vibrio cholerae an update

8/2/2019 Vibrio cholerae an update

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Vibrio cholerae

updateDr.T.V.Rao MD

Dr.T.V.Rao MD 1


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Cholera affects Millions

CHOLERA AFFECTS millions, in endemic areasand causes thousands of deaths especiallyduring seasonal epidemics. Robert Koch, the

famous microbiologist of Germany, discoveredVibrio cholerae, the causative organism.Because of its characteristic shape heoriginally referred to it as comma bacilli.

Nearly 7-8 epidemics caused by the pathogen`Vibrio cholerae affected different parts of theworld.

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Modes of Transmission

Water (infectiousdose = 109)

Food (infectiousdose = 103)

Person-to-person

The 19th-century illustration depicting the spirit of death at a pump was taken from

http://news.nationalgeographic.com/news/2004/06/0614_040614_tvcholera.htmlDr.T.V.Rao MD 3


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Discovery of Cholera Organisms

Cholera came to Florence in 1854 duringthe Asiatic Cholera Pandemic of 1846-63.Pacini became very interested in the

disease. Immediately following the deathof cholera patients, he performed anautopsy and with his microscope,conducted histological examinations of

the intestinal mucosa. During suchstudies, Pacini first discovered a comma-shaped bacillus which he described as aVibrio.

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Spread of Cholera Pandemics

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Filippo Pacini - 1854

Filippo Pacini, would gain

prominence for his

discovery ofVibrio cholera,

but not until 82 years after

his death, when theinternational committee on

nomenclature in 1965

adopted Vibrio cholerae

Pacini 1854 as the correctname of the cholera-causing

organism. Until then, many

credited Robert Koch (1843-

1910) with this seminal

discovery.Dr.T.V.Rao MD 6


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Robert Koch Isolates V.cholrae1883

The German physicianRobert Koch, like most ofthe scientific community,was unaware of Pacino'swork at the University of

Florence. Yet bothindependently came to asimilar conclusion. SinceKoch's findings eventuallybecame accepted by his

scientific peers, and werewidely know in the popularpress, he became theacknowledged discoverer ofthe cholera organism.

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Cholerae Outbreaks

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Vibrio's in Nature Vibrio's are among the most common bacteria

in surface water worldwide.

They appear curved aerobic rods and are

motile,possesing a polar flagellum

V.cholrae serogroups O1 and O139 cause

cholera in humans, while other vibrio's may

cause sepsis or enteritis

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Vibrio cholerae

Epidemiology and spread of

Cholera closely parallels therecognition of V.cholrae

transmission in water and thedevelopment of sanitary system

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Morphology and Identification

V.cholrae is a commashaped curved rod 2 4m long

It is actively motile bymeans of polarflagellum.

On prolonged

cultivation, vibrio's maybecome straight rodsthat resemble the gram-negative entericbacteria.

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Vibrio Cholerae

Vibrio spp.

V. cholerae

V. choleraeNon-O1

V. cholerae O1

BiotypeClassicalEl Tor

SerotypeOgawaInaba

ToxinToxigenicNon-toxigenic

Other vibrios

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Vibrio cholerae O1

Salt resistant

Heat and acid sensitive

El Tor biotype Asymptomatic infections common

75% asymptomatic

18% mild diarrhea

1-5% severe-cholera gravis

Fast growing in food

Lengthy survival in environment

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Vibrio cholerae O1

Infectious dose:

106 108

Varies with vehicleof transmission

Gastric acidity

Incubation period 1-3 days (-5

days)

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Transmission-worldwide

Contaminated water

Contaminated food

Raw or undercooked seafood

Rice, cereals, gruels left at ambient

temperature Person to person transmission not common

Fecal-oral transmission is possible

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Known Virulence Factors

Integrons

Toxins CT

HA Protease

RTX Toxin ACE and Zot

Adherence/Adhesins Accessory Colonization Factors (ACF)

OmpU & other Omp Proteins - outer membraneproteins

Mannose-fucose-resistant cell hemagglutinin &Mannose sensitivev hemagglutinin (Faruque,2002)

Toxin Co-regulated Pilus (TCP)Image from: http://www.amnh.org/nationalcenter/infection/01_mic/01a_bac.htmlDr.T.V.Rao MD 16


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Enrichment Medium

Enrichment of thefecal specimens are

done on Alkalinepeptone water

VenkataramanRamakrishan Meidum

is simple medium canbe used as transportmedium

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Culture V.cholrae produces convex,

smooth, round colonies thatare opaque and granular intransmitted light;

Grow well at 370c on manydefined media.

Vibrio's grow at a very highpH ( 8-5 9-5 ) and are

rapidly killed by acid In resource poor

laboratories MacConkeyagar can be used.

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Selective Medium - TCBS

V.cholrae grows well

on Thiosulphate

citrate bile sucrose(TCBS ) agar, on

which it produces

yellow colonies thatare readily visible

against the dark

green background ofthe a ar.Dr.T.V.Rao MD 19


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Growth Characteristics V.cholerae ferments sucrose and

mannose but not arabinose

A positive Oxidase test is keystep in preliminary identificationof V.cholerae and other Vibrio's

Vibrio species are susceptible tocompound 0/129 anddifferentiates from Aeromonas

Vibrio's usually grow on mediumcontaining 6% Nacl

Halophilic vibrios need and grow

in the presence of > 6% Nacl On Blood agar Vibrios show

hemodigesion

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Antigenic Structure and

Biological Classification

Many Vibrio's share a single healable H antigen.Antibodies to H antigen are probably not involved inthe protection of susceptible hosts.

V.cholrae has O lipopolysaccharide that conferserologic specificity

There are at least 139 O antigen groups

V.cholrae strains of O group 1 and O group 139 cause

classic cholera. Occasionally non 01/non 0139 V.cholrae cause

Cholera like disease

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V.cholrae - typing

V.cholrae 01 hasdeterminants that makepossible futher typing

Serotypes areOgawa, Inaba and

Hikojima.

Epidemic V.cholerae is

biotyped into1 Classic

2 El Tor

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El Tor Vibrio's

The El Tor vibrios producea Hemolysin and positiveresults with Voges-Proskauer test

Resistant to Polymyxin B

Molecular techniques canalso be used to typeV.cholrae

Tests can be donereference laboratoriesand technicallydemanding

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New Epidemic of

V. cholrae 0139 The epidemic of cholera caused by V cholerae 0139

has affected at least 1 1 countries in southern Asia. Vcholerae 0139 produces severe watery diarrhea anddehydration that is indistinguishable from the illness

caused by V cholerae 01, and appears to be closelyrelated to V cholerae 01 biotype El Tor strains.Specific totals for numbers of V cholerae 0139 casesare unknown because affected countries do not

report infections caused by 01 and 0139 separately;however, >100,000 cases of cholera caused by Vcholerae 0139 may have occurred.

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V.cholrae- 0-139

V.cholrae 0139 is verysimilar to V.cholrae 01 ElTor biotype

V.cholrae 0139 do not

produce 01 typelipopolysaccharide anddoes not have all the genesnecessary to make thisantigen

V.cholrae 0139 make apolysaccharide capsule likeother non 01 V.cholraestrains while V.cholrae 01does not make a capsule

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V.cholrae 0139

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V.cholrae 0139

Epidemic

The epidemic of cholera caused by V

cholerae 0139 has affected at least 1 1

countries in southern Asia. V cholerae

0139 produces severe watery diarrhoea

and dehydration that is indistinguishable

from the illness caused by V cholerae 01and appears to be closely related to V

cholerae 01 biotype El Tor strains.

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Vibrio cholerae - Enterotoxin V.cholrae produce heat labile enterotoxin with a Moll wt. of

about 84,000 consisting of sub units A ( MW 28,000 ) and B

Ganglioside GM1 serves as a mucosal receptor for subunit B,which promotes entry of subunit A into the cell

Activation of subunit A1 yields increased levels of intracellularcAMP and results in prolonged hyper secretion of water andelectrolytes

There is increased sodium dependent chloride secretion, andabsorption of sodium and chloride is inhibited

The genes for V.cholrae Enterotoxin are on the bacterialchromosome

Cholerae Enterotoxin is antigenically related to LT ofEscherichia

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Mechanism of Action of Cholera Toxin


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Mechanism of Action of Cholera Toxin

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1 2


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Mechanism

of Action of

Cholera

Toxin

1

43

2

NOTE: In step #4,uptake of Na+ and Cl-

from the lumen is alsoblocked.

HCO3- = bicarbonate

which provides

buffering capacity. Dr.T.V.Rao MD 30


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Pathology and pathogenesis

V.cholrae is pathogenic to humans.

When bacteria are consumed with food feworganism as much as 102 104 organisms areadequate to cause an attack because of the bufferingcapacity of the food

Any medication or conditions that decreasesstomach acidity makes a person more susceptible toinfection with V.cholrae

But a person with normal gastric acidity has toconsume 1010 or more V.cholrae are when ingestedwith water

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Clinical events inCholera

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Pathology

Cholera is not an invasive infection

Organisms do not reach blood, only actlocally

Virulent V.cholrae organism attach to themicrovillus of the brush border ofepithelial cells

They multiply and liberate cholera toxinand perhaps Mucinase and Endotoxin.

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Clinical manifestations

Diarrhea occurs as much as20 30 Liters/Day fluids arelost.

Results in dehydration

Shock

Acidosis

Can lead to death.

About 60% of infections arecaused with classicV.cholrae and areasymptomatic, about 75%of infections are caused by

El Tor biotype Dr.T.V.Rao MD 35


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Clinical features

The incubation period is 1 4 days for personwho develop symptoms, depends on the sizeof the inoculums ingested

Manifest withNausea , vomiting,

profuse diarrhea, and abdominal cramps

Rice water stool characteristic of choleraloss of fluid leads to profound dehydration

Circulatory collapse and anuria.

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Presentation- Rice water diarrheal

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Stool Examination

Stool specimen appear asRice water

On Microscopy contain

Mucus, epithelial cells andlarge number of Vibrio's.

Milder cases difficult todifferentiate from otherdiarrheal diseases.

El Tor vibrio's cause milderdisease than classicbiotypes.

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Specimen Collections

Stool specimens arecollected in acute stageof the disease. before

the antibiotics areadministered.

Simple collection ofstool in a wideMouthed container issafe and hygienic.

Specimens should notbe collected from bedpans.

Vomitus not advised. Dr.T.V.Rao MD 39


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Diagnosis

Stool culture: Toxigenic Vibrio cholerae O1

Use Cary Blair Transport media if available

Viable for many days at room temperature

Use TCBS media for culture

Use V. cholerae serogroup O1 antisera

Confirm presence of cholera toxin

Cholera Rapid Test Dipsticks

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Laboratory Diagnosis

Mucus flecks from

stool are cultured.

Smears are not

useful for diagnosis.

Dark field

microscopy shows

rapidly motilevibrio's.

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Laboratory Diagnosis

Culture

Growth is rapid onBlood agar,

On TCBS mediumtypical colonies can be

picked in 18 hours. The stool specimens

can be transported inVenkataraman

Ramakrishnan

medium Alkaline peptone water

is ideal enrichmentmedium

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Bio Chemical Reactions

V.cholrae( Classical )

Hemolysis -ve

Voges -proskauer test -ve

Polymyxin sensitivity +ve

Group IV phage

Susceptibility +ve

Chick erythrocyte

Agglutination -ve

V.cholrae (El Tor)

+ve

+ve

-ve

-ve

+ve

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Confirmatory Tests for V.cholrae V.cholrae organisms

are further identified

by slideagglutination tests

using anti -0 group 1

or group 139Antisera and by

Biochemical

reactions Dr.T.V.Rao MD 44


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Immunity in Cholerae Gastric acid produces some protection against

cholera vibrios.

An attack of cholera if followed by immunity to

reinfection but the duration and degree of immunityare not known.

In experimental animals specific IgA antibodies occurin the lumen of intestine

Vibriocidal antibodies in the serum titer > 1:20 havebeen associated with protection against colonizationand disease

The presence of antitoxin antibodies have not beenasociated with protection

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When you suspect Cholera

The diagnosis of cholera should be

considered in patients with watery

diarrheal who have recently (i.e.,within 7 days) returned from

cholera-affected countries. Patients

with suspected cholera should bereported immediately to local and

state health departments.Dr.T.V.Rao MD 46


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Treatment

Treat all cases / suspect cases promptly

Assess degree of dehydration

Determine if rehydration should be oralor IV

Dont wait for laboratory confirmation to

treat

Death rates from severe cholera can be

decreased from ~50% to


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Treatment The most important part of

therapy consists ofcorrection water andelectrolyte imbalance tocorrect severe dehydration

and salt depletion. Oral Tetracycline tends to

reduce stool output incholera and shortens theperiod of excretion of

vibrio's In some endemic areastetracycline resistance hasemerged the genes arecarried by transmissibleplasmids

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Vaccines for Cholera

The licensed parenteral cholera vaccine

provides only limited and brief protection

against V cholerae 01, may not provide

any protection against V cholerae 0139,

and has a high cost-benefit ratio;

therefore, the vaccine is not

recommended for travellers. New oral

cholera vaccines are being developed.

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Newer Vaccines

New oral cholera vaccines are being

developed and provide more reliable

protection, although still at a high cost per

case averted. None of these vaccines haveattained the combination of high efficacy, long

duration of protection, simplicity of

administration, and low cost necessary tomake mass vaccination feasible in cholera-

affected countries.

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CDC On Vaccination for CholeraeCholera vaccine is no longerrequired, nor recommendedfor the vast majority oftravellers by the Centresfor Disease Control andPrevention (CDC).

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Epidemiology of Cholerae

Six Pandemics of Cholera occurred between

1817 1923.

Most likely V.cholrae 01 of Classical type

contributed to pandemics.

All pandemics originated in Indian continent.

The seventh pandemic originated in Celebes

Islands in Indonesia on 1961.

Spread far and wide

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8th P d i ?


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8th Pandemic ?

Spread of 0-139

Several identify that onset of 0-139 is considered as

8th pandemic started in India.

Cholerae is spread by contact with persons in early oreven mild illness.

By contaminated water, food, flies

Only 1 -5% of exposed will get effected

Carrier stage seldom exceeds 3- 4 weeks.

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Control of Cholerae

Needs improvement of

Sanitation associated

with water treatment

and food.

Patients infected

preferably isolated .

Excreta disinfected

All contacts to be

followed upDr.T.V.Rao MD 54

Use of Vaccine and


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Use of Vaccine andChemoprophylaxis

Chemoprophylaxis with antibiotics is

effective.

Repated injection of vaccine containing

either Lipopolysaccharides extracted

from Vibrio's or dense Vibrio'ssuspension can offer limited prevention

to heavily exposed persons.

Vaccines not useful in Epidemic controlsDr.T.V.Rao MD 55


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Prevention of Cholera

Although cholera can be life-threatening, it is

easily prevented and treated. In the United

States, because of advanced water and

sanitation systems, cholera is not a majorthreat; however, everyone, especially

travellers, should be aware of how the disease

is transmitted and what can be done toprevent it.

Several regions in the Developing countries

continue to be endemic locations.Dr.T.V.Rao MD 56


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Cholera vaccines and vaccination

Two types of oral

cholera vaccines are

available: (i) Dukoral

and (ii) Shanchol andmORCVAX. The latter

two are identical

vaccines in terms of

strains but formulatedby different

manufacturers using

different methodsDr.T.V.Rao MD 57

Ch t i ti f D k l Sh h l


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Characteristics of Dukoral, Shancholand mORCVAX

The available oral cholera vaccines are

safe29 and provide sustained protection

of >50% that lasts for 2 years in endemic

populations.30 Shanchol and mORCVAX

have demonstrated longer term

protection in children aged


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Potential use of oral cholera vaccines

Use of oral cholera vaccines in

emergency situations is accepted but

remains a challenge. To date, there is no

specific indication for use of oral cholera

vaccines in endemic situations, and

intervention studies are being performed

to prove their effectiveness as a public

health tool.

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Other Bacteria resembling Vibrio

cholerae

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Characteristics of Aeromonas and

Plesiomonas Gastroenteritis

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Virulence Factors Associated with


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Virulence Factors Associated with

Non-cholerae Vibrio's

(Kanagawa positive)

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EpidemiologicalFeatures Aero monas Plesiomonas

Natural Habitat

Source ofInfection

Fresh or brackishwater

Contaminated water

or food

Fresh or brackishwater Contaminated

water or food

ClinicalFeatures

Diarrhea

VomitingAbdominal

Cramps

Fever

Blood/WBCs in

Stool

Present

PresentPresent

Absent

Absent

Present

PresentPresent

Absent

Present


Plesiomonas Gastroenteritis

Dr.T.V.Rao MD 63

Wish to Know More About


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Wish to Know More About

Cholerae

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Programme created by Dr.T.V.Rao MD

for Medical and Health care workers in

the Developing World

Email

[email protected]

vibrio cholerae an update

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