LECTURE OUTLINE. The genus Vibrio Vibrio cholerae Epidemiology of cholera Pathogenesis. Vaccine development Ecology of V. cholerae

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1 LECTURE OUTLINE The genus Vibrio Vibrio cholerae Epidemiology of cholera Pathogenesis Disease Virulence factors Vaccine development Ecology of V. cholerae Environmental survival and persistence Phase variation Biofilm formation

2 The 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 acid Growth temperature varies 10-37C

3 Vary in metabolic versatility The genus Vibrio 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 invertebrates

4 Clinically 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 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 NorthWest (209 cases, 1 death)

5 Clinically Significant Vibrio species V. parahemolyticus Diarrhea Consumption of contaminated sea food Infective dose is one million cells Most people develop symptoms (12-24 hours) will recover in 2-3 days Can cause large outbreaks Common pathogen in Asia (Taiwan, Japan) Strains endemic to Asia introduced to US by ballast water discharged from ships which traveled to Asia Is microbial traffic important in emergence of pathogens?

6 Clinically Significant Vibrio species V. vulnificus Diarrhea/Sepsis (microorganism in blood stream) Consumption of contaminated sea food Exposure to brackish or salt water containing the pathogen if the skin is broken Infective dose is 100 cells Healthy people with intact immune system develop gastroenteritis People with liver disease, cancer,aids, diabetes are at high risk Symptoms develop from 7 hours to several days after exposure Does not cause large outbreaks but cause severe and often fatal infections

7 Vibrio cholerae Isolated in pure culture by Robert Koch in 1883 Gram negative, facultative anaerobe Curved rod shaped Motile, single polar flagellum Over 200 serotype is identified based on LPS O-antige Disease cholera is caused by O1 and O139 Inhabitants of brackish and estuarine waters Can grow in fresh and salt water Facultative human pathogen M. Waldor

8 DEFINITIONS FROM (Bacterial Pathogenesis of Salyers and Whitt) Epidemiology study of incidence, transmission and prevention of disease Endemic--disease continually present at low levels in the community Epidemic--disease that appears sporadically and affects many individuals in a community Pandemic epidemic involving different countries

9 Epidemiology of Cholera Cholera is endemic to India, Bangladesh, regions of South 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 strains The last one started in Indonesia and caused by El Tor strains Since 1992, a new serotype has emerged, O139 which may represent the eight pandemic strain

10 Epidemiology of Cholera High degree of clustering of cases by location and season Highest rate of infection in children 1-5 years of age Protection against the disease is improved by sanitation and preexisting immunity Cholerae has been categorized as an emerging and re-emerging infections

11 WHO REPORT The seventh pandemic is still ongoing and the number of affected countries continues to increase especially in Africa. We do not know whether epidemic strain V. cholerae O139 Bengal will continue to be restricted to its present geographical area, or spread further. The threat of a new pandemic caused by V. cholerae O139 Bengal cannot be ruled out. An increasing number of geographic areas are becoming endemic for cholera reflecting a failure of effective epidemic control. Case fatality rates in Africa remain unacceptably high.

12 Water-borne illness Disease CHOLERA Transmitted by ingestion of food or water contaminated with V. cholerae The Infectious Dose- ID 50 varies depending on the ph of the stomach. In healthy volunteers bacteria produces infection, after neutralization of stomach acid 10 4 bacteria can cause disease The small intestine is the primary site of infection Clinical symptoms are voluminous diarrhea and dehydration

13 Infection Cycle of Vibrio cholerae Reidl and Klose, FEMS Microbiology Reviews, 26:125-

14 Motility and chemotaxis Virulence Factors Non-motile mutants are reduced for virulence Little is known about chemotaxis and signals that lead to colonization of intestine Adherence Long filamentous pili termed the toxin-coregulated pilus (TCP) that form bundles on bacterial surface is essential for colonization Mutants lacking TCP are avirulent in human volunteers and in animal models Genes necessary for production of TCP are organized in an operon that contains 15 genes which is part of Vibrio pathogenicity island (VPI) Major pilin subunit Assembly Secretion

15 Cholera toxin(ctx) Virulence Factors The most critical virulence factor, all the disease symptoms are caused by the action of cholerae toxin Structure It is composed of two polypeptides, A subunit and B subunit The ratio of B:A is 5:1 Mode 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 transported 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)

16 Virulence Factors G protein regulates activity of adenylate cyclase 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 CTX genetic element Cholera toxin genes are encoded on a lysogenic filamentous bacteriophage The phage can infect strains of V. cholerae missing toxin genes The receptor for the phage is TCP Transfer can occur in the gastrointestinal tract Transfer can occur in the aquatic environment Is evolution/generation of pathogens important for re-emergence of pathogens?

17 Vaccine Development A single clinical exposure to V. cholerae O1 confers protective immunity Live attenuated strain of V. cholerae as a vaccine Ctx deletion strains still caused mild diarrhea TCP mutants failed to induce significant protection Vaccine trials have been unsuccessful in clinical trials Rehydration therapy remains essential in cholera treatment

18 Environmental Factors and Survival of Vibrio cholerae Physical and Chemical Properties of Aquatic Habitat Temperature ph Salinity Nutrients Climate Rainfall Sunlight Temperature Vibrio cholerae Biological Properties of Aquatic Habitat Phytoplankton bloom Zooplankton bloom

19 Environmental Survival Entry into viable but non culturable (VBNC) state Phase variation (turning on/off selected genes via genetic re-arrangements) Biofilm formation

20 What Is a Biofilm? Biofilms are surface attached microbial communites formed when bacteria adhere to surfaces in aqueous environments and begin to excrete a slimy, glue-like substance that can anchor them to all kinds of material such as metals, plastics, soil particles, medical implant materials, and tissue. A biofilm can be formed by a single bacterial species, but more often biofilms consist of many species of bacteria, as well as fungi, algae, protozoa.

21 Advantageous of Biofilm Growth Mode Surfaces adsorb and concentrate nutrients Attached bacteria can degrade biotic surfaces (i.e chitin) In mixed species biofilms bacteria engage in metabolic interactions with other members Protection from toxic compounds