7/30/2019 Cholera Study Sheet

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Cholera

Causative agent: Vibrio cholerae

*Gram negative, curved rods*Small (0.5-1.5 x 3 micrometers)

*Facultative anaerobes*Fermentative*Require salt for growth (V. cholerae can grow on most media without added salt)

*Susceptible to stomach acids*Strains subdivided into 140 serogroups (O cell wall antigens)*Serogroup O1 subdivided into serotypes (Inaba, Ogawa, and Hikojima) and

biotypes (Classic and El Tor---classified based on certain enzymes produced) Epidemiology: *Serotype O1 responsible for major pandemics with significant mortality in

developing countries*Serotype O139 can cause similar diseases*Organism found in estuarine and marine environments worldwide*associated with chitinous shellfish

*organisms can multiple freely in water*bacterial levels in contaminated waters increase during the warm months*spread by consumption of contaminated food or water

*Direct person-person spread is rare b/c the infectious does is so high (more than108 organisms) because most organisms are killed by stomach acids

Virulence Factors:1) Cholera Toxin (CTX) encoded for by bacteriophage CTXΦ

*two subuints: ctxA (A1 and A2 portion connected by a disulfide bond) and ctxB (5 identical Bsubunits assembled to form a pentamer)

*the bacteriophage binds to the toxin-coregulated pilus (tcp) and moves into the bacterial cell,where it becomes integrated into the V. cholerae genome

*toxin finishes assembly in the periplasm and is then released into the gut lumen*binds to its receptor: the GM1 ganglioside on the luminal surface of the gut epithelial cells*several B subunits must bind to GM1 gangliosides to assure tigh attachment of the toxin

*the entire cholera toxin is endocytosed in caveolae in a clathrin-independent fashion

*caveloin addresses the vesicle to the trans-Golgi network--> vesicle fuses with outer cisternae*pH of the Golgi causes separation of A from B subunits—B pentamers remain in Golgi with no

further action*A subunit contains KDEL sequence transported to cis-Golgi (COPI-dependent)--> pinched→  

off into a vesicle that is moved to the RER--> fuses to get inside RER A subunit reduced by→  disulfide isomerase A1 enters cytoplasm via sec61 avoids ubiquitnylation (has very few lysine→ →  residues) activates ARF by enzymatic transfer of myristic acid to one of the amino acid R groups→  in ARF (the ARF refolds) and by ARF binding to GTP A1-ARF complex moves exits Golgi and→  enters baolateral membrane of the cell A1 subunit binds to the alpha subunit of the→  heterotrimeric G protein A1 subunit catalyzes the hydrolysis/transfer of ADP-ribose from NAD to→  the alpha subunit (releasing nicontinamide) this subunit now blocks the alpha subunit from→  hydrolyzing GTP (it's permamently activated) activated G-alpha subunit activates adenylate→  cyclase, which cleaves ATP to produce cAMP (potent intracellular messenger) cAMP diffuses→  

throughout cell: 4 molecules bind to the regulatory subunit of protein kinase A, which allows thecatalytic subunit to drift away and phosphorylate target proteins like CFTR at the expense of ATP → Ser813 of CFTR phosphorylated chloride transported out of cell into extracellular fluid sodium→ →  ions also are ejected out of the cell by the Na+/K+-ATPase into the lumen ---> water follows saltout of the cells

*Cholera toxin causes the crypt cells of the epithelium to secrete chloride ions (with Na+ andwater following)*In the presence of the toxin, the cells lining the villus are blocked from resorbing sodium andchloride as they normally do*Cholera toxin causes the goblet cells of the epithelium to secrete abnormally large amounts of 

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mucin into the gut lumen

2) Toxin co-regulated pilus* binding site for CTXΦ*mediates adherence to intestinal mucosal cells*nonadherent strains are unable to establish infection*encoded by the tcp gene complexing

3) Chemotaxis Protein

*adhesion factor*encoded for by the cep genes

4) Accessory Cholera Enterotoxin*increases intestinal fluid secretion

5) Zonlua Occludens Toxin*Increases intestinal permeability by loosening the tight junctions of the small intestine

6) Neuraminidase*modifies cell surface to increase GM1 binding sites for cholera toxin

Clinical Summary: begins as an abrupt onset of watery diarrhea and vomiting and can progressto severe dehydration, metabolic acidosis and hypokalemia, and hypolemic shock

*Most exposed have aymptomatic infections or self-limited diarrhea*Some develop severe, rapidly fatal diarrhea

-incubation period 2-3 days after ingestion of V. cholerae cells-abrupt onset of watery diarrhea and vomiting-“rice-water” stools-severe fluid and electrolyte loss can lead to dehydration, painful muscle cramps, metabolic

acidosis (due to bicarbonate loss), cardiac arrhythmia, and renal failure-mortality is 60% in untreated patients; less than 1% if rehydration started promptly

 Immunological Response:

-mostly non-inflammatory infection; toxin is causing illness, not the foreign cells themselves-usually no gross changes to intestinal epithelia or small bowel, but upregulated expression of 

some cytokines (NOT from primary reading, from a Nature paper)<http://www.nature.com/nrmicro/journal/v7/n10/box/nrmicro2204_BX1.html >

Diagnosis:-microscopic examination of stool is generally nonproductive because the organism is diluted inthe large volume of watery diarrhea-also, the organisms cannot be differentiated from other enteric organisms-culture should be performed early in the course of disease with fresh stool specimens maintainedin a neutral to alkaline pH (because Virbio organisms survive poorly in an acidic or dryenvironment)-special selective agar for Vibrios can be used to recover them from specimens with a mixture of organisms -----thiosulfate citrate bile salts sucrose (TCBS) agar; alkaline peptone broth (anenrichment broth, pH 8.6)-isolates can be serotyped using polyvalent antisera

For clinical diagnosis, history is important: recent consumption of shellfish? Recent visit to anendemic area? (Or are you in an endemic area?)

Treatment:-Must treat promptly with fluid and electrolyte replacement (before the massive fluid loss leads tohypovolemic shock)-Oral Rehydration Solution (ORS) is preferred-IV fluid replacement can be used if the patient is unable to drink the fluids ( e.g, our case, theHaiti case study)-antibiotic therapy can reduce toxin production and can help to eliminate the organism morerapidly

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(although of secondary value)-Azithromycin is the drug of choice for children and adults-Resistance to previously recommended drugs such as ciprofloxacin, furazolidone, andtrimethoprim-sulfamethoxazole) has emerged, rendering them uneffective

Public Health Aspects of Cholera:

*People infected with V. cholerae can shed bacteria for the first few days of acute illness and

represent important sources of new infections*Long-term carriage of V. cholerae does not occur, but vibrios are free-living in estuarine andmarine reservoirs*Only improvements in sanitation can lead to effective control of cholera**A variety of vaccines have been developed, but non have provided long-term immunity*One oral vaccine conferred immunity for 62% of trial participants at one year and showed someevidence for herd immunity, but multiple doses are required and protection fades 2-3 years afterimmunization*No vaccine for O139 strains*Because the infectious does of organisms is high, antibiotic prophylaxis is generally unneccesaryin people who use appropriate hygiene