Vibrio cholerae

MUHAMMAD MOAZZAM GULZAR

INTRODUCTION

• Comma shaped • Curved rod.• 2 – 4 µm long• Actively motile.• Polar flagellum.

• On prolonged Cultivationvibrios may become straight rods that resemble the gram-negative enteric bacteria.

Colonies are • Convex, • Smooth, • Round • opaque and granular

in transmitted light;

• Grow well at 370c on many defined media.

• Grow at pH ( 8.5 – 9.5 ) • Rapidly killed by acid

• In resource poor laboratories

MacConkey’s agarcan be used.

Culture

Selective Medium - TCBS

•Grows well on

Thiosulphate Citrate

Bile Sucrose (TCBS )agar,

•Yellow coloniesreadily visible against the dark greenbackground of theagar.

Classification: O1 Antigen

TransmissionContaminated food or

waterInadequate sewage treatmentLack of water treatmentImproperly cooked shellfish

Transmission by casual contact unlikely

Hanging latrine on Meghna River, Nepal

People with low gastric acid levels

Children: 10x more susceptible than adults

Elderly

Blood types

O>> B > A > AB

People Most at Risk

V. Cholerae Afflicted Areas (2000)

PATHOGENESIS

Infectious Dose106-1011 colony-forming units

Why such a high dosage?

Series of changes as moves from aquatic environment to intestine

Acidic environment of stomach

Intestinal environment

Pathology

• Cholera is NOT an invasive infection

• Organisms DO NOT reach blood, only act locally

• Virulent V.cholerae organism attach to the microvillus of the brush border of epithelial cells

• They multiply and liberate cholera toxin (CT) and perhaps Mucinase and Endotoxin.

Clinical events in Cholera

Clinical manifestations

• As much as 20 – 30 Liters/Day fluids are lost. (Diarrhea)

• Results in dehydration

• Shock

• Acidosis

• Can lead to death.

• About 60% of infections are caused with classic V.cholerae and are asymptomatic, about 75% of infections are caused by El Tor biotype

Pathogenesis: cont.

Colonization of the intestinal microvilliand the subsequent production and release of cholera toxin, lead to the purging diarrhea.

Cholera Toxin (CT) Structure

• The A subunit contains an intracellular ADP-ribosyl-transferase activity.

• The mature A subunit is proteolytically cleaved to produce a 21.8kDa A1 polypeptide, which contains the intracellular enzymatic activity, and a 5.4kDa A2 polypeptide

• The subunit B is composed of pentamer of similar protein.

• The crystal structure of CT revealed that the A and B subunits are connected through the C-terminus of the A2 subunit, which is inserted through the central pore of the B pentamer.

Cholera toxin consist if two subunits; A & B.

Mechanism of Action cont.

CT binds to ganglioside (GM1) on cell membrane of epithelial cell.

After binding of CT subunit A cleaves into A1 and A2

After cleavage, the A1 and A2 polypeptides remain linked by a disulphide bond.

Internalization is initiated once CT-GM1 complexes cluster which then invaginate to form apical endocytic vesicles.

Mechanism of Action cont.

Adenylate cyclase (AC) is activated normally by a regulatory protein (Gs) and GTP; however activation is normally brief because another regulatory protein (Gi), hydrolyzes GTP.

NORMAL CONDITION

Mechanism of Action cont.

A1 catalyzes the transfer of the ADP-ribosyl moiety of NAD to a component of the adenylate cyclase system.

So, the ADP-Ribose (ADPR) got attach to the Gs forming Gs-ADPR from which GTP cannot be hydrolyzed.

Since GTP hydrolysis is the event that inactivates the adenylate cyclase, the enzyme remains continually activated.

CHOLERA

Mechanism of Action cont.Overview

Normally, the epithelial cells transfer sodium and chloride ions from the inside of the intestines to the blood stream.

Once inside the cell the "A" subunit causes a change in the regulation of the cells genes and as a result, the flow of ions and water is reversed.

Thus, the toxin-damaged cells become pumps for water and electrolytes causing the diarrhea, loss of electrolytes, and dehydration that

are characteristic of cholera.

Pathogenesis: Mechanism of Action: Overview

How Enterotoxin acts

LABORATORY DIAGNOSIS

Diagnosis: Visible SymptomsAbdominal cramping

Vomiting, frequently watery,

Sunken eyes, cheeks

Almost no urine production

Dry mucous membranes

Watery diarrhea consists of:

fluid without RBC, proteins

electrolytes

enormous numbers of vibriocholerae (107 vibrios/mL)

RICE WATER STOOLS

Laboratory Diagnosis

Visualization by dark field or phase microscopy

Look like “shooting stars”

Gram Stain

Red, curved rods

Isolate V. cholerae from patient’s stool

Plate on TCBS agar

Yellow colonies form

Laboratory DiagnosisCulture

• Grwoth is rapid on Blood agar,

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

• The stool specimens can be transported in VenkatramanRamakrishnan medium

• Alkaline peptone water is ideal enrichment medium

Growth Characteristics

• Sucrose and mannose fermenterbut NOT arabinose

• Oxidase positve (differentiates b/w V.cholerae and other Vibrios)

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

• Usually grow on medium containing 6% NaCl (differentiates from Halophilic vibrios that need > 6% Nacl)

• On Blood agar Vibrios show hemodigesion

Bio Chemical Reactions

V.cholerae ( Classical )

Hemolysis -ve

Voges-proskauer test -ve

Polymyxin sensitivity +ve

Group IV phage

Susceptibility +ve

Chick erythrocyte

Agglutination -ve

(El Tor)

+ve

+ve

-ve

-ve

+ve

Confirmatory Tests for V.cholerae

• V.chlorae organisms are further identified by slide agglutination tests using anti -0 group 1 or group 139 Antisera and by Biochemical reactions

O8081 08083

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