Vibrio cholerae

Morphological and Cultural Properties,

Clinical Manifestation,
Pathogenesis, Virulence Factors,
Prevention and Treatment
 Vibrio cholerae
• Staining properties: V.
cholerae is Gram-negative
and comma-shaped.
• Initial isolates are slightly
curved, whereas they can
appear as straight rods
upon laboratory
culturing.
• The bacterium has a
flagellum at one cell pole
as well as pili.
 Ecology
• V. cholerae thrives in a water ecology,
particularly surface water. The primary
connection between humans and pathogenic
strains is through water, particularly in
economically reduced areas that don't have
good water purification systems. Non-
pathogenic strains are also present in water
ecologies.
 Cultural properties
• V. cholerae is a facultative anaerobe, and can
undergo fermentative metabolism.
• Cary Blair media is ideal for transport, and the
selective thiosulfate–citrate–bile salts agar
(TCBS) is ideal for isolation and identification.
Cary Blair Medium

• The low nutrient content of the medium

prevents bacterial overgrowth and ensures
bacterial survival over long periods.
 TCBS agar
Thiosulfate-citrate-bile salts-
sucrose agar or TCBS agar is
a type of selective agar
culture plate that is used in
microbiology laboratories to
isolate Vibrio spp. TCBS Agar
is highly selective for the
isolation of V. cholerae and
other vibrios.
 TCBS agar
• TCBS agar contains high concentrations of Sodium thiosulfate and
Sodium citrate to inhibit the growth of Enterobacteriaceae.
• Inhibition of Gram-positive bacteria is achieved by the incorporation
of ox gall, which is a naturally occurring substance containing a
mixture of bile salts, and sodium cholate, a pure bile salt.
• Sodium thiosulfate also serves as a sulfur source and, in combination
with ferric citrate, detects hydrogen sulfide production.
• Saccharose (sucrose) is included as a fermentable carbohydrate for
the metabolism of vibrios.
• The alkaline pH of the medium enhances the recovery of V. cholerae
and inhibits the growth of others.
• Thymol blue and bromothymol blue are included as indicators of pH
changes.
 Taxonomy
• Domain: Bacteria
• Phylum: Proteobacteria
• Class: Gamma-proteobacteria
• Family: Vibrionaceae
• Order: Vibrionales
• Genus: Vibrio
• Species: Vibrio cholerae
 Clinical Manifestations
Most cholera infections are asymptomatic in
nature. Only 5-10% of those infected will
display the severe symptoms that are
characteristic of cholera . Cholera can still
be spread from asymptomatic carriers, as
the bacteria will be shed for 7-14 days.
Two case types of cholera:
1. Mild cases are difficult to distinguish
from normal diarrheal diseases.
2. Severe cases (10%) are associated
with painless, watery diarrhea, with
as much as 20 L day-1 fluid loss,
leading to hypovolemic shock.
 Clinical Manifestations
The typical symptoms of cholera
include diarrhea, nausea and
vomiting, and dehydration. The
most serious of these symptoms
is the diarrhea, which leads to
dehydration.
The diarrhea associated with
cholera is very distinct and often
called "rice water stool" because
of its pale, milky white
appearance, which is what water
looks like after having washed
rice with it.
 Severe Dehydration
• The symptoms of severe
dehydration include
irritability, lethargy, sunken
eyes, a dry mouth, extreme
thirst, dry and shriveled skin
that's slow to bounce back
when pinched into a fold,
little or no urine output, low
blood pressure, and an
irregular heartbeat
(arrhythmia).
 Infectious dose
• Cholera is contracted by fecal-oral vehicle
transmission. This is usually due to
contaminated food or water. Person-to-person
contact is infrequent because of the large
infectious dose.
• Infectious dose is 103-106 cells if contaminated
water is the source and 102-104 cells if
contaminated food is the source.
• Humans are the primary reservoirs.
 Cases per year
• Cholera is an acute diarrhoeal disease that can
kill within hours if left untreated.
• Researchers have estimated that every year,
there are roughly 1.4 to 4.3 million cases, and
28 000 to 142 000 deaths per year worldwide.
• Up to 80% of cases can be successfully treated
with oral rehydration salts.
Molecular biology and virulence
factors
V. cholerae Classification Scheme
NON-TOXIGENIC TOXIGENIC

I may not be O1, I define Vibrios!

Or O139! I’m an
(but I can still O1 or O139 Strain
stir up trouble)

www.forth.go.jp/tourist/ panf/cholerae.html
 Classification Scheme
Division into ribotypes Toxigenic V. cholerae
Division into 2 epidemic serotypes

O1
Division into 2 biotypes O139

Classical El Tor
Each O1 biotype can have 3 serotypes

inaba ogawa hikojima

A&B
A&C A, B, C
(a little C) Antigens

Designed using information presented in review by NS Crowcroft. 1994. Cholera: Current Epidemiology. The Communicable
Disease Report. 4(13): R158-R163.
 Genome
• Aug 2000: published complete DNA sequence
of V. cholerae, El Tor strain
• Unusual - 2 distinct chromosomes
 Genomic Structure

 The cholera genome

contains 2 circular
chromosomes.
 The genome is approximately
4.0Mb, in which the classical
strain is divided between a
2.4Mb large chromosome
and a 1.6 Mb small
chromosome.
 In the El Tor strain, the large
chromosome contains
2.96Mb and the small
chromosome contains
1.07Mb
 Genome function
• The entire genome of the virulent strain V. cholerae El Tor N16961 has been
sequenced, and contains two circular chromosomes.
• Chromosome 1 has 2M (2,961,149) base pairs with 2,770 open reading
frames (ORF’s).
• It is the larger first chromosome that contains the crucial genes for toxicity,
regulation of toxicity and important cellular functions, such as transcription
and translation.
• Contains a genomic island of pathogenicity and is lysogenized with phage
DNA. That means that the genes of a virus has become integrated into the
bacterial genome and made the bacteria pathogenic.
• Chromosome 2 has 1MB (1,072,315) base pairs, 1,115 ORF’s. , including
essential genes for metabolism, heat-shock proteins and 16S rRNA genes.
 Chromosome 2, Is it a
plasmid/megaplasmid?
• The second chromosome is determined to be different from a plasmid or
megaplasmid due to the inclusion of housekeeping and other essential genes
in the genome.
• Also relevant in determining if the replicon is a chromosome is whether it
represents a significant percentage of the genome, and chromosome 2 is 40%
by size of the entire genome.
• And, unlike plasmids, chromosomes are not self-transmissable.

*** However it is believed that the second chromosome may have once been a
megaplasmid because it contains some genes that are usually found on
plasmids.
 Genomic Structure: Mobile Elements
(PLASMIDS)

 Several plasmids have been isolated.

 A 4.7Kb cryptic plasmid is present in all ctx-positive
strains.
 A 6.8Kb plasmid (P factor) is capable of mobilizing
chromosomal genes but less efficiently than the F
factor in E. Coli.
 Genomic Structure:
Bacteriophage
 In 1996 Matthew K. Waldor  The major virulence factor
and John J. Mekalanos of cholera, CTx (cholera
reported a stunning toxin) is encoded on the
discovery about the toxin. phage PAI that is capable of
 The toxin was for the first transducing the ctx gene
time shown to be not a part into other cholera strains.
of the bacterium but  The released phages
actually that of a specifically attach to the
filamentous bacteriophage bacterium and enter it.
(ctxΦ) that got integrated  Vigorous viral
into the V. cholerae genome. multiplication results in the
 Normally this virus remains production of large
silent within V. cholerae but amounts of toxin causing
during infection it gets severe diarrhea.
activated.
 Genomic Structure:
Pathogenicity Islands (PAI)
 Upon transduction, the
bacteriophage (ctxΦ) brings the
 toxin (Ctx = toxin gene),
 a specific pilus called toxin-co-
regulated pilus (TCP) [aids
intestinal mobilization and
colonization] and
 virulence gene regulation (toxT)
encoded in a 40Kb pathogenicity
island.
 This PAI is present in pathogenic
cholera strains. It is a large
genetic element (about 40 kb)
flanked by two repetitive
regions (att-like sites).
Virulence factors & pathogenesis
 Major Virulence Factors
• Integrons
- Allow V. cholerae to acquire genes from other organisms and
convert them into functional genes.
• Flagella (motility)
• Toxins
- CT; cholera toxin. It is the toxin responsible for inducing
diarrhea in patients with cholera.
• Adherence/Adhesins
– Toxin Co-regulated Pilus (TCP); located on the polar ends of the cell and
likely contribute to colonization via adherence.
– Accessory Colonization Factors (ACF); Involved in signaling between the
chemotaxis motility system and the pilus assembly system.
– OmpU & other Omp Proteins - outer membrane proteins; may be an
adhesin, although the role of the protein has not been definitively
assigned.
Pathogenesis: Cholera Toxin (CT) Structure
 CT is a prototype A/B
subunit toxin, consisting
of one A subunit and 5
B subunits.
 The B subunit weighs
11.6kDa each and
multimerize to form a
pentameric ring, which
binds the holotoxin to a
eukaryotic cell surface
receptor.
Pathogenesis: Cholera Toxin (CT) Structure
cont.
• The A subunit contains an
intracellular ADP-
ribosyltransferase activity.
• The mature A subunit is
proteolytically cleaved to
produce a 21.8kDa A1
polypeptide, which contains
the intracellular enzymatic
activity, and a 5.4kDa A2
polypeptide.
• After cleavage, the A1 and
A2 polypeptides remain
linked by a disulphide bond.
• The crystal structure of CT
revealed that the A and B
subunits are connected
through the C-terminus of
the A2 subunit, which is
inserted through the central
pore of the B pentamer.
Pathogenesis: Cholera Toxin (CT) Structure
cont.
 CT must be assembled for activity, as neither the A nor B
subunit individually can cause secretory diarrhea.
 CT holotoxin is assembled in the periplasmic space.
 The subunits are exported individually into the periplasm
through the cytoplasmic membrane via the general secretion
pathway
Pathogenesis: Mechanism of Action cont.

 The biological activity of

CT is dependent on
binding of the holotoxin
B pentamer to specific
receptors on the
eukaryotic cell.
 The B oligomer binds
with high affinity
exclusively to GM1
ganglioside.

B subunits bind to GM1 Receptor

Pathogenesis: Mechanism of Action cont.

 Internalization is
initiated once CT-GM1
complexes cluster which
then invaginate to form
apical endocytic
vesicles.
Pathogenesis: Mechanism of Action cont.

 These vesicles enter cellular

trafficking pathways leading
to the trans-Golgi network
(TGN).
 The toxin then moves
retrograde via the Golgi
cistern to the ER.
 Once in the ER, CT is
processed to activate the A1
peptide, which then targets
the membrane protein Gs
and adenylate cyclase
(AC)).
Pathogenesis: Mechanism of Action cont.

 Adenylate cyclase (AC)

is activated normally by NORMAL CONDITION
a regulatory protein
(GS) and GTP; however
activation is normally
brief because another
regulatory protein (Gi),
hydrolyzes GTP.
 Pathogenesis: Mechanism of Action cont
 Enzymatically, fragment A1 catalyzes
the transfer of the ADP-ribosyl
moiety of NAD to a component of
the adenylate cyclase system. CHOLERA
 The A1 fragment catalyzes the
attachment of ADP-Ribose (ADPR) to
the regulatory protein forming Gs-
ADPR from which GTP cannot be
hydrolyzed.
 Since GTP hydrolysis is the event GTP can not be
that inactivates the adenylate hydrolized

cyclase, the enzyme remains

continually activated.
 Thus, the net effect of the toxin is to
cause cAMP to be produced at an
abnormally high rate which
stimulates mucosal cells to pump
large amounts of Cl- into the
intestinal contents.
 Pathogenesis: Mechanism of Action cont.

 H2O, Na+ and other  Thus, the toxin-

electrolytes follow due damaged cells become
to the osmotic and pumps for water and
electrical gradients electrolytes causing the
caused by the loss of Cl-. diarrhea, loss of
 The lost H2O and electrolytes, and
electrolytes in mucosal dehydration that are
cells are replaced from characteristic of
the blood. cholera.
 So What’s Out
There?
Virulence Potential

Faruque, Shah et al. 2004. Genetic diversity & virulence potential of environmental Vibrio cholerae population in a cholera
endemic area. PNAS. 101(7); 2123-2129.
 Diagnosis: Visible Symptoms
 Decreased skin turgor
 Sunken eyes, cheeks
 Almost no urine production
 Dry mucous membranes
 Rice 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 TCBS agar
Microbiological & Molecular Methods
of Detection

• Microbiological culture-based methods

using fecal or water samples
• Rapid Tests
– Dark-field microscopy
– Rapid immunoassays
– Molecular methods - PCR
& DNA probes

www.city.niigata.niigata.jp/ info/sikenjo/521s...
 Biotyping
• Biotyping (distinguishing between the Classical and El Tor
biotypes) can be performed by the following tests:
 Polymyxin B sensitivity - Classical biotypes show a 12 to 15 mm zone of
growth inhibition when subjected to polymixin B whereas El Tor
biotypes show only a 1 to 2 mm zone.
 Hemolysin production - most El Tor biotypes will produce hemolysin
and will lyse sheep red blood cells. Classical biotypes do not produce
hemolysin and so will not lyse red blood cells.
 Phage sensitivity - El Tor biotypes are not sensitive to phage IV and will
not be lysed. Classical biotypes are sensitive to phage IV and will be
lysed.
 Agglutination with chicken red blood cells - El Tor strains will
agglutinate while the Classical biotypes will not.
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: 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 - Antibiotics
 Antibiotics are only prescribed to patients with
severe dehydration. Adjunct to oral rehydration
 Reduce fluid loss by half
 Reduce recovery time by half; 2-3 days instead of 4-6
 In normal condition it is not recommended -
 Short duration of illness
 Antibiotic resistance
 Limited gain from usage
 Treatment: Antibiotics
The first choice of antibiotic is doxycycline;
tetracycline and azithromycin are also preferred.
Erythromycin is approved for use in pregnant
women and children.
 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
 Vaccines: Live-Attenuated
 Eliminates need for multiple doses of non-living
antigens
 Ensures that crucial antigens 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
Thank you