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Anaerobic Culture When, What, How, and How Far to Test
4/13/11
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What is an anaerobe? Obligate Moderate obligate Aerotolerant
“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
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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 soil, marshes, lake and river sediments, ocean, sewage, food and animals Endogenous flora oral cavity (gingival crevice, tonsillar crypts) gastrointestinal tract (exp. colon) genitourinary tract skin Think about normal habitat and how we acquire anaerobic infection
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Endogenous flora – Gram negatives
Oral Large bowel Female GU Male GU Skin B. fragilis group √ Prevotella and Bacteroides spp. Porphyromonas Fusobacterium Veillonella
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Endogenous flora – Gram positives
Oral Large bowel Female GU Male GU Skin Anaerobic cocci √ Propionibacterium Actinomyces Lactobacillus Eubacterium Bifidobacterium
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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 GU Endometritis, post-abortal, tuboovarian abscess, post hysterectomy Skin CNS shunts, post orthopedic surgery Any Sepsis, endocarditis
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Characteristics of anaerobic infections
adjacent to mucous membranes mixed flora on Gram stain foul odor gas in tissue
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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) C. perf 80-95% C. novyi 10-40% C. septicum 5-20%
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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: , 2010
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Inappropriate sites (not all inclusive)
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, brain heart infusion, w/5% Sheep, YE, Vitamin K1, hemin Nonselective (enriched) 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 broths
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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 F. mortiferum F. varium
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Lipase
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Anaerobic gram-positive cocci
Former Current Peptostreptococcus anaerobius P. magnus Finegoldia magna P. micros Micromonas micros P. asaccharolyticus Peptoniphilus asaccharlyoticus P. prevotii Anaerococcus prevotii P. tetradius Anaerococcus tetradius Peptococcus niger Staphylococcus saccharlyoticus Peptococcus saccharolyticus Other genera: Atopobium, Coprococcus, Gallicola
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Identification of anaerobic gram-positive 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 Other C. bifermentans + - C. perfringens DZ hemolysis C. sordelii
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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) report MIC and interpretation
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Antibiotics with anti-anaerobe activity
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
agar dilution broth dilution (for only B. fragilis group) Etest β-lactamase Cefinase NOT disk diffusion disk elution Etest and metronidazole
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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 mcg/mL clindamycin mcg/mL metronidazole 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
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