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 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.,

histolyticum, C. carnis C. tertium, C.

2

How to recognize the anaerobes

 Enriched (Columbia blood agar)  Chocolate agar  Reducible blood agar + + O 2 + + + CO 2 + + + ANO 2 3

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 4

Endogenous flora – Gram negatives

B. fragilis

group

Prevotella

and

Bacteroides

spp.

Porphyromonas Fusobacterium Veillonella

Oral √ Large bowel √ Female GU Male GU Skin √ √ √ √ √ √ √ √ √ √ √ 5

Endogenous flora – Gram positives

Anaerobic cocci

Propionibacterium Actinomyces Lactobacillus Eubacterium Bifidobacterium

Oral Large bowel √ √ Female GU √ Male GU √ Skin √ √ √ √ √ √ √ √ √ √ √ 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 GU Endometritis, post-abortal, tuboovarian abscess, post hysterectomy Skin Any CNS shunts, post orthopedic surgery 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) 9

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?

10

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.

11 CID 50:133-164, 2010

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 12

Media for isolation of anaerobes

Anaerobic blood agar

Bacteroides Bile Esculin

Laked blood kanamycin vancomycin Phenylethyl alcohol Egg-yolk agar Chopped meat/ thioglycollate Columbia, Schaedler, CDC,

Brucella

, brain heart infusion, w/5% Sheep, YE, Vitamin K 1 , hemin Nonselective (enriched) TSA, ferric ammonium citrate, hemin, bile salts, gentamicin selective & differential for

B. fragilis

group

Brucella

base with 5% laked blood, kanamycin & vancomycin nutrient agar, 5% blood, PEA egg yolk base meat particles/casein, soy, glucose, agar, vitamin K 1 , hemin selective for

Bacteroides

and some

Prevotella

inhibits enteric GNR and swarming of some clostridia lipase & lecithinase production nonselective, enrichment broths 13

Anaerobic gram-negative rods



Bacteroides fragilis

group 

Bacteroides ureolyticus



Bilophila wadsworthia



Fusobacterium nucleatum



Fusobacterium necrophorum

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Anaerobic gram-negative rods

ALN, L-alanyl-L-alanyl β-naphthylamide

Sutterella

15

Bacteroides fragilis

group

16

B. fragilis

group

 resistant to penicillin, colistin, bile, kanamycin, vancomycin BBE 17

Pigmented

Prevotella

and

Porphyromonas

species

 as above  brick red fluorescence  black pigment 18

Bacteroides urealyticus

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

Bilophila wadsworthia

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

Fusobacterium

species

Bile S Lipase Indole + S R + + R R + Nitrate Gram stain Pointed rod FIID

F. nucleatum

Pleo rod Pleo rod Pleo rod

F. necrophorum F. mortiferum F. varium

21

Lipase

22

Anaerobic gram-positive cocci

Former

Peptostreptococcus anaerobius P. magnus P. micros P. asaccharolyticus P. prevotii P. tetradius Peptococcus niger Staphylococcus saccharlyoticus

Current

Peptostreptococcus anaerobius Finegoldia magna Micromonas micros Peptoniphilus asaccharlyoticus Anaerococcus prevotii Anaerococcus tetradius Peptococcus niger 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.’ 24

Anaerobic gram-negative cocci

  

Veillonella

species  susceptible to kanamycin, colistin  and bile brick red fluorescence

Acidaminococcus Megasphaera

Nitrate + + Vanco R S/R S Identification

Veillonella

species probable Possible

Prevotella/Porphyromonas

Possible overdecolorized gram-positive cocci 25

Identification of common nonswarming clostridia

Species C. bifermentans C. perfringens C. sordelii Lecithinase + + Lipase Indole + + + Urease Other DZ hemolysis + 26

Clostridium perfringens

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

Identification of swarming clostridia

Species Lecithinase Lipase Indole Urease Other C. novyi A C. septicum C. sporogenes C. tetani + + + w V + β-hemolytic Rare spores Abundant oval spores Terminal spores 28

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 29

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) 30

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 31

Susceptibility testing methods

 agar dilution  broth dilution (for only

B. fragilis

group)   Etest β-lactamase  Cefinase

NOT

 disk diffusion  disk elution 32

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 33

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 34

Identification

 ALN negative  indole positive  lipase positive 

Fusobacterium necrophorum

 penicillin 0.032 mcg/mL  clindamycin 0.016 mcg/mL  metronidazole 0.032 mcg/mL 35

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 36

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 -

Clostridium septicum E. coli, Bacteroides fragilis,

37

Clostridium septicum

 Clostridia are 2 nd to

Bacteroides

anaerobic blood culture isolates among  risk factors for

C. septicum

: neoplastic disease especially in ileocecal region or leukemia; inflammatory bowel disease (e.g. Crohn’s); cyclic neutropenia; cirrhosis 38

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 [email protected]

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