Vibrio cholerae update

Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

Cholera affects Millions• CHOLERA AFFECTS millions, in endemic areas

and causes thousands of deaths especially during seasonal epidemics. Robert Koch, the famous microbiologist of Germany, discovered Vibrio cholerae, the causative organism. Because of its characteristic shape he originally referred to it as comma bacilli. Nearly 7-8 epidemics caused by the pathogen `Vibrio cholerae affected different parts of the world.

Dr.T.V.Rao MD 2

Modes of Transmission• Water (infectious

dose = 109)• Food (infectious

dose = 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

Discovery of Cholera Organisms

• Cholera came to Florence in 1854 during the Asiatic Cholera Pandemic of 1846-63. Pacini became very interested in the disease.  Immediately following the death of cholera patients, he performed an autopsy and with his microscope, conducted histological examinations of the intestinal mucosa. During such studies, Pacini first discovered a comma-shaped bacillus which he described as a Vibrio. 

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Spread of Cholera Pandemics

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Filippo Pacini - 1854• Filippo Pacini, would gain

prominence for his discovery of Vibrio cholera, but not until 82 years after his death, when the international committee on nomenclature in 1965 adopted Vibrio cholerae Pacini 1854 as the correct name of the cholera-causing organism. Until then, many credited Robert Koch (1843-1910) with this seminal discovery. Dr.T.V.Rao MD 6

Robert Koch Isolates V.cholrae

1883• The German physician Robert

Koch, like most of the scientific community, was unaware of Pacino's work at the University of Florence. Yet both independently came to a similar conclusion. Since Koch's findings eventually became accepted by his scientific peers, and were widely know in the popular press, he became the acknowledged discoverer of the 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 the

recognition of V.cholrae transmission in water and the

development of sanitary system

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Morphology and Identification

• V.cholrae is a comma shaped curved rod 2 – 4 µm long’

• It is actively motile by means of polar flagellum.

• On prolonged cultivation, vibrio's may become straight rods that resemble the gram-negative enteric bacteria.

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Vibrio CholeraeVibrio 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 vehicle of transmission

– Gastric acidity• Incubation period

– 1-3 days (½-5 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 membrane

proteins• 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

Enrichment Medium

• Enrichment of the fecal specimens are done on Alkaline peptone water

• Venkataraman Ramakrishan Meidum is simple medium can be used as transport medium

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Culture• V.cholrae produces convex,

smooth, round colonies that are opaque and granular in transmitted light;

• Grow well at 370c on many defined media.

• Vibrio's grow at a very high pH ( 8-5 – 9-5 ) and are rapidly killed by acid

• In resource poor laboratories MacConkey agar 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 that are readily visible against the dark green background of the agar.

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Growth Characteristics

• V.cholerae ferments sucrose and mannose but not arabinose

• A positive Oxidase test is key step in preliminary identification of V.cholerae and other Vibrio's

• Vibrio species are susceptible to compound 0/129 and differentiates from Aeromonas

• Vibrio's usually grow on medium containing 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 in the protection of susceptible hosts.

• V.cholrae has O lipopolysaccharide that confer serologic 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 has determinants that make possible futher typing

• Serotypes are Ogawa, Inaba and Hikojima. Epidemic V.cholerae is

biotyped into 1 Classic 2 El Tor

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El Tor Vibrio's• The El Tor vibrios produce

a Hemolysin and positive results with Voges- Proskauer test

• Resistant to Polymyxin B• Molecular techniques can

also be used to type V.cholrae

• Tests can be done reference laboratories and technically demanding

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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. V cholerae 0139 produces severe watery diarrhea and dehydration that is indistinguishable from the illness caused by V cholerae 01, and appears to be closely related to V cholerae 01 biotype El Tor strains. Specific totals for numbers of V cholerae 0139 cases are unknown because affected countries do not report infections caused by 01 and 0139 separately; however, >100,000 cases of cholera caused by V cholerae 0139 may have occurred.

Dr.T.V.Rao MD 24

V.cholrae- 0-139• V.cholrae 0139 is very

similar to V.cholrae 01 El Tor biotype

• V.cholrae 0139 do not produce 01 type lipopolysaccharide and does not have all the genes necessary to make this antigen

• V.cholrae 0139 make a polysaccharide capsule like other non 01 V.cholrae strains while V.cholrae 01 does 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 01 and 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 intracellular cAMP and results in prolonged hyper secretion of water and electrolytes

• There is increased sodium dependent chloride secretion, and absorption of sodium and chloride is inhibited

• The genes for V.cholrae Enterotoxin are on the bacterial chromosome

• Cholerae Enterotoxin is antigenically related to LT of Escherichia

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Mechanism of Action of Cholera Toxin

REVIEWDr.T.V.Rao MD 29

Mechanism of Action of

Cholera Toxin

1

43

2

NOTE: In step #4, uptake of Na+ and Cl- from the lumen is also blocked.

HCO3- = bicarbonate

which provides buffering capacity.

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Pathology and pathogenesis

• V.cholrae is pathogenic to humans.• When bacteria are consumed with food few

organism as much as 102 – 104 organisms are adequate to cause an attack because of the buffering capacity of the food

• Any medication or conditions that decreases stomach acidity makes a person more susceptible to infection with V.cholrae

• But a person with normal gastric acidity has to consume 1010 or more V.cholrae are when ingested with water

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Clinical events in Cholera

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Pathology

• Cholera is not an invasive infection• Organisms do not reach blood, only act

locally• Virulent V.cholrae organism attach to the

microvillus of the brush border of epithelial cells

• They multiply and liberate cholera toxin and perhaps Mucinase and Endotoxin.

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Clinical manifestations

• Diarrhea occurs as much as 20 – 30 Liters/Day fluids are lost.

• Results in dehydration• Shock• Acidosis• Can lead to death.• About 60% of infections are

caused with classic V.cholrae and are asymptomatic, about 75% of infections are caused by El Tor biotype Dr.T.V.Rao MD 35

Clinical features• The incubation period is 1 – 4 days for person

who develop symptoms, depends on the size of the inoculums ingested

• Manifest with Nausea , vomiting, profuse diarrhea, and abdominal cramps Rice water stool characteristic of cholera loss 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 as Rice water

• On Microscopy contain Mucus, epithelial cells and large number of Vibrio's.

• Milder cases difficult to differentiate from other diarrheal diseases.

• El Tor vibrio's cause milder disease than classic biotypes.

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Specimen Collections

• Stool specimens are collected in acute stage of the disease. before the antibiotics are administered.

• Simple collection of stool in a wide Mouthed container is safe and hygienic.

• Specimens should not be collected from bed pans.

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

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 motile vibrio's.

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Laboratory DiagnosisCulture

• Growth is rapid on Blood agar,

• On TCBS medium typical colonies can be picked in 18 hours.

• The stool specimens can be transported in Venkataraman Ramakrishnan medium

• Alkaline peptone water is ideal enrichment medium

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Bio Chemical Reactions

V.cholrae( Classical ) Hemolysis -ve Voges -proskauer test -vePolymyxin sensitivity +veGroup IV phage Susceptibility +veChick erythrocyteAgglutination -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 slide agglutination tests using anti -0 group 1 or group 139 Antisera and by Biochemical reactions Dr.T.V.Rao MD 44

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 immunity are not known.

• In experimental animals specific IgA antibodies occur in the lumen of intestine

• Vibriocidal antibodies in the serum titer > 1:20 have been associated with protection against colonization and disease

• The presence of antitoxin antibodies have not been asociated 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 be reported immediately to local and state health departments.

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Treatment• Treat all cases / suspect cases promptly• Assess degree of dehydration• Determine if rehydration should be oral

or IV• Don’t wait for laboratory confirmation to

treat• Death rates from severe cholera can be

decreased from ~50% to <1%Dr.T.V.Rao MD 47

Treatment• The most important part of

therapy consists of correction water and electrolyte imbalance to correct severe dehydration and salt depletion.

• Oral Tetracycline tends to reduce stool output in cholera and shortens the period of excretion of vibrio's

• In some endemic areas tetracycline resistance has emerged the genes are carried by transmissible plasmids

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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 have attained the combination of high efficacy, long duration of protection, simplicity of administration, and low cost necessary to make mass vaccination feasible in cholera-affected countries.

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CDC – On Vaccination for Cholerae

Cholera vaccine is no longer required, nor recommended for the vast majority of travellers by the

Centres for Disease Control and Prevention

(CDC). Dr.T.V.Rao MD 51

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 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 or even 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 Chemoprophylaxis

• Chemoprophylaxis with antibiotics is effective.

• Repated injection of vaccine containing either Lipopolysaccharides extracted from Vibrio's or dense Vibrio's suspension can offer limited prevention to heavily exposed persons.

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

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 major threat; however, everyone, especially travellers, should be aware of how the disease is transmitted and what can be done to prevent it.

• Several regions in the Developing countries continue to be endemic locations. Dr.T.V.Rao MD 56

Cholera vaccines and vaccination

• Two types of oral cholera vaccines are available: (i) Dukoral and (ii) Shanchol and mORCVAX. The latter two are identical vaccines in terms of strains but formulated by different manufacturers using different methods

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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 <5 years and do not require booster doses every 6 months.

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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

REVIEWDr.T.V.Rao MD 61

Virulence Factors Associated with Non-cholerae Vibrio's

(Kanagawa positive)

REVIEWDr.T.V.Rao MD 62

Epidemiological Features Aero monas Plesiomonas

Natural HabitatSource of Infection

Fresh or brackish water

Contaminated water or food

Fresh or brackish water Contaminated

water or food

Clinical Features

DiarrheaVomiting

Abdominal CrampsFever

Blood/WBCs in Stool

PresentPresentPresentAbsentAbsent

PresentPresentPresentAbsentPresent

Pathogenesis Enterotoxin (??) Invasiveness

Characteristics of Aeromonas and Plesiomonas Gastroenteritis

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Wish to Know More About Cholerae

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