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Anaerobic Culture When, What, How, and How Far to Test 4/13/11

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Presentation on theme: "Anaerobic Culture When, What, How, and How Far to Test 4/13/11"— Presentation transcript:

1 Anaerobic Culture When, What, How, and How Far to Test 4/13/11

2 2 What is an anaerobe? Obligate “growth in the absence of O 2 but fail to multiply in the presence of O 2 on the surface of nutritionally adequate solid media incubated in room air or in a CO 2 incubator (5-10% CO 2 in air), e.g., C. haemolyticum, C. novyi type B, oral treponemes Moderate obligate can grow at O 2 level averaging 3%, e.g., B. fragilis group, C. perfringens > Prevotella, Porphyromonas, Fusobacterium Aerotolerant can grow in a CO 2 incubator, e.g., C. tertium, C. histolyticum, C. carnis

3 3 How to recognize the anaerobes Enriched (Columbia blood agar) Chocolate agar Reducible blood agar + O2O2 CO 2 ANO

4 4 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

5 5 Endogenous flora – Gram negatives OralLarge bowel Female GU Male GU Skin B. fragilis group√ Prevotella and Bacteroides spp. √√√ Porphyromonas√√√√ Fusobacterium√√√ Veillonella√√

6 6 Endogenous flora – Gram positives OralLarge bowel Female GU Male GU Skin Anaerobic cocci√√√√√ Propionibacterium√√√√√ Actinomyces√ Lactobacillus√ Eubacterium√√ Bifidobacterium√

7 7 Sources of infection by endogenous flora OralBrain abscess, chronic sinusitis, lung abscess (aspiration) GISub 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 SkinCNS shunts, post orthopedic surgery AnySepsis, endocarditis

8 8 Characteristics of anaerobic infections adjacent to mucous membranes mixed flora on Gram stain foul odor gas in tissue

9 9 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)

10 10 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?

11 11 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

12 12 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

13 13 Media for isolation of anaerobes Anaerobic blood agar Columbia, Schaedler, CDC, Brucella, brain heart infusion, w/5% Sheep, YE, Vitamin K 1, 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, PEAinhibits enteric GNR and swarming of some clostridia Egg-yolk agaregg yolk baselipase & lecithinase production Chopped meat/ thioglycollate meat particles/casein, soy, glucose, agar, vitamin K 1, hemin nonselective, enrichment broths

14 14 Anaerobic gram-negative rods Bacteroides fragilis group Bacteroides ureolyticus Bilophila wadsworthia Fusobacterium nucleatum Fusobacterium necrophorum

15 15 Anaerobic gram-negative rods ALN, L-alanyl-L-alanyl-β-naphthylamide Sutterella

16 16 Bacteroides fragilis group

17 17 B. fragilis group resistant to penicillin, colistin, bile, kanamycin, vancomycin BBE

18 18 Pigmented Prevotella and Porphyromonas species as above brick red fluorescence  black pigment

19 19 Bacteroides urealyticus kanamycin, colistin and bile susceptible pits the agar urease positive asaccharolytic (requires formate-fumarate)

20 20 Bilophila wadsworthia bile resistant kanamycin and colistin susceptible vancomycin resistant black center in colony on BBE urease positive

21 21 Fusobacterium species BileLipaseIndoleNitrateGram stainFIID S-+-Pointed rodF. nucleatum SRSR Pleo rodF. necrophorum R---Pleo rodF. mortiferum R-+-Pleo rodF. varium

22 22 Lipase

23 23 Anaerobic gram-positive cocci FormerCurrent Peptostreptococcus anaerobius P. magnusFinegoldia magna P. microsMicromonas micros P. asaccharolyticusPeptoniphilus asaccharlyoticus P. prevotiiAnaerococcus prevotii P. tetradiusAnaerococcus tetradius Peptococcus niger Staphylococcus saccharlyoticusPeptococcus saccharolyticus Other genera: Atopobium, Coprococcus, Gallicola

24 24 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.’

25 25 Anaerobic gram-negative cocci NitrateVancoIdentification +RVeillonella species probable -S/RPossible Prevotella/Porphyromonas +SPossible overdecolorized gram-positive cocci Veillonella species susceptible to kanamycin, colistin and bile brick red fluorescence Acidaminococcus Megasphaera

26 26 Identification of common nonswarming clostridia SpeciesLecithinaseLipaseIndoleUreaseOther C. bifermentans+-+- C. perfringens+---DZ hemolysis C. sordelii+-++

27 27 Clostridium perfringens box car-like gram-positive rods DZ β-hemolysis no WBC α-toxin (an exotoxin) is lecithinase

28 28 Identification of swarming clostridia SpeciesLecithinaseLipaseIndoleUreaseOther C. novyi A++--β-hemolytic C. septicum----Rare spores C. sporogenes Abundant oval spores C. tetani--w-w V-Terminal spores

29 29 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

30 30 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)

31 31 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

32 32 Susceptibility testing methods agar dilution broth dilution (for only B. fragilis group) Etest β-lactamase Cefinase NOT disk diffusion disk elution

33 33 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

34 34 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

35 35 Identification ALN negative indole positive lipase positive Fusobacterium necrophorum penicillin mcg/mL clindamycin mcg/mL metronidazole mcg/mL

36 36 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

37 37 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

38 38 Clostridium septicum Clostridia are 2 nd 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

39 39 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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