MICROBIOLOGY Module 3

Campylobacter, Helicobacter, and Vibrio 3.3

Dr. Meliton Bato September 13, 2018

Outline Taxonomy
o I. Campylobacter spp.
A. Campylobacter jejuni
 Kingdom: Bacteria
B. Other Campylobacter spp.  Phylum: Proteobacteria
1. C. fetus
2. C. concisus
 Class: Epsilon
3. C. fetussubspvenerealis  Order:Campylobacteriales
4. C. jejunisubsdoylei
5. C. coli  Family: Campylobacteriaceae
6. C. lari  Genus: Campylobacter
7. C. hyointestinalis
8. C. upsaliensis A. Campylobacter jejuni
9. C. soutorum  Representative of the genus
II. Helicobacter spp.  Genus Campylobacter means “curved rods”
A. Helicobacter pylori
B. Other Helicobacter spp.
(campylos and baktron)
1. H. cinaedi  Small, comma-shaped
III. Vibrio spp.
2. H. fennelliae
 Bipolar flagella at both ends
A. Vibrio cholerae  0.2-0.9 micron in width and 0.5-5.0 micron in length
B. Halophilic Vibrios
1. V. parahaemolyticus  Gram (-) bacilli
2. V. alginolyticus
3. V. vulfinicus
 Motile
 Resemble Vibrio species
LEGEND  Pleomorphic- different shapes; if culture is old, become
 Book coccoid in shape
Transers Notes  Non-spore former
CAMPYLOBACTER SPP.  Microaerophilic (require small amounts of oxygen,
Clinical Case # 1 usually 5 to 10 %)
A.M., a 24- year old female was brought to the ER for  Thermophilic at 42° C but can also live at mesophilic
bilateral, ascending lower extremity weakness and temp.
associated dyspnea. She was conversant but began to  Selective media: Butzler’s medium, Preston
breathe heavily after 2 days since hospital admission and her Campylobacter selective medium, and Blaser’s
oxygen saturation dropped from 97% to 60%. Immediately, medium (Campy-BAP) , Skirrow’s medium
she was intubated. Laboratory results revealed normal CBC  Plates incubated for 48 hours
without leukocytosis and her CXR revealed normal results. A  Circular and convex but can be flat and tend to swarm on
neurologist was brought into the team and assessed the case moist agar
as Guillain- Barre Syndrome (GBS). A month ago, the
 Non CHO fermenters
patient complained of a 4-day history of acute diarrhea
 Oxidase (+)
associated with headache, nausea, vomiting, body malaise
and undocumented fever. Previously, she was brought to the  Catalase (+)
same ER but was sent home and was given probiotics. There  Nitrate Reducer
was resolution of the diarrhea. What could have caused her  Sodium hippurate hydrolyzer only for C. jejuni
GBS?  Campylobacter jejuni: 90-95% of all human
Campylobacter infections,
Discovery
 C. coli is highly homologous to C. jejuni
Tip: dahil classified sya under enterobacteriaceae, this
 1886: Theodore Escherich described a non-culturable organism is oxidase, catalase positive and a nitrate reducer
spiral-shaped bacteria
How can humans be infected by C. jejuni
 1909: T. Smith demonstrated sheep abortions were
associated with this infection hence named Vibrio fetus -  Ingestion –fecal oral route
gastrointestinal complication would be septic abortion in  Jejunum and ileum are first colonized
sheep  Colon and rectum are also infected
 1947: V. fetus was isolated in blood culture from a  Invasive and cytopathic
pregnant patient  Can cause Mesenteric adenitis- inflammation of lymph
o Developed influenza-like symptoms nodes in the mesentery; can cause appendicitis, can pass
o Stillborn fetus with necrotic and infarcted through portal vein circulation then cause sepsis
placenta  Occult bacteremia- can develop into sepsis
 1957: King hypothesized V. fetus can cause human
enteric disease- through zoonotic infections
o Pregnancy, gastrectomy, tooth extraction, heart
disease, DM, cirrhosis were predisposing factors
seen in association studies of 15 patients
o Butzler et al showed V. fetus in diarrheic
children’s stools
 1967: Sebald and Veron separated Campylobacter from
Vibrio species and reclassified V. fetus as Campylobacter

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MICROBIOLOGY Campylobacter, Helicobacter, and Vibrio MODULE 3, LECTURE 3

Culture
o Stool sample or rectal swabs usually; blood
possible
0
o Campylobacter spp survive 1-2 weeks at 4 C in
Cary-Blair transport medium
o Selective media: Butzler’s selective medium,
Skirrow’s, Campylobacter selective medium, Preston
Campylobacter selective medium and Blaser’s
medium (Campy-BAP)
o Skirrow’s medium: vancomycin, polymyxin B,
trimethoprim
0 0
o Plates incubated at 42 C but C. fetus at 37 C
o O2 5%; CO2: 10%; N2:85% (microaero)
o Flat and effuse, tendency to spread on moist agar
o Nonhemolytic, colorless, moist, flat, convex D
Biochemical testing
o C. Jejuni is the only one that can hydrolize
Hippurate (Mnemonic: Camp John Hay: C. jejuni:
Virulence Factors hippurate)
Serology
Heat-labile Enterotoxin
o Complement fixation test
o Resembling cholera toxin
o ELISA
o Raises intracellular cAMP
o Causes diarrhea
Capsular polysaccharides
o Increase adherence to human enterocytes
o Provides resistance against epithelial microbial
peptides ((β-defensins, lysozymes) and lysis and
phagocytosis
o Needed in biofilm production
Flagellin A (flaA) – for motility
o Adhesion and invasion
o Overcome intestinal peristalsis
Cytolethal distending toxin (CDT)
o Best characterized toxin
o 3 subunits: cdtA and cdtC binds to host cell- trimeric
o cdtB has DNAse I-like activity- minsan nag a-
apoptosis yung cells mo
o Host cell destruction- can cause also apoptosis
Type IV (T4SS) and VI (T6SS) secretion systems
o Similar to conjugation
o Resembles the tail spike of the T4 phage

Clinical Manifestations
• Acute onset
• Crampy abdominal pain
• Profuse diarrhea- Majority of patients have this, may be
Dysenteric (bloody diarrhea)
• Headache
• Malaise Treatment
• Fever
• 5-8 days  Gastroenteritis is self-limiting
• Prolonged carriage in immunodeficient patients  Fluid and electrolyte management (center of treatment
• Complications: Aseptic arthritis and Guillain-Barre for general diarrhea)- to prevent hypovolemic shock
syndrome  Severe gastroenteritis and sepsis treated with
• Major clinical features of campylobacter that have a erythromycin, tetracyclines, quinolones
median frequency of 84% manifest diarrhea, followed by  Proper food preparation
abdominal pain then fever, myalgia, headache and lastly  Pasteurization of milk
vomiting and blood in feces  Prevention of contaminated water supply
Diagnosis
Clinical Microscopy
o Gram staining = gull wing shaped rods
o Dark field/phase contrast = darting or tumbling
motile spiral rods
o Stool direct smear
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MICROBIOLOGY Campylobacter, Helicobacter, and Vibrio MODULE 3, LECTURE 3

Causes of antibiotic resistance of C. jejuni

B. Campylobacter fetus
 Veterinary pathogen Differentiation between C. jejuni and C. fetus
 Infective abortion in cattle and sheep
 Opportunistic in systemic infections of
immunocompromised patients
 Occasionally cause diarrhea
• Bacteremia, septic thrombophlebitis, arthritis, septic
abortion, meningitis
C. Campylobacter concisus
 Gingivitis
 Periodontal disease
 Can be isolated in stool
D. Campylobacter fetus subsp. venerealis
 Enzootic infertility in cattle
 Not zoonotic
E. Campylobacter jejuni subsp. doylei
 Does not reduce nitrate to nitrite but hydrolyzes
hippurate
 Pathogenicity still unknown
 Isolated from human gastric epithelium biopsy and stool
F. Campylobacter coli
 Clinical infection indistinguishable to C. jejuni
 3-5 % of Campylobacter diarrhea
 Healthy pigs as reservoir
 Hippurate hydrolysis (-)
G. Campylobacter lari
 Formerly known as C. laridis
 found in birds and dogs HELICOBACTER SPP.
 Zoonotic Clinical Case # 2
H. Campylobacter hyointestinalis
 Swine ileitis M.L., A 27- year old male from Quezon City came to
 Previously nonzoonotic but reportedly found in humans your clinic complaining of an acute abdominal pain. He said
with diarrhea and homosexual men with proctitis that the pain was emanating from the epigastric area, the pain
I. Campylobacter upsalienses severity was 10/10, associated with recurrent vomiting. The
 From dogs abdominal pain started 3 weeks ago when he started to be
 Occasionally causes human diarrhea stressed from his PRC board exams. So, he drank a lot of
J. Campylobacter sputorum coffee to be able to study in the wee hours of night. After being
 Normal flora of the respiratory tract and gingival seen by you at the clinic, he was immediately brought into the
crevices ER. CXR was done and revealed normal results. He was
 Isolated in 2% of healthy people referred to GI and an EGD with gastric biopsy was done a day
 Diarrhea, abscess, and sepsis after admission. The scope revealed multiple ulcerations in
the duodenum. Histopathologic biopsy specimens revealed a
spiral shaped rod bacteria near the gastric epithelium. He
said that he had recurrent abdominal pain for 5 years

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MICROBIOLOGY Campylobacter, Helicobacter, and Vibrio MODULE 3, LECTURE 3

already but this time, it was the worst ever he had experienced. Bacterial antigen
What could be this bacterium? o Cross-reacts with anthral gastric antigens stimulating
an autoimmune response
Discovery
 1983: Warren and Marshall observed campylobacter-like Protease
bacteria in gastric mucosa of patients with gastritis and o Degrades gastric mucosa
peptic ulcer
 Originally called C. pyloridis Ability to cause hypergastrinemia
 Different from other campylobacters because of o Upsets gastrin-HCl homeostasis
multisheathed flagella, strong urea hydrolyis, unique fatty
acid profile
 Colonized stomachs of half the world’s population
Taxonomy
 Kingdom: Bacteria
 Phylum: Proteobacteria
 Class: Epsilonproteobacteria
 Order: Campylobacterales
 Family: Helicobacteraceae
 Genus: Helicobacter

A. Helicobacter pylori
 Gram (-) spiral-shaped
 0.5 – 0.9 microns wide x 2 – 4 microns long
 Motile due to tuft of sheathed unipolar flagella
 Nonspore former
 Pleiomorphic, old cultures are coccoid
 Microaerophilic
 Mesophilic (35 -37)
H. pylori Media
o Can be grown in chocolate agar or Skirrow’s
Campylobacter selective medium
o Circular, convex, translucent colonies
o 3-5 days incubation
o Biochemically, produces urease (used for rapid
Pathogenesis
diagnostic test), oxidase (+), catalase (+),
phosphatase (+), H2S (+)  Gastric mucosa colonization
o Noncarbohydrate metabolizer  Antrum
o Nitrate reducer (-)  Present in large numbers in the mucus overlying the
mucosa at pH 7
 Extends into the gastric glands
 Gastric juices, bile, saliva
 Bacterial protease, toxins, ammonia
 H. pylori is toxic to gastric acid
 Urease produces ammonia that buffers and neutralizes
the acid near the periphery of the bacterium
 Chronic active or type B gastritis
 Antral gastritis, duodenal and gastric ulcers, gastric
malignancies: gastric adenoCA and MALT lymphoma

H.pylori causes GI Ulcers

o Associated with anthral gastritis, duodenal ulcer,
gastric ulcer, gastric carcinoma
o Present in less than 20% under age 30 but increases
in prevalence to 40 – 60% at age 60
o In developing countries, infection maybe 80 or higher
Virulence factors
Vacuolation-causing cytotoxin
o Coded by the CagA gene
o Found in the cag pathogenicity island (cag PAI)

Urease
o Produces ammonia that neutralized HCl
o Favors bacterial growth

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MICROBIOLOGY Campylobacter, Helicobacter, and Vibrio MODULE 3, LECTURE 3

Laboratory Diagnosis Pros and Cons of the different H. pylori tests

Noninvasive Tests
 Serology
o Abs to H. pylori or to its products are
detected in the serum via ELISA
o Ab titer falls several months if infection is
eradicated
 Urea Breath Test
o Detects bacterial urease in the stomach by
measuring CO2 resulting from the splitting of
urea into CO2 and ammonia
o Urea tagged with C-14 and C-13 is fed to the
patient
o If the pt’s stomach is colonized, urea is
converted into ammonia and CO2
o High CO2 readings
o Procedure:
a. Take urea capsule with water
b. set for 15 mins. Blow into a collection card
for 3 -5 minutes
c. Test the collection card with helicobacter
detector to obtain results
 Fecal Antigen Test
o Polyclonal abs are used to detect the
antigens in feces
o Potential to replace serology as a routine
screening
 Polymerase Chain Reaction
o Various DNA probes developed for direct
detection in gastric juice, feces, dental
plaque, water supplies

Invasive Tests
 Microscopy (of BIOPSY)
o Gram staining, silver staining, H and E
staining, Giemsa staining, IF
o Warthin-Starry silver stain: most
sensitive
 Culture
o Done on nonselective medium (chocolate
agar) and selective medium (Skirrow)
o Incubated from 2-7 days in a moist.
Microaerophilic atmosphere at 35 – 37
degrees Celsius in the presence of 5 – 10
CO2; high humidity is essential
o Morphology, gram staining, biochemical
properties
 Biopsy Urease Test
o Biopsy specimen is placed into a small
quantity of urea solution
o An indicator of alkalinity results in ammonia
formation via urease
o Specimen is crushed in 0.5 ml urea with
indicator and incubated at 37 degrees
Celsius
o If bacteria are present, pH changes within a
few minutes to 2 hours due to ammonia
production
o Detects allkalinity within 2 hours
Treatment in ADULTS
 Triple therapy with metronidazole and either bismuth
subsalicylate or bismuth subcitrate plus either amoxicillin
or tetracycline for 14 days eradicates H pylori infection in
70–95% of patients. An acid-suppressing agent given for 4
to 6 weeks enhances ulcer healing. Proton pump inhibitors
(PPIs) directly inhibit H pylori and appear to be potent
urease inhibitors. The preferred initial therapy is 7–10
days of a PPI plus amoxicillin and clarithromycin or a
quadruple regimen of a PPI metronidazole, tetracycline,
and bismuth for 10 days
 Bismuth salicylate
 Tetracycline
 Amoxicillin
 Metronidazole
 Clarithromycin o 2 weeks at least
 Proton pump inhibitor

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MICROBIOLOGY Campylobacter, Helicobacter, and Vibrio MODULE 3, LECTURE 3

 Fish in the stream appearance in stained films of acute

cholera patients
 Actively motile: single polar sheathed flagellum
 Darting motility
 Swarm of gnats in acute cholera stool
 Nonspore former, noncapsulated, nonacidfast

Cultural Characteristics
 Aerobic
 Grows in 13-40 degrees Celsius (optimum 37)
 Better in alkaline medium (pH 7.4-9.6) with optimum pH
Treatment in CHILDREN at 8.2
*almost the same , varies with dosage only  Nonhalophilic (does not require salt)
B. Helicobacter cinaedi  Grow them in ordinary and special media
 Proctitis in homosexual males Types of Media
 Bacteremia in MSM with concurrent TB associated with Ordinary Media
HIV and AIDS a. Nutrient Agar
C. Helicobacter fennelliae  Moist, translucent, round disks
 Proctitis in homosexual males  1-2 mm, bluish tinge
D. Helicobacter heilmanii  Distinctive odor
 Spirochetal in morphology b. Mac Conkey Agar
 Strongly urease (+)  Colonies are smaller
 Occurs in 1% in the gastric mucosa  Colorless but become reddish due to late
VIBRIO SPP. lactose fermentation
Clinical Case # 3 c. Blood Agar
C.17-year old male who was severely emaciated  Colonies are surrounded by green zone due
and had diarrhea for only a day but had 25 bouts of to hemodigestion
diarrhea. The volume of every stool was approximately half a d. Gelatin Slab
pail. The stool looked like washings of rice. The senior doctor  White line of growth along the track of the
with you in the medical mission ordered for an IV drip and an inoculating wire
IV antibiotic. A week after, the patient was already well and
 Liquefaction of gelatin begins at the top which
was discharged uneventfully. What could have caused this
spreads in a funnel-shaped (infundibuliform)
patient’s diarrhea?
or turnip shaped (napiform) manner in 3 days
Introduction at 22 degrees Celsius
 12 out of the 35 Vibrio spp. are pathologic e. Peptone Water
 Halophilic and nonhalophilic  Incubated at 37 degrees Celsius
 Grow with added salt (HALOPHILIC)  Fine surface pellicle which breaks into
 Vibrio cholerae: representative of the genus membranous pieces after shaking
– causes cholera  Turbidity and a powdery deposit
– 1883: Isolated in Egypt by Robert Koch
 V. parahaemolyticus, V. vulnificus, V. minicus, V. Special Media
alginolyticus f. Holding or Transport Media
A. Vibrio cholera  Venkatraman-Ramakrishnan (VR)
Morphology o 20 g crude sea salt + 5 g peptone in 1 L
 Gram (-), short, curved, cylindrical rods distilled water with pH 8.6-8.8
 1.5 micron x 0.2-0.4 microns in size o Screw capped im 10-15 ml
o 1-30 ml stool added
 Rounded or slightly pointed ends
o Vibrio does not multiply but remain
 V. comma
viable for several weeks
 S-shaped or spiral forms seen when 2 or more cells lie o Prevents overgrowth
end to end
 Upon serial transfers: become straight
 Pleiomorphic in old cultures

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MICROBIOLOGY Campylobacter, Helicobacter, and Vibrio
 Cary-Blair
o Buffered solution of disodium phosphate
(1.1 g) sodium thioglycollate (1.5 g),
NaCl (5.0g) , agar (5.0 g) to 1 L distilled
water at pH 8.4
o Can be used for Salmonella, Shigella
 Autoclaved Sea Water
o Holding medium
Enrichment media
 Alkaline Peptone Water
o pH 8.6
o preliminary enrichment from stool
 Monsur’s Taurocholate Tellurite Peptone
Water
o pH 9.2
Plating Media
 Alkaline Bile Salt Agar (BSA)
o pH 8.2
o Modified nutrient agar
o Contains 0.5 percent sodium
taurocholate
o Colonies similar to nutrient agar
 Monsur’s Taurocholate Trypticase Tellurite
(GTTA)
o pH 8.5
o Useful for isolation of Vibrio in stool
o High pH and K tellurite are inhibitory to
other enterobacteria except Proteus and
GPB
o After 24 hours, 1-2 mm translucent
colonies with greyish-black cente and
turbid halo due to hydrolysis and gelatin
denaturation
o After 48 hrs, colonies become 3-4 mm
 Thiosulphate-Citrate-Bile-Sucrose (TCBS)
Agar*
o PH 8.6
o Most commonly used selective plating
o Na thiosulphate, Na citrate, ox bile,
sucrose, yeast extract, peptone, NaCl,
ferric citrate, thymol blue, bromothymol
blue (indicator), and water
o Medium resembles DCA except pH 8.6
and sucrose rather than lactose
o V. Cholerae are yellow;
parahaemolyticus (sucrose
nonfermenting Vibrios) green
Biochemical Reactions
 Sugar Fermentation
o Ferments glucose, maltose, mannose, sucrose,
mannitol
o Ferments lactose only after several days (late
lactose fermenter)
o Arabinose and dulcitol not fermented
 Cholera Red Reaction
o Strongly indole positive and reduces nitrates to
nitrites
o These 2 processes provide for the reaction
o Sulfuric acid is added to a 24-hr peptone water
culture at 37 degrees Celsius
o Reddish-pink color due to nitroso-indole
 Catalase, Oxidase
o Positive
 Methyl Red, Urease- Negative
Cruz, De Leon
MODULE 3, LECTURE 3
 Amino acid Decarboxylation
o Lysine and ornithine (+)
o Arginine (-)
 Gelatin Liquefaction
o Positive
 Voges-Proskauer
o (+) in El Tor biotype
o (-) classical biotype
 Sheep RBC hemolysis
o (+) in El Tor biotype
o (-) in classical biotype
 String Test
o A loopful of colonies is mixed with 0.5% sodium
deoxycholate on a slide
o (+) if suspension loses turbidity, becomes
mucoid, forms a string when the loop is drawn
away from suspension
Survival
 Susceptible to heat, drying and acids
 Resists high alkalinity

0
Heat at 56 C for 30 mins or within a few seconds of
boiling
 Die quickly on dry fomites and in sewage-polluted water
 Survive 1-2 weeks in clean, nonacidic fresh or sea water
 In general, El Tor survives longer than the classic
 Survive in clean tap water for 30 days
 Fruits: 1- 5days at room temperature; refrigerator: a week
 A few days: moist fruit, vegetables, fish, cooked foods
 Easily killed by disinfectants and short exposure to gastric
juices
 Survive within 24 hours in achlorhydric gastric juice
 Sensitive to tetracyclines, aminoglycosides, TMP-SMX
Antigenic Structure
 Flagellar antigen (H antigen)
o Many Vibrios share a heat-labile flagellar antigen
o Abs against this agn are not protective
 LPS/ endotoxin (O antigen)
o Confer serological specificity
o Protective in mice
o Plays no role in cholera pathogenesis
o Responsible for immunity by killed vaccines
o May cause fatal illness when inoculated
peritoneally into mice
Classification
V.  Heidelberg (1934) – 6 groups based on mannose,
sucrose, arabinose fermentation
 Serological classification
o Gardner and Venkatraman (1935)
o Possessing common flagellar (H antigen) = group
A vibrios while the rest as group B
o Bases on O antigen, group A vibrios are
classified into subgroups called O
serogroups/serovars
o More than 200
o All isolates from epidemic cholera (until 1992) are
serogroup 0-1
o Cholera nondifferential serum (group 0-1
antiserum) agglutinates pathogenic cholera
hence agglutinable vibrios
o Nonagglutinable vibrios (NAG) are
nonpathogenic and are called noncholera vibrios
(NCV) but recent epidemics suggest otherwise
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MICROBIOLOGY Campylobacter, Helicobacter, and Vibrio MODULE 3, LECTURE 3

Biotypes Pathogenesis
 2 biotypes: classical and El Tor  Fecal-oral route

Chick Red Cell Agglutination Test

• A loopful emulsified into a saline drop
with 2.5 % chick erythrocyte suspension
• Clumping is +

Polymyxin B Sensitivity Test

• Diffusion disk method
• 50 units of polymyxin B

 Highly susceptible to acids

Cholera Phage Sensitivity Test  Achlorhydria is a risk factor
• Mukherjee’s group IV phage routine test
dilution
 Multiply in the relatively alkaline intestines
• Phage typing schemes have been  Once in the small intestines, migrate to epithelium
standardized for El Tor and classical  Actively motile
• Classical has 5 types based on 4 phages  Hemagglutinin-protease (cholera lectin) cleaves mucus
• El Tor has 6 types based on 5 phages
and fibronectin which frees the other vibrio bound to the
bowel mucosa
Subtypes  Adhesion facilitated by toxin coregulated pilus (TCP)
 Do not invade or damage cells
* Based on minor O antigen
 Ogawa Virulence Factors
o A and B  Cholera toxin (ctxA, ctxB)
o Southeast Asian epidemic in 1961  Toxin coregulated pilus (tcp)
 Inaba  Accessory colonization factor (acf)
o A and C  Hemagglutinin-protease (hap)
o South American epidemic  Note: uses repressor genes to control pathogenicity (toxR)
 Hikojiima Cholera Toxin
o AB and C  Choleragen, cholera enterotoxin, CT, CTX
o Could be agglutinated by both Ogawa and Inaba  Structurally, biochemically, serologically similar to heat-
antiserums labile enterotoxin of E. Coli
Non-agglutinating (NAG) Vibrios  Far more potent than LT biologically
 Not agglutinable by 0-1 antiserum  CT production determined by filamentous phage
 Called noncholera vibrios integration within its chromosome
 Misnomer o Plasmids dictate whether toxin will be produced
 In general, nonpathogenic  Non-toxigenic strains become toxigenic when introduced
 Commonly isolated from environment and healthy human as a plasmid
intestines  84,000 MW
 More than 200 serogroups  1 A and 5 B subunits
 1992: serogroup0-139 caused a cholera epidemic  B (binding) subunits bind to ganglioside GM1
 Some NAG cause cholera-like diseases receptors found in enterocytes
Vibrio cholera: 0139  A (active) subunit enters into the cell and fragments into
 1992 epidemic A1 and A2
 Seen in Chennai  A2 fragment links A1 to the B subunit
 Mid-January 1993: spreads into Bangladesh and northern  A1 activates adenylyl cyclase via transfer of ADP from
India NAD to a regulatory protein of adenylyl cyclase
 Follows the course of rivers  Causes irreversible activation and generation of cAMP
 Resembled EL Tor biochemically  cAMP signals cell to stop Na and Cl ion uptake and
 May evolved from 01 but with modified LPS hypersecretion of Cl and HCO3
 Makes a polysaccharide capsule in contrast to other  There is passive outflow of water from cells and blocks
vibrio uptake of water from the lumen, resulting in watery
diarrhea
 More invasive
 Other biological effects:
 Possesses novel antigens
o Lipolysis activation in rat testicular tissue
 01 vaccine unprotective
o Elongation of Chinese hamster ovary cells
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MICROBIOLOGY Campylobacter, Helicobacter, and Vibrio MODULE 3, LECTURE 3

o Histological changes in the adrenal tumor cells Epidemiology

and vero cell  Both an epidemic and endemic disease
o Increases skin capillary permeability  7 major pandemics since 1817
 Exclusively human disease
 Spread by contaminated water or uncooked food
 Source of contamination: infected stools
 Climate has an effect on the formation of epidemic waves
 November to February: coincides with the monsoon rains
in Bangladesh
 May to July: during hot and dry season before onset of the
rainy season in Calcutta

th
1961: 7 pandemic caused by El Tor
o EL TOR
• Originated from Sulawesi (Celebes),
Indonesia but the cholera microbe was
isolated by Gotschlich at the El Tor quarantine
station in Egypt
• Encircled the globe in 30 years
• Affected all South American countries except
for Uruguay
• By 1994, became endemic in Central and
South America
 1992: V. cholerae O139 seen as outbreaks in Madras
Cholera: Laboratory Diagnosis
Specimens:
 watery stools and rectal swabs
 Stool should be collected before patient is treated with
empiric antibiotic
Rubber catheters
* Pathogenesis of cholera: Ganglioside GM1 serves as the o Used with early acute cases
mucosal receptor for subunit B, which promotes entry of o Collected into sterile container
subunit A into the cell. Activation of subunit A1 yields increased o Introducing the catheter into the rectum and letting
levels of intracellular cyclic adenosine monophosphate (cAMP) liquid flow into the screw-capped container
and results in prolonged hypersecretion of water and Rectal swab
electrolytes. There is increased sodiumdependent chloride o Can be collected on convalescent patients without
secretion, and absorption of sodium and chloride by the diarrhea
microvilli is inhibited. Electrolyte-rich diarrhea occurs—as much o Cotton swabs are moistened with transport medium
as 20–30 L/day—with resulting dehydration, shock, acidosis,  Vomitus not helpful
and death.
 Microscopy
Disease: Cholera  Culture
 Acute diarrheal disease  Serology
 Incubation period:  24 hr – 5 days Cholera: Treatment
 Clinically begin slowly with mild diarrhea and vomiting in 1- 1. Oral Rehydration Therapy (ORT)
3 days or abruptly with sudden massive diarrhea
 Absolute priority
 60% infections of classical cholera are ASYMPTOMATIC
 Often sufficient
 75% of El Tor also
 Severe cholera warrants IV
 Severe form: profuse painless watery diarrhea and 2. Antibacterial Therapy
copious effortless vomiting
 Oral tetracycline (Doxycycline)
 Leads to hypovolemic shock and death in less than 24
 Multidrug resistance can occur
hrs.
Cholera: Immunity
 Last for 4-6 days
 Symptoms due to massive loss of fluid and electrolytes  Gastric acid provides some protection against cholera
 Cholera stool: colorless watery fluid with mucoid vibrio
flecks (rice, watery stools)  Confined to the intestine
• Inoffensive, sweetish odor  Natural immunity at 6-12 months
• HCO3-rich isotonic electrolyte solution with  Reinfection is possible
little protein  Specifc IgA abs occur in the lumen (animal models)
 ECF diminution, hemoconcentration, hypoK, base-deficit  Abs at titer 1:20 confer protection against vibrio
acidosis, shock colonization
 Muscular cramps, renal failure, pulmonary edema, cardiac  Antitoxin abs has not been shown protective
arrhythmias, paralytic ileus  Immunity may be local or systemic
 Mortality rate 25-50%  Coproantibodies + antibodies within stool and found in
 El Tor causes milder disease lumen

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MICROBIOLOGY Campylobacter, Helicobacter, and Vibrio
Cholera: Prophylaxis
General Measures
o Provision of safe drinking water
o Proper human excrement disposal
o Patient education
o Environmental and Food Sanitation
Specific Measures-Vaccines
o Vaccines
 Killed Whole Organism Vaccine
 Killed suspension containing 8
trillion V. cholerae per ml
 Both Ogawa and Inaba
serotypes
 Can increase to 12 T per ml
 2 equal doses , SQ
 4- 6 week interval
 showed 60% protection for 3-
6months
 Single dose is ineffective in
children below 5 yrs old while 2
doses at 1-4 week interval is
immunogenic
 In adults, act as booster
 Cell somatic antigen are parts
of the bacterium with adjuvants
such as Aluminum hydroxide
and phosphate
 Oral Vaccine
o Nonliving oral B subunit-whole cell (BS-
WC) vaccine

10
Killed whole cell (2.5 x 10 heat-
killed vibrios of Ogawa and Inaba) +
recombinant B subunit of the CT
 2 dose schedule: 10-14 days
 50-60% protection for 3 years
o Live Oral vaccine
 Recombinat DNA vaccine with
expression of V. cholerae01 in
attenuated S. typhi Ty21 as carrier
 Live salmonella colonized the
Peyer’s patches and induces igA
response
 Single dose vaccine
 80% protection to El Tor
HALOPHILIC VIBRIOS
 There are vibrios that have high salt requirement
 Natural habitat is sea water
 Adapted to marine life
 V. parahaemolyticus, V. alginolyticus, V. vulfinicus
B. Vibrio parahaemolyticus
Morphology
 Morphologically resembles V. cholera but capsulated
 Bipolar staining
 Pleomorphism (when grown in 3% salt agar and old
cultures)
 Does not grow in peptone water w/o salt or on cysteine-
lactose electrolyte deficient (CLED) agar
 Grows in peptone water w/ 8% (but not 10%) NaCl
 Grows well on blood agar
 On McConkey: pale, nonlactose-fermenting colonies
 On sheep blood agar: produces beta-hemolysis
 On TCBS: green (nonsucrose-fermenting)
 Has peritrichous flagella when grown on solid media
Cruz, De Leon Checked by: YAO
MODULE 3, LECTURE 3
 Polar flagella on liquid media
Biochemical Reactions
 Oxidase, catalase, indole, citrate (+)
 Reduces nitrate to nitrite
 Ferments glucose, maltose, mannose, arabinose
 Does no ferment lactose, sucrose, dulcitol, inositol
 VP (+) (voges proskauer)
 Lysine and ornithine (not arginine) decarboxylator
Pathogenesis
 Not all strains pathogenic
 Strains isolated from water sources are nonhemolytic in
high salt blood agar called WAGATSUMA’S AGAR but
those isolated from human patients are (Kanagawa
phenomenon)
 This is due to heat stable hemolysin
 Kanagawa (+) are pathogenic
 Causes enteritis via intestinal invasion
 AGE after eating contaminated improperly cooked seafood
 Incubation period: 12-24 hrs
 Nausea/vomiting, dysentery, watery diarrhea, abdominal
cramps
 Fecal leukocytes
 Subsides spontaneously
 Fluid replacement
Epidemiology
 Occurs worldwide
 Highest incidence in people who eat raw seafood
 Common in summer and in adults
Laboratory Diagnosis
 Isolated from stool
 Enrichment culture in alkaline peptone water with 1%
NaCl is used for food inspection
C. Vibrio vulnificus
 Previously known as L+ vibrio or Beneckea vulnifica
 Marine vibrio
 Second most serious type of Vibrio-associated illness
 Green, nonsucrose fermenting on TCBS
 VP (-)
 Lactose fermenter (lactose fermenting vibrio)= key
biochemical identification
 Salt tolerance of 8%
 Can grow in a 300 unit disk of polymyxin
 Cause wound infections, bacteremia and gastroenteritis
 Skin: metastatic hemorrhagic bullae
 Sepsis
 Infections are high in patients with hepatic disease,
hematologic disease, CKD, immunodeficient and
immunosuppressed pts
 Lab diag: culture
 Tx: Tetracycline and Ciprofloxacin
D. Vibrio alginolyticus
 Halophilic
 Regarded previously as biotype 2 of V. parahaemolyticus
 Higher salt tolerance
 VP(+)
 Sucrose fermenter
 Forms large yellow colonies
 Pronounced swarming on nonselective solid media
 Eye, ear, wound infection in humans exposed to salt water
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