Chapter 19 –
Anaerobes of Clinical
Importance
MLAB 2434 – Clinical Microbiology
Cecile 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 anaerobes

If 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

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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 gramstained 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


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
 Procedures

Macroscopic exam of specimen
 Gram stain (methanol fixation
instead of heating)
 Inoculation of anaerobic media
 Anaerobic 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

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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
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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 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
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

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.
