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Lecture-22Water Borne Human Pathogens• Topics:• The genus Vibrio•Vibrio choleraeVibrio cholerae– In vivo life cycle of V. cholerae• Disease• Virulence factors– Evolution of pathogenic V. cholerae• Legionella pneumophila• Disease• Virulence factorsThe genus Vibrio• Vibrios are aquatic organisms• They occur in both marine and fresh water habitats • Have relatively simple growth factor requirements– grow in synthetic media with glucose as a carbon and energy source– require salt or sea-water based medium for optimal growth–tolerate alkaline media and sensitive to acidtolerate alkaline media and sensitive to acid– Growth temperature varies 10-37CThe genus Vibrio• Vary in metabolic versatility– Some species can grow on more than 150 different organic compounds as a carbon and energy source– Capable of both respiratory and fermentative metabolism– Some species can grow very fast (double in 10 minutes)• Gram negative straight or curved rods• Motile– In liquid media by means of single polar flagellum– On solid media they may synthesize lateral flagella• Free living or in association with aquatic animals– Live in mutualistic association with fish and other marine life– Pathogenic to fish, vertebrates and invertebratesClinically important Vibrio species• V. vulnificus and V. parahaemolyticus– Halophiles (require salt water environment)– Normal residents of coastal waters– Temperature and salinity of water influences their abundance (cell number increases during the summer)– In the US they are the most common vibrios associated with seafood borne illness•Oyster beds in Texas closed in the summer of 1998 (416 people•Oyster beds in Texas closed in the summer of 1998 (416 people in 13 states fell ill after eating oysters harvested from this location)• In the summer of 1997, V. paraheamlyticus caused large outbreak in the Pacific North West (209 cases, 1 death)Vibrio cholerae•Isolated in pure culture by Robert Koch in 1883•Curved rod shaped•Motile, single polar flagellum•Inhabitants of brackish and estuarine watersFlih h•Gram negative, facultative anaerobe•Can grow in fresh and salt waterM. Waldor•Facultative human pathogenEpidemiology of Cholera• Cholera is endemic to India, Bangladesh, regions of South America Africa Australia and Gulf coast ofSouth America, Africa, Australia and Gulf coast of the US• There has been seven pandemics since 1817– First six started in Indian subcontinent (Ganges delta) and caused by Classical strainsy– The last one started in Indonesia and caused by El Tor strains.Cholera• Water-borne illness • Transmitted by ingestion of food or water contaminated with V. cholerae• The Infectious Dose- ID50varies depending on the pH of the stomach. In healthy volunteers 108bacteria produces infection, after neutralization of stomach acid 104bacteria can cause disease• The small intestine is the primary site of infectionpy• V. cholerae colonize the epithelium without invasion or apparent damage; i.e., This is an extracellular pathogen that does not invade host tissue.• Clinical symptoms are voluminous diarrhea and dehydrationInfection Cycle of Vibrio choleraeReidl and Klose, FEMS Microbiology Reviews, 26:125-139Animal models• Human volunteers: Since Cholera is only a disease of humans, this is the only true model. The Center for Vaccine Development (CVD) at the University of Maryland is the site for many human trials. • Rabbit ligated ileal loop: Adult rabbit ileum is divided into 5-10-cm segments by suture and injected with live bacteria or test material. After 12-24h, animal is sacrificed; loop length and fluid volume measured as ml/cm readout. This assay is useful to quantitate toxin. • Infant mouse (most common current model): Newborn mice are inoculated with bacteria; at sacrifice stomach and entire gut are removed and weighed;with bacteria; at sacrifice, stomach and entire gut are removed and weighed; gut wt/carcass wt = fluid accumulation ratio. • Bacteria can be cultured and competitive index determined. – What are the pros and cons of using a competitive index?Virulence Factors Discovery of Cholera Toxin (CT)• In 1959 De and Chatterjee, injected living V. cholerae or cell free filtrates into the lumen of ligated rabbit ileal loop and observed large amount fluid accumulation.observed large amount fluid accumulation.• In 1969 Finkelstein et al. purified and characterized the toxin.• In 1983, by administering purified CT to volunteers, Levin et al. were able to conclusively demonstrate that the toxin is the major mediator of the cholera syndrome.ajo edato o t ec oeasy do e• Ingestion of only 5μg of purified toxin resulted in production of 1-6L of diarrheal stool.Cholera Toxin• Structure– It is composed of two polypeptides, A subunit and B subunitsubunit– The ratio of B:A is 5:1• The biological activity of CT is dependent on binding of the B pentamer to specific receptors on the eukaryotic cell.• The B oligomer binds with high affinity exclusively to GM1 ganglioside.Pathogenesis: Mechanism of Action• Normally, the epithelial cells of the inner lining of the intestines (lumen) transfer sodium and chloride ions from the inside of the intestines to the blood stream. • The "B" subunit of cholera toxin is bound by a host receptor (like a specific "landing pad") allowing the "A" subunit to enter the cell. • 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. Cholera toxinMode of Action– Catalyzes an ADP-ribosylation reaction– CTX binds to surface sugar on the intestinal cells via B subunits–After binding A subunit which has ADP-ribosylating enzymatic activity is gygy ytransported into host cell– Inside the cell, the A subunit ADP ribosylates (attaches an ADP-ribose) a G protein (largest family of mammalian cell-surface receptors, they mediate cellular response to signaling molecules)– G protein regulates activity of adenylate cyclase–ADP ribosylation leads to increased adenylate cyclase activity–ADP ribosylation leads to increased adenylate cyclase activity– Increased cAMP levels– Increased cAMP within intestinal epitelial cells leads to increased Cl-secretion– Osmotic imbalance causes water flow into the intestinal lumen---DIARRHEA—Virulence Factors• Adherence– Long filamentous pili termed the toxin-coregulated pilus


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