Dr.T.V.Rao MD
Dr.T.V.Rao MD 1
Dr.T.V.Rao MD 2
Campylobacters causes Important
Zoonotic Infections
Dr.T.V.Rao MD 3
History of Campylobacter
First isolated as Vibrio fetus in 1909 from
spontaneous abortions in livestock
Campylobacter enteritis was not
recognized until the mid-1970s when
selective isolation media were developed
for culturing campylobacters from human
feces
Most common form of acute infectious
diarrhea in developed countries; Higher
incidence than Salmonella & Shigella
combined
Dr.T.V.Rao MD 4
General Characteristics
Common to Superfamily
Gram-negative
Helical (spiral or curved) morphology; Tend to be
pleomorphic
Characteristics that facilitate penetration and
colonization of mucosal environments (e.g.,
motile by polar flagella; corkscrew shape)
Microaerophilic atmospheric requirements
Become coccoid when exposed to oxygen or
upon prolonged culture
Neither ferment nor oxidize carbohydrates
Dr.T.V.Rao MD 5
Campylobacter jejuni
Bacteria commonly found in animal feces. It
is one of the most common causes of human
gastroenteritis in the world. Food poisoning
caused by Campylobacter species can be
severely debilitating, but is rarely life-
threatening. It has been linked with
subsequent development of Guillain-Barre
syndrome (GBS), which usually develops
two to three weeks after the initial illness
Dr.T.V.Rao MD 6
Morphology & Physiology of Campylobacter
Small, thin (0.2 - 0.5 um X 0.5 - 5.0 um),
helical (spiral or curved) cells with typical
gram-negative cell wall; “Gull-winged”
appearance
• Tendency to form coccoid & elongated
forms on prolonged culture or when
exposed to O2
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Motility – A Darting type
Distinctive rapid
darting motility
• Long sheathed
polar flagellum
at one (polar) or
both (bipolar)
ends of the cell
• Motility slows
quickly in wet
mount
preparation
Dr.T.V.Rao MD 8
Growth Requirements
Microaerophilic &
capnophilic
5%O2,10%CO2,85%N2
Thermophilic (42-43C)
(except C. fetus)
• Body
temperature of
natural avian
reservoir
May become
nonculturable in nature
Dr.T.V.Rao MD 9
Important Zoonotic
Infection
Campylobacter remains the
leading cause worldwide of
zoonotic disease in humans,
surpassing Salmonella
infections. Moreover, cases
are still believed to be
underreported. Often
causing the same symptoms
as Salmonella, such as mild to
severe diarrhea,
Campylobacter infections can
also be an infectious trigger
for the more serious
Guillain-Barre Syndrome.
Dr.T.V.Rao MD 10
C.jejuni carried in ….
Although C. jejuni is
not carried by healthy
individuals in the
United States or
Europe, it is often
isolated from healthy
cattle, chickens, birds
and even flies. It is
sometimes present in
non-chlorinated water
sources such as streams
and ponds.
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Laboratory Diagnosis
Feces refrigerated & examined within few hours
Rectal swabs in semisolid transport medium
Blood drawn for C. fetus
Care to avoid oxygen exposure
Selective isolation by filtration of stool specimen
Enrichment broth & selective media
Filtration: pass through 0.45 μm filters
Dr.T.V.Rao MD 12
Laboratory Identification
Microscopy:
Gull-wing appearance in gram stain
Darting motility in fresh stool (rarely done in
clinical lab)
Fecal leukocytes are commonly present
Identification:
Growth at 25o, 37o, or 42-43oC
Hippurate hydrolysis (C. jejuni is positive)
Susceptibility to nalidixic acid & cephalothin
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Culture
An atmosphere with reduced O2 (5% O2)
with added CO2 (10% CO2)
At 42 ℃ (for selection)
Several selective media can be used (eg,
Skirrow’s medium)
Two types of colonies:
watery and spreading
round and convex
Dr.T.V.Rao MD 14
Selective Medium
• Blood-free,
charcoal-based
selective
medium agar
(CSM) for
isolation of
Campylobacter
jejuni
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Characteristics of C.jejuni
Characteristic
Growth at 25 °C Result
-
Growth at 35-37 °C -
Growth at 42 °C +
Nitrate reduction +
Catalase test +
Oxidase test +
Growth on MacConkey agar +
Motility (wet mount) +
Glucose utilization -
Hippurate hydrolysis +
Resistance to nalidixic acid -
Resistance to cephalothin +
Dr.T.V.Rao MD 16
Pathogenesis & Immunity
Infectious dose and host immunity determine
whether gastro enteric disease develops
• Some people infected with as few as 500 organisms
while others need >106 CFU
Pathogenesis not fully characterized
• No good animal model
• Damage (ulcerated, edematous and bloody) to the
mucosal surfaces of the jejunum, ileum, colon
• Inflammatory process consistent with invasion of the
organisms into the intestinal tissue; M-cell (Payer's
patches) uptake and presentation of antigen to
underlying lymphatic system
Non-motile & adhesion-lacking strains are a
virulent Dr.T.V.Rao MD 17
Putative Virulence Factors
Cellular components:
Endotoxin
Flagellum: Motility
Adhesins: Mediate attachment to mucosa
Invasins
GBS is associated with C. jejuni Serogroup O19
S-layer protein “microcapsule” in C. fetus:
Extracellular components:
Enterotoxins
Cytopathic toxins
Dr.T.V.Rao MD 18
Other Species of Campylobacters
C jejuni infections may
also produce serious
bacteremic conditions
in individuals with
AIDS. Most reported
bacteremia's have been
due to Campylobacter
fetus fetus infection.
Campylobacter lari,
which is found in
healthy seagulls, has
also been reported to
produce mild recurrent Dr.T.V.Rao MD 19
diarrhea in children
Other Species of
Campylobacters
Campylobacter
upsaliensis may cause
diarrhea or bacteremia,
while Campylobacter
hyointestinalis, which
has biochemical
characteristics similar
to those of C fetus,
causes occasional
bacteremia in
immunocompromised
Dr.T.V.Rao MD individuals. 20
Treatment, Prevention & Control
Gastroenteritis:
•Self-limiting; Replace fluids and electrolytes
•Antibiotic treatment can shorten the excretion period;
Erythromycin is drug of choice for severe or complicated
enteritis & bacteremia; Fluoroquinolones are highly active
(e.g., ciprofloxacin was becoming drug of choice) but
fluoroquinolones resistance has developed rapidly since
the mid-1980s apparently related to unrestricted use and
the use of enrofloxacin in poultry
•Azithromycin was effective in recent human clinical trials
•Control should be directed at domestic animal reservoirs
and interrupting transmission to humans
Dr.T.V.Rao MD 21
Complication are rare
but occurs occasionally
Complications are relatively rare, but infections have
been associated with reactive arthritis, hemolytic
uremic syndrome, and following septicemia,
infections of nearly any organ. The estimated
case/fatality ratio for all C. jejuni infections is 0.1,
meaning one death per 1,000 cases. Fatalities are rare
in healthy individuals and usually occur in cancer
patients or in the otherwise debilitated. Only 20
reported cases of septic abortion induced by C. jejuni
have been recorded in the literature.
Dr.T.V.Rao MD 22
Sequelae to Infection
Guillain-Barre syndrome (GBS), a demy elating disorder
resulting in acute neuromuscular paralysis, is a serious
sequelae of Campylobacter infection . An estimated one case of
GBS occurs for every 1,000 cases of Campylobacteriosis Up to
40% of patients with the syndrome have evidence of recent
Campylobacter infection . Approximately 20% of patients with
GBS are left with some disability, and approximately 5% die
despite advances in respiratory care. Campylobacteriosis is also
associated with Reiter syndrome, a reactive arthropathy. In
approximately 1% of patients with campylobacteriosis, the
sterile postinfection process occurs 7 to 10 days after onset of
diarrhea . Multiple joints can be affected, particularly the knee
joint. Pain and incapacitation can last for months or become
chronic. Dr.T.V.Rao MD 23
Complications with
C.jejuni
Guillain-Barre
Syndrome (GBS)
• Favorable
prognosis with
optimal
supportive care
Intensive-care unit
for 33% of cases
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Dr.T.V.Rao MD 25
A Tribute to Warren and
Marshall
for Discovery of H.pylori
Dr.T.V.Rao MD 26
Helicobacter pylori
Helicobacter pylori (H. pylori) is a type of bacteria.
Researchers believe that H. pylori is responsible for the
majority of peptic ulcers.
H. pylori infection is common in the United States.
About 20 per cent of people under 40 years old and half
of those over 60 years have it. Most infected
people, however, do not develop ulcers. Why H.
pylori does not cause ulcers in every infected person
is not known. Most likely, infection depends on
characteristics of the infected person, the type of H.
pylori, and other factors yet to be discovered
Dr.T.V.Rao MD 27
Helicobacter pylori
Helicobacter pylori is the prototype organism in this
gastritis,
group. It is associated with antral
gastric ulcers, and gastric
carcinoma.
Dr.T.V.Rao MD 28
Helicobacter pylori
Helicobacter
pylori is a spiral
gram negative
bacteria.
It has a multiple
polar flagella
above the pole
and motile
Dr.T.V.Rao MD 29
Beginning of Scientific
understanding
Peptic ulcers have plagued men throughout the
centuries, but the exact cause of the condition was
uncertain. In 1940, Dr. A. Stone Freedberg of
Harvard Medical School identified unusual curved
bacteria in the stomachs of ulcer victims; he
suspected that they might be responsible for ulcers
but abandoned the research when his team was
unable to grow the bacteria in the laboratory
Dr.T.V.Rao MD 30
History of H.pylori
for short) was first
Helicobacter pylori (H.pylori
discovered in the stomachs of patients with
gastritis & stomach ulcers nearly 25 years ago by Dr
Barry J. Marshall and Dr J. Robin Warren of Perth,
Western Australia. At the time (1982/83) the
conventional thinking was that no bacterium can
live in the human stomach as the stomach produced
extensive amounts of acid which was similar in
strength to the acid found in a car-battery. Marshall
& Warren literally “re-wrote” the text-books with
reference to what causes gastritis & gastric
ulcers.
Dr.T.V.Rao MD 31
Global prevalence of H. Pylori
Land Mark Changes in
H.pylori
The name of the
bacterium was
grammatically
corrected in 1987 to
Campylobacter pylori
and, in 1989 the
bacterium was renamed
Helicobacter pylori and
assigned as the type
species of a novel genus
due to its 16s rRNA
sequence.
Dr.T.V.Rao MD 33
How is it transmitted?
Believed to be transmitted orally due to tainted food
or water.
Also believed to be passed through belching, or
gastro-esophageal reflux, which is when small
amount of stomachs contents is forced up the
esophagus.
After this process, it’s believed that the H. Pylori is
passed orally.
Genes Contribute to
Pathogenicity
CAG –
Cytotoxin
associated gene
Vac –
Vacuolating
cytotoxin gene
Dr.T.V.Rao MD 35
Pathogenic Mechanism
It is well established that urease, Vacuolating
cytotoxic VacA, and the pathogenicity island (cag
PAI) gene products, are the main factors of virulence
of this organism. Thus, individuals infected with
strains that express these virulence factors probably
develop a severe local inflammation that may induce
the development of peptic ulcer and gastric cancer.
The way the infection spreads throughout the world
suggests the possibility that there are multiple
pathways of transmission.
Dr.T.V.Rao MD 36
Culturing H.pylori
H.pylori grows on
Skirrow”s medium
with 1Vancomycin,
2 Polymyxin
3 Trimethoprim
Grows in 3 -6 days at
370c
Colonies appear
Translucent 1-2 mm in
diameter
Optimal growth occurs
in Microaerophic
environment
Dr.T.V.Rao MD 37
Biochemical Characters
Motile
Catalase +
Oxidase +
Strong producer
of
Urease
Dr.T.V.Rao MD 38
Urea Hydrolysis
Urease
C=O(NH2)2 + H+ + 2H2O HCO3- + 2 (NH4+)
Urea Bicarbonate Ammonium
ions
And then…
HCO3- CO2 + OH-
Dr.T.V.Rao MD 39
Pathology and
Pathogenesis
H.pylori is found in the deep mucus layer
Grows optimally at pH 6.0 to 7.0
But gastric mucosa has a strong buffering in spite of
lower pH on the lumen side of stomach
H.pylori also produces a protease that modifies the
gastric mucus and further reduces the ability of acid
through the mucus
Dr.T.V.Rao MD 40
Mechanisms in
Pathogenicity
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Localization of H.pylori
Dr.T.V.Rao MD 42
Pathogenesis
The potential character of H.pylori lie with
production of potent Urease activity which yields
production of Ammonia and further buffering acid.
H.pylori is quite motile even in mucus finds its way
to epithelial surface
H.pylori overlies the gastric type but not intestinal
epithelial cells.
Dr.T.V.Rao MD 43
Factors contributing to Peptic
ulceration
There is a strong
association between
presence of H.pylori
infection and peptic
ulceration
Mucosal inflammation
and damage involves
both bacterial and host
factors
Dr.T.V.Rao MD 44
H.pylori causes Peptic ulcers in the
Stomach
Dr.T.V.Rao MD 45
Factors influencing
Pathogenicity
Lipopolysaccharides - damage mucosal cells
and Ammonia produced by Urease activity may
directly damage cells.
Gastritis – Chronic and active inflammation
establishes Polymorph nuclear and Mononuclear
cell infiltration within the Epithelial and Lamina
propria
Events lead to Destruction of epithelium is
common.
Glandular atrophy is common.
Dr.T.V.Rao MD 46
Clinical Manifestations
Acute infection
Upper Gastrointestinal illness
Nausea
Pain
Fever – very occasionally
Acute symptoms lasts for < 1 week,
May extend up to 2 weeks
Infection last for years, decades or even lifetime
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Consequences of H.pylori Infection
Dr.T.V.Rao MD 48
Association of Duodenal and
Gastric ulcers in H.pylori
About 90 % of patients with Duodenal
ulcer, and 50- 80 % of gastric ulcers are
associated with H.pylori infection.
H.pylori may have greater role in
Gastric carcinoma and Lymphomas
Dr.T.V.Rao MD 49
Mechanism of Cancer in
H.pylori
Dr.T.V.Rao MD 50
Laboratory Diagnosis
Specimens for histopathology – Gastric
biopsy specimens can be used for
Histological examination
Specimens obtained after Gastroscopy,
Biopsy, routine stains will demonstrate
Gastritis and special stains show curved
spiral organisms
Specimens collected in sterile saline mixed
are used for culturing
Dr.T.V.Rao MD 51
Endoscopy – Gastric
Biopsy
Dr.T.V.Rao MD 52
Serology
The detection of
Antibodies in active
infection is useful
But the tests are limited
utility as antibodies
persist even after
H.pylori infection is
eradicated.
Several commercial kits
are available, but lacks
the role in identifying
acute infections.
Dr.T.V.Rao MD 53
Special Tests for H.pylori
Rapid tests for detection of
Urease activity are widely
used in presumptive
identification of Gastric
Biopsy specimens.
Gastric Biopsy can be
placed into urea containing
medium with color
indicator.
If H.pylori is present the
Urease rapidly splits urea
and resulting shift in pH
yields a color change in the
medium
Dr.T.V.Rao MD 54
Urea Breath Test
H. pylori infection can
be detected in the
exhaled breath using
this special test. This
test is positive only if
the person has a
current infection.
Sensitivity and
specificity of this test
ranges from 94-98%.
Dr.T.V.Rao MD 55
Carbon-14-urea Breath Test
Dr.T.V.Rao MD 56
Urea Breath Test
In this test 13C or 14C
labeled urea is
ingested by patients
If H.pylori is present
the urease activity
generates labeled
Co2 that can be
detected in the
patients exhaled
breath
Dr.T.V.Rao MD 57
Antigen Detection Test in
Stool
Detection of H.pylori
antigen in stool is
appropriate test in
patients with
H.pylori infection
Absence of antigen
indicates cure of
Infection after
Chemotherapy.
Dr.T.V.Rao MD 58
H.pylori and Cancer
.If a person has had an H.pylori
infection constantly for 20-30
years, it can lead to cancer of the
stomach. This is the reason that
the World Health Organization's
(WHO) International Agency for
Research into Cancer (IARC) has
classified H.pylori as a “Class- I-
Carcinogen” i.e. in the same
category as cigarette smoking is
to cancer of the lung &
respiratory tract.
Dr.T.V.Rao MD 59
Treatment
Triple therapy has prompt response, contain a
combination of following drugs
1 Metronidazole
2 Bismuth subsalicylate or Bismuth sub citrate
3 Amoxicillin or Teracycles
administered up to 14 days
Eradicates H.pylori
In 70 – 95 % of patients
Acid suppressing agent is supporting
Dr.T.V.Rao MD 60
Other Drug Combinations
Other alternatives
Proton pump inhibitor directly inhibit
H.pylori
Combined with
Amoxicillin
Clarithromycin or Amoxicillin
And Metronidazole
Dr.T.V.Rao MD 61
Vaccines trails for H.pylori
are in Progress
pylori may
A vaccination against Helicobacter
represent both prophylactic and therapeutic
approaches to the control of H. pylori infection.
Different protective H. pylori-derived antigens, such
as urease, vacuolating cytotoxin A, cytotoxin-
associated antigen, neutrophil-activating protein and
others can be produced at low cost in prokaryote
expression systems and most of these antigens have
already been administered to humans and shown to
be safe.
Dr.T.V.Rao MD 62
Epidemiology
In Developed countries H.pylori are present in <20 %
of the persons below 30 years.
By 60 years prevalence increases to 40 – 60 %
In Developing countries prevalence of infection is
higher to 80 % even in younger individuals
Person to person transmission of H.pylori is likely
because of interfamilial clustering of infection
Acute epidemics of Gastritis suggest a common
source of H.pylori.
Dr.T.V.Rao MD 63
Helicobacter pylori changes the
future of Gastroenterology
Dr.T.V.Rao MD 64
Barry J. Marshall and J. Robin
Warren have been awarded the
2005 Nobel Prize in medicine
Dr.T.V.Rao MD 65
Helicobacter Foundation
The "Helicobacter
Foundation" was
founded by Prof. Barry
J. Marshall in early
1994, and is dedicated
to providing you with
the latest information
about Helicobacter pylori,
its diagnosis, treatment
and clinical
perspectives.
Dr.T.V.Rao MD 66
Created by Dr.T.V.Rao MD for ‘e’
learning resources for Microbiologists
in Developing World
Email
doctortvrao@gmail.com
Dr.T.V.Rao MD 67