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MICRO-PARA
Controlling Microbial Growth in Vitro Using Antimicrobial Agents to Control Microbial Growth Microbial Ecology Epidemiology and Public Health Health Care Epidemiology: Nosocomial Infections and Infection Control Diagnosing Infectious Disease Pathogenesis of Infectious Diseases
various chemical compounds to sustain life For energy sources Sources of C-H-O-N-S + Phosphorus and other elements
Moisture
Cells consist of 70 95% water, most will die in environments with too little water. Other microbial stages (endospores and protozoan cysts) can survive desiccation in their dormancy or resting states
Temperature
pH
Most microorganisms prefer a neutral or slightly alkaline growth medium (7.0 7.4) Acidophiles survive acid stomach (2.0-5.0) Alkalophiles found in the intestines (9.0)
V. cholerae only human pathogen that grows well above pH 8.0
Pressure exerted on a cell membrane by solution both inside and outside the cell.
Types of solutions:
Isotonic equal concentration of solutes outside and inside the cell Hypertonic - concentration of solutes outside the cell is greater than inside the cell Hypotonic - concentration of solutes outside the cell is lesser than inside the cell
Osmosis movement of a solvent from lower concentration of solute to a higher concentration of solute
Isotonic solution
- Balance solution - No movement of water
Hypotonic solution
If sufficient water enters the cell, it will swell, then eventually lyse or burst. Hemolysis bursting of red blood cells. Plasmoptysis cell rupture of bacterial cell.
Hypertonic solution
If sufficient water leaves the cell, it will shrink. Crenation shrinkage of red blood cell due to loss of water. Plasmolysis shrinking away of bacterial cell membrane and cytoplasm from the cell wall. (Application :Use of salt and sugar in food preservation)
Haloduric organisms do not prefer to live in salty environments but are capable of surviving there (such as Staphylococcus aureus)
Dead Sea Salt Beds, Israel The Dead Sea, between Jordan and Israel, has grown smaller over the last 10,000 years due to evaporation, which removes water faster than precipitation can replenish it. The resulting salt deposits form an enormous salt reserve.
Barometric Pressure
Barophiles thrive deep in the ocean where the atmospheric pressure is very high
Gaseous Atmosphere
JUST ASKING. . .
Identification of pathogens Learn more about them Harvest antibiotics and other microbial products Produce vaccines Used in industries
Culture medium
Culture
Culture Types
Pure only one specie is present (important for the isolation of pathogenic organism) Mixed different species Stock pure culture of microorganisms used as a source of supply for industry, research and students use
Solid- 2- 3% agar
BAP, Citrate, EMB, MacConkey
According to Composition
Tissue Culture- from living cells- for culture of viruses and rickettsia (WI 38 from normal human skin)
( Hela cells from human cervical cancer cells)
Tubed
According to Use
Simple- with nutrients to support growth allows most non-fastidious organisms to grow at their natural rate used for routine culture Nutrient Agar
Enriched- with nutritive supplements needed for bacterial growth Blood Agar Plate (+ 5% sheep red blood cells) Chocolate Agar Plate N. gonorrheae
Differential - organisms growing together with differences in their cultural characteristics EMB and Mac Conkey lactose fermenters (red) non-lactose fermenters(colorless)
Blood Agar Plate alpha (partial/incomplete hemolysis) beta (complete hemolysis/ clear) gamma (no hemolysis)
Selective- with one or more agents inhibitory to all organisms EXCEPT organisms being sought
SSA - BSA (S. typhi) PEA Gram (+) cocci CNA Columbia Colistin Nalidixic Acid Gram (+) streptococci Mannitol Salt Agar for Salt tolerant bacteria
INOCULATION
Adding a portion of the specimen to the medium
STERILE TECHNIQUE
Techniques used in an attempt to create an environment that is sterile (devoid of microorganisms)
INCUBATION
Holding a culture at a particular temperature for a certain length of time
Anaerobic incubator
With atmosphere devoid of oxygen
Human growth refers to increase in size;going from a tiny baby to a large adult.
BACTERIAL GROWTH
Examples:
E. coli, Staphylococcus, Streptococcus, V. cholerae 20 - 30 minutes Pseudomonas and Clostridium 10 minutes M. tuberculosae 18 24 hours
when bacteria are grown in suitable media and samples taken at intervals, plotting of results will yield a characteristic growth curve.
It tends to increase cell mass and number in an exponential
Bacterial Growth:
The bacterial growth curve:
Stationary phase
Death phase
B. Exponential/Logarithmic
maximum rates of cell division and increase in cell mass most metabolically active
FUNGI
Brain-Heart Infusion Agar Brain-Heart Infusion Agar + Blood Sabouraud Dextrose Agar
PROTOZOA
Not usually done Example of protozoa that can be cultured in vitro are Amebae
Microbistatic agent Bacteriostatic agents Lyophilization Sepsis Asepsis Antisepsic technique Thermal Death Point Thermal Death Time
Mechanism of Action
Comment
Preferred Use
FILTRATION
GASEOUS ATMOSPHERE
Sterilization
Disinfection
Disinfectants
Chemicals used to disinfect inanimate objects (bedside equipments and operating rooms
Antiseptics
Solutions used to disinfect skin and other living tissues
Sanitization
Reduction of microbial populations to levels considered safe by public standards.
Microbistatic agent Drug/chemical that inhibit the growth of microorganisms Bacteriostatic agents One that specifically inhibits the metabolism and reproduction of bacteria Lyophilization Process that combines dehydration (drying) and freezing
HEAT
DRY HEAT
160 165 C for 2 hours 170 180 C for 1 hour Use of Oven Incineration Flaming Using electric heating devices
MOIST HEAT
Heat applied in the presence of moisture (boiling/steaming) Autoclave
like a metal pressure cooker that uses steam under pressure to completely destroy all microbial life. 121.5 C, 15 psi, 20 minutes Kills veratative microorganisms, bacterial endospores, viruses
COLD
Inhibits bacterial growth Refrigeration (slow freezing) Liquid Nitrogen (rapid freezing) * Refreezing of thawed food is an unsafe practice.
DESICCATION
Process of being thoroughly dried
RADIATION
Suns rays include infrared (heat) rays and ultraviolet (UV) rays Penetrate cells and damage DNA UV lamps are used as germicidal lamps:
Nurseries Operating rooms Elevators Entryways
Caution: when working with UV lamps avoid exposure or eyes or skin to the rays (can cause serious burns and cellular damage)
Skin cancer can be caused by excessive exposure to UV
X-rays, gamma rays, beta rays may be lethal or cause mutations to microorganisms
Damage DNA and proteins within the cell
ULTRASONIC WAVES
Used in cleaning and sterilizing equipment delicate equipments in hospitals, medical clinics and dental clinics. Sound waves mechanically dislodge organic debris on instruments and glass wares.
FILTRATION
Micropore filters are used in the laboratories to filter bacteria and viruses out of liquids Variety of filters include plastic films, unglazed porcelein, asbestos, diatomaceous earth, and cellulose membrane filters. Biologic Safety cabinets contain High-Efficiency Particulate Air (HEPA) filters.
GASEOUS ATMOSPHERE
Aerobes and microphiles are killed by placing them in atmosphere devoid of oxygen. Obligate anaerobes are killed by placing them into an atmosphere containing oxygen
Prior cleaning of the object or surface to be disinfected The organic load present in the material to be treated (feces, blood, vomitus, pus) Bioburden (type and level of microbial contamination
Concentration of disinfectant
Contact time (amount of time disinfectant must remain in contact with the organism in order to kill them Physical nature of the object being disinfected(smooth,rough,crevices,hinges) Temperature and pH
CHEMOTHERAPY
Use of drug to treat any disease or condition These drugs are called CHEMOTHERAPUETIC AGENT
ANTIMICROBIAL AGENT
A drug used to treat an infectious disease, either by inhibiting or killing pathogens in vivo.
Antibacterial Antifungal Antiprotozoal Antiviral
ANTIBIOTICS
Antimicrobial substance produced by a microorganism that is effective in killing or inhibiting the growth of other microorganisms. Semisynthetic antibiotics chemically modified to kill a wider variety of pathogens or reduce side effects
Unfortunately
JUST ASKING. . .
COMPETITIVE INHIBITION
Sulfonamide molecule is similar in shape to PABA (para-amino benzaldehyde) PABA is converted to folic acid which is essential in the synthesis of some bacterial proteins If there is no conversion of PABA to folic acid to essential proteins, the bacterial cell will eventually die Sulfa drugs are BACTERIOSTATIC AGENTS
JUST ASKING. . .
Vancomycin
SOMETHING TO REMEMBER
Antimicrobial agents work well against bacterial pathogens because the bacteria (being procaryotic) have different cellular structures and metabolic pathways that can be disrupted or destroyed by drugs that do not damage the eucaryotic hosts cell.
MULTIDRUG THERAPY
Two or ore drugs are used simultaneously to kill all the pathogens and to prevent resistant mutant strains from emerging.
Four drugs used in M. tuberculosae infection
Synergism VS Antagonism
SYNERGISM
Two antimicrobial agents are used to treat an infectious disease a greater degree (effect) than that achieved by either drug alone.
Trimethoprim + Sulfamethoxazole =
Co-trimoxazole (Bactrim and Septra)
ANTAGONISM
Two drugs working against each other The extent of pathogen killing is less than that achieved by either drug alone.
ANTIFUNGAL AGENTS
How do they work ?
ANTIPROTOZOAL AGENTS
How do they work ?
ANTIVIRAL AGENTS
ZIDOVUDINE (AZT)
First antiviral drug effective against HIV (1987) Coctails (1990s) a combination of antiviral drugs
SUPERBUGS
MRSA (Methicillin-resistant S. aureus) MRSE (Methicillin-resistant S. epidermidis) VISA (Vancomycin-Intermediate S. aureus) VRSA (Vancomycin-Resistant S. aureus)-very common in nosocomial infection VRE (Vancomycin Resistant Enterococcus spp.) MRTB (Multidrug-Resistant M. tuberculosis)
INTRINSIC RESISTANCE
Lack specific target site for the drug (Mycoplama cellwalless) Drug cannot cross the bacterial cell wall or cell membrane
ACQUIRED RESISTANCE
Alteration of drug- binding sites due to chromosomal mutation Alteration of the structure of the cell mebrane
Ability of organism to produce enzymes that destroys the drug (R-factor is passed on to other bacteria via conjugation)
Ability to develop Multidrug- Resistance pumps (MDR transporters)-pumps drug out of the cell before it causes damage
BETA-LACTAMASES
Penicillinases destroys the B- lactam rings of Penicillins Cephalosporinases destroys the Blactam ring of Cephalosporins
EMPIRIC THERAPY
Factors to consider. . .
Laboratory result of pathogens identity refer to pocket chart. Is patient allergic to any antimicrobials? Age of the patient? Is patient pregnant? In-patient or out-patient?
Availability of the drug What is the site of infection? Medications received by the patient. Other medical problems? Is patient immunocompromised? Cost of the drug?
Side Effects. . .
Allergies Toxicity (Chloramphenicol aplastic anemia) Superinfection (opportunists and secondary invaders overgrowth)
Antibiotic Associated Diarrhea (AAD) and Pseudomembranous Colotis (PMC) caused by C. difficile Candida albicans infection
What Can Clinicians, Paramedical Professionals and Patients Do To Help in the War Against Drug Resistance? (pp154-155)
Editorial Cartoon Poster Slogan Jingle Radio Advertisement Poem
MICROBIAL ECOLOGY
Study of the numerous interrelationships between microorganisms and the world around them. Microbes interact with. . .
other microbes _ organisms other than microbes non-living world around them
Symbiosis
Living together or close association of two dissimilar organisms (symbionts)
Neutralism
A symbiotic relationship in which neither symbiont is affected by the relationship Different microorganisms occupy the same etiologic niche, but have absolutely no effect on each other.
Commensalism
Beneficial to one symbiont and neither beneficial nor harmful to the other. Many organisms in the indigenous flora are considered commensals.
Mutualism
Beneficial to both symbionts E. coli obtains nutrients from food materials ingested by the host and produces vitamin K which is a blood clotting factor
Parasitism
Beneficial to one symbiont and detrimental to the other organism
SYNERGISTIC RELATIONSHIP
Microorganisms may team up to produce a disease that neither could cause by itself Synergistic Infection
INDIGENOUS MICROFLORA
TABLE 10 - 1
Anatomic Locations of Bacteria and Yeasts Found As Indigenous Microflora of Humans
Coughing, sneezing, blowing of nose can carry these microbes along the eustacian tube to the middle ear
External surface of the eye is lubricated, cleansed and protected by tears, and the presence of enzyme lysozyme and other substances
Nasal passages and throat have an abundant and varied population of microorganisms Healthy carriers harbor virulent pathogens in their nasal passages or throats
MICROFLORA OF THE ORAL CAVITY (MOUTH) Shelter for numerous anaerobic and aerobic bacteria
Thrive well in particles of food and in the debris of dead epithelial cells around the teeth
Poor dental hygiene results in the development of dental caries, gingivitis and other periodontal diseases
Species include: Actinomyces, Bacteroides, Lactobacillus, Streptococcus, Neisseria and Veillonella
MICROFLORA OF THE GASTROINTESTINAL TRACT Gastric enzymes and extremely acidic pH of the stomach usually prevent growth of indigenous microflora and most transient microbes
Helicobacter pylori lives in some people s stomachs and is a common cause of ulcer
Many of the microflora of the colon are opportunists like E. coli E. coli is the most common cause of UTIs (Urinary Tract Infection)
Frequent urination prevents UTI Most common cause of urethritis: Chlamydia trachomatis, Neisseria gonorrheae and Mycoplasmas through sexual intercourse Vaginal secretions are acidic, encouraging the growth of lactobacilli that produces lactic acid (inhibit growth of bacteria associated with bacterial vaginosis)
MICROBIAL ANTAGONISM
microbes against microbes
Microflora competes for space and nutrients against newcomers Effects of antibiotics produced by some organisms against other microorganisms Some produce protein bacteriocins which kill other bacteria
Bacteria and yeasts that are used to reestablish and stabilize microbial balance Example: Use of Lactobacillus in yogurt or in medications
MICROBIAL COMMUNITIES
BIOFILMS
Complex communities of assorted organisms Examples: dental plaque, slippery coating on rocks, slime that acculmilates
Medical Significance:
Form on urinary catherter, implants Implicated in endocarditis, middle ear infection, kidney stones, etc. Examples: C. albicans, S. aureus, Enterococcus spp,Klebsiella and Pseudomonas
BIOTECHNOLOGY
industrial use of microbes in the production of certain foods and beverages, food additives, chemicals, amino acids, enzymes, etc. as well as refining of ores to obtain copper, uranium and gold
BIOREMEDIATION
Use of microbes to dispose of industrial and toxic wastes and other environmental pollutants pesticides, herbicides, oil spills
1976
Legionnaires Disease
severe form of pneumonia, characterized by headache, chest pain, lung congestion, and high fever. The name is derived from an outbreak at an American Legion convention in a Philadelphia hotel in July 1976.
1992 - 1993
1993
Hantaviruses are carried by specific rodent hosts and are transmitted directly from host to host by virus-laden saliva, urine, and feces. Humans are infected through exposure to the dried excretions from infected rodents. Hantaviruses cause two different human diseases: hemorrhagic fever with renal syndrome, in which damage to the kidneys is common, and acute respiratory distress syndrome, in which damage to the lungs is common.
1993
Cryptosporidiosis
A diarrheal disease which resulted from drinking water that was contaminated with Cryptospridium parvum
2002
EPIDEMIOLOGY
The study of disease, basically the factors that determine the following:
Frequency Distribution Determinants in human population
Characteristics of various pathogens Susceptibility of the population resulting from overcrowding Lack of immunization Nutritional status Inadequate situation Location (reservoir) Various ways it is transmitted
What pathogens are causing the infection? Where do the pathogens come from? When do certain diseases occur Why do some diseases occur in some places but not in others How are pathogens transmitted? Do some diseases occur only at certain time of the year? If so, why?
TERMINOLOGIES
COMMUNICABLE DISEASE
Transmission : person to person
CONTAGIOUS
Communicable disease that is easily transmitted from one person to another
ZOONOTIC DISEASES
Infectious diseases that human acquire from animal sources
INCIDENCE
Number of new cases of that disease in a defined population during a specific time period
PANDEMIC DISEASES
A disease occurring worldwide
HIV / AIDS (pp.179 180) Tuberculosis (p. 180) Malaria (p. 180)
Health status Nutritional status Socioeconomic, hygiene, travel,immune status, substance abuse
Physical factors
Location, climate, heat, cold, humidity, season of the year
Presence of a pathogen Source of the pathogen (reservoir) Portal of exit Mode of transmission Portal of entry Susceptible host
Communicable diseases are transmitted from person to person in the following ways:
Indirectly by Fomites
Stethoscope Gloves
MORBIDITY RATE MORTALITY RATE PREVALENCE SPORADIC DISEASE ENDEMIC DISEASE EPIDEMIC DISEASE RESERVOIR OF INFECTION
PASSIVE CARRIERS INCUBATORY CARRIERS CONVALESCENT CARRIERS ACTIVE CARRIERS FOMITES PARENTERAL INJECTION BIOLOGICAL WARFARE BIOTERRORISM AGENTS
CARRY-OVER ACTIVITY
TRACE THE CHAIN OF INFECTION IN A FORM OF A DIAGRAM
HEALTHCARE EPIDEMIOLOGY
-
Any activity designed to study and / or improve patient care outcomes in any type of healthcare institution or setting.
Includes a variety of disciplines and activities directed at enhancing the quality of healthcare and preventing and controlling adverse outcomes. (SHEA)
Whether they are working in a hospital, nursing home, medical or dental clinic, or caring for a sick person they MUST follow standardized procedures to prevent the spread of communicable diseases.
Iatrogenic Infections
physician induced
Result of medical or surgical treatment (surgeons, physicians,healthcare personnel) Examples: post - surgical wound infections and urinary tract infections (catheterization)
Urinary catheters provide a superhighway for indigenous normal flora to access the urinary bladder
70% of nosocomial infections involved drug resistant bacteria
Urinary Tract Infections Surgical Wound Infections Lower Respiratory Tract Infections Bloodstream Infections
Elderly patients Women in labor and delivery Premature infants and newborns Surgical and burn patients Diabetic and cancer patients Patients receiving treatment with steroids, anticancer drugs, anti-lymphocyte serum and radiation Immunosuppressed patients Patients who are paralyzed and undergoing renal dialysis or catheterization
Increasing number of drug resistant pathogens Failure of personnel to follow infection control guidelines Increased number of immunocompromised patients
OTHER FACTORS
Increase use of less-highly trained healthcare workers Increase use of antiimflammatory and immunosuppressed agents Overuse and improper use of indwelling medical devices
TABLE 12 1 (p.201)
Roughly 1/3 of adults seem to have forgotten one of the most basic lessons their mothers taught them: wash your hands properly. Although 95% of people say that they scrub after using public toilets, researchers from the American Society of Microbiology found that only 67 % actually do.
INFECTION CONTROL
Numerous measures that are taken to prevent infections from occurring in healthcare settings
MEDICAL ASEPSIS
clean technique involves procedures and practices that reduce the number and transmission of pathogens
Hand washing Personal grooming Proper cleaning of supplies and equipments Disinfection
SURGICAL ASEPSIS
sterile technique practices use to keep objects and areas sterile
Scrubbing hands and fingernails Sterile gloves , masks, gowns, shoe cover Using sterile solutions and dressing
designed to reduce the risk of transmission of blood borne and other pathogens in hospitals contains guidelines for:
Hand washing Wearing of gloves, masks, eye protection Cleaning of patient care equipment Others
CONTACT PRECAUTIONS
WORD TO PONDER. . .
All healthcare workers MUST fully comprehend the problem of nosocomial infections, MUST be completely knowledgeable about infection control practices, and MUST personally do everything in their power to prevent nosocomial infections from occurring.
(1/2 CW)
Interview doctors and paramedical practitioners of the effective ways employed in the hospital to reduce nosocomial infections.
Implementing the proper selection, collection, transport and processing of appropriate clinical specimens
When an attending physician suspects that a patient has a particular infectious disease, appropriate clinical specimens must be obtained and certain diagnostic tests may be requested.
The doctor or any qualified healthcare professional must select the appropriate specimen, collect it properly, and then properly transport it to the laboratory where it is processed.
All specimens should be collected or transferred into a leakproof primary container with a secure closure. Care should be taken by the person collecting the specimen not to contaminate the outside of the primary container within the institution , the primary container should be placed into a second container, which will contain the specimen if the primary container breaks or leaks in transit to the laboratory.
Specimen should be collected at the right site, where the least contamination is likely to occur
Specimen should be taken before antimicrobial therapy has begun
The acute stage of the disease is the most appropriate time to collect most specimens
Exercise care and tact, avoid harming patient or causing discomfort or undue embarrassment to the patient Collect sufficient quantity of the specimen
Use sterile, disposable specimen containers Properly label specimen container, submit with appropriate request slip
Specimen should be delivered to the lab ASAP.
Patients name, age, sex Hospital identification number Name of the requesting physician Info about the type of specimen Site from where it is collected Date and time of collection Initials of the person who colleted the specimen Information about any antimicrobial agents taken by the patient
The proper specimen to diagnose UTI is a clean-catch, mid-stream urine (CCMS). It must be refrigerated until it can be transported to the laboratory
Cerebrospinal specimen should be submitted to the laboratory and processed immediately
Routine throat swabs are collected to determine whether a patient has strep throat.
Clinicians and people in the paramedical field like nurses are aware that something is wrong with a patient when they see some changes in the person - like his skin color
(eg. yellowish discoloration in jaundice, paleness in anemia or bluish black discoloration in the case of hematoma)
SIGN OR SYMPTOM?
When we study the structural and functional manifestations of disease we are interested in PATHOLOGY.
Microbes land in anatomic sites where it is unable to multiply .(eg. H. influeanzae on skin) Microbes land on sites with no receptors Antibacterial factors like lysozymes in tears, saliva, perspiration Indigenous microflora (microbial antgonism)
Indigenous microflora produce antibacterial factors like bacteriocin Individuals health status Immunity to particular pathogen (vaccination) Presence of phagocytic wbc
Note : When all the mechanical barriers and cellular protection fail to do their job we get sick of infectious diseases
down with something but are not yet sure what is it.
period (but for certain infectious diseases permanent damage may be caused by destruction of tissues. Eg. Deafness my follow ear infection)
Terminologies:
which go symptomatic to asymptomatic, and then go back to being symptomatic eg. Shingles
Entry skin (bite of an arthropod), inhalation, ingestion, introduction thru the GUT, introduction directly into the blood)
Attachment to some tissues in the body Multiplication result in local infection (abscess) to systemic (throughout the body)
Virulence factors
(attributes that enable pathogens to attach, escape destruction and cause disease)
Attachment
Receptors ( molecules in host cell that a pathogen recognize and attach to) and adhesions (molecules in the pathogen that is able to recognize and bind to a particular receptor) Fimbriae (pili) enable bacteria to attach to surfaces, hair-like
Collagenases-destroys collagen in tissues thus enables pathogens to invade tissues (C. perfringens)
Hemolysins cause damage to hosts rbc (hemolysis) Lecithinase destruction of muscle tissues (C. perfringens)
Toxins
Endotoxin released by Gram (-) organisms cauring fevers, chills and eventully shock (low bp and inadequate blood supply to brain and kidneys)
Neurotoxins target CNS (C. tetani) Enterotoxins affect GIT (B. cereus) Exfolitive toxin affects layers of the skin (S. aureus scalded skin syndrome) Leukocidins destroy wbc (Stph, Strep, Clostridia)
Antigenic variation pathogens able to change their surface antigens - host cell cannot recognize (eg, influenza virus)
Camouflage and Molecular mimicry some coat themselves with host protein so as not to be recognized as foreign (eg. N. gonorrheae)
Destruction of Antibodies produce enzymes that destroy IgA antibodies (H. influenza)