Persistance of Vibrio cholerae and other vibrios in the aquatic environment and link with pathogenicity for humans

In the early 80's, evidences rapidly accumulated showing unequivocally that pathogenic Vibrio species are naturally occurring in the aquatic environment. Since then, significant effort has been devoted to the study of the aquatic ecology of vibrios, the conclusion of which is that the aquatic environment plays a pivotal role in persistence, dissemination and evolution, as well as transmission to humans, of these bacteria. In water, Vibrio abundance is related to temperature, salinity, and concentration of organic matter, and it is tightly dependent on the presence of environmental reservoirs (e.g., zooplankton crustacea) and concentration sites (e.g., tissues of filter feeding organisms), which are in turn controlled by climate variability. The identification of environmental niches hosting vibrios in the environment as well as the knowledge of the factors controlling their occurrence are thus of fundamental importance to the study of Vibrio ecology. Based on these observations, the principal aims of my study have been: to evaluate the role of different environmental matrices as reservoir of Vibrio spp. and pathogenic vibrios in Ligurian Sea coastal waters, and to reveal the major environmental factors controlling their presence and distribution. Moreover, considering the importance of both chitin-containing organisms (e.g., copepods) in Vibrio persistence inside the water, and bivalves as a route of pathogen concentration and transmission to humans, special attention has been given to the study of factors affecting their interactions with chitin and bivalves, using Vibrio cholerae as a study model. The possible influence of persistence in bivalves in pathogenicity for humans of this microrganism has also been studied. To investigate the role of different marine matrices as reservoirs of Vibrio spp. and pathogenic species (V. cholerae, Vibrio vulnificus and Vibrio parahaemolitycus), we have carried out a 16 months study in the La Spezia Gulf and have analysed the major environmental factors controlling their presence, abundance and distribution in the aquatic environment. Microbiological analysis has been performed on water, mussel, sediment and zooplankton samples and the considered environmental variables were Sea Surface Temperature (SST), salinity, concentration of chlorophyll-a and concentration of proteins. The obtained data, while confirming the dependence of Vibrio presence and culturability on environmental variables, have also shown that the sediment can act as an additional reservoir of vibrios which, in this compartment, are less sensitive to the environmental factors. This is probably due to sediment intrinsic characteristics that make this matrix less sensitive to season environmental variations. Considering the strong link between Vibrio presence in water and environmental factors, we have evaluated the effect of temperature and salinity on V. cholerae binding to chitin. The obtained results have shown that V. cholerae binding to chitin is affected by temperature, while salinity does not have any effect. Similar results have been obtained by Real Time RT PCR analysis of Gbp (gbpA) and MSHA (mshaA) gene expression in different environmental conditions. In fact, we have found that gbpA and mshaA expression varies with temperature but is not affected by salinity and nutrient concentration. These findings suggest that low Vibrio concentrations in coastal water in winter period can be ascribed, at least in part, to temperature effect on expression of genes encoding for surface ligands involved in adhesion to chitin surfaces. Concerning the results on V. cholerae interactions with mussels, it must be recalled that previous studies suggested that bacterial persistence in bivalve tissues depends, at least in part, on the capability to survive to the bactericidal activity of the mussel blood, the hemolymph. It has been previously shown that MSHA (Mannose Sensitive Hemagglutinin) expressed by V. cholerae El Tor strains mediates adherence to, and killing by Mytilus galloprovincialis hemocytes; such interactions occur via specific opsonins(s) present in mussel hemolymph serum. In order to identify the opsonizing serum component binding the MSHA ligand, mussel serum has been applied to a V. cholerae (MSHA-positive) - agarose affinity column and the bound material has been eluted with 0.5 M D-mannose. The resulting eluate, analyzed by 12% SDS-PAGE, yielded a single fraction of approximately 42 kDa which has beenshown to mediate MSHA-positive V. cholerae interactions with hemocytes and, surprisingly, also adherence to cultured intestinal cells. Addition of D-mannose has abolished such increase. Moreover, our studies have shown that binding of p42 fraction to V. cholerae cell wall induces a protective effect to acid shock. This effect can play a fundamental role in the increase of V. cholerae survival during infection of human host, i.e., after the organism has passed through the stomach. These results suggest that V. cholerae capability to colonize human intestine depends on both bacterial intrinsic properties and factors acquired during its persistence in the aquatic environment. In conclusion, data presented in this thesis can contribute to a better comprehension of Vibrio ecology and represent the basis for the development of new strategies to control the diffusion of pathogenic vibrios in coastal marine environment and to prevent their transmission to humans.