Anaerobic Culture
When, What, How, and How Far to Test
4/13/11
http://www.slh.wisc.edu/outreach-data/event-detail.php?id=204
What is an anaerobe?
 Obligate
 “growth in the absence of O2 but fail to multiply in the
presence of O2 on the surface of nutritionally adequate
solid media incubated in room air or in a CO2 incubator
(5-10% CO2 in air), e.g., C. haemolyticum, C. novyi
type B, oral treponemes
 Moderate obligate
 can grow at O2 level averaging 3%, e.g., B. fragilis
group, C. perfringens > Prevotella, Porphyromonas,
Fusobacterium
 Aerotolerant
 can grow in a CO2 incubator, e.g., C. tertium, C.
histolyticum, C. carnis
2
How to recognize the anaerobes
 Enriched (Columbia blood agar)
 Chocolate agar
 Reducible blood agar
+
+
+
+
+
O2
CO2
+
+
+
ANO2
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What specimens are appropriate for
anaerobic culture?
 Sources of anaerobes

Environment
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soil, marshes, lake and river sediments, ocean,
sewage, food and animals
Endogenous flora
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oral cavity (gingival crevice, tonsillar crypts)
gastrointestinal tract (exp. colon)
genitourinary tract
skin
4
Endogenous flora – Gram negatives
Oral
B. fragilis group
Prevotella and
Bacteroides spp.
√
Porphyromonas
√
Fusobacterium
√
Veillonella
√
Large Female Male Skin
bowel GU
GU
√
√
√
√
√
√
√
√
√
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Endogenous flora – Gram positives
Oral Large Female Male Skin
bowel GU
GU
Anaerobic cocci
√
√
√
√
√
Propionibacterium
√
√
√
√
√
Actinomyces
√
√
Lactobacillus
Eubacterium
√
Bifidobacterium
√
√
6
Sources of infection by endogenous
flora
Oral
Brain abscess, chronic sinusitis, lung abscess
(aspiration)
GI
Sub diaphragmatic, hepatic, and sub hepatic
abscess. Abdominal post trauma, post surgery,
malignancy, ruptured viscus. Perirectal,
necrotizing fasciitis, gas gangrene, C. difficile
colitis
Female Endometritis, post-abortal, tuboovarian
GU
abscess, post hysterectomy
Skin
CNS shunts, post orthopedic surgery
Any
Sepsis, endocarditis
7
Characteristics of anaerobic infections
 adjacent to mucous membranes
 mixed flora on Gram stain
 foul odor
 gas in tissue
8
Sources of infection by exogenous
flora
Culture plays a role in diagnosis
 Gas gangrene
 C. perfringens, C. novyi, C. septicum and others
 bite wounds
 intravenous drug abuse
 septic abortion
Culture not generally used in diagnosis
 C. difficile
 nosocomial
 C. botulinum
 foodborne, wound, infant
 C. tetani
 tetanus
 C. perfringens
 gastroenteritis (food poisoning)
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Appropriate sites
 Specimens from sites uncontaminated by skin or
mucous membrane flora should be cultured for
anaerobes



sterile body fluids (blood, synovial, pleural, pericardial,
CSF shunts, etc. Urine only if suprapubic tap.)
aspirates from closed abscesses
FNA and tissue biopsies
 AND
 antimicrobial therapy will be given
 Intraabdominal sepsis?
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Intraabdominal sepsis
 Community acquired, mild-moderate

do not culture
 Community acquired, severe/high
risk/immunocompromised

order Gram stain and aerobic culture
Anaerobic culture not recommended as empiric
therapy is used and TAT too long.
CID 50:133-164, 2010
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Inappropriate sites (not all inclusive)
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throat or NP swabs
gingival or tongue swabs or extracted teeth
sputum or BAL
GI contents (gastric, small bowel, feces, rectal
swabs, fistulae, stoma), perirectal abscesses
surface swabs, e.g. decubitus ulcers
sites adjacent to skin or mucous membranes that
have not been properly decontaminated.
urine (voided, catheter collected, cystoscopy)
vaginal, cervical, urethral secretions
IUD
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Media for isolation of anaerobes
Anaerobic
blood
agar
Columbia, Schaedler, CDC, Brucella, Nonselective
brain heart infusion, w/5% Sheep, YE, (enriched)
Vitamin K1, hemin
Bacteroides
Bile Esculin
TSA, ferric ammonium citrate, hemin,
bile salts, gentamicin
selective & differential
for B. fragilis group
Laked blood
kanamycin
vancomycin
Brucella base with 5% laked blood,
kanamycin & vancomycin
selective for
Bacteroides and
some Prevotella
Phenylethyl
alcohol
nutrient agar, 5% blood, PEA
inhibits enteric GNR
and swarming of
some clostridia
Egg-yolk agar
egg yolk base
lipase & lecithinase
production
Chopped meat/
thioglycollate
meat particles/casein, soy, glucose,
agar, vitamin K1, hemin
nonselective,
enrichment broths13
Anaerobic gram-negative rods
 Bacteroides fragilis group
 Bacteroides ureolyticus
 Bilophila wadsworthia
 Fusobacterium nucleatum
 Fusobacterium necrophorum
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Anaerobic gram-negative rods
Sutterella
ALN, L-alanyl-L-alanyl-β-naphthylamide
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Bacteroides fragilis group
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B. fragilis group
 resistant to penicillin,
colistin, bile, kanamycin,
vancomycin
BBE
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Pigmented Prevotella and
Porphyromonas species
 as above
 brick red fluorescence black pigment
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Bacteroides urealyticus
 kanamycin, colistin and bile susceptible
 pits the agar
 urease positive
 asaccharolytic (requires formate-fumarate)
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Bilophila wadsworthia
 bile resistant
 kanamycin and colistin susceptible
 vancomycin resistant
 black center in colony on BBE
 urease positive
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Fusobacterium species
Bile
Lipase Indole
Nitrate
Gram stain
FIID
S
-
+
-
Pointed rod F. nucleatum
SR
+-
+
-
Pleo rod
F. necrophorum
R
-
-
-
Pleo rod
F. mortiferum
R
-
+
-
Pleo rod
F. varium
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Lipase
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Anaerobic gram-positive cocci
Former
Current
Peptostreptococcus anaerobius
Peptostreptococcus anaerobius
P. magnus
Finegoldia magna
P. micros
Micromonas micros
P. asaccharolyticus
Peptoniphilus asaccharlyoticus
P. prevotii
Anaerococcus prevotii
P. tetradius
Anaerococcus tetradius
Peptococcus niger
Peptococcus niger
Staphylococcus saccharlyoticus
Peptococcus saccharolyticus
Other genera: Atopobium, Coprococcus, Gallicola
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Identification of anaerobic grampositive cocci
 If susceptible (>12 mm) to sodium
polyanethol sulfonate (SPS) = P. anaerobius
 Generic identification

Peptostreptococcus species or ‘anaerobic
gram-positive coccus formerly known as
Peptostreptococcus species.’
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Anaerobic gram-negative cocci
 Veillonella species
susceptible to kanamycin, colistin
and bile
 brick red fluorescence
 Acidaminococcus
 Megasphaera

Nitrate
Vanco
Identification
+
R
Veillonella species probable
-
S/R
Possible Prevotella/Porphyromonas
+
S
Possible overdecolorized gram-positive
cocci
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Identification of common
nonswarming clostridia
Species
Lecithinase
Lipase
Indole
Urease
C. bifermentans
+
-
+
-
C. perfringens
+
-
-
-
C. sordelii
+
-
+
+
Other
DZ
hemolysis
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Clostridium perfringens
 box car-like gram-positive
rods
 DZ β-hemolysis
 no WBC
 α-toxin (an exotoxin)

is lecithinase
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Identification of swarming
clostridia
Species
Lecithinase
Lipase
Indole Urease
Other
C. novyi A
+
+
-
-
β-hemolytic
C. septicum
-
-
-
-
Rare spores
C. sporogenes
-
+
-
-+
Abundant
oval spores
C. tetani
-
-w
V
-
Terminal
spores
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Nonsporing gram-positive rods
 Actinomyces

only A. viscosus may be catalase positive
 Bifidobacterium
 Eggerthella lenta
 Eubacterium
 Lactobacillus
 Propionibacterium

P. acnes – indole (most) and catalase positive
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When to perform susceptibility testing
(CLSI M11-A7, 2007)
 Sites
 brain abscess
 endocarditis
 osteomyelitis
 joint infection
 prosthetic devices
 bacteremia
 Organisms
 B. fragilis group
 Bacteroides
 Prevotella
 Fusobacterium
 Clostridium
 Bilophila
 Sutterella (formerly B.
gracilis and others)
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Antibiotics with anti-anaerobe activity
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ampicillin
amoxacillin-clavulanic acid; ampicillin-sulbactam
cefoxitin, cefotetan
cefotaxime, ceftriaxone, cefoperazone, cefmetazole, ceftizoxime
chloramphenicol
clindamycin
imipenem, meropenem, ertapenem
metronidazole
mezlocillin
moxifloxacin
penicillin
piperacillin, piperacillin-tazobactam
tetracycline
ticarcillin, ticarcillin-clavulanic acid
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Susceptibility testing methods
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
agar dilution
broth dilution (for only B. fragilis group)
Etest
β-lactamase

Cefinase
NOT
 disk diffusion
 disk elution
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Case #1
 16 year old girl who developed sore throat
and fever 5 days prior to admission (PTA)
 3 d PTA – headache, temp 104 F
 over the next 2 days abdominal pain,
vomiting, leg pain, decreased urine output
 lives in Madison; traveled to CA, VE, ME;
spends time with grandmother in Spring
Green; no known tick bites
 2 cats
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Case #1 cont
 WBC 7.5 (45 N/12L/36B/3





meta)
hemoglobin 11.8
platelets 56,000
monospot negative
1 blood culture drawn
gram-negative rod growing
only in anaerobic bottle at
12.8 hr
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Identification
 ALN negative
 indole positive
 lipase positive
 Fusobacterium necrophorum



penicillin 0.032 mcg/mL
clindamycin 0.016 mcg/mL
metronidazole 0.032 mcg/mL
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Lemierre’s Disease
 acute jugular vein septic thrombophlebitis
often complicated by sepsis and metastatic
abscesses, especially lungs, pleural space,
liver and large joints
 life-threatening infection
 prolonged treatment
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Case #2
 53 y/o man with HCV, peripheral vascular disease,
portal hypertension, hyperlipidemia and rectosigmoid
carcinoma
 had resection of CA with anastamosis and was
discharged home on hospital day 7
 8 days later he developed fever, sweats and LLQ
pain. CT scan showed an abscess in the posterior
pelvis. The abscess was drained and aerobic and
anaerobic cultures performed.
 Organisms recovered - E. coli, Bacteroides fragilis,
Clostridium septicum
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Clostridium septicum
 Clostridia are 2nd to Bacteroides among
anaerobic blood culture isolates
 risk factors for C. septicum: neoplastic
disease especially in ileocecal region or
leukemia; inflammatory bowel disease (e.g.
Crohn’s); cyclic neutropenia; cirrhosis
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Also see CLSI M35-A2 Abbreviated identification of
bacteria and yeast; Approved guideline, second
edition
Thank you!
Carol A. Spiegel, Ph.D.
Director, Clinical Microbiology
University of Wisconsin Hospital & Clinics
ca.spiegel@hosp.wisc.edu
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