2. Introduction
• Cholera is a well-known disease caused by intestinal infection with the toxin-
producing bacteria Vibrio cholerae.
• It is fatal diarrheal disease results in large volumes of watery stool, causing rapid
dehydration.
• In many developing and underdeveloped countries, cholera remains a major public
health problem.
• Since its endemic origins in Asia, different serotypes of V. cholerae have reached the
pandemic level 7 times.
• Cholera was first recorded in 1563 in a medical report in India and around 1817 it
was found to the rest of the world.
• Historically, this disease is well-known for being associated with several large
epidemics and pandemics.
3. • V
. cholerae is a facultative anaerobe, highly
motile, comma-shaped gram-negative, curved
rod bacteria belonging
family Vibrionaceae with a
to
single
the
polar
flagellum.
• normal inhabitant of aquatic environments
such as drinking water, freshwater,
wastewater, brackish water and sea water.
• It has over 200 identified serotypes based on
antigen and only o1 and o139 are toxigenic
and cause cholera disease.
• It is endemic where socioeconomic
conditions are poor, sanitary systems and
public hygiene are
drinking water
rudimentary
is not
and safe
available.
Conti….
4. Pathogenicity
• Upon entry, the bacterial sheds its outer membrane vesicles, containing all the
membrane modifications that make it vulnerable for the host attack.
• The organisms produce a powerful enterotoxin (cholera toxin) which comprises two
subunitsAand B.
• B subunit binds to receptors on the intestinal cell, enabling the A subunit to enter the
cells.
• A subunit inside cells activates enzyme adenylate cyclase that helps to increase the
level of cAMP ( cyclicAdenosine Monophosphate) in cells.
• Large volume of fluid and electrolytes is secreted into the lumen of intestine causing
watery diarrhea.
5. Mode of Transmission
• The bacterium as are found especially in salt water where they attach themselves
easily to the chitin containing shells of crabs, shrimps and other shellfish.
• Consumption of undercooked or raw marine life species.
• Humans are the only natural host for V. cholerae, and transmission is by the fecal-
oral route.
• Eating contaminated food or drinking contaminated water.
• Spread through skin contact with contaminated water from human feces.
6. Contin…..
Incubation period
• It is the period between the exposure to an infection and the appearance of the first
symptom
• Few hours to 5 days
• Its commonly seen in 1-2 days
People most at risk
• People with low gastric acid levels
• Children 10x more susceptible than adult
• Blood types O>>B>A>AB
7. Symptom
• This bacteria infects the intestine where it then causes diarrhea
• If serious may lead to kidney failure and possible coma
• Some symptoms include :
watery diarrhea
Vomiting
Rapid heart rate
Loss of skin elasticity
Low blood pressure
Thirst and muscle cramps
11. Clinical manifestations
• Cholera is an extremely virulent disease that can cause severe acute watery diarrhea
• Cholera toxin is multimeric protein of 84000 Da
• It is composed of two major regions
• RegionA(28000 Da) : responsible for biological activity of enterotoxin
• Region B(56000 Da) : composed of 5 identical non covalently associated peptide
chain of 11500 Da
• The two region are non covalently bounded
• TheAsubunit require nicking by protease to activate it and it is made up of two
peptide linked by a single disulphide bridge
• Incubation period is 1-4 days
13. Previous TU Question
What is the drug of choice and mechanism of action for the treatment of vibrio cholerae ? (2076, 2074)
• Cholera toxin, released from the bacterium Vibrio cholerae, consists of an ‘A’
subunit coupled to a ‘B’subunit that contains five identical peptides assembled in
a pentameric ring.
• Cholera toxin binds to intestinal enterocytes through interaction of the B subunit
with the GM1 ganglioside receptor. It is then internalized through retrograde
endocytosis.
• The internalizedAsubunit causes constitutive activation of adenylyl cyclase
(AC),
resulting in elevated levels of intracellular cAMP , which causes active secretion
of salt and fluid through activation of the cystic fibrosis transmembrane
conductance regulator (CFTR) ClK channel in the apical plasma membrane of
enterocytes, in addition to inhibition of electroneutral Na absorption .
14. Prevention of diarrhea in cholera can be achieved by :
(a) elimination of V. cholera bacteria through host immunity and/or antibiotics;
(b) inhibition of cholera toxin binding to intestinal epithelial cells;
(c)inhibition of increased cAMP levels by the action of enkephalins on opioid
receptors; and
(d) inhibition of ClK secretion by CFTR ClK channels.
15. Inhibition of cholera toxin receptor binding
Enkepha linase inhibitors
• The enkephalins are endogenous relating substances that prevent fluid secretion by
enterocytes through binding to delta opioid peptide receptors.
• On receptor binding, enkephalins cause activation of inhibitory G proteins (Gi),
resulting in reduced levels of intracellular cAMP and consequent deactivation of
apical membrane CFTR Cl K channels and basolateral KC channels
• Enkephalinase inhibitors such as racecadotril increase the levels of intestinal
enkephalin, resulting in reduced secretion of salt and fluid.
16. CFTR inhibitors
• CFTR is the final rate limiting step for intestinal ClK secretion and thus fluid
secretion in cholera and other enterotoxin-mediated secretory diarrheas.
• small-molecule thiazolidinone, glycine and malonic acid hydrazides and
quinoxalinediones inhibitors of CFTR ClK conductance with sub micromolar
inhibitory potency were discovered by high-throughput screening, and shown to
be effective in preventing ClK and fluid secretion by CT in human intestinal cells
and rodent models
• glycine hydrazide class and proanthocyanidin oligomer of CFTR inhibitors that
also prevent CT-induced fluid secretion in vivo
17. • These targets include determinants of its virulence factor expression and motility.
ToxT gene is a transcriptional activator that induces expression of two important
virulence factors, CT and toxin-coregulated pilus (TCP).
• Virstatin and capsaicin were identified as inhibitors of Vibrio cholerae virulence
factor expression. Virstatin inhibits ToxT dimerization, which is a process required
for ToxT transcription activation of genes encoded for CT and TCP.
• Capsaicin reduces CT and TCP expression by suppressing the expression of ToxT.
• High-throughput screening identified quinazoline-2,4-diamino analog as an
inhibitor of V
. cholerae motility.
Targeting bacterial virulence and motility
18. Diagnosis
Serogrouping
V
. cholerae colonies from gelatin agar (GA) plates were tested to identify their
serogroups using slide agglutination with polyvalent antiserum, followed by
monoclonal and V. cholerae serogroup O1- and O139-specific antisera
Dipstick assay (antigen detection)
The dipstick test was performed by simultaneously introducing both O1 and O139
dipsticks into 200 μl of freshly collected stool sample contained in a tube. A positive
result appeared as two pink lines and a negative result was a single upper pink control
line. The results were discernible within 10 min.
19. Other lab finding
• Dehydration leads to high blood urea
• WBC will be high due to hemoconcentration
• It lead to metabolic acidosis with wide anion gap
20. Alkaline bile salt agar
• Thiosulphate citrate bile salt agar v. Cholerae o1 &
0139 incubate at 37 degree for 24 hours
• Visualization by dark field or phase microscopy
• Gram stain : red curved rods bacteria
• Isolation v. cholerae from patients stools
• Yellow colonies on thiosulphate bile salt sucrose
Molecular test
• PCR
