Vibrio cholerae

Vibrio cholerae for masters level students.
Content is about its introduction, causes symptoms, diagnosis its mode of transmission and treatment.

1. VIBRIO CHOLERAE

2. Presentation on
Vibrio Cholerae
Bishal Panth
DipeshThapa
National College, Kathmandu
3
rd
Semester
Presented by :

3. Content:
•Introduction
•Pathogenicity
•Mode of Transmission
•Symptom
•Determinant factors of infection
•Clinical Perceptive
•Diagnosis
•Drugs
•Prevention
}
}
Bishal Panth
DipeshThapa

4. Introduction
•Choleraisawell-knowndiseasecausedbyintestinalinfectionwiththetoxin-
producingbacteriaVibriocholerae.
•Itisfataldiarrhealdiseaseresultsinlargevolumesofwaterystool,causingrapid
dehydration.
•Inmanydevelopingandunderdevelopedcountries,choleraremainsamajorpublic
healthproblem.
•SinceitsendemicoriginsinAsia,differentserotypesofV.choleraehavereachedthe
pandemiclevel7times.
•Cholerawasfirstrecordedin1563inamedicalreportinIndiaandaround1817it
wasfoundtotherestoftheworld.
•Historically,thisdiseaseiswell-knownforbeingassociatedwithseverallarge
epidemicsandpandemics.

5. •V.choleraeisafacultativeanaerobe,highly
motile,comma-shapedgram-negative,curved
rodbacteriabelongingtothe
familyVibrionaceaewithasinglepolar
flagellum.
•normalinhabitantofaquaticenvironments
suchasdrinkingwater,freshwater,
wastewater,brackishwaterandseawater.
•Ithasover200identifiedserotypesbasedon
antigenandonlyo1ando139aretoxigenic
andcausecholeradisease.
•Itisendemicwheresocioeconomic
conditionsarepoor,sanitarysystemsand
publichygienearerudimentaryandsafe
drinkingwaterisnotavailable.
Conti….

6. Pathogenicity
•Uponentry,thebacterialshedsitsoutermembranevesicles,containingallthe
membranemodificationsthatmakeitvulnerableforthehostattack.
•Theorganismsproduceapowerfulenterotoxin(choleratoxin)whichcomprisestwo
subunitsAandB.
•Bsubunitbindstoreceptorsontheintestinalcell,enablingtheAsubunittoenterthe
cells.
•Asubunitinsidecellsactivatesenzymeadenylatecyclasethathelpstoincreasethe
levelofcAMP(cyclicAdenosineMonophosphate)incells.
•Largevolumeoffluidandelectrolytesissecretedintothelumenofintestinecausing
waterydiarrhea.

7. Mode of Transmission
•Thebacteriumasarefoundespeciallyinsaltwaterwheretheyattachthemselves
easilytothechitincontainingshellsofcrabs,shrimpsandothershellfish.
•Consumptionofundercookedorrawmarinelifespecies.
•HumansaretheonlynaturalhostforV.cholerae,andtransmissionisbythefecal-
oralroute.
•Eatingcontaminatedfoodordrinkingcontaminatedwater.
•Spreadthroughskincontactwithcontaminatedwaterfromhumanfeces.

8. Contin…..
Incubation period
•Itistheperiodbetweentheexposuretoaninfectionandtheappearanceofthefirst
symptom
•Fewhoursto5days
•Itscommonlyseenin1-2days
Peoplemostatrisk
•People with low gastric acid levels
•Children 10x more susceptible than adult
•Blood types O>>B>A>AB

9. Symptom
•Thisbacteriainfectstheintestinewhereitthencausesdiarrhea
•Ifseriousmayleadtokidneyfailureandpossiblecoma
•Somesymptomsinclude:
waterydiarrhea
Vomiting
Rapidheartrate
Lossofskinelasticity
Lowbloodpressure
Thirstandmusclecramps

10. Situation of cholera in Nepal

11. Situation of cholera in World wide

12. Virulence Factors
Major virulence factors
•Cholera toxin (CT)
•Toxin co-regulated pilus (TCP)
Minor virulence factors
•Accessory colonization factor (ACF)
•Haemagglutination(HA) protease
•Potential virulence factors
•Outer membrane proteins (OMP)
Previous TU Question
Write down any 5 virulence factors of vibrio cholerae ? (2070)

13. 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
•Region A(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
•The A subunit require nicking by protease to activate it and it is made up of two
peptide linked by a single disulphidebridge
•Incubation period is 1-4 days

14. Strategies for drug therapy of cholera

15. 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 internalized A subunit 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) ClKchannel in the apical plasma membrane of
enterocytes, in addition to inhibition of electroneutral Na absorption .

16. 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 ClKsecretion by CFTR ClKchannels.

17. Inhibition of cholera toxin receptor binding
Enkephalinaseinhibitors
•Theenkephalinsareendogenousrelatingsubstancesthatpreventfluidsecretionby
enterocytesthroughbindingtodeltaopioidpeptidereceptors.
•Onreceptorbinding,enkephalinscauseactivationofinhibitoryGproteins(Gi),
resultinginreducedlevelsofintracellularcAMPandconsequentdeactivationof
apicalmembraneCFTRClKchannelsandbasolateralKCchannels
•Enkephalinaseinhibitorssuchasracecadotrilincreasethelevelsofintestinal
enkephalin,resultinginreducedsecretionofsaltandfluid.

18. CFTR inhibitors
•CFTRisthefinalratelimitingstepforintestinalClKsecretionandthusfluid
secretionincholeraandotherenterotoxin-mediatedsecretorydiarrheas.
•small-moleculethiazolidinone,glycineandmalonicacidhydrazidesand
quinoxalinedionesinhibitorsofCFTRClKconductancewithsubmicromolar
inhibitorypotencywerediscoveredbyhigh-throughputscreening,andshownto
beeffectiveinpreventingClKandfluidsecretionbyCTinhumanintestinalcells
androdentmodels
•glycinehydrazideclassandproanthocyanidinoligomerofCFTRinhibitorsthat
alsopreventCT-inducedfluidsecretioninvivo

19. •These targets include determinants of its virulence factor expression and motility.
ToxTgene is a transcriptional activator that induces expression of two important
virulence factors, CT and toxin-coregulated pilus (TCP).
•Virstatinand capsaicinwere identified as inhibitors of Vibrio cholerae virulence
factor expression. Virstatininhibits ToxTdimerization, which is a process required
for ToxTtranscription activation of genes encoded for CT and TCP.
•Capsaicinreduces CT and TCP expression by suppressing the expression of ToxT.
•High-throughput screening identified quinazoline-2,4-diaminoanalog as an
inhibitor of V. cholerae motility.
Targeting bacterial virulence and motility

20. Diagnosis
Serogrouping
V.choleraecoloniesfromgelatinagar(GA)platesweretestedtoidentifytheir
serogroupsusingslideagglutinationwithpolyvalentantiserum,followedby
monoclonalandV.choleraeserogroupO1-andO139-specificantisera
Dipstickassay(antigendetection)
ThedipsticktestwasperformedbysimultaneouslyintroducingbothO1andO139
dipsticksinto200μloffreshlycollectedstoolsamplecontainedinatube.Apositive
resultappearedastwopinklinesandanegativeresultwasasingleupperpinkcontrol
line.Theresultswerediscerniblewithin10min.

21. 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

22. 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

23. Treatments
Eg: Tetracyclines
Glucose + salt+ water
Eg: dukoral

25. Prevention