Chapter 19 – Anaerobes of Clinical Importance

MLAB 2434 – Clinical MicrobiologyCecile Sanders & 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 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 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

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 syringeSpecimen 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

ProceduresMacroscopic exam of specimenGram stain (methanol fixation

instead of heating)Inoculation of anaerobic mediaAnaerobic 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 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

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 bacilliClostridium

• 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 bacilliActinomyces, Bifidobacterium,

Eubacterium, Mobiluncus, Lactobacillus, and Propionibacterium

Most are from endogenous sources and are therefore opportunists

Frequently Encountered Anaerobes (cont’d) Anaerobic gram-negative bacilli

EndogenousInclude Bacteroides fragilis group,

Porphyromonas spp., Prevotella spp., and Fusobacterium spp.

Anaerobic cocci (usually endogenous)Gram-positive – PeptostreptococcusGram-negative – Veillonella spp.