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Summary.A collection of 28 strains of Vibrio cholerae non-0 1 isolated during a 3-year period (1989-199 1) from hospitalised patients with acute diarrhoea in Calcutta, India, were examined with regard to virulence-associated factors. Of the 28 isolates (each representing a case), 18 were isolated as the sole infecting agent; the remaining 10 were recovered as cocultures from cases infected with V. cholerae 01. Of the strains isolated in this study, 82 YO could be serotyped, with serovars 0 5 (32.1 %), 0 1 1 and 034 (14.3 YO each) predominant. Serovars 0 7 , 014, 034, 039 and 097 were associated exclusively with sole infections. Two strains of V. cholerae non-0 1 produced anti-cholera toxin IgG-absorbable cholera toxin (CT). Both CT-producing V. cholerae non-01 strains hybridised with the DNA probe specific for the zonula occludens toxin (ZOT) but none of the remaining 26 strains hybridised with the ZOT probe. The majority of the strains were cytotoxic for CHO, HeLa and Vero cells, with end-point titres of 4-512. Fewer strains produced a cytotonic effect, with end-point titres of 2-16. Of the 28 strains of V. cholerae non-01 examined, 7 5 % , 75Y0, 25% and 14.3% produced haemolysin that was active against erythrocytes of rabbit, sheep (Eltor haemolysin), chicken and man, respectively. Strains that produced a haemolysin active against both rabbit and sheep erythrocytes were dominant (35.7%). Ten (35.7%) of the 28 strains examined showed cell-associated haemagglutinating activity on human blood. Of the 10 strains, nine were isolated as sole pathogen and only one strain was associated with mixed infection. Three distinct patterns of inhibition by sugars were detected ; inhibition of haemagglutination by mannose 1 YO but not by fucose and galactose 1 YO was the dominant haemagglutination inhibition pattern. Six different virulence phenotypes were encountered among strains of V. cholerae non-01 in this study. The prominent phenotype, which was associated commonly with isolates from patients solely infected by V. cholerae non-0 1, was exhibited by strains that produced the Eltor haemolysin, a cytotoxin and a cell-associated haemagglutinin. The production 9f cell-associated haemagglutinin appeared to be the only distinctive phenotype that could distinguish between isolates from patients solely infected with V. cholerae non-0 1 and those associated with mixed infections. From this study, it is apparent that the virulence of V. cholerae non-01 is multifactorial and mediated by several traits functioning in an integrated fashion. The clinical significance of V. cholerae non-01 must be assessed in its totality; the presence of a single factor should not be construed as the cause of en teropa t hogenici t y .

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (>100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.Vibrio cholerae non-serogroup O1 strains are ubiquitous in aquatic environments (35) and have been recognized as the causative agents of sporadic cholera-like disease and outbreaks (5,34,39,43). In addition, environmental nontoxigenic, non-O1 strains play an important role in the evolution of toxigenic V. cholerae (19). However, only a minority of the strains of V. cholerae non-O1 seem to be enteropathogenic. The pathogenicity markers of vibrios are lacking in many species, including V. cholerae non-O1, non-O139 strains. The nature of virulence and the infective doses need to be determined for the establishment of guidelines for risk assessment of each species in surface water and food material earmarked for hu...

The genomes of the O3:K6 strains of Vibrio parahaemolyticus which abruptly emerged in Calcutta, India, in February 1996 and which demonstrated an unusual potential to spread and an enhanced propensity to cause infections were examined by different molecular techniques to determine clonality. No restriction fragment length polymorphism (RFLP) in the gene encoding the thermostable direct hemolysin was observed among the O3:K6 isolates of V. parahaemolyticus. Clonal diversity among the O3:K6 strains became evident by examining the RFLPs of the rrn operons and by the use of pulsed-field gel electrophoresis. Five ribotypes were distinguished among the O3:K6 strains examined, with ribotype R4 constituting the major type. Strains of O3:K6 isolated between June and August 1996 showed different pulsotypes compared to the pulsotypes of strains isolated before and after this period, indicating genetic reassortment among these strains, but those isolated between August 1996 and March 1998 showed identical or nearly similar pulsotypes. It is clear that there is a certain degree of genomic reassortment among the O3:K6 clones but that these strains are predominantly one clone.

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