BACTERIAL INFECTION ON

UPPER RESPIRATION TRACT

Ike Irmawati P.A, MSi Med
Mikrobiologi FK Yarsi

Infections of the Respiratory tract

Most common entry point for infections
Upper respiratory tract
nose, nasal cavity, sinuses, mouth,
throat
Lower respiratory tract
Trachea, bronchi, bronchioles, &
alveoli in the lungs
2

Upper & Lower Respiratory Tract

Infection
Type microorganism:
- restricted to surface
- spread through body
Two groups of microbes can
distinguished :
professional & secondary
invaders
4

Protective Mechanisms
Normal flora: Commensal organisms
Limited to the upper tract
Mostly Gram positive or anaeorbic
Microbial antagonist (competition)

Protective Mechanisms
Clearance of particles
and organisms from the respiratory tract
For the upper respiratory
tract :
The mucociliary system
in the nasopharynx
The flushing action of
saliva in the oropharynk

Bacterial Infections
Specific areas of the upper respiratory
system can become infected to produce
pharyngitis, laryngitis, tonsillitis, sinusitis, and
epiglottis.
These infections may be caused by several
bacteria and viruses, often in combination.

Sore Throat
Clinical features: pharyngitis tonsillitis
Causative organisms
mostly viral
bacterial causes:
Streptococcus pyogenes
(a.k.a. Group A beta-haemolytic streptococci)

Less commonly: Corynebacterium diphtheriae,

Group C and G beta-haemolytic streptococci,
Arcanobacterium haemolyticum, Fusobacterium
necrophorum, Haemophylus influenzae type B,
Borrelia vincenti

Otitis Media & Sinusitis

Local spread of organisms from URT , e.g.
Streptococcus pneumoniae
Haemophilus influenzae
viruses probably commonly involved
Clinical features
Fever, local pain, dizziness, deafness

Acute Epiglottitis
Infective emergency
Caused by
Haemophilus
influenzae capsular
type B (Hib)
Now very rare due to
Hib vaccine program
Can lead to acute
respiratory obstruction

Diagnosis
take blood cultures
H. influenzae requires rich
medium
(e.g. lysed blood or
chocolate agar)
Requires X and V
growth factors on
nutrient agar
Management
Intravenous cefotaxime or
ceftriaxone
Isolate the patient
Prevention
Hib vaccine

Diphtheria

Acute toxin-mediated disease

caused by Corynebacterium
diphtheriae
Gram-positive aerobic bacillus

Incubation period 2-5 days

Typically involves pharynx and
tonsils
leathery adherent membrane,
which can cause respiratory
obstruction

Toxin effects
Myocarditis
Neuropathy

Streptococcal infection on
Respiratory Tract

Streptococci
Characters of Streptococci

Gram positive cocci

1m in diameter
Chains or pairs
Usually capsulated
Non motile
Non spore forming
Facultative anaerobes
Catalase negative
Of these organism considered in this chapter:
S.pyogenes,S.pneumoniae,S.viridans

Classification of Streptococci
Streptococci can be classified according to:
Oxygen requirements

Anaerobic (Peptostreptococcus)
Aerobic or facultative anaerobic (Streptococcus)

Serology (Lanciefield Classification)

Hemolysis on Blood Agar (BA)

Serology: Lanciefield Classification

Streptococci

Lanciefield classification

Group A
S. pyogenes

Group B
S. agalactiae

Group C
S. equisimitis

Group D
Enterococcus

Streptococci classified into many groups from A-K & H-V

One or more species per group
Classification based on C- carbohydrate antigen of cell wall
Groupable streptococci
A, B and D (more frequent)
C, G and F (Less frequent)
Non-groupable streptococci
S. pneumoniae (pneumonia)
viridans streptococci
e.g. S. mutans
Causing dental carries

Other groups
(E-U)

Classification of Streptococci Based on

Hemolysis on Blood Agar
Hemolysis on BA
-hemolysis

Partial hemolysis
Green discoloration around the colonies
e.g. non-groupable streptococci (S. pneumoniae & S. viridans)

-hemolysis

Complete hemolysis
Clear zone of hemolysis around the colonies
e.g. Group A & B (S. pyogenes & S. agalactiae)

-hemolysis

No lysis
e.g. Group D (Enterococcus spp)

Hemolysis patterns on blood agar

17

Hemolysis on Blood agar

-hemolysis

-hemolysis

-hemolysis

19

Pathogenesis and Spectrum of Disease

Organism
S.pyogenes

S.pneumoniae

S.viridans

Virulence Factors
Protein F & lipoteichoic acid mediates
epithelial cell attachment &adhesion
M protein as antiphagositic
The M protein has many antigenic varieties
and thus, different strain of S.pyogenes
cause repeat infections
Hyaluronic acid capsule, which acts to
camouflage the bacteria
Produce enzyme and hemolysins-contribute
tissue invasion and destructions,i.e: streptolysin
O,S,streptokinase,DNAse,&hyaluronidase
Streptococcal Pyrogenic exotoxins that
stimulate macrophages and helper T cells to
Polysaccharide
release
cytokines
capsule
inhibit phagocitosis
Pneumolysin
Production of extracellular complex
pollysaccharide-enhanced attachment to host
cellular surface,such as cardiac endothel

Spectrum of
respiratory infections
Pharyngitis,pneumonia,scarlet
fever,Streptococcal toxic
shock syndrome
Rheumatic fever

pneumonia

Subacute bacterial
endocarditis

Pathogenesis S.pyogenes
Pathogenesis of group A streptococci

Adherence to the epithelial cells;

>10 adhesion molecules
invasion into the epithelial cells;
mediated by M protein and F protein
important for persistent infections and invasion into deep
tissues
avoiding opsonization and phagocytosis;
M protein, M-like proteins, and C5a peptidase
producing enzymes and toxins

Complication that result from S. pyogenes

infection (poststreptococcal disease)
Rheumatic fever: Life threatening inflammatory disease that
leads to damage of heart valves muscle
Rheumatic fever: most commonly preceded by infection of the
respiratory tract. Inflammation of heart (pancarditis), joints,
blood vessels, and subcutaneous tissue. Results from cross
reactivity of anti-M protein Ab and the human heart tissue. This
disease can be reactivated by recurrent streptococcal infections
Deposition of antibody-streptococcal Ag complexes in kidneys
results in damagee to glomeruli--------------------->glomerulonephritis

S. pneumoniae
Morphology and Physiology
Gram-positive lancet-shaped diplococci for typical organisms.
alpha-hemolytic
Growth is enhanced by 5-10% CO2.
*Quellung reaction (for rapid identification or typing of the bacteria)
These organism may harmlessly inhabit the upper respiratory tract
When it gain access to the lungs by aspiration -acute suppurative
pneumoniae
When it accesses the bloodstream & meninges-acute,supurative often lifethreatening infections

Contd S. pneumoniae
Pathogenesis and Immunity
Pneumococci produce disease through their ability to multiply in the tissues
(invasiveness).
Virulence factors: capsule, cell wall polysaccharide, phosphorylcholine,
pneumolysin, IgA protease, etc.
40-70% of humans are at sometimes carrier of virulent pneumococci. Major
host defense mechanisms: ciliated cells of respiratory tract and spleen. The
normal respiratory tract has natural resistance to the pneumococcus.
Loss of natural resistance may be due to:
1. Abnormalities of the respiratory tract (e.g. viral RT infections).
2. Alcohol or drug intoxication; abnormal circulatory dynamics.
3. Patients undergone renal transplant; chronic renal diseases.
4. Malnutrition, general debility, sickle cell anemia, hyposplenism
or splenectomy, nephrosis or complement deficiency.
5. Young children and the elderly.

S. pneumoniae virulence factors

S. pneumoniae
Clinical diseases
Pneumococcal pneumonia develops when the bacteria
multiply rapidly in the alveolar space after aspiration. The
affected area is generally localized in the lower lobes of
the lungs (lobar pneumonia). Children and the elderly can
have a more generalized bronchopneumonia. Resolution
occurs when specific anticapsular antibodies develop.
Sudden onset with fever, chills and sharp chest pain.
Bloody, rusty sputum. Empyema (mostly caused by type 3) is
a rare but significant complication.
Complications caused by spreading of pneumococci to other
organs: sinusitis, meningitis, endocarditis, septic arthritis,
middle ear infection.

Streptococcus viridans
Alpha hemolytic or nonhemolytic
Nongrupable
S.viridans colonizes in the oropharynx,GI tract,urinary tract,
and rareli on skin surface.
Generally considered to be of low virulence
Production of extracellular complex polysacarides (such as
glucans&dextrans) enhance attachment to host cell
surface,such as cardiac endothelial cells & tooth surface in the
case of dental caries
Disease:
subacut bacterial endocarditis (particularly patients with
previosly damaged heart valves)
Intra abdominal infection
caries dentis

Diagnosis of streptococcal
disease

Laboratory diagnosis S. pyogenes

Antigen detection methode:
Antigen detection tests: commercial kits for rapid detection of
group A streptococcal antigen from throat swabs, using latex
agglutination,coagglutination & Elisa tecnology.
Latex agglutination able to detect Capsuler pollysacharide
antigen of pneumococcus
Serological test/antibody detection methode:

ASO titration for respiratory infections.

Anti-DNase B and antihyaluronidase titration for skin
infections.
Antistreptokinase; anti-M type-specific antibodies.

Culture: Specimens are cultured on blood agar plates in air.

Identification

Laboratory diagnosis S. pneumoniae

Examination of sputum
Stained smears of sputum: a rapid diagnosis.
Quellung test with multivalent anticapsular antibodies.
Culture
Specimen: sputum, aspirates from sinus or middle ear, CSF.
cultured on blood agar plate in 5-10% CO2.
Identification: bile solubility, optochin sensitivity, etc. for
differentiation
from
other
a-hemolytic
streptococci.
Additional biochemical, serologic or molecular diagnostic tests
for a definitive identification.
Antigen detection: detect capsular polysaccharide in body
fluids.

Outline of differentiation between GramPositive cocci

18.05.09

32

Identification of Gram-positive cocci

None

Differentiation between -hemolytic Streptococci

Hemolysis

Bacitracin

CAMP test

S. pyogenes

sensitivity
Susceptible

Negative

S.
agalactiae

Resistant

Positive

Differentiation between -hemolytic Streptococci

Hemolysis

Optochin
Bile
Inulin
sensitivity solubility Fermentation

S.
pneumoniae

Sensitive (
14 mm)

Soluble

Not ferment

S. viridans

Resistant

Insoluble

Ferment

(13 mm)

Prevention and Control

Most streptococci are normal flora of the human body
Source of S. pyogenes and S. agalactiae is a person harboring
these organisms (carrier).
Control:
1. Prompt eradication of streptococci from early infections.
2. Prophylactic antibiotic treatment for rheumatic fever
patients.
3. Eradication of S. pyogenes from carriers.
4. Dust control, ventilation, air filtration, UV irradiation and
aerosol mists are of doubtful efficacy.
5. Intrapartum penicillin to mother at risk of giving birth to an
infant with invasive group B disease.

Haemophilus

Characteristics of Haemophilus
Small, pleomorphic gram-negative
coccobacilli, non motile
(+) cytochrome oxidase
Growth in culture requires exogenous
hemin (oxidized ferroprotoporphyrin)
(X factor) and/or nicotinamide adenine
dinucleotide (NAD) (V factor)

Haemophilus: Natural Habitats

Normal inhabitant of the upper
respiratory, gastrointestinal, and
genital tracts of humans except
Haemophilus ducreyi (not normal
microbial flora)

Haemophilus species of
clinical importance
1. H. influenzae
-type b is an important human pathogen

2. H. ducreyi
-sexually transmitted pathogen (chancroid)

3. Other Haemophilus are normal flora

- H. parainfluenzae

- H. aphrophilus
- H. aegyptius

Haemophilus: Modes of Infection

Encapsulated strains of Haemophilus
influenzae associated with invasive infection
caused by person-to-person spread of H.
influenzae due to inhalation of infectious
respiratory droplets
Type b H. influenzae was most commonly
associated with disease prior to conjugate
vaccine but prevalence has declined with
advent of vaccination

Haemophilus: Types of
Infectious Disease
Encapsulated (types a-f) strains of H.
influenzae produce invasive infection
(pneumonia, meningitis, epiglottitis, and
bacteremia)
Unencapsulated (non-typeable) strains of H.
influenzae cause otitis media in children, and
lower respiratory tract infections (acute
tracheobronchitis, pneumonia) in children
and adults

Haemophilus: Species
Identification
Preliminary findings: small faintly-staining
(with safranin) gram-negative coccobacillary
to filamentous rods that grow on chocolate
agar but not sheep blood agar (except H.
aphrophilus that with passage in culture
grows on both)
X and V factor requirements determined
using X, V, and XV factor impregnated paper
strips on Mueller-Hinton agar

Haemophilus influenzae

Differentiation of Species
Hemolysis

Growth
Factor
X

Haemophilus influenzae
Aerobic gram-negative bacteria
Polysaccharide capsule
Six different serotypes (a-f) of
polysaccharide capsule
95% of invasive disease caused
by type b

Haemophilus influenzae type b

Clinical Features*

*prevaccination era

Haemophilus influenzae type b

Epidemiology
Reservoir

Human
Asymptomatic carriers

Transmission

Respiratory droplets

Haemophilus influenzae type b

Pathogenesis
Organism colonizes nasopharynx
In some persons organism invades
bloodstream and cause infection at distant
site
Antecedent upper respiratory tract
infection may be a contributing factor

Pathogenic Mechanisms
H. influenzae
Antiphagocytic polysaccharide capsule is the
major pathogenesis factor
Lipopolysaccharide lipid A component from the
cell wall (major role in non capsule strains)
All virulent strains produce neuraminidase and
an IgA protease
No exotoxins

Pathogenesis Host Factors

Hib conjugate vaccine (Poliribitol phosphate ( PRP)
capsule)
The Hib conjugate vaccine does not protect against
nontypeable strains
Persons at risk for invasive H influenzae disease
Asplenia
Immunocompromised

H. influenzae serotype b:
diagnosis and treatment timeline
Incubation

Signs or
symptoms

Exposure
Incubation period unknown
(~ 2-4d)

Infectiousness

Lab
Specimens

Prophylaxis

Onset*
Sudden
Onset

Management of
sequelae

Infectious while organisms are

present, or until 24-48h of antimicrobial
therapy
Specimens from sterile site, for culture (cerebrospinal
fluid, blood, pus, middle ear fluid). Gram Stain is
Immunize contacts aged <5y
presumptive
Antimicrobial prophylaxis

*Invasive disease includes meningitis, epiglottitis, pneumonia, septic arthritis, and cellulitis (less commonly osteomyelitis and pericarditis).
Modified from Michigan Health Department http://www.michigan.gov/documents/mdch/2Hflu_Rev2008_231415_7.pdf

Haemophilus influenzae
IsoVitaleX-enriched
chocolate agar
Requires 2 erythrocyte
factors for growth: X
(hemin) and V (NAD).
X & V factors are released
following lysis of red blood
cells
5% CO2 enhances growth

Satellite Phenomenon
H. influenzae

Grows near S. aureus on blood agar:

satellite phenomenon.

Public Health AspectsH. influenzae

Typing based on capsule polysaccharide a f
Polyribose-ribitol phosphate (PRP) capsule (type b)
Nonencapsulated (nontypeable) organisms are part
of normal flora of the respiratory tract
95% of invasive disease caused by type b

Polysaccharide Conjugate
Vaccines
Stimulates T-dependent immunity
Enhanced antibody production,
especially in young children
Repeat doses elicit booster response

Corynebacterium

Corynebacterium
Classification
Corynebacterium diphtheriae and
diphtheroids(look like C.diphtheriae)
Some are saprophytic
Some produce disease in animals.
C. diphtheriae is the most important
pathogen in the group.
- Other Corynebacterium are part of the normal
flora of the skin and URT.

Corynebacterium
Are called diphtheroids and may occasionally cause
disease, particularly in immunocompromised
individuals.
C. ulcerans toxigenic strains may produce a disease
similar to, but less severe than diphtheria.
J-K Group commonly cause infections in those with
underlying disease.
Diseases include bacteremia, meningitis, peritonitis, wound
infections, etc.
It is becoming more and more of a problem.

C. pseudotuberculosis found in those with exposure to

animals.
Can cause pneumonia or lymphadenitis.
Produces a different exotoxin than C. diphtheriae.

Biological
Features
Aerobic, Gram+, Noncapsulated, rods, arrangement=palisade or
Chinese
letters

Gray-black colonies on tellurite medium

Metachromatic granules

Chinese-letter morphology in Gram stain

Diphtheroids Gram stain

Arrangement of C. diphtheria

Corynebacterium
Loefflers agar slant contains serum and egg
that enhance the formation of metachromatic
granules (polymerized
polyphosphoric acid) in C. diphtheriae.
Also called Babes-Ernst granules.
They are visualized by staining with
methylene blue.

Corynebacterium
A medium containing tellurite should be used to
select for Corynebacterium and other G+
organisms -it inhibits G organisms.
Two kinds are used:
Cystine tellurite has a longer shelf life
Tinsdale helps to differentiate amongst the
Corynebacterium.
Colonies on either appear black or gray due
to tellurite reduction.
S. aureus and Listeria also grow as black
colonies.
On Tinsdale C. diphtheriae, ulcerans, and
pseudotuberculosis form brown halos around
the colonies due to formation of ferric sulfide.

Corynebacterium
3 morphological types of C. diphtheriae are found on
tellurite containing media:

Mitis black colonies with a gray periphery

Gravis large, gray colonies
Intermedius small, dull gray to black.
All produce an immunologically identical toxin.

Incubation -35-370 C for 24 hours.

They prefer a pH of 7.8-8.0 for good growth.
They require access to oxygen (poor An O2 growth).

Biochemistry
Catalase +
Nonmotile
C. ulcerans is urease +, C. diphtheriae is -, and C.
pseudotuberculosis is usually +

Corynebacterium
Virulence factors C. diphtheriae
For C. diphtherias to cause diphtheria an exotoxin must
be produced.
Is a heat-labile polypeptide produced during
lysogeny of a phage that carries the "tox gene.
Alkaline pH of 7.8- 8.0, aerobic conditions, and a
low environmental iron level are essential for toxin
production (occurs late in the growth of the
organism).
The toxin inhibits protein synthesis by ADPribosylating elongation factor 2.

Corynebacterium
Trypsin cleaves the toxin into 2 fragments, A and
B, that are linked together by a disulfide bridge.
Fragment B is required for toxin binding to tissue
cells and fragment A contains the toxic activity.
One molecule of toxin can inhibit 90% of the
protein synthesis in a cell.
C. ulcerans and C. pseudotuberculosis sometimes
make a diphtheria-like toxin.

Diphtheria Toxin
(DT)

Cleaved to yield A/B fragment, joined by S-S bond

- A (catalytic domain)
- B (transmembrane and receptor binding domains)
Receptor: heparin-binding epidermal growth factor - rich on
cardiac cells and nerve cells
Toxin diffuses throughout body via blood
- Cardiac, neurologic complications
- Heart/respiratory damage, paralysis

C. diphtheria toxin
Toxin enters through
receptor mediated
endocytosis
Acidification of
endocytic vesicle
allows A to
dissociate from B
A enters cycoplasm

C. diphtheria toxin

Corynebacterium
To prove that an isolate can cause diphtheria, one must
demonstrate toxin production.
This is most often done on an Elek plate:
The organism is streaked on a plate containing
low iron.
A filter strip containing anti-toxin antibody is placed
perpendicular to the streak of the organism.
Diffusion of the antibody into the medium and
secretion of the toxin into the medium occur.
At the zone of equivalence, a precipitate will form.

Elek plate

Pathogenesis of diphtheria

Early

stages: Sore throat. Low fever. Swollen

neck glands.
Late stages: Airway obstruction and breathing
difficulty. Shock

Corynebacterium
Clinical Significance (C. diphtheria)
Is normally found in the throats of healthy carriers.
The organism infects only man and it has a limited
capacity to invade.
Diphtheria - Disease usually starts as a local infection
of the mucous membranes causing a membranous
pharyngitis
Local toxin effects result in degeneration of
epithelial cells.
Inflammation, edema, and production of a
pseudomembrane composed of fibrin clots,
leukocytes, and dead epithelial cells and
microorganisms occurs in the throat.

Diphtheria - pseudomembrane
This may obstruct the airway and result in
suffocation.

Corynebacterium
The more dangerous effects occur when the toxin
becomes systemic and attacks the heart (heart
failure), peripheral nerves (paralysis), and the
adrenal glands (hypofunction).
Cutaneous diphtheria More common in tropical
and subtropical areas.
Necrotic lesions with occasional formation of a
local pseudomembrane occur.

DIAGNOSIS
Clinical: Muscle weakness, edema and a
pseudomembranous material in the upper
respiratory tract characterizes diphtheria.
Laboratory: Tellurite media is the agar of
choice for isolation of Corynebacteria,
which produce jet black colonies

Corynebacterium
Shick skin test like the Dick test in that it tests
for circulating antibody to the toxin by
injecting a small amount of toxin
intradermally and observing for a local
erythematous and necrotic reaction.
If this occurs it indicates that the person
has no anti-toxin antibodies and is,
therefore, susceptible to diphtheria.

Control
Sanitary: Reduce carrier rate by use
of vaccine.

Immunological: A vaccine (DPT)

prepared from an alkaline formaldehyde

inactivated toxin (i.e. toxoid) is required.
Passive immunization with antitoxin can
be used for patients.
Chemotherapeutic: Penicillin,
erythromycin or gentamicin are drugs of
choice.