Characterization of Vibrios isolated from coastal regions with emphasis on V. cholerae's quantification

Abstract

Microorganisms are essential for the environment and contribute to the stability of ecosystems. In the marine ecosystem, bacteria are dominant species in terms of relative abundance and contribute to biogeochemical processes. Bacteria of the Vibrio genus are abundant in this environment and, eventually, some of them can be pathogenic, offering hazard for the human and animal health. The main species associated to food and waterborne disease outbreaks, according to Food and Agriculture Organization of the Unites Nations, are V. parahaemolyticus, V. cholerae e V. vulnificus. At the Brazilian coastal regions, a little is known about the distribution of these populations and the hazards that represent. Thus, it is necessary to conduct appropriate studies to characterize and quantify the microbiological hazards present in marine ecosystems for food safety and for the recreational use of the marine coastal water, as well as understanding their ecological relationships and better define their taxonomy. The study aims to identify and characterize Vibrio spp. isolated from water, plankton and shellfish samples collected from the coastal region of São Paulo and from ballast water samples, Brazilian port regions and clinical and environmental strains from the collection of our laboratory of Molecular Microbial Ecology lab at the ICB-USP using phenotypic and genotypic methods, and correlate the results to the spatial differences and the presence of anthropogenic activities found at the locations analyzed. Thus, the isolates will be evaluated for the presence of virulence genes (ctxA, tcpA, TDH, TRH, VSP-I and VSP-II), susceptibility to antibiotics and characterized by MLSA (Multilocus Sequence Analysis) technique using six genes (recA, pyrH, rpoA, toxR, rpoD and dnaJ). Moreover, the technique of total DNA extraction from seawater samples will be evaluated and optimized and the real time PCR technique, for V. cholerae quantification using hlyA, rbf O1, rbf O139 and ctxA genes, will be standardized in seawater samples. This study, will allow to characterize and to verify the distribution of vibrios species in coastal regions of São Paulo state, in ballast water and water from port areas, as well as to standardize the methodology for total DNA extraction from seawater samples and for the pathogenic and non-pathogenic quantification of V. cholerae in marine ecosystems that can be applied in environmental monitoring. (AU)