1. Anaerobes of Clinical Importance Part 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 Anaerobic jars
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 Hyperbaric oxygen Antitoxins
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
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.