Anaerobes of Clinical ImportancePart One
MLAB 2434 – Microbiology Keri Brophy-Martinez
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
Concepts in Anaerobic Bacteriology
Obligate (strict) anaerobesGrow ONLY in the absence of
molecular oxygenKilled by exposure to air
Aerotolerate (moderate) anaerobesCan tolerate exposure to air for
several hours but perform metabolic activities
Concepts in Anaerobic Bacteriology
Facultative anaerobesDo not require O2, but use it if
available.
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
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
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” 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
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 anaerobesIf 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 Prior antibiotic therapy when organism
was resistant Immunosuppresion
Virulence Factors Polysaccharide capsule Adherence factors Clostridial toxins/exoenzymes Hyaluronidase Lipases Proteases/Proteinases/
Phospholipases/ Permeases Necrotizing toxins
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
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
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
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
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
Processing Clinical Samples for Anaerobic Culture Inoculation of anaerobic media
Require enriched media for growthUtilize pre-reduced media
•Eliminates dissolved O2 in media•Reducing agents lower redox
potential•Inoculate nonselective, selective
and liquid enrichment mediaAnaerobic incubation
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
o Cycloserine-cefoxitin-fructose agar (CCFA)o 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
Anaerobic MediaBacteroides fragilis on KVLB (left) and BBE agar (right)
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
Anaerobic Incubation Anaerobic chambers
Anaerobic Incubation Anaerobic
GasPak System Anaerobic bags/
containers
Interpretation of Cultures Primary cultures are examined after
48 hours of incubation If no growth, reincubate for up to 5
days before discarding
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
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
Interpretation of Cultures If the aerotolerance test confirms an
anaerobe, evaluate colony morphology Consider:
1. 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
2. Number of different types of colonies3. Quantitation4. Type of media supporting growth
Interpretation of Cultures Gram stain suspicious colonies
Note gram reaction, shape, presence of spores, filamentous etc
Location of SporesTerminal Subterminal
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 Fatty acid analysis Alcohols Molecular testing
Antimicrobial Susceptibility Not routinely performed
Drugs of choice• Chloramphenicol, metronidazole,
cephalosporins Perform beta-lactamase testing
Gram negative rods
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
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.