Chapter 19 – Anaerobes of Clinical Importance

1. Chapter 19 – Anaerobes of Clinical Importance MLAB 2434 – Clinical Microbiology Cecile Sanders & Keri Brophy-Martinez

2. 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

3. 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

4. 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

5. 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)

6. 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

7. 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

8. 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

9. 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”

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. Presumptive Identification of Anaerobes • Aerotolerance • Fluorescence • Special-potency antimicrobial disks • Catalase test • Spot indole test • Motility test • Lecithinase and lipase reactions • Presumpto plates

21. 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

22. 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

23. 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

24. 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.