Vibrio fluvialis: an emerging human pathogen

The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, including cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the toxR, ompU, and ompT genes are apparently in the ancestral Vibrio chromosome. The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner. In both classical and El Tor biotypes of V. cholerae, a toxR mutant strain has a reduced minimum bactericidal concentration (MBC) of bile, the bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and toxT mutant strains. Classical and El Tor toxR mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS. Growth of either V. cholerae biotype in subinhibitory concentrations of bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot. Measurement of transcription of a ompUp-lacZ fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent ompU transcription by the presence of bile or DC, suggesting that ToxR may respond to the presence of bile. The toxR mutant strains of three additional human intestinal pathogenic Vibrio species, V. mimicus, V. fluvialis, and V. parahaemolyticus, display lower MBCs of bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains. Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and bile resistance of pathogenic Vibrio species and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced resistance to bile. We speculate that ToxR-mediated bile resistance was an early step in the evolution of V. cholerae as an intestinal pathogen.

Circulation of mobile genetic elements linked to drug resistance spread was studied in Vibrio strains isolated from surface urban water (river and sea) and shellfish samples in 2002-2003 in Maputo, Mozambique. Class 1 integrons and integrating conjugative elements (ICE) were investigated by PCR and mating experiments in strains of major health interest: 10 Vibrio cholerae, six Vibrio parahaemolyticus, two Vibrio alginolyticus and one Vibrio fluvialis. Resistance to at least two antibiotics (predominantly beta-lactams) was detected in all the strains, with additional resistances to sulfamethoxazole, spectinomycin, streptomycin and/or trimethoprim. Class 1 integrons contributed partially to the expression of drug resistance and were found in five isolates: four V. cholerae (blaP1 cassette, one strain also contained the dfrA15 cassette) and one V. alginolyticus (aadA2 cassette). ICEs, apparently devoid of resistance genes, were found in eight V. cholerae, three V. parahaemolyticus and one V. fluvialis isolates. A wide variability was observed by molecular characterization of ICEs. Five ICEs were included in the SXT/R391 family and seven ICEs were not classified. Our results indicate that the SXT/R391 family and related ICEs comprise a large class of polymorphic genetic elements widely circulating in environmental Vibrio strains in Africa, beside those evidently linked to drug resistance in clinical isolates.

We assessed the occurrence of Vibrio pathogens in the final effluents of a rural wastewater treatment facility in the Eastern Cape Province of South Africa as free or plankton-associated (180 microm, 60 microm and 20 microm plankton sizes) entities using standard culture-based and molecular techniques. The free-living Vibrio densities varied from 0 to 3.45 x 10(1) cfu ml(-1), while the plankton-associated Vibrio densities vary with plankton sizes as follows: 180 microm (0-4.50 x 10(3) cfu ml(-1)); 60 microm (0-4.86 x 10(3) cfu ml(-1)); 20 microm (0-1.9 x 10(5) cfu ml(-1)). The seasonal variations in the Vibrio densities in the 180 and 60 microm plankton size samples were significant (p < 0.05), while the 20 microm plankton size and free-living Vibrio densities were not. Molecular confirmation of the presumptive vibrios isolates revealed fluvialis (36.5%), as the predominant species, followed by Vibrio vulnificus (34.6%), and Vibrio parahaemolyticus (23.1%); only API 20NE was employed to detect Vibrio metschnikovii (5.8%), suggesting a high incidence of pathogenic Vibrio species in the final effluent of the rural wastewater facility. Analysis suggested that the concentration of Vibrio species correlated negatively with salinity and temperature (p < 0.001 and p < 0.002 respectively) as well as with pH and turbidity (p < 0.001) in the final effluent. We conclude that rural wastewater treatment facilities in the Eastern Cape Province of South Africa are potential sources of Vibrio pathogens in the aquatic environment of the communities.