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Anaerobes (Gram Neg)

Victor S. Flauta, M.D.
March 30, 2011

Epidemiology
GNAR

Mucosa of animals and humans Predominant species: oral cavity, GIT, vagina Infections acquired: Endogenously (breached by trauma or disease)

Exception: clenched-fist wounds & bite wounds

Iatrogenically (by surgery) Aspiration pneumonia: mixed anaerobes

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Source: Uptodate

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GN Anaerobes
Organism B. fragilis F. nucleatum B. wadsworthia Prev. intermedia ANA BAP large Bread crumb or opalescent Small, transluscent Small BBE Gray/black NG Central black dot (H2S) Black on LKV Comment Regular GNR Indole +; pointed ends Cat + Coccobacilli, IND +; lipase +; red fluorescence

Porphyromonas
Bacteroides ureolyticus Veilonella

uniform
Flat transparent w/ pitting Small, transparent

NG
NG NG

Red fluorescence
Urea +, cat GNDC; red fluorescence

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Clues to probability of anaerobic

bacteria at infection site

Infection adjacent to surfaces that normally harbor anaerobes as normal flora Abscess formation or tissue necrosis Putrid odor Gas formation Gram stain of exudate showing polymicrobial flora Organisms with morphologic features of anaerobes Classic features of histotoxic clostridial syndromes: tetanus, botulism, C. perfringens food poisoning, gas gangrene, C. difficile-induced diarrhea or colitis, enteritis necroticans Infections that, by prior experience, usually involve anaerobic bacteria

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Collection & Transport Lower Resp Tract & endometrial samples are hard to obtain w/out contamination of resident flora Swab: least desirable and should be discouraged

small sample, prone to drying, intrinsically aerobic, can't be quantitated

Transport immediately in proper container If transported in glass, it can stay at room temp or refrigerated

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Isolation
Keep plates for 5 days to isolate Porphyromonas & Bilophila Media:

Brucella base is superior to CDC base & Schaedler base for GNAR CDC is better for GPAC Fastidious anaerobe agar (Lab M): good for Fusobacterium Ideal: 2 different basal media to maximize isolation

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Isolation
Media:

LKV: for Bacteroides & Prevotella (rapid pigmentation) For Porphyromonas reduce LKV vancomycin concentration (from 7.5 to 2 ug/ml) BBE: for Bacteroies, Bilophila, F. mortiferum/varium PEA: to prevent aerobic GNR & Clostridial swarming For Fusobacterium: neomycin-vancomycin agar metronidazole disk: R/O GPAR & facultative anaerobes since theyll show resistance

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Isolation
Must not expose to air during first 48 hrs Total incubation period of at least 5 days is recommended for primary plates. If shorter, may not detect Porphyromonas & Bilophila Blood culture:

Controversial: only <5% of strict anaerobes Still used since some facultative anaerobes grow faster in ANA blood culture media

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Identification
Vancomycin Resistant but Colistin & Kanamycin sensitive:

Fusobacterium B. ureolyticus NO3+ Bilophila NO3+ Sutterella Leptotrichia (brain surface texture)

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Antibiotic

B. fragilis

B. frag. grp

Prevotella

Pophyromonas

F. nucleatum

F. mortiferum/ varium

Carbapenem

B lactamase inhibitor Penicillins

R (50%)

Cephalosporins
(Cefoxitin;Ceftizoxime)

S (85%)

S (70%) S

Chloramphenicol

Tetracycline
(except tigecycline~ S to all)

Lincosamides (Clindamycin) Fluoroquinolones (Moxifloxacin)

S (80%)

R (50%) S (80%)

S (80%)

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The susceptibility trends for the species of the Bacteroides fragilis group against various antibiotics from 1997 to 2004 were determined by using data for 5,225 isolates referred by 10 medical centers. The antibiotic test panel included ertapenem, imipenem, meropenem, ampicillin-sulbactam, piperacillintazobactam, cefoxitin, clindamycin, moxifloxacin, tigecycline, chloramphenicol, and metronidazole. From 1997 to 2004 there were decreases in the geometric mean (GM) MICs of imipenem, meropenem, piperacillin-tazobactam, and cefoxitin for many of the species within the group. B. distasonis showed the highest rates of resistance to most of the -lactams. B. fragilis, B. ovatus, and B. thetaiotaomicron showed significantly higher GM MICs and rates of resistance to clindamycin over time. The rate of resistance to moxifloxacin of B. vulgatus was very high (MIC range for the 8-year study period, 38% to 66%). B. fragilis, B. ovatus, and B. distasonis and other Bacteroides spp. exhibited significant increases in the rates of resistance to moxifloxacin over the 8 years. Resistance rates and GM MICs for tigecycline were low and stable during the 5-year period over which this agent was studied. All isolates were susceptible to chloramphenicol (MICs < 16 g/ml). In 2002, one isolate resistant to metronidazole (MIC = 64 g/ml) was noted. These data indicate changes in susceptibility over time; surprisingly, some antimicrobial agents are more active now than they were 5 years ago.

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New GNAR
Alistipes putredinis: appendicitis Alistipes finegoldii: appendicitis Bacteroides coprocola: human feces Bacteroides goldsteinii: infection (intestinal origin) Bacteroides nordii: infection (intestinal origin) Bacteroies salyersiae: infection (intestinal origin) Bacteroides plebeius: human feces Cetobacterium somerae: childrens feces Desulfovibrio piger: infection (intestinal origin) Dialister micraerophilus: human clinical samples Dialister propionicifaciens: human clinical smaples Faecalibacterium prausnitzii: feces Fusobacterium canifelinum: bite infections (dogs, cats) Fusobacterium equinum: oral cavity of horses

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New GNAR
Pophyromonas gulae: gingival sulcus of animals Porphyromonas somerae: human skin, soft tissue & bone infections Porphyromonas uenonsis: non-oral human infections Prevotella baroniae: human oral cavity Prevotella marshii: human oral cavity Prevotella multiformis: human subgingival plaque Prevotella multisaccharivorax: human subgingival plaque Prevotella salivae: human oral cavity Prevotella shahii: human oral cavity Sneathia sanguinegens: human blood Tannerella forsythensis: human periodontal pockets

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B. fragilis

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B. fragilis

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Bacteroides fragilis
95% Confidence:

Most common species in clinical specimens Nonmotile GNR with rounded ends Broth: pleomorphic with vacuoles CDC: nonhemolytic, gray with concentric whorls Significance of capsules are still unclear Key characteristics:

Large colonies on ANA BAP Gray or black colonies on BBE GNR

Growth enhanced by bile Resistant to KVC (kanamycin, vanc, colistin) & Penicillin Sensitive to Rifampin

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B. fragilis
Ability to tolerate oxygen :

contain superoxide dismutase (protects against the toxic effects of oxygen) the ability to survive exposure to oxygen facilitates the survival and pathogenicity of the organism.

Bergan, T. Pathogenicity of anaerobic bacteria. Scand J Gastroenterol Suppl 1984; 91:1.

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B. fragilis
Normal constituent of colonic flora (abnormal in mouth, genital tract or URT) Found as mixed infections in abscess Produce enterotoxin induces IL-8 inflammatory diarrhea Also produces metalloproteases, LPS, capsular polysaccharides periodontal disease and abscess formation 1st Rx: Metronidazole 2nd Rx: Clindamycin

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B. thetaiotamicron

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B. thetaiotamicron
2nd most common species isolated in B. fragilis group Infections:

Peritonitis Intraabdominal abscess Hepatic abscess

Indole + (B. fragilis is indole neg)

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B. distasonis

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B. distasonis
B. distasonis showed the highest rates of resistance to most of the -lactams. B. fragilis, B. ovatus, and B. distasonis and other Bacteroides spp. exhibited significant increases in the rates of resistance to moxifloxacin.

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B. vulgatus

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B. vulgatus
The rate of resistance to moxifloxacin of B. vulgatus was very high (MIC range for the 8-year study period, 38% to 66%).

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B. ureolyticus

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Bacteroides ureolyticus
Thin GNAR with rounded ends Small colonies, flat transparent w/ pitting Greening of the agar May resemble Bilophila phenotypically (NO3+) but:

Bilophila is strongly catalase + & resistant to bile Bacteroides ureolyticus is catalase neg & urea +

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B. ovatus

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Porphyromonas spp.
P. gingivalis

Root canal infections, odontogenic sinusitis Produces phenylacetic acid Agglutinates sheep RBC Produces B-galactose-6-phosphate Produces N-acetyl-B-glucosaminidase Produces a-fucosidase Prevalent in urogenital or intestinal tract (important in infections arising from these sources) Root canal infections, odontogenic sinusitis Not as either of the foregoing species

P. asaccharolytica

P. endodontalis

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Porphyromonas spp.
P. somerae: pleuropulmonary infections, skin & soft tissue infections, bacterial vaginosis Pophyromonas gulae: gingival sulcus of animals Porphyromonas somerae: human skin, soft tissue & bone infections Porphyromonas uenonsis: non-oral human infections

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Bilophila wadsworthia
Reported by EJ Baron & colleagues in 1989 from appendicitis specimens and human feces Present in small number in bowel flora 3rd most common anaerobe recovered from gangrenous or perforated appendix Common constituent of the microbiota of intraabdominal infections Isolated from various clinical specimens Easily overlooked because of its fastidious growth

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Bilophila spp.
GNR, non-spore-forming, nonmotile, pleomorphic Grows after 4 days on BBE as opaque black colonies Grows on Brucella agar in 4-7 days as translucent gray colonies Catalase +, asaccharolytic, urease +, H2S +, NO3 +, Negative: indole, esculin, oxidase, B lactamase

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Bilophila spp.
Major metabolic product: acetate Like B. fragilis, it grows in 20% bile Unlike B. fragilis, it does not ferment CHO Unlike Fusobacterium, it is strongly CAT + & neg for butyric acid Resistant to beta lactams

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Prevotella melaninogenica

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Prevotella melaninogenica P. intermedia & others Normal oral flora Brackish brown hematin pigment Aka P. melaninogenica group Infections:

Aspiration pneumonia Pulmonary abscess Pleural empyema Cerebral abscess

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Other Prevotellas
P. bivia, P. disiens, P. buccae, P. oralis, P. buccalis Normal flora of the urogenital tract & oropharynx AKA P. oralis group Infections:

Chronic otitis media & sinusitis Dental abscesses Ulcerating gingivostomatitis Female genital tract infections Cerebral abscesses

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Fusobacterium nucleatum

Slender, pointed ends

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F. nucleatum
Thin rod with tapering ends (needle-shaped)

Capnocytophaga & Leptotrichia may also look like these but both are indole neg.

Indole + Greening of agar when exposed to air (due to production of H2O2) Has at least 3 different colony morphotypes Pleuropulmonary infections

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F. necrophorum

Large, pleomorphic

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F. necrophorum
Lipase positive fusobacterium Bile sensitive Long rod with round ends, pleomorphic with bizarre forms Indole + Fluoresces chartreuse Beta hemolysis around gray-yellow colonies

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F. necrophorum
Peritonsilar abscess: most common ANA Pharyngotonsilitis in children or young adults (as often as S. pyogenes)

May be associated with infectious mono

Lemierres Disease

Jugular vein septic thrombophlebitis Often complicated by sepsis & metastatic abscess

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F. mortiferum
Bizarre, round bodies

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F. mortiferum
Indole neg Extremely pleomorphic Filaments with swollen areas, round bodies & irregular staining

F. necrophorum may look similar but fewer round bodies

A bile resistant fusobacterium isolated from BBE is F. mortiferum or F. varium ONPG + (F. varium is ONPG neg) Intraabdominal infections

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F. varium

Large, rounded ends

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F. varium
Intraabdominal infections

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Veilonella spp.

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Veilonella spp.
Small percentage isolated in human specimens Rare infections:

Meningitis, osteomyelitis, prosthetic joint infections, pleuropulmonary infection, endocarditis, bacteremia

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Organism Veilonella spp

NO3 +

Catalase V

Glucose -

Acidaminococcus fermentans

Megasphaera elsdenii

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