Anaerobes (Gram Neg)

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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

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Source Uptodate
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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) Cat
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
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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 lt5 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 S S S S S S
B lactamase inhibitor S S S S S S
Penicillins R R R (50) S S S
Cephalosporins (CefoxitinCeftizoxime) 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
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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, 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 lt 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.
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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
GS from broth
GS from plate
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B. fragilis
BRU
CDC ANA
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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

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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 911.
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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
GS
GS
CDC Ana
BRU
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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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(No Transcript)
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B. distasonis
CDC Ana
GS from plate
BRU
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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.

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B. vulgatus
GS
CDC Ana
CDC Ana
BRU
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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
GS
CDC Ana
BRU
CDC Ana
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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
CDC Ana
GS
CDC Ana
BRU
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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
• 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

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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
  intra-abdominal 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
GS
CDC Ana
CDC Ana
BRU
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Prevotella melaninogenicaP. 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
GS
CDC Ana
Slender, pointed ends
CDC Ana
BRU
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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
GS
GS
Large, pleomorphic
BRU
CDC Ana
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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
GS
GS
Bizarre, round bodies
BRU
CDC Ana
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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
CDC Ana
GS
BRU
Large, rounded ends
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F. varium

• Intraabdominal infections

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Veilonella spp.
CDC Ana
GS
BRU
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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 NO3 Catalase Glucose
Veilonella spp V -
Acidaminococcus fermentans - - -
Megasphaera elsdenii - -
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