Chapter 19

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Chapter 19 Anaerobes of Clinical Importance

• MLAB 2434 Clinical Microbiology
• Cecile Sanders Keri Brophy-Martinez

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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

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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

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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

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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)

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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

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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

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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

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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

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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 2/3s of all bacteria
  are in the stool only cultured anaerobically if
  Clostridium difficle is suspected

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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

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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

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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

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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

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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

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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

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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

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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

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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

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Presumptive Identification of Anaerobes

• Aerotolerance
• Fluorescence
• Special-potency antimicrobial disks
• Catalase test
• Spot indole test
• Motility test
• Lecithinase and lipase reactions
• Presumpto plates

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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

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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

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Frequently Encountered Anaerobes (contd)

• Gram-positive non-spore-forming anaerobic bacilli
• Actinomyces, Bifidobacterium, Eubacterium,
  Mobiluncus, Lactobacillus, and Propionibacterium
• Most are from endogenous sources and are
  therefore opportunists

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Frequently Encountered Anaerobes (contd)

• 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.