VIBRIO

CHOLERAE
 Introduction

 Family: Vibrionaceae
Genus: Vibrio
Species: V. cholerae
 Gram-negative bacteria
 curved rod, comma shaped
 On prolonged incubation tend to become
straight rods
 2-4 um
 actively motile with a single, polar flagellum
Culture properties
 Colonies are smooth, convex, round and opaque
 Grow well at 37C at most media

 Thisulfate Citrate Bile Sucrose (TCBS) is used on

which it produces yellow colonies

 Oxidase positive

 Grows at high pH (8.5-9.5)

 sensitive to low pH

and die rapidly in solutions below pH 6

 Most species are halotolerant
 Found in both fresh and brackish water
 Live in close contact with algae and marine
invertebrates
 Not much known about how bacteria spend the
time between the outbreaks
 Transmission
 Transmitted by fecal-oral route
Contaminated food or water by feces of
asymptomatic and sick human carriers
Inadequate sewage treatment
Lack of water treatment
Improperly cooked food (fish, shellfish)
 no person-to-person transmission
 May persist in shellfish or plankton
Infection
 For person with normal stomach acidity the
infectious dose ranges is 1010 or more, when
transmitted by water
 If the vehicle is food, 102 – 104 is enough
 Any condition that decreases the stomach acidity
increases susceptibility
Incubation period
 Ranging from a 1-4 days
 the incubation period is shortest when
• highest dose of organism ingested
• High gastric pH
 Symptoms
Usually mild, or no symptoms at all
60-75% asymptomatic depending on the biotype
Nausea and Vomiting
watery vomiting is common and may begin before
or after diarrhea
Abdominal cramps
Diarrhea may be sudden or gradual
Profuse watery diarrhea (1L/hour)
Fecal material is known as rice water stool
 A person with cholera can lose 20literes of water
per day (losing about 50lb)
 Evident from-
 Loss of skin pasticity

 Sunken eyes

Without treatment, death in 18 hours-several days

 Death results from dehydration, collapse of the
circulatory system
 Disease is self limiting if the person survives the
acute phase
 Diarrhea washes out the bacteria
 Mortality
• In untreated patients, mortality can reach 25-50%
• Risk much higher in children
• 10x greater than adults

• As well as pregnant women

• 50% risk of fetal death in 3rd trimester
 People who recover acquire long-term immunity
to re-infection
 During an epidemic they may become chronic
asymptomatic carriers
 Can transmit the disease to susceptible members
and contaminate water
Pathogenesis
Ingestion of V. cholerae
Bacteria survive passage through the gastric barrier
of the stomach
On reaching the lumen of the small intestine, the
bacteria overcome the clearing mechanism of the
intestine (peristalsis)
Adhere and colonize to the intestinal mucosa
Produce and release of cholera toxin
Results in diarrhea leading to ion imbalance, fluid
loss, hypotension and death
Virulence factors
Colonization of the small intestinal mucosa
1. Motility and chemotaxis
 Has a polar flagellum

 Non motile mutants are less virulent

2. Adherence
 Contain long filamentous pili that form bundles at the

end
 Known as Toxin Coregulated Pili (Tcp pili)

 Contribute in the colonization

 Mutants lacking pili are avirulent

 Host cell receptor is not identified

 Purified Tcp pili do not bind to intestinal
epithelial cells
 Might not be needed for binding to intestinal
epithelial cells
 May instead mediate bacterium-to-bacterium
attachment
 Bacterial producing Tcp pili aggregate
Cholera toxin

 A-B ADP ribosylating toxin

 Consists of
 One (enzymatic) subunit

 Five B (binding) subunits

 Genes encoding are ctxA and ctxB, which are part of

the same operon
 The mature A subunit is proteolytically cleaved to
produce a A1 and A2 fragments
 remain linked by a disulphide bond
 the A2 subunit, is inserted through the central pore
of the B pentamer
Mechanism of Action
1. the B subunit binding to GM1 ganglioside
Some host cells contain gangliosides with longer sialic
acid chains, which is removed by Neuraminidase or
Sialidase
Makes the gangliosides structurally more similar to GM1
2. A1 subunit released from the toxin
Possibly by reduction of the disulfide bond
3. Toxin enters into host cell
 endocytosis
 A1 subunit passes through a pore formed by
the B subunits
 The GTP-bound form of Gs
increases Adenylate cyclase
activity
regulatory protein (Gi),
hydrolyzes GTP and
inactivates AC
 ADP-ribosylated Gs is always
active and cAMP levels are
uncontrolled
 Na and Cl transporter
activities are altered
 In normal condition net flow of ion is from lumen to
tissue
 Results in uptake of water from lumen to tissue
 Effect of cholera toxin on mucosal cells alter the
balance
Other toxins
 ZOT toxin
 Enterotoxin

 Affects the tight junctions or Zonula occludens

that bind mucosal cells together

 preserves the integrity of the mucosal

membrane
 Ion balance is lost

 Contents of the lumen diffuse to the

underlying tissue
 Gene lies immediately upstream of the ctxAB

operon on the CTX phage

ACE toxin (Accessory cholera enterotoxin)
 Produces fluid accumulation in the rabbit ileal loop

 Role in human disease is controversial

 The ace gene is closely linked genetically to the zot

gene
 Part of the CTX phage

 These toxins do not make a major contribution to

virulence
Vibrio serotypes
 Vibrio comprises 206 serogroups (O1-O206) based on
antigenic diversity of their outer membrane
lipopolysaccharides
 Strains of the O1 serogroups are divided into two
biotypes according to their phenotypic differences
 Classical

 El Tor

 Each of the V. cholerae O1 biotype can be divided

into three serotypes

 Ogawa

 Inaba

 Hikojima
Classification: Serogroups and
Biotypes
 Clinical manifestations of cholera caused by classical
V. cholerae are more severe and prolonged than those
caused by the El Tor
 Non-O1 and non-O139 V. cholerae can cause mild
diarrhoea but do not generate epidemics
 New V. cholerae O1 variants (Matlab variants)
carrying mixed classical and El Tor phenotypes were
isolated from hospitalized patients with severe watery
diarrhoea in Matlab, Bangladesh, in 2002
 V. cholerae O1 El Tor variants have been reported
from several Asian countries including China, Japan,
Hong Kong, Sri Lanka, and Vietnam and Africa
(Zambia)
 Lab diagnosis
 Specimen: stool
 Smear:
 microscopic appearance is not distinctive
 Dark field or phase contrast microscopy may show
the rapidly motile vibrios
 Culture:
 TCBS agar
 Blood agar
 Peptone agar
 For enrichment, a few drops of stool can be
incubated for 6-8h in taurocholate peptone broth
Specific tests:
 Slide agglutination tests using antigroup O1 and

O139 antiserum
 Biochemical tests
Treatment
 Untreated: 60% fatality
 Treated: <1% fatality
 Oral Rehydration & supportive therapy
 Antibiotics can be used
 to hasten the clearing of bacteria
 To prevent more toxin production
 Doxycycline or tetracycline (Tet resistance
may be developing) of secondary value
 For patients in coma and are suffering from
extreme dehydration, intravenous therapy
is needed
 Vaccines
• Two types of cholera vaccines are currently approved
for use in humans:
• Killed-whole-cell formulation:
 killed bacterial cells from both biovars of serovar
01 and purified B subunit of the cholera toxin
 Provides lower rates of protection to infants and
children than in adults
 Live-attenuated vaccine, genetically engineered
 ctxA gene is inactivated
 Provides >90% protection against classical
biovar and 65-80% against E1Tor biovar
 References
 Bbb - vibrio cholerae serogroup o1. (2009, May 04). Retrieved from
http://www.fda.gov/Food/FoodSafety/FoodborneIllness/FoodborneIllnessFoodbornePathogensNatural
Toxins/BadBugBook/ucm070071.htm
 Cholera. (2010). Centers for diease control and preventation . Retrieved November 23, 2010, from
http://www.cdc.gov/cholera/
 Cholera. (2010). World health organization. Retrieved November 23, 2010, from
http://www.who.int/topics/cholera/en/
 Cholera in Haiti. (2010). World health organziation . Retrieved November 23, 2010, from
http://www.who.int/csr/don/2010_10_26/en/index.html
 Handa, Sajeev. (2010). Cholera. Emedicine, Retrieved from
http://emedicine.medscape.com/article/214911-overview
 Leinwand, Donna. (2010). Haiti's cascading crises come down to h0. USA TODAY, Retrieved from
http://web.ebscohost.com/ehost/detail?vid=8&hid=106&sid=ecaf75f6-1139-45c6-8206-
2b75a950b422%40sessionmgr115&bdata=JnNpdGU9ZWhvc3QtbGl2ZQ%3d
%3d#db=f5h&AN=J0E145805781310
 Todar, Kenneth. (2009). Vibrio cholerae and asiatic cholera. Retrieved from
http://www.textbookofbacteriology.net/cholera.html
Diagnosis
No clinical manifestations help
distinguish cholera from other causes
of severe diarrhea:
Enterotoxigenic e. coli
Viral gastroenteritis
Bacterial food poisoning
Diagnosis: Visible
Symptoms
Decreased skin turgor
Sunken eyes, cheeks
Almost no urine production
Dry mucous membranes
Watery diarrhea consists of:
fluid without RBC, proteins
electrolytes
enormous numbers of vibrio
cholera (107 vibrios/mL)
Laboratory Diagnosis
Visualization by dark field or phase
microscopy
Look like “shooting stars”
Gram Stain
Red, curved rods of bacteria
Isolate V. cholerae from patient’s stool
Plate on sucrose agar
Yellow colonies form
Treatment
*Even before identifying cause of
disease, rehydration therapy must
begin Immediately because death can
occur within hours*

Oral rehydration
Intravenous rehydration
Antimicrobial therapy
Treatment: Oral
Rehydration
Reduces mortality rate from over 50%
to less than 1%
Recover within 3-6 days
Should administer at least 1.5x amount
of liquid lost in stools
Use when less than 10% of bodyweight
lost in dehydration
Treatment: Oral Rehydration
Salts (ORS)
Reduces mortality from
over 50% to less than 1%
Packets of Oral
Rehydration Salts
Distributed by WHO,
UNICEF
Dissolve in 1 L water
NaCl, KCl, NaHCO3, glucose
Treatment: How ORS
Works
Na+ transport
coupled to glucose
transport in small
intestine
Glucose enables
more efficient
absorption of fluids
and salts
Potassium
passively absorbed
Treatment: ORS in United
States?
American doctors skeptical of such
simple, inexpensive treatment
Cost
ORS: $270/infant
IV: $2,300/infant
$1 billion/year for IV treatment for rehydrating
children
Insurance companies do not reimburse for
ORS
600 American children die unnecessarily
from dehydration each year
Hospitals consider IV more time efficient
Less personal attention required
Treatment: Intravenous
Rehydration
Used when patients have lost more than
10% bodyweight from dehydration
Unable to drink due to vomiting
Only treatment for severe dehydration
Treatment: Intravenous
Rehydration
Ringer’s Lactate
Commercial product
Has necessary
concentrations of
electrolytes
Alternative options
Saline
Sugar and water
Do not replace
potassium, sodium,
bicarbonate
Treatment: Antibiotics
Adjunct to oral rehydration
Reduce fluid loss by half
Reduce recovery time by half
2-3 days instead of 4-6
Tetracycline, Doxycycline
Not recommended
Short duration of illness
Antibiotic resistance
Limited gain from usage
Traveling Precautions
Boil or treat water with chlorine or
iodine
No ice
Cook everything
Rule of thumb: “Boil it, cook it, peel it,
or forget it.”
Wash hands frequently
Vaccines
Need localized mucosal immune response
Oral Vaccine
Not recommended
Travelers have very low risk of contracting disease:
1-2 cases per million international trips
Not cost-effective to administer vaccines in
endemic regions
Brief and incomplete immunity
Two types approved for humans:
Killed whole-cell
Live-attenuated
Vaccines: Killed Whole-
cell Vaccines
Provides antigens to evoke
protective antitoxic and antibacterial
immunity
Contains:
1 x 1011 heat inactivated bacteria
Mixture of V. cholerae O1 El Tor and
classical strains
1 mg of B subunit of cholera toxin
Killed Whole-cell
Vaccines: Disadvantages
50% protection for 6 months to
adults
Gives less than 25% protection to
children aged 2-5
Need for multiple doses of nonliving
antigens
Vaccines: Live-
Attenuated
Eliminates need for multiple doses of
non-living antigens
Ensures that crucial antigens
potentially altered during killing
process would be retained
Expected to mimic broad immunity
conferred by natural infection
85-90% protection against classical
biovar
65-80% protection against El Tor biovar