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 Anaerobic System – 0% O 2

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 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 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 syringe Specimen 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 Procedures Macroscopic exam of specimen Gram stain (methanol fixation instead of heating) Inoculation of anaerobic media Anaerobic 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 CO 2 and H 2, which combines with O 2 H 2 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 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) 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’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.