it\’s a brief presentation on Vibrio cholerae, its pathogenesis and lab. diagnosis.

2. Vibriocholerae MUHAMMAD MOAZZAM GULZAR

3. INTRODUCTION Comma shaped Curved rod. 2 – 4 µm long Actively motile. Polar flagellum. On prolonged Cultivation vibrios may become straight rods that resemble the gram-negative enteric bacteria.

4. Culture 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 agar can be used.

6. Classification: O1 Antigen

7. Transmission Contaminated food or water Inadequate sewage treatment Lack of water treatment Improperly cooked shellfish Transmission by casual contact unlikely

8. Hanging latrine on Meghna River, Nepal

9. People with low gastric acid levels Children: 10x more susceptible than adults Elderly Blood types O>> B > A > AB People Most at Risk

11. V. Cholerae Afflicted Areas (2000)

13. PATHOGENESIS

14. Infectious Dose 106-1011 colony-forming units Why such a high dosage? Series of changes as moves from aquatic environment to intestine Acidic environment of stomach Intestinal environment

15. Pathology Cholera is NOTan invasive infection Organisms DO NOT reach blood, only act locally Virulent V.cholerae organism attach to the microvillus of the brush borderof epithelial cells They multiply and liberate cholera toxin (CT) and perhaps Mucinase and Endotoxin.

16. Clinical events in Cholera

17. 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

18. Pathogenesis: cont. Colonization of the intestinal microvilli and the subsequent production and release of cholera toxin, lead to the purging diarrhea.

19. Cholera Toxin (CT) Structure Cholera toxin consist if two subunits; A & B. The A subunit contains an intracellular ADP-ribosyl-transferaseactivity. 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.

20. 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.

21. Mechanism of Action cont. Adenylatecyclase (AC) is activated normally by a regulatory protein (Gs) and GTP; however activation is normally brief because another regulatory protein (Gi), hydrolyzes GTP. NORMALCONDITION

22. Mechanism of Action cont. A1 catalyzes the transfer of the ADP-ribosyl moiety of NAD to a component of the adenylatecyclase system. So, the ADP-Ribose (ADPR) got attach to the Gs forming Gs-ADPRfrom which GTP cannot be hydrolyzed. Since GTP hydrolysis is the event that inactivates the adenylatecyclase, the enzyme remains continually activated. CHOLERA

23. How Does Cholera Toxin Work? Inactivates GTPase function of Gi-protein. Gsproteins stuck in “On” position 100 fold increase in cAMP production Activation of ion channels which lead to pumping of large amounts of Cl- into the intestinal lumen. H2O, and electrolytes follow Cl- due to the osmotic and electrical gradients.

24. 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.

25. Pathogenesis: Mechanism of Action: Overview

27. How Enterotoxin acts

28. LABORATORY DIAGNOSIS

29. Diagnosis: Visible Symptoms Abdominal 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(107vibrios/mL) RICE WATER STOOLS

31. 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

32. Laboratory DiagnosisCulture Grwoth is rapid on Blood agar, On TCBS medium typical colonies can be picked in 18 hours. The stool specimens can be transported in VenkatramanRamakrishnan medium Alkaline peptone water is ideal enrichment medium

33. Growth Characteristics Sucrose and mannose fermenterbut NOTarabinose Oxidasepositve(differentiates b/w V.choleraeand other Vibrios) Vibriospecies are susceptible to compound 0/129 (differentiates from Aeromonas) Usually grow on medium containing 6% NaCl(differentiates from Halophilicvibrios that need > 6% Nacl) On Blood agar Vibrios show hemodigesion

34. 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

35. 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

36. THANKS O8081 08083