Anaerobes of Clinical Importance Part One

1. Anaerobes of Clinical ImportancePart One MLAB 2434 – Microbiology 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 (strict) anaerobes • Grow ONLY in the absence of molecular oxygen • Killed by exposure to air • Aerotolerate (moderate) anaerobes • Can tolerate exposure to air for several hours but perform metabolic activities

4. Concepts in Anaerobic Bacteriology • Facultative anaerobes • Do not require O2, but use it if available.

5. Why Anaerobes? • Oxygen is toxic because it combines with enzymes, proteins, nucleic acids, vitamins and lipids that are vital to cell reproduction • 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

6. Why Anaerobes? • Substances produced when oxygen becomes reduced are even more toxic, producing such things as hydrogen peroxide and superoxide anion • 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” • Advantages: barrier to colonization by pathogenic organisms, source of fatty acids, vitamins and cofactors, help mature neonate immune system • Disadvantages: opportunists for immunocompromised • 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 • Prior antibiotic therapy when organism was resistant • Immunosuppresion

12. Virulence Factors • Polysaccharide capsule • Adherence factors • Clostridial toxins/exoenzymes • Hyaluronidase • Lipases • Proteases/Proteinases/ Phospholipases/ Permeases • Necrotizing toxins

13. 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 • Distinct morphologic characteristics in gram-stained preparation

14. 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

15. 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

16. Specimens for Anaerobic Culture • Tissue • must be placed in an oxygen-free transport bag or vial • Usually ground for best results • Blood • aerobic AND anaerobic bottles are collected for most blood culture requests

17. Unacceptable Specimens for Culture • Swabs collected from throat, nasopharyngeal, gingival, rectal, vaginal, cervical, urethral, decubitus ulcers, feet and other exposed wounds • Sputum • Voided or catheterized urine

18. Processing Clinical Samples for Anaerobic Culture • Must be placed in an anaerobic chamber or holding device while awaiting processing • Procedures • Macroscopic exam of specimen • Foul odor • Presence of “sulfur granules” • Black pigmentation • Gram stain • Distinct morphology • Increased WBCs

19. Processing Clinical Samples for Anaerobic Culture • Inoculation of anaerobic media • Require enriched media for growth • Utilize pre-reduced media • Eliminates dissolved O2 in media • Reducing agents lower redox potential • Inoculate nonselective, selective and liquid enrichment media • Anaerobic incubation

20. Typical Anaerobic Media • Anaerobic blood agar (BRU/BA) • Supports growth of all obligate and facultative anaerobes • Bacteroides bile esculin agar (BBE) • Supports growth of bile-tolerant anaerobes, such as Bacteroides, Prevotella, Porphyromonas, Fusobacterium species • Kanamycin-vancomycin-laked blood agar (KVLB) • Supports growth of Bacteroides and Prevotella spp.; certain facultative gnr will also grow • Phenylethyl alcohol agar (PEA) • Supports growth of all obligate and gram positive facultative anaerobes, inhibits enteric gnr • Cycloserine-cefoxitin-fructose agar (CCFA) • Selective for C. difficile • Anaerobic broth, such as thioglycollate (THIO) or chopped meat • Supports growth of all types of bacteria; obligate aerobes near the top, obligate anaerobes at the bottom and facultative anaerobes throughout

21. Anaerobic Media Bacteroides fragilis on KVLB (left) and BBE agar (right)

22. Anaerobic Incubation • Anaerobic chambers • Sealed box which provides an oxygen-free environment for inoculation and incubation of culture • 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 • Methylene blue strips • Resazurin

23. Anaerobic Incubation • Anaerobic chambers

24. Anaerobic Incubation • Anaerobic GasPak System • Anaerobic bags/ containers

25. Interpretation of Cultures • Primary cultures are examined after 48 hours of incubation • If no growth, reincubate for up to 5 days before discarding

26. 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

27. Anaerobic Culture Workup • If observe growth on media or liquid media • Check aerotolerance • Subculture a colony to BAP, incubate in ambient air and subculture a colony to Ana BAP, incubate anaerobically • After 24 hours, determine if organism is obligate anaerobe or facultative anaerobe

28. Interpretation of Cultures • If the aerotolerance test confirms an anaerobe, evaluate colony morphology • Consider: • Color/pigment, surface, density, consistency, form, elevation, margins, fluorescence, pitting of agar, double zone of beta hemolysis, odor, swarming, molar tooth/breadcrumb, ground glass/fried egg • Number of different types of colonies • Quantitation • Type of media supporting growth

29. Interpretation of Cultures • Gram stain suspicious colonies • Note gram reaction, shape, presence of spores, filamentous etc

30. Location of Spores Terminal Subterminal

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

32. 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 • Fatty acid analysis • Alcohols • Molecular testing

33. Antimicrobial Susceptibility • Not routinely performed • Drugs of choice • Chloramphenicol, metronidazole, cephalosporins • Perform beta-lactamase testing • Gram negative rods

34. Treatment Protocols • Surgical therapy • Draining abscesses, removing dead tissue, eliminating obstructions • Hyperbaric oxygen • Oxygen is forced into necrotic tissues, killing anaerobes • Antitoxins • Used in cases of tetanus and botulism to neutralize the neurotoxins produced by C.tetani and C. botulinum

35. References • http://www.labsupplyoutlaws.com/products/Lab-Equipment/Microbiology-Apparatus/Environmental-Systems-for-Microbiology/BD-BBL-Bio-Bag-Environmental-Chambers.htm • Kiser, K. M., Payne, W. C., & Taff, T. A. (2011). Clinical Laboratory Microbiology: A Practical Approach . Upper Saddle River, NJ: Pearson Education. • Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.