Upload
muhammad-gulzar
View
827
Download
1
Tags:
Embed Size (px)
Citation preview
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
THANKS