Anaerobes Part I UT

Anaerobes(Gram Neg)Anaerobes(Gram Neg)Victor S. Flauta, M.D.March 30, 2011

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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

Source: Uptodate

GN Anaerobes

Organism ANA BAP BBE Comment

B. fragilis large Gray/black Regular GNR

F. nucleatum Bread crumb or opalescent

NG Indole +; pointed ends

B. wadsworthia Small, transluscent

Central black dot (H2S)

Prev. intermedia Small Black on LKV Coccobacilli, IND +; lipase +; red fluorescence

Porphyromonas uniform NG Red fluorescence

Bacteroides ureolyticus

Flat transparent w/ pitting

NG Urea +, cat -

Veilonella Small, transparent

NG GNDC; red fluorescence

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

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

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

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 they’ll show resistance

Isolation

Must not expose to air during first 48 hrsTotal incubation period of at least 5 days

is recommended for primary plates.If shorter, may not detect Porphyromonas

& BilophilaBlood culture:

Controversial: only <5% of strict anaerobes Still used since some facultative anaerobes

grow faster in ANA blood culture media

Identification

Vancomycin Resistant but Colistin & Kanamycin sensitive: Fusobacterium B. ureolyticus – NO3+ Bilophila – NO3+ Sutterella Leptotrichia (“brain surface” texture)

Antibiotic B. fragilis B. frag. grp

Prevotella Pophyromonas F. nucleatum

F. mortiferum/ varium

Carbapenem S S S S S S

B lactamase inhibitor

S S S S S S

Penicillins R R R (50%) S S S

Cephalosporins(Cefoxitin;Ceftizoxime)

S (85%) S (70%)

S S S S

Chloramphenicol S S S S S S

Tetracycline(except tigecycline~ S to all)

R R R S S S

Lincosamides(Clindamycin)

S (80%) R (50%)

S S S S (80%)

Fluoroquinolones(Moxifloxacin)

S S (80%)

S S S S

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, piperacillin-tazobactam, 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 ß-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 µg/ml). In 2002, one isolate resistant to metronidazole (MIC = 64 µg/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.

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: children’s 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

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

B. fragilis

Bacteroides fragilis

95% Confidence: Large colonies on ANA BAP Gray or black colonies on BBE GNR

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:

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

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.

B. fragilis

Normal constituent of colonic flora (abnormal in mouth, genital tract or URT)

Found as mixed infections in abscessProduce enterotoxin induces IL-8

inflammatory diarrheaAlso produces metalloproteases, LPS, capsular

polysaccharides periodontal disease and abscess formation

1st Rx: Metronidazole2nd Rx: Clindamycin

B. thetaiotamicron

2nd most common species isolated in B. fragilis group

Infections: Peritonitis Intraabdominal abscess Hepatic abscess

Indole + (B. fragilis is indole neg)

B. distasonis

B. distasonis showed the highest rates of resistance to most of the ß-lactams.

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

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%).

B. ureolyticus

Bacteroides ureolyticus

Thin GNAR with rounded endsSmall colonies, flat transparent w/ pittingGreening of the agarMay resemble Bilophila phenotypically

(NO3+) but: Bilophila is strongly catalase + & resistant to bile Bacteroides ureolyticus is catalase neg & urea +

B. ovatus

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

P. asaccharolytica Produces a-fucosidase Prevalent in urogenital or intestinal tract (important in

infections arising from these sources) P. endodontalis

Root canal infections, odontogenic sinusitis Not as either of the foregoing species

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

Bilophila wadsworthia

Reported by EJ Baron & colleagues in 1989 from appendicitis specimens and human feces

Present in small number in bowel flora3rd most common anaerobe recovered from

gangrenous or perforated appendixCommon constituent of the microbiota of intra-

abdominal infections Isolated from various clinical specimensEasily overlooked because of it’s fastidious

growth

Bilophila spp.

GNR, non-spore-forming, nonmotile, pleomorphicGrows after 4 days on BBE as opaque black

coloniesGrows on Brucella agar in 4-7 days as translucent

gray coloniesCatalase +, asaccharolytic, urease +, H2S +, NO3

+, Negative: indole, esculin, oxidase, B lactamase

Bilophila spp.

Major metabolic product: acetateLike B. fragilis, it grows in 20% bileUnlike B. fragilis, it does not ferment CHOUnlike Fusobacterium, it is strongly CAT +

& neg for butyric acidResistant to beta lactams

Prevotella melaninogenica

Prevotella melaninogenicaP. intermedia & others

Normal oral floraBrackish brown hematin pigmentAka P. melaninogenica group Infections:

Aspiration pneumonia Pulmonary abscess Pleural empyema Cerebral abscess

Other Prevotellas

P. bivia, P. disiens, P. buccae, P. oralis, P. buccalisNormal flora of the urogenital tract & oropharynxAKA P. oralis group Infections:

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

Fusobacterium nucleatum

Slender, pointed ends

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 morphotypesPleuropulmonary infections

F. necrophorum

Large, pleomorphic

F. necrophorum

“Lipase positive fusobacterium”Bile sensitiveLong rod with round ends, pleomorphic

with bizarre formsIndole +Fluoresces chartreuseBeta hemolysis around gray-yellow

colonies

F. necrophorum

Peritonsilar abscess: most common ANAPharyngotonsilitis in children or young

adults (as often as S. pyogenes) May be associated with infectious mono

Lemierre’s Disease Jugular vein septic thrombophlebitis Often complicated by sepsis & metastatic

abscess

F. mortiferum

Bizarre, round bodies

F. mortiferum

Indole negExtremely pleomorphicFilaments 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

F. varium

Large, rounded ends

F. varium

Intraabdominal infections

Veilonella spp.

Small percentage isolated in human specimens

Rare infections: Meningitis, osteomyelitis, prosthetic joint

infections, pleuropulmonary infection, endocarditis, bacteremia

Organism NO3 Catalase Glucose

Veilonella spp + V -

Acidaminococcusfermentans

Megasphaeraelsdenii

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Anaerobes Part I UT

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