Obligately anaerobic Gram negative nonsporeforming rods

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Obligately anaerobic Gram negative
non-spore-forming rods

• Bacteroides, Prevotella Fusobacterium
• Refer to Introduction to anaerobes

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Introduction

• Anaerobic Gram-negative bacilli are common flora
  of mucous membranes throughout the body. As
  said in Intro to anaerobes, some are mutualistic
  but others are opportunistic pathogens. They are
  the most common anaerobes involved in infection
  and include some of the most antibiotic-resistant
  species.
• There are over 2 dozen genera in this group, but
  Bacteroides, Prevotella Fusobacterium are the
  most important clinically
• The key characters for classification are
  motility, arrangement of flagella, organic and
  volatile fatty acid metabolic end products and
  cellular fatty acid patterns
• Common pathology includes oral/dental, pulmonary,
  GI female genitourinary tract and skin, soft
  tissue and bone infections. They may play a role
  in such diverse pathologic processes as
  periodontal disease and colon cancer.

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Bacteroides fragilis Group

• B. fragilis is a mysterious organism. Much
  remains to be learned. It is a wonderful
  mutualist in the colon, but a serious pathogen
  outside of the colon. First lets discuss the
  good things that it does.
• It dominates the normal human gut flora. It
  outnumbers E. coli 1000 to 1, and makes up 30-50
  of the weight of feces. Here, it is critical in
  intestinal homeostasis gut function, environment
  and community stability Not surprisingly, it is
  bile-resistant, and often present in mixed
  culture with facultative anaerobes, especially
  enteric Gram-negative rods
• Via production of organic acids it provides about
  70 of the energy supply of the colonic mucosal
  epithelial cells
• Competes with opportunistic colon residents such
  as Salmonella and Shigella limiting their
  resources and making the environment unfavourable
  for their growth by acid production and
  deconjugating bile salts (good or bad?).
• Involved in intestinal vitamin K and folic acid
  production

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Pathology - the bad things

• The most frequently isolated anaerobe from
  clinical samples (80), and considered the 1
  most clinically important obligate anaerobe due
  to frequency of infection and antibiotic
  resistance. It is a problem when it becomes
  extra-colonic.
• Common routes through the colon mucosa include GI
  surgery, perforated appendicitis or ulcer,
  diverticulitis, inflammatory bowel disease, and
  other colon trauma
• Associated with colitis and deep abscesses (big
  problem with B. fragilis) with an offensive odor,
  and an untreated mortality rate of 60. Abscess
  can progress ? intestinal obstruction, fistula
  formation, and septic infection often in the form
  of non-Clostridial gas gangrene
• Implicated in initiating (or at least
  contributing to) colon cancer (remember the
  deconjugation of bile acids?).
• Diarrhea in children of 1-5 years old (and some
  adults), especially in those with inflammatory
  bowel disease (involved in Crohns disease?).

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continued

• Several toxins produced including neuraminidase
  and hyaluronidase, both aid in invasion
• Prominent capsule involved in pathogenesis it is
  anti-phagocytic and directly involved in abscess
  formation instigates cytolytic immune response.
• Often functions as part of a pathogenic
  community. Perhaps the other members decrease
  pO2 and Eh
• Unique among anaerobes, some strains of B.
  fragilis are resistant to penicillin. This is
  diagnostic, and commonly determined using
  Cefinase discs for beta-lactamase.

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B. fragilis capsule
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B. fragilis ID

• Presumptive ID obligately anaerobic non
  spore-forming Gram negative rod
• Next 3 things production of acid from glucose
  fermentation, grow in presence of 20 bile, and
  hydrolyze esculin
• Cell morphology is distinctive
• pale-staining, pleomorphic rods with dark and
  light staining areas within a single cell
• swollen areas with what looks like vacuoles
  inside (can be misconstrued as endospores)
• enormous capsule similar to Klebsiella
• Bacteroides fragilis can be grown anaerobically
  on SBA, but is commonly cultured on bile esculin
  agar (BBE), a selective medium containing oxgall
  and gentamycin to inhibit enteric bacteria.
  Esculin with ferric ammonium citrate allows
  detection of esculin hydrolysis which produces
  blackening around colonies.

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B. fragilis note the irregular staining and
swollen areas
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B. fragilis on AnBAP (left) and bile esculin
agar (right)
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ID continued

• Resistant to or even enhanced growth by bile vs
  Fusobacterium
• Non-fluorescent in UV light and non-pigmented on
  prolonged incubation vs Prevotella Porphyrmonas
• Production of large amounts of acetate and
  succinate from glucose fermentation
• Non-distinctive colony morphology - similar to
  enterics
• Semi-automated / automated ID methods
• Antibiotic susceptibility testing Penicillin and
  Kanamycin resistant but rifampin sensitive
• Rapid enzyme test (4 hr)
• Chomatographic metabolite profiles
• Nucleic acid probes
• ELISA

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Prevotella

• Prevotella are normal symbiotic colon flora of
  cows, sheep pigs, but can function as diarrheic
  pathogens.
• The genus Prevotella includes former Bacteroides
  species. They constitute the 2nd most common
  isolates of anaerobic Gram-negative rods. P.
  melaninogenica and P. intermedia are the primary
  pathogens in the group
• Both species (in humans) are normal oral and
  vaginal flora, and are the predominant AGNR
  isolated from all manner of RT infections and
  their complications. Both are primarily involved
  in periodontal disease
• Cells appear as short rod to coccoid Gram
  negative
• Growth of pathogenic strains often requires
  vitamin K, or similar compounds, as well as heme.
  
• Smears taken from agar plates tend to display
  cocci or coccobacilli (with rods in the minority)

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P. intermedia
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P. melaninogenica
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Prevotella

• Both species produce red fluorescence on primary
  isolation media (usually BAP) when subjected to
  UV light.
• Colonies of both species grown on blood produce a
  distinctive pigment (a brown to black pigment
  from the heme protoporphyrin) if incubated for
  several day. The protoporphyrin is converted to
  melanin (think about the name).
• A related (but perhaps less significant genus)
  organism, Porphyrmonas, is also significant as a
  pathogen in dental infections
• These organisms can be differentiated
  presumptively based on vancomycin sensitivity
• Porphyrmonas is vancomycin sensitive
• Prevotella is vancomycin resistant

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Fusobacterium

• The genus Fusobacterium are fusiform or
  spindle-shaped cells with sharpened ends. They
  are long and thin, often appearing filamentous if
  cell density is high
• Fusobacteria are commonly found in the GI,
  genitourinary, URT and the oral cavity. Like the
  other AGNRs, they may exist as commensals or may
  be pathogenic. They can cause serious infections
  in many body sites.
• F. nucleatum is the most common clinical isolate
  in the genus. It is most frequently associated
  with pleuropulminary infections and disease.
• Other pathologies include brain abscess, septic
  arthritis, liver abscess, intra-abdominal
  infections, and oral pathology (as well as
  halitosis) in all but toothless infants
  (contributes to dental caries and gingivitis).
  Recently attention has been called to septicemia
  with high mortality (30-35?).

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continued

• F. necrophorum is less common but more virulent.
  It forms abscesses involved in pulmonary
  pathology, meningitis, jugular veinous
  thrombosis, and liver pathology. It is of
  veterinary importance as the cause of foot rot
  in cows, as well as the human pathologies.
• Presumptive ID
• non-fluorescing, bile sensitive, fusiform, AGNRs
• colonies on blood are small, white, alpha
  hemolytic and have a distinctive bad odor.
• Fusobacteria produce butyric acid as a major
  fermentation by product as opposed to the other
  AGNRs

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Multi-focal necrosis of lambs liver by
Fusobacteium necrophorum
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Presumptive Identification
Obligate Anaerobic Gram Negative Bacteria
Red Fluorescence UV Light
(Rod)
Yes
No
Rod
Coccus
Bile Resistant
No
Yes
Veillonella
Porphyrmonas Prevotella
Bacteroides fragilis Group
Fusiform
Vancomycin sensitive Vancomycin resistant
Fusobacterium
Other
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Presumptive Identification
Obligate Anaerobic Gram Negative Bacteria
Red Fluorescence UV Light
(Rod)
Yes
No
Rod
Coccus
Bile Resistant
No
Yes
Veillonella
Porphyrmonas Prevotella
Bacteroides fragilis Group
Fusiform
Vancomycin sensitive Vancomycin resistant
Fusobacterium
Other