Obligate Anaerobes and Microaerophilic Bacteria

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Obligate Anaerobes and Microaerophilic Bacteria

• Dr. John R. Warren
• Department of Pathology
• Northwestern University
• Feinberg School of Medicine
• June 2007

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Anaerobic Bacteria Modes of Infection

• Normally present on mucosal surfaces of the
  gastrointestinal tract, genitourinary tract, and
  oral cavity
• Heavily colonized mucosal surfaces portal of
  entry into tissue and blood
• Breach in a normal mucosal barrier (cancer,
  inflammation, surgery) provides access of
  anaerobic bacteria to sterile tissue sites
• Aspiration of necrotic colonized tissue from oral
  cavity provides access of anaerobic bacteria to
  deep normally sterile lung tissue
• In above settings anaerobic bacteria become
  opportunistic pathogens, producing serious
  sometimes fatal infection

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Anaerobic Bacteria Specimens

• Sterile aspirates or tissue preferred, swabs as
  alternative if aspiration or tissue sampling not
  possible (wounds)
• Immediate transport of specimens under anaerobic
  conditions (anaerobic transport bags and tubes)
• Fetid or putrid odor, gas in specimen, sulfur
  granules, and black discoloration of
  blood-containing exudates clues to anaerobic
  infection

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Anaerobic Bacteria Specimen Gram Stains

• Polymicrobial infection characteristic of
  anaerobic bacteria, and multiple distinct
  morphotypes of gram-negative and gram-positive
  bacteria suggestive of anaerobic infection
• Presumptive anaerobic identification occasionally
  possible based on Gram stain morphology

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Anaerobic Bacteria Specimen Gram Stains

• Clostridium perfringes Large boxcar-shaped
  gram-positive bacilli with blunt ends
• Actinomyces Branching filamentous bacilli with
  beaded gram-positivity
• Bacteroides, Porphyromonas, Prevotella Faintly
  staining gram-negative coccobacilli with safranin
  as counterstain and enhanced staining with carbol
  fuchsin as counterstain
• Fusobacterium nucleatum Thin gram-negative
  bacteria with tapered (pointed) ends
• Fusobacterium necrophorum Pleomorphic, long
  gram-negative rod with round ends and bizarre
  shapes (filaments, coccoid forms or round bodies)
• Veillonella Tiny gram-negative cocci with gram
  variability

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Anaerobic Bacteria Culture Media for Primary
Isolation

• Brucella blood agar (BRU) (CDC anaerobic blood
  agar, Schaedler blood agar, enriched brain heart
  infusion agar) Nonselective for isolation of
  obligate and facultative anaerobes, enriched
  with vitamin K1 and hemin
• Bacteroides bile esculin agar (BBE) Selective
  and differential, gentamicin inhibits aerobic
  organisms, 20 bile inhibits most anaerobes,
  esculin hydrolysis turns medium brown
  (presumptive for Bacteroides fragilis group)

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Anaerobic Bacteria Culture Media for Primary
Isolation

• Kanamycin-vancomycin-laked blood agar (KVLB)
  (Kanamycin-vancomycin blood agar,
  paromomycin-vancomycin blood agar) Selective,
  kanamycin inhibits facultative gram-negatives,
  vancomycin inhibits gram-positives and
  Porphyromonas, laked blood allows early detection
  (with 48 hr) of pigmented Prevotella
• Phenylethyl alcohol agar (PEA) Selective for
  growth of gram-positive and gram-negative
  anaerobes, inhibits Enterobacteriaceae including
  swarming Proteus
• Thioglycolate broth (THIO) Growth of aerobic and
  anaerobic bacteria, a backup culture

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Anaerobic Incubation

• 85 nitrogen
• 10 hydrogen
• 5 carbon dioxide

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Anaerobic Bacteria Aerotolerance Testing

• Anaerobic blood agar plate (Brucella blood agar)
  incubated anaerobically
• Chocolate agar plate incubated in carbon
  dioxide-enriched air
• Growth on anaerobic and chocolate agar
  facultative anaerobe
• Growth on anaerobic agar alone obligate
  anaerobe

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Anaerobic Bacteria Presumptive Identification1

• Gram stain morphology
• Colony morphology on non-selective agar medium
• Colony pigmentation
• Reactions on selective agar mediuim (Bacteroides
  bile esculin, kanamycin-vancomycin laked blood
  agar)
• Susceptibility to special potency disks (5-µg
  vancomycin, 1-mg kanamycin, and 10-µg colistin)
  (inhibition zone gt 10 mm indicates susceptibility
  for purposes of identification)
• Spot indole test
• 1Sufficient for most anaerobic isolates
  (exceptions isolates from blood and other
  normally sterile body fluids, intraoperative
  tissue, and pure wound isolates

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Obligate Anaerobic Gram-Negative Bacilli

• Bacteroides fragilis group
• Prevotella species
• Porphyromonas species
• Fusobacterium nucleatum and Fusobacterium
  necrophorum

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Bacteroides fragilis group

• Major component of normal colonic flora, smaller
  numbers in female genital tract, but virtually
  never found in the oropharyngeal flora
• Bacteroides fragilis group most commonly
  encountered anaerobe in clinical infection
• Consists of 12 species of which two (B. fragilis,
  B. thetaiotamicron) prevalent in human infections
• Associated with intra-abdominal, perineal, and
  perirectal infections, as well as soft tissue
  infections (foot and decubitus ulcers)
• Most common anaerobic organism causing bacteremia
• ß-lactamase production prevalent with resistance
  to cefoxitin and other broad spectrum
  cephalosporins
• Resistance to clindamycin, imipenem, and
  metronidazole also encountered

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Pigmented Prevotella andPorphyromonas1

• Important components of normal oral flora, also
  present as intestinal and female genital tract
  flora
• Pathogens in oral, dental, and bite infections,
  and produce infections of the head, neck, and
  lower respiratory tract
• Prevotella (P. melaninogenica) associated with
  pelvic abscesses, septic abortion, endometritis,
  tuboovarian abscess, and pelvic inflammatory
  disease, often in mixed infection with B.
  fragilis group
• Uniform (gt95) susceptibility to cefoxitin,
  clindamycin, imipenem, and metronidazole, but
  susceptibility testing still necessary for fully
  identified isolates
• 1Brown to black pigment (within 2-3 d appears as
  brick-red fluorescence by exposure to long-wave
  UV light)

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Non-Pigmented Prevotella

• Prevotella bivia and P. disens associated with
  polymicrobial female genital tract infections
  (endometritis, pelvic inflammatory disease)
• P. bivia and P. disens frequently resistant to
  ß-lactam antibiotics
• Less frequently recovered from oral or
  pleuropulmonary infections

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Fusobacterium nucleatum and Fusobacterium
necrophorum

• Fusobacterium nucleatum most common in clinical
  infections, including oral, head, and neck
  infections, and as the sole cause of
  pleuropulmonary infection
• F. necrophorum highly virulent (potent endotoxin)
  causing severe pharyngotonsillits in children
  with local complications (neck space infection,
  jugular vein septic thrombophlebitis)
• Lemierre syndrome (postanginal septicemia)
  suppurative infection of the lateral pharyngeal
  space associated with F. necrophorum bacteremia
  and septic jugular vein thrombophlebitis which
  can progress to septic embolization to the lung
  and pulmonary abscess formation
• Polymicrobial necrotizing (cavitary) pneumonia
  due to Fusobacterium and Prevotella

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Presumptive Identification of Anaerobic
Gram-Negative Bacilli1

• Van Kan Col BIL IND
• B. fragilis group R R R
  v2
• Pig Prevotella R Rs v
  v
• Non-Pig Prevotella R R v
  v
• Pig Porphyromonas S R R
  v
• Fusobacterium R S S
  v3
• 1BILgrowth on bile esculin agar, INDspot
  indole, Rresistant, Rsresistant rarely
  susceptible, Ssusceptible, vvariable
• 2B. fragilis indole , B. thetaiotamicron indole
  
• 3F. nucleatum , F. necrophorum v

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Obligate Anaerobic Gram-Negative Cocci

• Veillonella

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Veillonella

• Component of normal oral and fecal flora
• Produce infection of oral, bite-wound, head and
  neck, and soft tissue
• In addition to Gram stain, presumptive
  identification obtained by the following panel of
  results vancomycin R, kanamycin S, colistin S,
  no growth on Bacteroides bile esculin agar,
  indole negative
• Presumptive identification confirmed by reduction
  of nitrate to nitrite (positive nitrate disk
  test)

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Obligate Anaerobic Gram-Positive Bacilli

• Clostridium perfringes1
• Clostridium septicum1
• Clostridium difficile1
• Actinomyces israelii
• Propionibacterium acnes
• Lactobacillus
• 1Spore-forming (spores rare for C. perfringes and
  C. difficile)

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Clostridium

• Prinicipal natural habitats soil and intestinal
  tracts of many animals including humans
• C. perfringens frequently isolated from soil
• Infant and adult feces yield similar numbers of
  C. perfringens (103-108 cfus/g), feces of
  infants frequently contain C. difficile, but
  feces of healthy adults less frequently positive
  for C. difficile (lt3)
• C. septicum commonly found in soil, and isolated
  from feces of humans although less commonly than
  C. perfringens (2 of normal adults, but with
  carriage rates of 10-63 in the appendix)

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Clostridium

• C. perfringens most frequently isolated from
  clinical specimens
• Alpha-toxin (phospholipase C) produced by C.
  perfringens may cause life-threatening
  myonecrosis due to infection of traumatic and
  non-traumatic wounds (gas gangrene)
• C. perfringens bacteremia occurs in 15 of
  myonecrosis cases and is characterized by
  intravascular hemolysis (drop in hematocrit by
  one-half in a few hours possible),
  hemoglobinuria, hypotension, renal failure, and
  metabolic acidosis
• Crepitant cellulitis due to C. perfringens
  infection of laceration-type wounds involving
  subcutaneous and retroperitoneal soft tissue
  infection can progress to systemic fulminant
  infection
• Polymicrobial abdominal abscess formation
• Polymicrobial bacteremia with C. perfringens and
  Bacteroides and/or Enterobacteriaceae indicates
  an intestinal source (devastating effect of
  clostridial toxins generally absent)

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Clostridium

• Enterotoxin-producing C. perfringens type A
  associated with gastroenteritis with nausea and
  vomiting due to ingestion of undercooked meat or
  meat products
• Beta toxin-producing C. perfringens causes
  enteritis necroticans, a severe small bowel
  disease of children
• Clostridium septicum bacteremia occurs most
  frequently in individuals with relapsing leukemia
  or colon carcinoma, and neutropenia secondary to
  cytotoxic drugs
• Recognition of C. septicum bacteremia urgent for
  adequate therapy (mortality 70) based on
  susceptibility testing (resistance to clindamycin
  and penicillins occurs), and for performance of
  imaging studies of lower intestine to rule out
  carcinoma in patients without a known underlying
  cause

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Clostridium

• Toxigenic strains of Clostridium difficile a
  major cause of antibiotic-associated diarrhea
  (pseudomembranous colitis)
• Clinical diagnosis primarily based on direct
  detection of C. difficile toxins A and B in
  diarrheal stool specimens
• Recovery of toxigenic C. difficile by stool
  culture required for molecular epidemiological
  investigation of nosocomial outbreaks of
  pseudomembranous colitis

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Presumptive Identification of Clostridium
perfringens and Clostridium septicum

• Boxcar-shaped gram-positive rods with blunt ends
  and rare or no sporulation, double zone of
  hemolysis on blood agar (smaller zone of complete
  hemolysis due to theta-toxin, outer zone of
  partial hemolysis due to phospholipase C), with
  opacification of egg yolk agar (due to
  phospholipase C) ? Clostridium perfringens
• Straight or curved gram-positive rods with
  subterminal oval spores swelling the cells, gray
  to translucent, markedly irregular swarming
  (rhizoid margins with Medusa head pattern) over
  the surface of blood agar with underlying
  ß-hemolysis ? Clostridium septicum

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Isolation and Toxin Testing of Clostridium
difficile

• Growth of yellow colonies on cycloserine-cefoxitin
  -fructose agar (CCFA)1 with horse-stable odor and
  constituted by straight gram-positive bacilli
  with short end-to-end chains and rare subterminal
  oval and/or free spores ? Clostridium difficile2
• 1Nutritive animal peptone base made selective by
  cycloserine and cefoxitin (inhibitory of normal
  intestinal flora), and differential by fructose
  and neutral red as pH indicator (C. difficile
  non-fermentative for fructose and alkalinizes
  medium by peptone catabolism)
• 2Isolates inoculated to brain heart infusion
  broth and toxin testing performed on supernatant

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Non-Spore Forming Anaerobic Gram-Positive Bacilli

• Actinomyces israelii Endogenous to mouth, upper
  respiratory tract. Actinomycosis chronic
  granulomatous infection with abscess formation
  and draining sinuses (head, neck, brain,
  pulmonary, genital tract). Sulfur granules in
  exudate with eosinophilic clubs and gram-positive
  filaments diagnostic. Molar tooth colonies
  develop in culture.
• Propiobacterium acnes Endogenous to skin,
  conjunctiva, oral cavity, and large intestine.
  Clinical infection of bone (osteomyelitis) and
  CNS secondary to ventriculoatrial shunt implants.
  Common blood culture contaminant. Presumptive
  identification as catalase-positive coryneform
  gram-positive rods with a positive indole
  reaction.
• Lactobacillus Endogenous to oral, intestinal,
  and genital mucosa. Cause endocarditis
  (monomicrobial bacteremia) and associated with
  abdominal and pelvic abscess formation
  (polymicrobial bacteremia). Presumptive
  identification as catalase-negative, slender
  gram-positive bacilli with parallel sides and
  boxy ends, non-hemolytic or a-hemolytic on blood
  agar (colony morphology resembles viridans
  streptococci).

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Obligate Anaerobic Gram-Positive Cocci

• Peptostreptococcus anaerobius
• Other (Finegoldia magna, Schleiferella
  asaccharolytica)

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Anaerobic Gram-Positive Cocci

• Three species most commonly isolated from
  clinical specimens
• gtPeptostreptococcus anaerobius
• gtFinegoldia magna
• gtSchleiferella asaccharolytica
• Anaerobic gram-positive cocci members of normal
  flora for skin, oropharynx, and the upper
  respiratory, gastrointestinal, and genitourinary
  tracts

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Anaerobic Gram-Positive Cocci

• Associated with head and neck infections
  (including chronic otitis media and sinusitis),
  pneumonia, brain abscess, post-partum
  endometritis, tubo-ovarian abscesses, pelvic
  inflammatory disease, septic abortion, and
  chorioamnionitis
• Intra-abominal abscess formation in mixed
  infections with other anaerobes,
  Enterobacteriaceae, and Enterococcus

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Anaerobic Gram-Positive Cocci Presumptive
Identification

• Gram-positive, gram-variable, or gram-negative
  cocci or cocci bacilli (confirm as gram-positive
  cocci by susceptibility to 5-µg vancomycin disk
  with inhibition zone gt10 mm)
• Peptostreptococcus anaerobius Growth inhibition
  by sodium polyanethol sulfonate (SPS) (zone of
  inhibition gt12 mm around a SPS disk)
• Finegoldia magna and Schleiferella
  asaccharolytica SPS resistant, S. asaccharolytica
  indole positive

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Definitive Species Identification of Anaerobic
Bacteria

• Biochemical reactions in prereduced,
  anaerobically sterilized (PRAS) liquid media
• Fermentation end-product analysis and/or cell
  wall fatty acid profiling by gas liquid
  chromatography (GLC)
• 16S rRNA gene sequencing

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Microaerophilic Bacteria

• Campylobacter jejuni
• Helicobacter pylori

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Campylobacter jejuni

• Campylobacter jejuni the most common enteric
  pathogen worldwide (2 million cases in US
  annually)
• Sporadic infections in summer and early fall due
  to ingestion of contaminated poultry products,
  raw milk, and water.
• Clinical presentation ranges from asymptomatic to
  severe with fever, abdominal cramps, and diarrhea
  (occasionally bloody) lasting several days to
  weeks
• Extraintestinal complications bacteremia,
  arthritis (Reiters syndrome), meningitis,
  endocarditis, abortion, and Guillain-Barré
  syndrome

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Campylobacter and Arcobacter

• Campylobacter coli accounts for 5-10 of diarrhea
  due to the campylobacters
• C. fetus subsp. fetus produces bacteremia and
  extraintestinal infection
• C. lari and C. upsaliensis associated with
  diarrhea and bacteremia
• Arcobacter butzleri and A. cryaerophilus are also
  associated with diarrhea and bacteremia

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Thermophilic Microaerophilic Incubation

• 42oC
• 5 oxygen, 10 carbon dioxide, 85 nitrogen

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Campylobacter jejuni

• Microaerophilic growth at 42oC (Campy BAP1)
• Curved, seagull-wing gram-negative rods
• Cytochrome oxidase positive, hippurate
  hydrolysis
• 1Brucella agar base (polypeptone and yeast
  extract, glucose) with trimethoprim, polymixin B,
  cephalothin, vancomycin, and amphotericin B, 10
  sheep blood

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Campylobacter and Arcobacter1

• 42o 25o hip iah
  cat
• C. jejuni
  
• C. coli
  
• C. fetus subsp. fetus /
  
• C. lari
  
• C. upsaliensis /
  
• Arcobacter /
  /
• 142ogrowth at 42o, 25ogrowth at 25o,
  hiphippurate hydrolysis, iahindoxyl acetatge
  hydrolysis, catcatalase, key reactions

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Helicobacter pylori

• Humans the major if not sole source of H. pylori
• Infection acquired during childhood by the
  salivary oral-oral or fecal-oral route
• In developed countries 30-50 of adult population
  colonized, in developing countries 70-90
• Persistent gastric infection associated with
  chronic gastritis, peptic ulcer, gastric
  adenocarcinoma, and B-cell gastric mucosa
  associated lymphoma

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Helicobacter pylori

• H. pylori a gram-negative, spiral, curved, or
  straight microaerophilic bacillus
• Gastric biopsy the specimen most frequently
  submitted
• Recovery of H. pylori in culture requires
  non-selective medium (Brucella, brain heart
  infusion, or Columbia agar) enriched with blood
  (5-7 horse blood) and incubation for minimum of
  7 days under microaerophilic conditions (5 O2,
  10 CO2) at 35o-37oC under conditions of high
  humidity
• Presumptive identification of H. pylori
  Cytochrome oxidase-positive and catalase-positive
  gram-negative curved to straight rod positive for
  rapid urease production and resistant to 30-µg
  nalidixic acid disk

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Helicobacter pylori

• Diagnosis of H. pylori usually by urease testing,
  histology, or serology, not by culture
• CLO test gastric biopsy placed in a
  urea-containing gel with a pH indicator that
  turns magenta if gel alkalinized by urease
  activity
• Histology Presence of curved bacilli by Giemsa
  or Warthin-Starry stain on mucosal surface of a
  gastric biopsy
• Serological test Detection of IgG antibodies to
  H. pylori by ELISA

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Recommended Reading

• Winn, W., Jr., Allen, S., Janda, W., Koneman,
• E., Procop, G., Schreckenberger, P., Woods,
• G.
• Konemans Color Atlas and Textbook of
• Diagnostic Microbiology, Sixth Edition,
• Lippincott Williams Wilkins, 2006
• Chapter 16. The Anaerobic Bacteria.

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Recommended Reading

• Murray, P., Baron, E., Jorgensen, J., Landry,
• M., Pfaller, M.
• Manual of Clinical Microbiology, 9th
• Edition, ASM Press, 2007
• Citron, D., Poxton, I.R., and Baron, E. J.
  Chapter 58. Bacteroides, Porphyromonas,
  Prevotella, Fusobacterium, and Other Anaerobic
  Gram-Negative Rods.
• Johnson, E.A., Summanen, P., and Finegold, S.M.
  Chapter 57. Clostridium.
• Koenoenen, E., and Wade, W.G. Chapter 56.
  Propionibacterium, Lactobacillus, Actinomyces,
  and Other Non-Spore-Forming Anaerobic
  Gram-Positive Rods.
• Song, Y., and Finegold, S.M. Chapter 55.
  Peptostreptococcus, Finegoldia, Anaerococcus,
  Peptoniphilus, Veillonella, and Other Anaerobic
  Cocci.
• Fitzgerald, C., and Nachamkin, I. Chapter 59.
  Campylobacter and Arcobacter.