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Approximately 30 years have elapsed since Dr. Fujino's original discovery that Vibrio parahaemolyticus (then termed Pasteurella parahemolytica) was the cause of "summer diarrhea" in Japan. Since that finding, V. parahaemolyticus has been established as a cause of gastroenteritis in numbers and places approaching global proportions. It has been isolated in marine and estuarine areas almost worldwide and despite its halophilic nature, V. parahaemolyticus has been isolated from saline-free waters. The relationship of this organism to the environment reveals a close association with other marine organisms especially copepods on which the Vibrios depend for survival in winter months and growth in summer months. There is a uniquely provocative disparity between human strains of V. parahaemolyticus which are Kanagawa phenomenon (KP) positive and the environmental strains which to a large extent are KP negative, the significance being that pathogenicity is measured according to the Kanagawa phenomenon (hemolytic activity) reaction. The hemolysin of the pathogenic strains is a thermostable, cardiotoxic protein, which thus far has not been implicated in the mechanism(s) which causes human gastroenteritis. The interest in this organism has been widened in recent years by the finding that similar organisms, V. alginolyticus, lactose positive vibrios and group F vibrios also cause serious disease in humans.

Vibrio cholerae was isolated at several locations in Chesapeake Bay in fall 1976 and spring 1977. Strains induced fluid accumulation in rabbit ileal loops and positive activity in Y-1 adrenal cells. Vibrio cholerae, Vibrio parahaemolyticus, and related vibrios show a spatial and temporal distribution characteristic of Vibrio species in an estuary. The Vibrio cholerae strains isolated from Chesapeake Bay represent serotypes other than O-group I--that is, so-called nonagglutinable vibrios--and are not recognized as a serious epidemic threat, although they have caused cholera-like diarrhea sporadically.

A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.

A microbiological survey of Aeromonas hydrophila in Chesapeake Bay and its tributaries showed that this species is ubiquitous, occurring in numbers ranging from <0.3/1 to 5 x 103/mI in the water column and cu. 4.6 x 102/g in sediment. It was recovered from water samples collected at several locations in Chesapeake Bay representing various salinity regimes, but the numbers of A . hydrophila in higher salinity water, i.e. 2 IS%,, were low. Results of stepwise multiple linear regression analysis showed that concentrations of A . hydrophila were correlated with total, aerobic, viable, heterotrophic, bacterial counts, and, in addition, were inversely related to salinity and to concentration of dissolved oxygen. Seasonal occurrence was recorded, with fewer strains of A . hydrophila encountered during the winter months. The potential pathogenicity o f A . hydrophila strains isolated from Chesapeake Bay was estimated by testing selected isolates for toxigenicity, using the Y-1 adrenal cell assay. Of 116 isolates tested, 83 (71%) produced a cytotoxic response, a characteristic found to be correlated with the lysine decarboxylase and Voges-Proskauer reactions. Eight of 11 strains tested, which elicited fluid accumulation in the rabbit ligated ileal loop assay, also provoked a cytotoxic reaction in the Y-I adrenal cell assay. Results of the study indicate that large numbers of toxigenic A . hydrophila can be found in an estuary and such strains may be pathogenic for man and/or animals.

A total of 65 isolates of Vibrio cholerae, serotypes other than 0-1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17%o, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was <4 or >17%o. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salnonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae 0 group 1 antiserum, the. majority were toxigenic; i.e., approximately 87% of the isolates exhibited toxicity for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.

iThe genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF.

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