH 3/28/09 DS
3. Check the AgCl level of the electrode. 1/21/09 AF 2/24/09 LH 3/28/09 DS

EYEWASHES: Flush.
(Rooms 332, 337, 339, 342, 351[2], 354) 1/21/09 AF 2/24/09 LH 3/28/09 DS

STAINING SINK: Clean. 1/20/09 AF 2/24/09 LH 3/28/09 DS

GROUNDING: Check annually.

REVIEWED BY: M Rausch Page 1 of 5

YEAR: 2009
Preventive maintenance is to be performed in the months highlighted for each item listed.

FIG. 5.1  Preventive maintenance log sheet.

Results of media observations must be recorded and include media should be tested with dilute suspensions of organisms,
lot numbers. Fig. 5.3 shows an example of a media observation whereas biochemical media can be tested with undiluted
log, which helps ensure that good-quality media are used on all organisms.
patient samples. Corrective action must be taken when a medium • QC testing should be performed according to CLSI recom-
does not meet standards. This can be documented on a separate mendations.
record known as a media failures log (Table 5.3). • Expiration dates must be established.
When a medium needs to be quality controlled because it was • Fig. 5.4 shows a log sheet used for testing media prepared in
prepared in house (in the laboratory) or because it is complex, house.
several basic rules must be followed:
• All media must be tested before use. Reagent Quality Control
• Each medium must be tested with organisms expected to grow With few exceptions, reagents should be tested on each day
or give a positive reaction as well as with organisms expected of use with both positive and negative controls. Reagents that
not to grow or to produce a negative reaction. are documented to have consistent and dependable results may
• The medium should be tested for sterility and pH. be tested less frequently. Some reagents may be tested more
• The organisms selected for QC should represent the most than once a day. Reagents that are opened and used repeatedly,
fastidious organisms for which the medium was designed. such as albumin, should be checked daily for sterility. Always
• Testing techniques should be different for primary plating media examine the manufacturer’s package insert for recommended QC
than those for biochemical or subculture media. Primary plating requirements.
94 PART 1  Introduction to Clinical Microbiology

MEDIUM TESTED MFG LOT NUMBER EXP. DATE STERILITY TEST ORGANISMS RESULT ACTION TAKEN DATE TECH.

Chocolate II BD L3RTPO 5/23/09 OK H. influenzae Pass None 3/8/09 MR


N. meningitidis

GC-Lect BD –

N. gonorrhoeae
N. meningitidis
Jembec-Neiss. BD A 3NENC 4/3/09 OK S. epidermidis Pass None 3/8/09 MR
C. albicans
E. coli

Campy bld BD LINDHK 4/28/09 OK C. jejuni Pass None 3/8/09 LM


E. coli

Campy thio BD H4EOAJ 2/1/09 OK C. jejuni Pass None 3/8/09 LM


E. coli

Reviewed by: Date:


FIG. 5.2  Commercial media that have been retested by the laboratory. The result is pass or fail.

DATE MEDIUM LOT NUMBER MOISTURE STERILITY APPEARANCE BREAKAGE INITIALS

3/4/09 BAP A2RUWO ✔ ✔ ✔ ✔ MR

3/4/09 MAC A4RUUH ✔ ✔ ✔ ✔ MR

3/4/09 CHOC A4RUUX ✔ ✔ ✔ ✔ MR

3/4/09 CNA A1RUWB ✔ ✔ ✔ ✔ MR

3/4/09 HE AZRCUF ✔ ✔ ✔ ✔ MR

3/4/09 CIN AZNEJE ✔ ✔ ✔ ✔ MR

3/4/09 PD OSU PREP: 1/6/09 ✔ ✔ ✔ ✔ MR

3/4/09 GC-LECT K3RTNZ ✔ ✔ ✔ ✔ MR

3/4/09 SCH A4NETG ✔ ✔ ✔ ✔ MR

3/4/09 SCH-GV A3NENN ✔ ✔ ✔ ✔ MR

3/4/09 CDC ANA AZRWAW ✔ ✔ ✔ ✔ MR

3/4/09 SMAC OSU PREP: 2/15/09 ✔ ✔ ✔ ✔ MR


FIG. 5.3  Media observation log. A check in each column indicates that the medium is acceptable.
CHAPTER 5  Performance Improvement in the Microbiology Laboratory 95

QC should be performed and documented on all staining • Optochin


procedures at least daily. For the Gram stain, known gram-positive • Oxidase
bacteria (Staphylococcus aureus ATCC 25923) and known gram- • L-Pyroglutamyl-β-naphthylamine
negative bacteria (Escherichia coli ATCC25922) are recommended. • Typing sera
All staining procedures must be verified before patient results • Voges-Proskauer broth
can be released. Some examples of reagents that should undergo • X and V strips
QC in microbiology are: Fig. 5.5 shows a variety of testing that might be performed
• All stains daily at an individual workstation.
• Bacitracin
• β-Lactamase Antimicrobial Susceptibility
• Catalase Quality Control
• Coagulase The CLSI provides guidelines for control of susceptibility testing.
• Gelatin The recommended control organisms are specific strains from the
• Germ tube solution American Type Culture Collection (ATCC; Table 5.4). In addition
• Hippurate to the organisms listed in Table 5.4, fastidious organisms such as
• Kovacs reagent Haemophilus influenzae and Neisseria gonorrhoeae are tested to
• Nitrate ensure that the best possible results are obtained when same type
of isolates recovered from patient samples are being tested.
In any susceptibility system, many variables can affect the
TABLE 5.3  Media Failures Log accuracy of results, including the following:
• Antimicrobial agent potency
Date 2/14/16 • Agar depth (Bauer-Kirby test)
• Evaporation (microtiter dilution)
Media TMS slants • Cation content
Lot no. In-house preparation 2/13/16
Expiration date 6 months from preparation
• pH
Quantity Two racks • Thymidine content
Failure Failure to give proper reaction with S. epidermidis, • Instrument failure
S. aureus, and other coagulase-negative staphs • Inoculum concentration
are OK • Temperature
Action taken QC repeated, S. epidermidis failed; memo sent to • Moisture (Bauer-Kirby test)
all technicians and all tubes discarded; new
• Difficulty in determining end points
TMS slants prepared
Technologist MAR Careful storage of degradable supplies and precision in the
implementation of recommended procedures are mandatory to
QC, Quality control; TMS, trimethylsilyl ester. obtain accurate and reproducible susceptibility results.

MEDIA AND ORGANISM QC PLATED QC READ QC


DATE LOT NUMBER PLATED DATE DATE PASSED/FAILED INITIALS

S. aureus
3/8/09 TMA 3/9/09 3/10/09 Pass MR
S. epidermidis

S. agalactiae +
3/8/09 Beta toxin 3/9/09 3/9/09 Pass MR
S. pyogenes –

3/8/09 PSE E. faecalis 3/9/09 3/10/09 Pass MR

P. aeruginosa
3/8/09 TSI C. freundii 3/9/09 3/10/09 Pass MR

FIG. 5.4  Log sheet for testing media prepared in house. QC, Quality control.
96 PART 1  Introduction to Clinical Microbiology

Month March
BLOOD CULTURE WORKSTATION QUALITY CONTROL
Year 2009
DATE INITIALS GASPAK HEAT CHANGE ACRIDINE NaDesoxy/ HEATING BLOCKS
JAR CATALYST DESICCANT ORANGE WELLCOGEN (35°- 37° C)
Anaerobic Date (weekly) Pos Neg Pos Neg #20 #17
1 LM OK ✔ N D + – 35° 36°
2 AD OK ✔ N D + – 35° 36°
3 AD OK x 2 ✔ N D N D 35° 36°
4 MCl OK ✔ + – N D 36° 36°
5 MCl OK ✔ N D N D 35° 36°
6 LH OK x 2 ✔ N D N D 35° 36°
7 AD OK x 2 ✔ ✔ N D + – 36° 36°
8 AD OK x 2 ✔ N D + – 36° 37°
9 LH OK ✔ N D + – 36° 36°
10 AD OK ✔ N D N D 36° 36°
11 CI OK ✔ + – N D 36° 36°
12 MG OK ✔ + – + – 36° 36°
13 DS OK x 2 ✔ N D N D 36° 36°
14 DS OK ✔ ✔ N D N D 36° 35°
REVIEWED BY: MBT
DATE: 4/4/09
FIG. 5.5  Various testing methods that can be performed daily at an individual workstation. Shown
is an example of a 2-week workstation quality control. ND, Not done.

accomplished, QC organisms may be tested weekly instead of


TABLE 5.4  Recommended Control Organisms for daily. All results from the 20- or 30-day evaluation should be
Susceptibility Testing kept as long as the antimicrobial agent is used or for at least 2
years after discontinuation of use of the agent.
Organism Susceptibility Test(s)
Personnel Competency
Escherichia coli, ATCC 25922 Gram-negative drugs
Escherichia coli, ATCC 35218 β-Lactamase inhibitor drugs Personnel competency, the ability of an individual to perform a
Staphylococcus aureus, ATCC 25923 Gram-positive drugs—Kirby- task accurately and effectively, is determined by use of a variety
Bauer test of techniques, such as direct observation, review of work sheets,
Staphylococcus aureus, ATCC 29213 Gram-positive drugs—minimal or written examination. A popular technique used to determine
inhibitory concentration competency is proficiency testing, in which carefully designed
Pseudomonas aeruginosa, ATCC Monitors Ca2+ and Mg2+
samples are given to laboratory scientists as unknowns for the
27853 contenta
Enterococcus faecalis, ATCC 29212 Monitors thymidineb
purpose of identifying them. Proficiency testing as it pertains to
the laboratory is discussed later in this chapter. Proficiency samples
a
As Ca2+ and Mg2+ concentrations increase, P. aeruginosa becomes more for demonstrating competency may be purchased commercially
resistant to the aminoglycosides.
b
or prepared internally. All tests performed on patients must be
Increases in thymidine concentration cause false resistance to certain drugs,
such as sulfonamides, trimethoprim, and trimethoprim-sulfamethoxazole.
subjected to proficiency testing twice a year, even if commercial
proficiency testing is not available.
Another form of personnel QC is to discourage all laboratory
Susceptibility testing of control organisms is usually conducted scientists from signing off, or finalizing, their own work. If each
daily until precision can be demonstrated with 20 or 30 consecutive laboratory scientist’s result is reviewed by another laboratorian,
days of susceptibility testing using CLSI guidelines. The control mistakes are likely to be caught before results are released.
organism results must be evaluated before end points are determined Employee competency has always played a large role in quality.
on patient isolates. Minimal inhibitory concentrations (MICs) The Clinical Laboratory Improvement Act of 1988 (CLIA ’88)
must be within one log dilution of the expected MIC according has mandated that the competency of each employee be determined
to CLSI guidelines. Once the 20- or 30-day evaluation has been and verified on employment. Reverification must take place
CHAPTER 5  Performance Improvement in the Microbiology Laboratory 97

annually. Proof of competency must be maintained in each


employee’s personnel file. A person may be qualified to prepare Quality Control Manual
a slide for staining but may not be able to stain it, or a person All rules and procedures for QC should be available to employees
may be qualified to prepare and stain a slide but not to read or at the workstation in written form in a QC manual. The manual
interpret the smear test results. As part of CLIA requirements, all must be reviewed, signed at least annually, and revised as needed
tests or analyses have been assigned a complexity rating. In by a supervisor.
microbiology, all tests are moderately complex or highly complex.
Personnel must meet certain educational requirements before being
permitted to perform at each level of complexity.
Performance Improvement
In addition to meeting educational requirements, a person’s Accrediting agencies such as TJC and the College of American
competency must be observed and documented for each test Pathologists (CAP) emphasize PI in their accreditation checklists.
performed. The many agencies involved in accreditation and The CLIA also mandates the use of written PI policies in labo-
inspection have different requirements and interpretations of ratories that include all three phases of testing. Every laboratory
competency verification, making this a complicated task for all labo- must have a plan for improvement. To effectively improve quality,
ratories. An example of a competency check-off form is shown in all employees must understand the plan and take active roles.
Fig. 5.6.
The requirement for ongoing continuing education (CE) Vision and Mission Statements
programs for all employees is yet another form of QC. These Creating a short vision or mission statement for all employees to
programs may teach theory or new techniques, present case studies, learn can be an effective tool for uniting everyone behind the
or simply provide training on new instrumentation. Documentation same cause. It can be as simple as the vision statement of the
that all CE programs have been completed is essential. Training Wilford Hall Ambulatory Center operated by the 59th Medical
for new instrumentation should also be documented in the individual Wing, “Exemplary Care, Global Response,” or the mission state-
employee’s personnel competency file. ment used at The Ohio State University Wexner Medical Center,
“to improve people’s lives through innovation in research, education
Use of Stock Cultures and patient care.” An organization’s vision and mission statements
To operate a QC program, all laboratories must maintain stock must be clearly emphasized so that all employees become involved
cultures. They are available from many sources: and understand that they each have a role in the mission, and they
• Commercial must strive to make PI part of their daily lives to achieve the
• Patient isolates organization’s vision. Problems are not to be viewed as real
• Proficiency testing isolates problems but as opportunities for improvements and a chance to
• ATCC excel.
When QC testing appears to have failed, it is usually the stock
culture rather than the test itself that has failed. With repeated Individualized Quality Control Plan
subculturing, organisms can mutate. For best results, a stock culture Individualized quality control plan (IQCP) is the policy name
should be grown in a large volume of broth and then divided for an alternative CLIA QC program that provides laboratories
among enough small freezer vials to last a year. With this technique, with the opportunity for equivalent quality testing. Laboratories
a new vial can be removed from the freezer weekly so that organ- have the options of following CLIA regulations or developing an
isms do not have to be subcultured continually. Before testing, IQCP. The three required parts of an IQCP are risk assessment, QC
an organism should be subcultured twice after thawing to return plan (QCP), and quality assessment. An IQCP will not necessarily
it to a healthy state. Media selection for freezing is at the discretion reduce QC, but it does permit laboratories to develop their own
of individual laboratories but the media should not contain sugars. QC protocols that recognize technology included in laboratory test
If organisms use sugars while being maintained, the acid products systems. An IQCP allows laboratories to customize QC on the basis
that result might kill the organisms over time. The following are of unique environments, test systems, etc. The laboratory must
popular media choices for stock cultures: document that the QCP is based on evidence collected in house
• Schaedler broth with glycerol but can incorporate test system manufacturers’ quality certificates.
• Skim milk If a laboratory chooses to implement an IQCP, the laboratory
• Chopped meat (anaerobes) director must review and approve the QCP. Once the QCP is in
• Tryptic soy agar deeps (at room temperature) place, the quality assessment plan monitors laboratory testing and
• Cystine tryptic agar without carbohydrates defines investigation and problem solving, leading to adjustment
Another popular method of storage is the use of storage beads. to the QCP as needed. In microbiology, for example, visual quality
Storage beads are contained in a vial containing 1 mL of thiogly- checks of media along with manufacturers’ quality certificates
colate broth. After the vial has been inoculated with the organism, could be considered in the risk assessment. A microbiology labora-
the broth is removed and the vial with the beads is stored at tory could adapt an IQCP that applies to all media or use separate
−70°C. The advantage of this system is that a single bead can be plans for individual media. In the case of laboratory instrumentation,
removed without the entire vial being thawed. Although this form for example, a continuous-monitoring blood culture system, the
of storage is more convenient, it is probably more expensive than laboratory director might choose to use the manufacturer’s instruc-
those mentioned earlier. Organisms stored frozen should be kept tions for QC if they meet or exceed CLIA regulations. The labora-
at −70°C; alternative storage methods include freezing in liquid tory must document that the test system results support the number
nitrogen and lyophilization. and frequency of the QCs specified in the QCP.
98 PART 1  Introduction to Clinical Microbiology

Carol Johnson
Employee name:___________________________________________________ Year___________________2009

Work station
Demonstrates following abilities:
#1 #2 #3
Respiratory Urines O & Ps

1. Handles specimens safely during testing, storing, and


discarding ✔ ✔ ✔

2. Prepares specimens for analysis according to laboratory policies


and procedures (parasitology and special procedure areas) — — ✔

3. Analyzes specimens according to laboratory procedures for the


workstation; knows theory and principles of the tests being performed ✔ ✔ ✔

4. Clearly records all work done so that another person could take
over the work station
✔ ✔ ✔

5. Reports results accurately and in a timely manner


✔ ✔ ✔

6. Makes appropriate critical value and courtesy calls


✔ ✔ ✔

7. Consistently performs and records quality control and documents


all remedial action ✔ ✔ ✔

8. Remedial action necessary (yes or no)


No No No

Remedial actions:____________________________________________________________________________

__________________________________________________________________________________________

__________________________________________________________________________________________

__________________________________________________________________________________________

__________________________________________________________________________________________

Date remediation completed:___________________________________________________________________

A Holbrook 2/11/09
Evaluator signature____________________________________ Date___________________ (Work station 1)

L Creme 6/9/09
Evaluator signature____________________________________ Date___________________ (Work station 2)

S Young 8/27/09
Evaluator signature____________________________________ Date___________________ (Work station 3)

Carol Johnson 8/28/09


Employee signature____________________________________ Date___________________

FIG. 5.6  Competency documentation.


CHAPTER 5  Performance Improvement in the Microbiology Laboratory 99

Proficiency Testing BOX 5.1  Measurable Processes


TJC and the CLIA require that laboratories enroll in a Centers Patient preparation
for Medicare and Medicaid Services (CMS)–approved proficiency Specimen handling
testing program for each regulated analyte tested. Laboratories Collection
are required to maintain successful performance on proficiency Labeling
testing. Unsuccessful performance is defined as a failure to achieve Preservation
satisfactory performance for two consecutive or two of three Transportation
Communication processes
consecutive testing events. When unsuccessful proficiency testing
Transfer of information
occurs, TJC will request a plan of action. If the problem cannot Completeness of requisitions
be resolved, an onsite evaluation may be conducted, which may Reporting timeliness
affect accreditation status and force the laboratory to stop Report accuracy
testing. Test appropriateness
Proficiency testing samples are available from several approved Patient needs and expectations
Risk management activities
providers, and a list is available at the CMS website. Proficiency
Quality control activities
testing samples are to be assayed in the same manner as patient
material, except that no proficiency testing sample shall be referred
to another laboratory for analysis. A laboratory is not to test
proficiency testing samples on more than one instrument or by
multiple methods unless that is how patient specimens are pro- identified than can be acted on, priorities must be established.
cessed. In microbiology, microorganisms in proficiency tests must Priorities are often based on risk, frequency, or high-volume
be identified in the same manner as clinical specimens. processes.

Indicators of Performance Problem-Action Form


Improvement: Process Versus Outcome A simpler approach to monitoring or documenting quality issues
Many types of monitors or indicators can be incorporated into a is a problem-action form. This approach is most commonly used
quality improvement program. Accrediting agencies generally to document issues that are quickly resolved, but could also be
check that a variety of types are used. Some monitors are ongoing used for long-term monitor summation. The form is a brief state-
data collections with no suspected problems. Results are compiled ment consisting of the following information:
and evaluated routinely. This procedure establishes a trend and • Date
makes problems easy to detect as disruptions in the trend. These • Problem
are often referred to as process monitors. • Evaluation and investigation
Outcome monitors are measurements of the result of a process, • Corrective action
such as complications that a patient experiences as the result of • Outcome
a process. Other monitors may be created in response to a suspected The form may be signed by the person submitting it, and
problem. Data may be collected for a short period to resolve a additional documentation may be attached as necessary. Table
specific issue. These monitors are called focused monitors. 5.5 illustrates the use of the problem-action form.

Establishing Performance Monitors


TJC makes recommendations as an accrediting agency for establish-
Case Check 5.1
ing performance monitors. The components of the recommendation
are to plan, design, measure, assess, and improve. Whereas direct antigen tests for strep throat have improved, they are
• Plan: The plan is not expected to be a single-department prone to false-negative results. However, specimen collection and processing
could have been performed incorrectly. Monitoring of false-negative results
approach. Rather, it should be a coordinated, organization-wide for group A streptococci could identify a pattern requiring intervention
approach for improving patient outcomes that includes inter- to improve results.
disciplinary collaborative actions.
• Design: Clear objectives are needed to describe a new process,
component, or service.
• Measure: Systematic data collection is necessary for improve- The Customer Concept
ments or for ongoing measurements. Box 5.1 lists suggested Laboratories must focus on the customer concept. Who are the
measurable processes. customers, and what is a customer’s perception of quality? Patients
• Assess: A review of the collected data should be systematic, are not the only customers. Anyone who looks to the laboratory
interdisciplinary, and interdepartmental, as well as statistically for a service is a customer. Physicians, nurses, insurers, and patients
based using analytic tools. Internal comparisons or comparisons are all customers. Each customer may view quality differently
with similar processes in other organizations are appropriate. and may have different expectations. Laboratory professionals
Guidelines for assessments might be accreditation standards, may judge quality in terms of accuracy, whereas a physician views
practice guidelines, or legal and regulatory requirements. it as turnaround time, the patient as compassion and relief from
• Improve: Current processes or a design of new processes may pain, and the insurance company as cost-effectiveness. Customer
need to be redesigned. Because more opportunities are usually satisfaction must be surveyed to determine perceptions.
100 PART 1  Introduction to Clinical Microbiology

improve. Benchmarking was initially practiced in business and


TABLE 5.5  Clinical Microbiology Problem- industry but has now become an important part of hospital quality
Action Report management programs. It is done willingly and openly. A hospital
may join a large group of other hospitals that all share operating
Date 11/15/16 statistics. Productivity and cost-effectiveness are two large catego-
Problem A blood culture was required from a patient
ries commonly used in a benchmarking comparison. The best
at the urgent care facility, but no staff performers in a group of hospitals are highlighted. Hospitals can
member was trained to draw blood individually and anonymously see where they are by comparison
cultures. The patient had to drive from the with other hospitals’ statistics and then contact the best performers
urgent care facility to the hospital to have to evaluate their differences. A code of conduct is followed when
the blood drawn by a phlebotomist who benchmarking is performed that includes ethics and etiquette.
was trained in the appropriate techniques
Although hospitals generally benchmark other hospitals, they
Evaluation No employees at the urgent care facility have
been trained to collect blood cultures. The eventually incorporate lessons from other successful industries.
same is true of the clinic outpatient
laboratory. This is the second occurrence in Commercially Purchased Monitors
about 2 months The CAP created a national PI assessment program called
Corrective action Seriously ill patients should not have to travel Q-probes. Laboratories throughout the country subscribe to an
from one facility to another to have their annual series of monitors. At least one of these monitors each
blood drawn. Laboratory administration
was informed of this situation
year has a microbiology focus. Q-probes have covered topics
Outcome All outpatient sites will receive training and such as adequacy of sputum cultures, turnaround time for spinal
written instructions for the proper fluid Gram-stain results, blood culture contamination rates, and
collection of blood for culture. Patients will appropriateness of ordering patterns for stool specimens. The
no longer have to drive to the hospital for method of data collection is precisely outlined, and all worksheets
this service if they are already at an and data forms are provided. Information is returned to subscribers
outp