VIBRIO CHOLERA AND LAB.DIAGNOSIS OF CHOLERA

GUIDED BY PRESENTED BYDr Preeti Srivastava Mr.SHEETAL SHARMAAssociate Professor Msc. Final Year

2Discovery of Cholera Organisms :

VIBRIO CHOLERAE is first isolated by Robert Koch in 1883 in Pond water during a cholera outbreak in Egypt, though it had been observed earlier by Filippo Pacini in 1854 in Florence in denuded epithelium of intestine of patient who had died of Cholera, Pacini give the name Vibrio Cholerae.

3A. FILIPPO PACINI B. ROBERT KOCH

Transmission-Worldwide :

Contaminated water. Contaminated food.

Raw or undercooked seafood. Person to person transmission also possible.

MORPHOLOGY :

VIBRIO CHOLERAE Is Gram –Negative nonsporing, non capsulated , curved or comma –shaped rod , about 1.5-2.4x0.2-0.4 µm in size, with rounded or slightly pointed ends. Because of its typically “comma shaped“ appearance it is also called comma vibrio.

The organism is very actively motile with a single sheathed polar flagellum and the motility is of the “darting type” . When acute cholera stool or young culture is examined microscopically the actively, motile bacteria appear like a “swarm of gnats”.

CULTURE CHARACTERISTICS :

Vibrio Cholerae is aerobic, growth being scanty and slow anaerobically. It grow with in a temperature range of 16-40*C (optimum 37*C) . Grow is better in alkaline medium, the range of pH being 7.4-9.6 (Optimum 8.2), Nacl (0.5-1%) is required for optimal growth, though high concentration ( 6% and above) are inhibitory.

ORDINARY MEDIA On Nutrient agar , after overnight growth, colonies are moist, translucent ,

round disc, about 1-2 mm . On MacConkey Agar colonies are colourless at first but become reddish on

prolonged incubation due to late fermentation of lactose. On Blood Agar colonies are initially surrounded by a zone of greening, which

later become clear due to hemodigestion.

SPECIAL MEDIA :

HOLDING OR TRANSPORT MEDIA Venkataraman-Ramakrishnan (VR) medium is prepared by dissolving 20 g

crude sea salt and 5g peptone in 1 ltr. distilled water and adjusting pH 8.6-8.8 . It dispensed in screw capped bottles in quantities of 10-15 ml . About 1-3 ml of stool must be added to each bottle , in this medium vibrios do not multiply but remain viable for several weeks.

Cary-Blair medium is buffered solution of sodium chloride , sodium thioglycollate, di sodium phosphate, calcium chloride at pH 8.4. It is suitable medium for Salmonella, Shigella and Vibrios.

Autoclaved sea water also serve as holding medium.

SPECIAL MEDIA :

Enrichment Media : Alkaline peptone water at a pH of 8.6. Monsur’s taurocholate tellurite peptone water at pH of 9.2. Both are good enrichment and transport media. Plating Media : Alkaline Bile salt agar (BSA) at pH 8.2 is a simple medium that has stood the test of time

and is widely used. In Monsur’s gelatin taurocholate trypticase tellurite agar (GTTA), cholera vibrio produces

small translucent colonies with greenish black center and turbid halo. The Thiosulphtae citrate bile salt sucrose (TCBS) medium containing thiosulphate, citratr,

bile salt and sucrose is available commercially and is very widely used at present, cholera vibrio produce large colonies which may become green on continued incubation.

Identification :

Vibrio Cholerae may be identified by the String test. A loopful of growth is mixed with a drop of 5% deoxycholate in saline on a slide. If the test is positive the suspension loses its turbidity, become mucoid and forms a “string” when the loop is drawn slowly away from the suspension.

String Test

Gardner and Vankataraman’s Classification Vibrio Group A Group B

(Cholera Vibrios, Biochemical ( Biochemically and antigenically hetrogeneous)

similarities, common H antigen) (Vibrio Cholerae)O subgroups O1(Serogroups)

Non-O1 (currently upto 0-139).

(Biotypes) ‘Classical’ ‘EL Tor’

Serotypes (A.) Ogawa (B.) Inaba (C.) Hikojima

Differentiation between Classical Cholera and El Tor Vibrios :

Test Classical cholera El Tor Haemolysis - + Voges-Proskaure - + Chick erythrocyte aggu. - + Polymyxin B sensitivity + - Gp. IV phage susceptibility + - El Tor phage 5 susceptibility - +

CHOLERA :

Vibrio cholerae is the bacterium that causes Cholera. As this is a food and water borne disease, a person gets infected by eating or drinking infected food or water. Once the bacteria enter the body, it reaches the small intestine. Here, it starts releasing toxins that leads to accumulation of fluids in the small intestine. This is the reason for passing watery stools. Water sources such as waterways, drinking water, groundwater, etc. contaminated with feces is the reason for passing of the cholera bacterium in healthy individuals. It can also be transmitted through unwashed and contaminated foods. Undercooked seafood or raw shellfish also lead to an infection.

PATHOGENESIS :

Cholera is an exclusively human disease. The incubation period varies from 3 hours to 6 days. The faeces and vomitus of human cases or carrier are the main source of infection. The human infection occurs by ingestion of contaminated foods and drinks.

V. Cholerae is non-invasive, and in the final analysis, cholera is caused by the action of an enterotoxin elabroated by the organism after it colonises the small intestine.

Secrete enterotoxin

Enterotoxin binds to intestinal cells

Chloride channels activated

Release Large quantities of electrolytes & bicarbonates

Fluid hypersecretion

Diarrhea

Dehydration

CLINICAL SYNDROME :

Clinical manifestation being on an average 24-48 hrs. after ingestion of bacteria, with sudden onset of painless watery diarrhoea and vomiting. In severe cases ,the volume of stool excreted in first 24 hrs. is 250 ml/kg body weight, with loss of more fluid, faeces- streaked stool specimens turn colourless and odourless, free of protein and speckled with mucus rice water stool.

LAB. DIAGNOSIS :

SPECIMENS : Faecal samples are the best source of culture material.

Specimen should be collected in the acute stage of illness and prior to administration of antibiotics. A lubricated catheter may be introduced into the rectum to collect the liquid stool into a screw capped container .

Rectal swabs made with good quality cotton wool, absorbing about 0.1- 0.2 ml of fluid, may be used. Since cholera vibrios die in few hours in hot climate , the specimen is to be preserved at 4* c or in holding medium.

Transport And Enrichment Media :

TRANSPORT MEDIA : If there are chances of delay, 2 – 3 gms of faeces are emulsified in 10-15

ml- Venkataraman –Ramakrishnan fluid medium or in bile peptone transport medium or in Cary-Blair transport medium.

Enrichment Media : In case of specimen reach the laboratory in a few hours, it may be

collected in enrichment media such as alkaline peptone water or Monsur’s medium which saves the time required for isolation of the organism.

Direct Microscopy :

Diagnosis by microscopy is not recommended as the results are not reliable. Organisms can also be detected directly by Dark field microscopy on a wet mount of fresh stool by demostrating the characteristic darting motility of the vibrio and its inhibition by antiserum.

CULTURE :

Stool sample id directly plated to one or more selective media like BSA (Bile Salt Agar), TCBS (Thiosulphate Citrate Bile salt Sucrose Agar), and Monsur’s GTTA medium.

Specimen sent in enrichment media are to be incubated for 6-8 hrs. Including transit time.

PLATING MEDIA Selective TCBS Agar and Bile salt agar are more frequently used as

platting media. Non selective media Blood Agar and MacConkey’s agar may also be used. The plates to be used should be as fresh as possible and should not be older than 3-5 days. All plates should be dried well before streaking . The inoculated plated generally examined after overnight incubation at 37*c.

COLONY MORPHOLOGY AND STANING :

After overnight incubation, media are examined for typical colonies of v. Cholerae. On MacConkey’s agar colonies are pale and on monsur’s medium the colonies has black centre with turbid halo around the colony. TCBS shows yellow colonies and on BSA translucent colonies are present.

Gram staining from colony show typical gram negative comma shaped bacilli. These show darting motility on hanging drop preparation.

V.Cholerae colonies in TCBS Agar

Biochemical Reactions :

V.Cholerae ferments glucose, mannitol, sucrose, maltose, mannose with acid production. Lactose is usually not fermented. Catalase, Oxidase are positive, VP also positive.

Characteristic V.Cholerae Catalase + Motility + Indole + Voges-Proskauer (VP) + Lactose - Sucrose + Mannitol + Peptone 1% with 0% Nacl +

Cholera Red Reaction :

Carbohydrate metabolism is fermentative, producing acid, but no gas. Cholera vibrios ferment glucose , mannitol, maltose, mannose and sucrose but not inositol and lactose, so lactose split very slowly. Indole is formed and nitrates are reduced to nitrites. These two properties contribute to the ‘cholera red reaction’ which is tested by adding few drops of concentrated sulphuric acid to a 24 hrs. peptone water culture. With cholera vibrios a reddish pink colour develop due to the fermentation of nitrose-indole.

Catalase and oxidase test are positive , methyl red and urease test are negative.

TREATMENT

The treatment of cholera consist essentially of the prompt and adequate replacement of fluid and electrolytes. Oral administration of fluid containing glucose and electrolyte, either alone or supplement of intravenous fluid, is highly successful and freely available method of treating cholera.

Antimicrobial therapy is of secondary importance. Oral tetracycline was recommended for reducing the period of vibrio excretion and need for parental fluid.

T H A N K Y O U