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Chapter 19 – Anaerobes of Clinical Importance. MLAB 2434 – Clinical Microbiology Cecile Sanders & Keri Brophy-Martinez. Concepts in Anaerobic Bacteriology. Air = about 21% O 2 and 0.03% CO 2 CO 2 Incubator = about 15% O 2 and 5%-10% CO 2 Microaerophilic System = 5% CO 2

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chapter 19 anaerobes of clinical importance

Chapter 19 – Anaerobes of Clinical Importance

MLAB 2434 – Clinical Microbiology

Cecile Sanders & Keri Brophy-Martinez

concepts in anaerobic bacteriology
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 bacteriology1
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 bacteriology2
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
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 anaerobes1
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
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 found1
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
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 anaerobes
    • If mucosal surfaces are disturbed, infections can occur in the oral cavity and in aspiration pneumonia
    • Sometimes cause “bad breath”
anaerobic anatomical sites for endogenous anaerobes1
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
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
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
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
Specimens for Anaerobic Culture
  • Aspirates
    • Should be collected with needle and syringe
    • Excess air expressed from syringe
    • Specimen injected into oxygen-free transport tube or vial
  • Swabs – if collected, must be transported in an anaerobic system
specimens for anaerobic culture1
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
Processing Clinical Samples for Anaerobic Culture
  • Must be placed in an anaerobic chamber or holding device while awaiting processing
  • Procedures
    • Macroscopic exam of specimen
    • Gram stain (methanol fixation instead of heating)
    • Inoculation of anaerobic media
    • Anaerobic incubation
typical anaerobic media
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 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
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
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
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
Frequently Encountered Anaerobes
  • Gram-positive spore-forming anaerobic bacilli
    • Clostridium
      • 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
Frequently Encountered Anaerobes (cont’d)
  • Gram-positive non-spore-forming anaerobic bacilli
    • Actinomyces, Bifidobacterium, Eubacterium, Mobiluncus, Lactobacillus, and Propionibacterium
    • Most are from endogenous sources and are therefore opportunists
frequently encountered anaerobes cont d1
Frequently Encountered Anaerobes (cont’d)
  • Anaerobic gram-negative bacilli
    • Endogenous
    • Include Bacteroides fragilis group, Porphyromonas spp., Prevotella spp., and Fusobacterium spp.
  • Anaerobic cocci (usually endogenous)
    • Gram-positive – Peptostreptococcus
    • Gram-negative – Veillonella spp.