Vibrio cholarae: Mode of transmission, pathogenesis, clinical disease, lab diagnosis, treatment and control

Vibrio spp

The name ‘vibrio’ is derived from the characteristics vibratory mortility.
Vibrio are Gram negative rigid, curved rods that are actively motile by means of a polar flagellum. They are non-sporing and non capsulated. They are natural inhabitants of sea water but are also found in fresh water worldwide.
The vibrio consists of at least 33 species of curved bacilli of which 12 species have been implicated in human infections. The most important member of this genus is – Vibrio cholorae, the causative agent of cholera. It was first isolated by Koch (1883) from cholera patient in Egypt.

Vibrio cholerae

General characteristics

Vibrio cholera are Gram negative, short curved, cylindrical rods shaped bacteria with rounded or slightly printed ends.
Size: about 1.5 mm * 0.2-0.4 mm
Shape: The cell is typically common shaped, it is also called ‘comma’ vibrio but the curvature is often lost on sub culture. Pleomorphism is frequent in old cultures.
Motility: It is actively motile with single sheathed polar flagellum showing darting type of motility.

Habitat:

V. cholera is a salt water bacterium. The primary habitat of the bacteria is the marine ecosystem in association with plankton. The bacteria can multiply freely in the water. The number of bacteria in contaminated water increase during the warm and hot months of the year. In infected humans, Vibrio are present in small intestine.

Mode of transmission of cholera:

Humans and water are the two main reservoirs of infections for cholera.
The infection is transmitted to humans by ingestion of contaminated food and water.
Person to person transmission is rare.
The infection rate is highest in the areas of poor sanitation and hygiene and where potable water is not available.
The infective dose of V. cholerae is usually high, more than 16/6^th orgm/ml because most of the organisms are killed by the high acidity in the stomach.

**Virulence factors of Vibrio cholerae:**

V. cholerae possess following virulence factors:

Cholera toxin (CT):

Cholera toxin also known as choleragen or cholera enterotoxin.
It is a protein (heat-labile) of mol.wt. 85.600 KDa and is destroyed by heating at 56 degree Celsius for 30 minutes.
It is structurally and functionally similar to the heat-labile enterotoxin of E coli through CT is far more potent in biological activity.
CT consists of two major non covalently associated sub units A and B. These sub units are expressed by the genes Cholera toxin A and Cholera toxin B.
A sub unit: The A (active) subunit has mol.wt of 27,215 kDa and exhibits biologic activity. Sub unit A consists of two fragments A1 and A2 connected together by a covalent disulfide bond. A2 fragment links active A1 fragment of the subunit B.
Sub unit B: The B subunit are 5 in number each of the mol.wt. 11,677 kDa. It facilitates the binding of A region to the intestinal cell.
Functions of cholera toxin:
The cholera toxin inhibits the absorption capacity and activates the excretory chloride transport in the intestinal enterocytes, eventually leading to loss on NaCl in the intestinal lumen. The osmotic concentration of the intestinal lumen is balanced by secretion of large quantities of water which is eventually over comes the absortive capacity of intestine and lends to diarrhea.
It also inhibits absorption of sodium and chloride in the intestine.
It also increase skin capillary pamneability. Hence it is also called as a permeability factor.

2. Toxin coagulated pillin (TCP):

The pilli helps in adherence of V. cholera to mucosal cells of the intestine.

3. Accessory colonization factor:

These also helps in adhesion of bacteria to the intestinal mucosa.

4. Hemagglutination protease (mucinase):

This enzymes formally known as cholera lectin is both agglutinin and zinc dependent protease.
The enzyme splits mucus and fibronectin as well as subunit of cholera toxin.
It induces intestinal inflammation and also helps in releasing free vibrios from the bound mucosa to the intestinal lumen.

5. Neuraminidase:

The enzyme destroys neuraminic acid thereby increasing toxin receptors for V. cholera.

6. Sidephores:

It is responsible for sequestration of iron.

Pathogenesis of vibrio cholera:

The infection due to V. cholera is acquired due to the ingestion of contaminated food and drinks. Vibrio are highly susceptible to acids and gastric acidity provides an effective barrier against small doses of cholera vibrios.
In the small intestine, vibrios are enabled to cross the protective layer of mucus and reach the epithelial cells by chemotaxic motility, mucinase and proteolytic enzymes. Adhesion to the epithelial cells surface and colonization is facilitated by toxin coagulated pillin (TCP). Throughout the course of infection the vibrios remain attached to the epithelium but do not damage or invade the cells. The pathological changes induced by Vibrio cholera are biochemical rather than histological.
Vibrio cholerae once attached to the intestinal wall, produce cholera toxin. The B sub unit of cholera toxin binds to the GM1 ganglioside receptors on the surface of jejunal epithelial cells.
Conformational alteration of cholera toxin occurs allowing the presentation of A subunit to cell surface. The A subunit enters the cell and activate adenyl cyclase enzyme system.
Adenyl cyclase activity of cells increases resulting in an increase and accumulation of intracellular cyclic, 3,5- adenosine monophosphate (cAMP).
The increased intracellular cAMP causes inhibition of reabsorption of Na+, K+ and CI- by cells lining the villi and hypersecretion of Cl- and HCO3-ions.
This causes a net loss of sodium, potassium and sodium bicarbonate into the intestine with a corresponding loss to maintain the isotonicity of the intestinal fluid. As a results there is purging diarrhea of ‘rice water stool’ with loss of water and electrolytes that are characteristic of cholera.
***V. cholerae 0139 strain shows a similar pathogenic mechanism to that of V. cholerae 01 except that it produces a unique 0139 LPS and an immunologically related O antigen capsule which enhance virulence of this organism.

Clinical manifestation of Vibrio cholerae:

Cholera:

Cholera frequently called Asiatic cholera or epidemic cholera is a severe diarrheal disease caused by V. cholera.
In severe forms, cholera is a dramatic and terrifying illness in which profuse painless watery diarrhea and effortless vomiting may lead to hypovolemic shock and death in less than 24 hours.
The incubation period is short and varies from 2 to 3 days after ingestion of the bacteria.
The disease may last 4-6 days during which period the patient may pass a total volume of liquid stool equal to twice his body weight.
Profuse, watery diarrhea is the most important manifestation of cholera.
Several abdominal cramp, vomiting, dehydrations are other common symptoms.
The complications are renal failure, pulmonary edema, cardiac arrhythemia, paralytic ileus, electrolyte imbalance hypoglycemia.
The cholera stool is profusely watery, colorless with flacks of mucus said to resemble water in which rise has been washed, hence called ‘rice water stools’. It has a characteristics inoffensive sweetish odor. It contains few leukocytes. In composition it is a bicarbonate rich electrolytic fluid, which contains little protein.

Laboratory diagnosis

Specimen:

Watery stool and mucous flakes from stool
Rectal swab from contact and carriers
Wound swab for isolation of V alginolyticus and V rulnificus

Collection of specimen

Stool should be collected in the acute stage of the disease before the administration of antibiotics
Faecal specimen should be collected in a sterile container
The specimen is best collected by introducing a lubricated catheter into the rectum and letting the liquid stool flow directly into screw capped container
Rectal swabs may be used, provided they are made with good quality cotton wool, absorbing about 0.1-0.2 ml of the fluid. They are useful in the collecting specimens from convalescent who no longer have watery diarrhea.
Collection from a bedpan should be avoided because of the risk of contamination or the presence of disinfectant

Transportation of specimen:

The sample should be processed without delay
If a delay of more than 6 hours is suspected specimen should be transported in enrichment media such as alkaline peptone water.
If transport medium are not available, strips of blotting paper may be soaked in the watery stool and sent to the laboratory packed in plastic envelops. Whenever possible specimens should be plated at the bedside and the incubated plates sent to the laboratory.

Microscopic examination:

Vibrio cholerae are gram negative slightly curved rods with a single flagellum at one end.
Microscopic examination of gram stained stool smear is not recommended for vibrios.
Dark field microscopy is a useful method for demonstrating characteristic motility of the bacilli and its inhibition by antisera. This is a rapid method of examination of stool collected from cases or after enrichment for 6 hours.
Direct immunofluorescence is another rapid method of demonstration of vibrios in the stool.

2. Culture

V. cholerae are strongly aerobic growth being scanty or slow in aerobic condition. They grow within a temperature range of 16-40 degree Celsius. Growth is better in alkaline medium of pH in the range of 6.4-9.6 NaCl (0.5-1%) is required for optimal growth through high concentration of Nacl are inhibitory. Vibrios grow on wide range of media such as

i. Non-selective media:

On nutrient agar– colonies are moist, transcend, 1-2 mm in diameter with a bluish tinge in transmitted light. The growth has a distinctive odor.
On macConkey’s agar- initially the colonies are colorless but may turn to on prolonged incubation due to late fermentation of lactose.
On blood agar– eltor biotypes produces hermolylic colonies. Biotype classical however produce greenish coloration around the colonies which later becomes clear due to hemo-digestion.
In gelatin stab culture: V. cholera produces dibuliform (funnel shape) or napiform liquefaction after 3 days of incubation at 22°.
In peptone water, Vibrio cholerae form a fine surface pellicle in about 6-9 hours of incubation.

**Transport medium for Vibrio cholerae:**

Venkatramen-ramakrishanna medium

It is a simple liquid medium prepared by dissolving 20 gm sea salt and 5 g peptone in 1 litre of distilled water.
The pH of medium is 8.6-8.8.
About 1-3 ml of stool is dispensed in screw capped bottles in volumes of 10-15 ml. Here the vibrios do not multiply but remains viable for several weeks.

Cary blair medium

It is a solid medium. It contains buffered solutions of sodium chloride, sodium thioglycollate disodium hydrogen phosphate, CaCl2 and agar has pH of 8.
It is also suitable medium for Salmonella and Shigella.

Enrichment media:

Alkaline peptone water- pH 8.6
Monsur’s taurocholate tellurite peptone water
Potassium tellurite solution added to make selective for V cholera
pH of 9

Selective media for Vibrio cholerae:

On Tthiosulphate-citrate bile salt sacrose agar (TCBS): V. cholerae produce large, yellow colonies due to the fermentation of sucrose. Non- sucrose fermenting V. parahaemolyticus produce blue green colonies pH of medium is 8.6
On monsur’s GITTA medium (pH 8.5) colonies of V cholera are small translucent with grayish black center and a turbid halo after 24 hours of incubation. Colonies become larger after a prolonged incubation of 48 hours.
On alkaline Bile salt Agar colonies are similar to that on NA. It is modified NA containing 0.5 % solid taurocholate.

3. Cholera red reaction

When V. cholerae are grown for 24 hours in peptone water medium containing adequate amount of tryptophan and nitrate, they produce indole and reduce nitrate to nitrite. On adding a few drops of sulphuric acid, nitroso-indole is formed which is red in color. This reaction was once thought to be diagnostic of cholera, however now it is established that indole producing organism can also reduce nitrates eg. E. coli thus giving positive reaction.

4. String test

A loopful of growth is mixed with a drop of 0.5 % sodium deoxycholate in saline on a slide. If the test is positive the suspensions loses its turbidity becomes mucoid and forms a string when the loop is drawn slowly away from the suspensions.

5. Distilled water motility test

A loopful of growth from a NA subculture is mixed in a drop of sterile distilled water on one end of a slide. On another end loopful of growth is mixed in a drop of peptone water. The preparations are covered with a cover glass, sealed and examined microscopically using 40X objective.
All Vibrios species are immobilized in distilled water but remain motile in the peptone water.

6. Sero-diagnosis:

The complement dependent vibriocidal assay and antitoxin assay using live and killed vibrio suspensions are useful for demonstration of vibrio antibodies in serum.

7. Serotyping of vibrio:

Serotyping of V. cholerae is done by slide agglutination, using specific V cholera 01 antisera. In this test the colonies are picked up with a straight wire and mixed with a drop of antisera on the slide.
Agglutination of the bacteria shows that the test is positive for V. cholerae O1. if positive agglutination of bacteria is repeated using specific Inaba and Ogawa sera for serotyping.
Hikojima strains agglutination well with both Inaba and Ogawa sera. If Agglutination is negative the test is repeated with at least five more colonies as both O1 and non O1 vibrios may co-exist in the same specimen

8. Biotyping of Vibrio:

If the slide agglutination is positive and the colony is identified as V cholera 01, then it is tested by various tests to determine the isolated V cholera 01 as classical or Eltor.
Vibrio colonies that are not agglutinated with V cholera 01 antisera are usually tested with antisera to the H antigen. Vibrio isolates that are agglutinated by H antisera but not by 01 antisera are identified as non-01 cholera vibrio. Such non cholera vibrios are tested for 0139 by using specific antisera against 0139 antigen.

Treatment of cholera:

Oral rehydration therapy: The most important aspect of therapy is prompt water and electrolyte replacement to correct the severe dehydration and salt depletion. This is achieved by oral dehydration therapy consisting of glucose, Nacl, KCl and sodium citrate. The glucose facilitates absorption of sodium in the small intestine and salts present in ORT restore the electrolyte balance and therapy reverse acidosis.
Antibiotics: Antibiotics are of secondary importance. It is used to distinguish the duration of illness. V.cholerae are sensitive to tetracycline, sulphonamides, ampicillin, kanamycin streptomycin, trimethoprim, tetracycline or doxycycline is the drug of choice for adults and trimethoprim, sulfamithoxazole for children. Pyrazolidone is usually recommended for pregnant women.

Prevention and control of cholera:

Prevention measures includes identification and case management improved water supply and sanitation, improved personal hygiene and health education.

Vaccination against Vibrio cholerae:

Killed cholera vaccines

It is a traditional vaccine used against cholera
It is a suspension of 8000 million V. cholerae per ml consisting of equal numbers of Inaba and Ogawa serotypes.
The rate of protection is relatively low and immunity is short lived 3-6 month.

2. Non-living oral B subunit whole cell vaccine

It consists of heat killed whole cell V. cholerae 01 in combination with the purified recombinant B subunit of cholera toxoid. This vaccine has shown to give overall protection of about 80% of vaccinated persons for 2 years.

3. Live oral cholera vaccine

It consist of an attenuated live oral genetically modified V cholera 01 strain.
A single dose confess high protection (95%) against V cholera classical and 65% protection against V cholera Eltor following booster dose given after 3 month.