Gram negative spiral or curved bacteria

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Gram negative spiral or curved bacteria

• Campylobacter
• and
• Helicobacter

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Characteristics of Campylobacter

• Small curved Gram negative bacteria in the genus
  Campylobacter and other similar genera inhabit
  the gastric and intestinal mucosa of warm blood
  animals causing various gastrointestinal
  syndromes
• The organism was likely first described in fetal
  tissues of aborted sheep in 1913, but the type
  strain (eventually C. jejuni) was not reported
  until 1931. The organism was initially named
  Vibrio fetus due to its curved nature. Vibrio
  was the only known curved rod at the time
• Years later Elizabeth King at the CDC described a
  group of microaerophilic curved bacteria from the
  blood of children with acute dysentery, the first
  report of this type bacteria in human clinical
  specimens.
• The bacteria were first isolated when fecal
  emulsions were filtered (0.65?m pore size) onto
  blood agar plates and incubated for a week in a
  moist carbon dioxide enriched environment

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Campylobacter
4
continued

• Genetics studies eventually demonstrated that
  these organisms were not related to Vibrio. The
  type strain was named Campylobacter jejuni since
  it is most abundant in the jejunal region of the
  small intestine
• From research conducted with human volunteers,
  it was shown that the bacteria were not strict
  anaerobes nor would they grow in ambient air.
  They were, in fact, microaerophilic, capnophilic
  (love elevated CO2), and required a humid
  environment
• It was further determined that their optimum
  growth temperature was not 37oC but 42oC (the
  body temperature of birds)
• A good selective medium for the C. jejuni is SBA
  supplemented with antibiotics. The medium is
  called Campy-BAP or CBA
• Nearly all Campylobacter clinical isolates found
  in the U.S. come from fecal samples

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Laboratory Methods

• Specimen collection and transport techniques that
  are useful for Salmonella, Shigella, and EHEC
  will suffice for Campylobacter
• Streak CBA for isolation and place in a Campy
  Pouch or other commercial product to give
  microaerophilic, capnophilic conditions.
  Incubation is at 42oC for 48 h
• Fecal samples are not routinely directly examined
  for Campylobacter, but rather are examined for
  fecal leukocytes. There is a strong association
  between intestinal Campylobacter infections and
  fecal leukocytes.
• Some microbiologists advocate performing a wet
  prep and observing for fecal leukocytes as well
  as the characteristic rapid darting motility of
  Campylobacter

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Laboratory Methods

• Others advocate a direct basic fuchsin or Gram
  stain of feces because of the claim that
  Campylobacter will be a predominant organism in
  the stool sample of intestinal Campy infections
• The characteristic cell morphology (S shapes,
  gull-wings and long spirals) can be seen via
  direct examination of feces
• Some microbiologists advocate using Campy thio
  media in addition to CBA plate for primary
  isolation and culturing. Campy thio is
  conventional thioglycollate (reducing agent)
  supplemented with antibiotics vs enterics.
• The inoculated Campy thio media is incubated in
  the refrigerator (for how long?) Campy can
  reach log phase growth at 4oC but enterics
  cannot.
• Growth is taken from 5-10 mm below the medium
  surface and subcultured, and these plates are
  incubated under the conditions appropriate for
  growing Campylobacter

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Laboratory Methods

• A few other bacterial species will occasionally
  grow on CBA at 42oC, especially Pseudomonas
  aeruginosa.
• This is potentially problematic since both
  organisms are oxidase and catalase positive,
  however they are easily distinguished via
• morphologically distinct
• motility pattern recall the rapid darting of
  Campylobacter
• Pseudomonas will grow on SBA in ambient air
• There are in fact many species of Campylobacter
  and other related curved and spiraled Gram
  negative bacteria
• Most symptomatic human infections, as far as we
  can tell at present, are caused by C. jejuni C.
  coli is the second most common species
• Not all of these related species will grow at
  42oC on CBA

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Laboratory Methods

• Few labs attempt definitive Campy ID unless the
  isolate is from blood or other sterile sites.
  The reported isolation of an enteric
  Campylobacter is considered adequate for the
  physician
• C. jejuni does have one unique characteristic
  among the Campylobacter-like bacteria it is
  hippurate positive. The test is based on the
  ability of a microbe to hydrolyze hippuric acid
  (with the enzyme hippuricase) releasing the amino
  acid serine. Serine is then detected with the
  reagent ninhydrin turns purple.
• The rapid hippurate hydrolysis test is a
  three-hour test.
• A positive hippurate hydrolysis test on a small,
  curved, Gram negative, catalase and oxidase
  positive rod that grows optimally under
  microaerophilic conditions at 42oC, and has
  darting motility is reported as C. jejuni.
• A negative test is reported as enteric
  Campylobacter-like organism isolated

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Campylobacter pathology

• Recent methods indicate that infections are
  frequent and widespread. It has a worldwide
  distribution. In most parts of the U.S. C. jejuni
  is isolated more frequently than Salmonella and
  Shigella. This is fairly new info., and was a
  big revelation.
• It is isolated from 4 to 35 of fecal samples
  from people with diarrheal disease.
• Associated with many animals, but especially
  large birds (those that we eat). The body
  temperature of birds is 42oC. Can pass to the
  genitourinary tract of infected non-human animals
  where it often causes spontaneous abortion.
• Gastroenteritis-dysentery can be moderate to
  severe, and is associated with fecal-contaminated
  meat, unpasteurized dairy and water. Symptoms
  include explosive bloody diarrhea, abdominal
  pain, fever, nausea, and vomiting. Extent of
  blood in feces generally surpasses most other
  invasive enterocolitic pathogens.

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continued

• For most people Campy dysentery is self-limiting
  in 3 to 7 days
• Septic arthritis and meningitis have been
  reported as complication of Campylobacter
  infections
• Recently found to be the most common pathogenic
  antecedent to a common neurological condition,
  Guillain-Barré syndrome. The connection is not
  yet understood
• It is also thought to be associated with
  demyelinating disease of the peripheral nerves

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Campylobacter jejuni on CBA
42oC 48h microaerophilic conditions
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Characteristics of Helicobacter

• Helicobacter pylori was discovered in 1982 in the
  gastric mucosa of humans, and was initially
  included in the genus Campylobacter because of
  its curved, spiraled shape
• Genetic, morphological, and biochemical
  differences lead to the establishment of the new
  genus. Perhaps the biggest difference between
  them is the strong urease produced by H. pylori
• Helicobacter moves by rapid corkscrew motility
  using one or more sheathed flagella sort of a
  hybrid motility
• The primary sources of the organism appear to be
  unpasteurized milk and contaminated surface
  water.
• Based upon limited serological testing, it is
  estimated that one-half of the worlds population
  is or has been infected with H. pylori.
  Fortunately, it appears that only 10 of those
  infected will develop GI pathology from the
  organism.

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Helicobacter pylori
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continued

• Incidence of infection is very low in U.S.
  children but infections in the elderly may be as
  high as 80.
• Once established in the gastric mucosa infection
  can persist for life unless treated with
  antibiotics. The majority of cases (90) are
  asymptomatic but there is overwhelming evidence
  that H. pylori is the leading cause of, or
  contributor to gastritis.
• Due to the long-standing conventional wisdom that
  peptic ulcers were caused by stress or
  environmental factors, the medical community
  was initially skeptical about the connection
  between H. pylori and gastritis
• As part of the evidence that H. pylori causes
  gastritis the discoverer infected himself and
  developed peptic ulcers. Barry Marshall Robbin
  Warren jointly won the 2005 Nobel prize for
  science for their role in linking H. pylori to
  peptic ulcers

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continued

• Circumstantial evidence of a bacterial etiology
  of gastritis included the fact that over the
  counter medications such as Pepto-Bismol,
  Tagamet and Zantac were shown to inhibit H.
  pylori in vitro
• Helicobacter cinnaedi and H. fennelliae are
  sexually transmitted species. Both are rarely
  encountered, and are isolated primarily from
  homosexual men.

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Clinical Significance

• H. pylori uses fimbriae for specific attachment
  to proteins in the stomach mucosa. Individuals
  with type O blood have the greatest chance of
  gastritis, apparently because their mucosal
  proteins have a high affinity for H. pylori
  fimbriae.
• Spiral motility allows penetration through the
  mucus into the epithelial side of the mucosa. A
  potent urease produced by H. pylori releases
  ammonia neutralizing the environment surrounding
  individual bacteria, thus protecting them from
  the HCL in gastric juice, but also contributing
  to the symptoms.
• H. pylori also induces a strong inflammatory
  response in the mucosa resulting in accumulation
  of PMNs and macrophages
• In these ways, H. pylori causes gastritis and
  peptic ulcers with recurrent pain, bleeding and
  inflammation.

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continued

• Infected individuals produce antibodies specific
  for H. pylori. Although they seem to afford
  little protection against the pathogen, they are
  the basis for a serological test.
• High titers of IgG can be demonstrated in
  chronically infected individuals. These
  individuals seem to be at a much higher risk of
  developing carcinoma of the stomach

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Diagnostic Techniques

• H. pylori was first detected using Warthin-Starry
  silver stained gastric biopsies. H. pylori does
  not Gram stain well
• The urease test is rapid and reliable for
  detection. A small piece of gastric biopsy
  placed in urea medium will yield the urease
  reaction within two hours if H. pylori is
  present. The down side is that a surgical
  procedure is needed to procure the specimen
• Another diagnostic test is the breath test.
  Patients swallow a pill containing radioactive
  urea (13C or 14C). H. pylori in the stomach
  hydrolyzes the radioactive urea releasing radio
  active CO2 which is detectable in their breath.
  The disadvantage of this procedure is the
  requirement of a special instrument to detect
  radio active carbon dioxide

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Urease test
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Diagnostic Techniques

• H. pylori can be cultured on a variety of
  selective or nonselective media containing blood,
  heme, or charcoal
• Culture is not usually performed because
  Helicobacter requires 3-7 days to form tiny
  colonies, and cultures often do not grow due
  apparently to inherent inhibitors in gastric
  biopsies
• As mentioned previously, serum antibodies can be
  detected in the chronically infected. ELISA
  based tests are commercially available

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Comparison of Helicobacter and Campylobacter
Reservoir Humans Humans Humans Birds and
other animals Pigs Cattle and sheep
Human Disease Gastritis and Peptic
ulcers Proctitis, enteritis, sepsis Proctitis,
enteritis, sepsis Explosive bloody
diarrhea Explosive bloody diarrhea Systemic
infections
H.pylori H. cinnaedi H. finnelliae C.
jejuni C. coli C. foetus