Chapter 19 – Anaerobes of Clinical Importance MLAB 2434 – Clinical Microbiology Cecile Sanders &...
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Transcript of Chapter 19 – Anaerobes of Clinical Importance MLAB 2434 – Clinical Microbiology Cecile Sanders &...
Chapter 19 – Anaerobes of Clinical Importance
MLAB 2434 – Clinical MicrobiologyCecile Sanders & Keri Brophy-Martinez
Concepts in Anaerobic Bacteriology
Air = about 21% O2 and 0.03% CO2
CO2 Incubator = about 15% O2 and 5%-10% CO2
Microaerophilic System = 5% CO2
Anaerobic System – 0% O2
Concepts in Anaerobic Bacteriology Obligate anaerobes grow ONLY
in the absence of molecular oxygen but vary in their sensitivity to oxygen and can be classified as moderate anaerobes or strict anaerobes
Moderate anaerobes can tolerate exposure to air for several hours but cannot multiply
Concepts in Anaerobic Bacteriology
Strict anaerobes are killed by only a few minutes’ exposure to air
Fortunately strict anaerobes are seldom associated with human infections
Why Anaerobes?
Oxygen is toxic because it combines with enzymes, proteins, nucleic acids, vitamins and lipids that are vital to cell reproduction
Substances produced when oxygen becomes reduced are even more toxic, producing such things as hydrogen peroxide and hydroxyl radicals (p. 568)
Why Anaerobes?
Anaerobes do not have enzymes for protection against the toxic effects of molecular oxygen, so oxygen can have a bacteriostatic or even bactericidal effect on them
Anaerobes require environments with low oxidation-reduction potential (redox), so they must live in areas where the redox potential is low
Where Anaerobes are Found Anaerobes are thought to be the
earliest forms of life All life on earth was anaerobic for
hundreds of millions of years Today they are found in soil,
fresh and salt water, and in normal flora of humans and animals
Where Anaerobes are Found Anaerobes that live outside the
body are called “exogenous anaerobes” (Example: Clostridium species)
Anaerobes that live inside the body are called “endogenous anaerobes”
Most anaerobic infections are from endogenous sources
Anaerobic Anatomical Sites for Endogenous Anaerobes
Mucosal surfaces such as linings of oral cavity, GI tract, and GU tract
Respiratory Tract – 90% of bacteria in the mouth are anaerobesIf mucosal surfaces are disturbed,
infections can occur in the oral cavity and in aspiration pneumonia
Sometimes cause “bad breath”
Anaerobic Anatomical Sites for Endogenous Anaerobes
Skin – frequently these normal skin anaerobes contaminate blood cultures
GU Tract – anaerobes rarely cause infection in the urinary tract, but cervical and vaginal areas have 50% anaerobes
GI Tract – Approximately 2/3’s of all bacteria are in the stool; only cultured anaerobically if Clostridium difficle is suspected
Factors that Predispose Patients for Anaerobic Infections Trauma to mucosal membranes
or skin Interruption of blood flow Tissue necrosis Decrease in redox potential in
tissues
Indications of Anaerobic Infections Usually purulent (pus-producing) Close proximity to a mucosal surface Infection persists despite antibiotic therapy Presence of foul odor Presence of large quantities of gas (bubbling or
cracking sound when tissue is pushed) Presence of black color or brick-red
fluorescence Presence of “sulfur granules” Distinct morphologic characteristics in gram-
stained preparation
Collection, Transport and Processing Specimens for Anaerobic Culture Any specimen collected on a
swab is usually not acceptable because of the possibility of having normal anaerobic organisms
Must be transported with minimum exposure to oxygen
Specimens for Anaerobic Culture Aspirates
Should be collected with needle and syringe
Excess air expressed from syringeSpecimen injected into oxygen-free
transport tube or vial Swabs – if collected, must be
transported in an anaerobic system
Specimens for Anaerobic Culture Tissue – must be placed in an
oxygen-free transport bag or vial Blood – aerobic AND anaerobic
bottles are collected for most blood culture requests
Processing Clinical Samples for Anaerobic Culture Must be placed in an anaerobic
chamber or holding device while awaiting processing
ProceduresMacroscopic exam of specimenGram stain (methanol fixation
instead of heating)Inoculation of anaerobic mediaAnaerobic incubation
Typical Anaerobic Media
Anaerobic blood agar (BRU/BA) Bacteroides bile esculin agar
(BBE) Kanamycin-vancomycin-laked
blood agar (KVLB) Phenylethyl alcohol agar (PEA) Anaerobic broth, such as
thioglycollate (THIO) or chopped meat
Anaerobic Incubation
Anaerobic chambers (p. 581) Anaerobic jars
Gas-Pak envelopes generate CO2 and H2, which combines with O2
H2 is explosive; palladium catalyst MUST be used
Anaerobic bags or pouches All systems must have an oxygen
indicator system in place
Indications of Anaerobes in Cultures Foul odor when opening anaerobic jar
or bag Colonies on anaerobically incubated
media but not on aerobic media Good growth on BBE Colonies on KVLB that are pigmented
or fluorescent Double zone of hemolysis on blood
agar
Presumptive Identification of Anaerobes Aerotolerance Fluorescence Special-potency antimicrobial disks Catalase test Spot indole test Motility test Lecithinase and lipase reactions Presumpto plates
Definitive Identification of Anaerobes PRAS (Pre-reduced Anaerobic System)
and non-PRAS biochemical test media Biochemical-based and preexisting
enzyme-based minisystems Gas-liquid chromatographic (GLC)
analysis of metabolic end products Cellular fatty acid analysis by GLC
Frequently Encountered Anaerobes Gram-positive spore-forming
anaerobic bacilliClostridium
• Most from exogenous sources• Examples: tetanus, gas gangrene,
botulism, food poisoning, pseudomembranous colitis (C. difficle)
• C. difficle is most often detected via direct stool antigen detection
Frequently Encountered Anaerobes (cont’d) Gram-positive non-spore-forming
anaerobic bacilliActinomyces, Bifidobacterium,
Eubacterium, Mobiluncus, Lactobacillus, and Propionibacterium
Most are from endogenous sources and are therefore opportunists
Frequently Encountered Anaerobes (cont’d) Anaerobic gram-negative bacilli
EndogenousInclude Bacteroides fragilis group,
Porphyromonas spp., Prevotella spp., and Fusobacterium spp.
Anaerobic cocci (usually endogenous)Gram-positive – PeptostreptococcusGram-negative – Veillonella spp.