Correa-Vélez, Karlen E.

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    Detection and microbiological and molecular characterization of Vibrio parahaemolyticus in the clam Phacoides (Lucina) pectinatus (Gmelin, 1791) and the oyster Crassostrea rhizophorae (Guilding, 1828) from the southwest coast of Puerto Rico
    (2017) Correa-Vélez, Karlen E.; Ríos-Velázquez, Carlos; College of Arts and Sciences - Sciences; Santos Flores, Carlos J.; Rodríguez Minguela, Carlos; Department of Biology; Palomera García, Rogelio
    Bivalves such as clams and oysters are filtering mollusks that inhabit aquatic environments. These organisms are relevant in the food industry for the potential presence of pathogenic bacteria, such as Vibrio parahaemolyticus, that these mollusks can accumulate during their filter feeding. Recently, the Centers for Disease Control and Prevention (CDC) estimated a higher incidence (7,880 cases/year) of Vibrio infections, where approximately 2,800 cases were associated with V. parahaemolyticus. The Food and Drug Administration (FDA) recognized this bacterium as a leading cause of human gastroenteritis associated with seafood consumption. However, the outbreaks caused by seafood contaminated with this pathogen have been also described in countries like United States, Spain, Japan, Taiwan and Brazil. Because the foodborne illness distribution, the PulseNet International network was created in order to track foodborne infections worldwide. In Puerto Rico, there are no laws or regulatory agencies that assess quality bivalve for sale. Therefore, the Island does not have statistics on foodborne disease incidence caused by consumption of raw bivalves. This research sought the detection of V. parahaemolyticus in both raw bivalves consumed in Puerto Rico, established the potential of pathogenicity of the isolates, and sought the molecular typing of the V. parahaemolyticus isolated strains using Pulse Field Gel Electrophoresis (PFGE). This study used standardized tests, including molecular protocols established by the FDA in the Bacteriological Analytical Manual (BAM), for the detection of V. parahaemolyticus strains in oyster and clam samples from the southwest coast. Finally, molecular analysis involved the use of Multiplex PCR to detect the presence of V. parahaemolyticus pathogenic associated genes and the use of a PFGE technique followed the standardized protocol established by the PulseNet International Network to molecularly subtyping of V. parahaemolyticus. To confirm the isolated strains as part of genus Vibrio, a PCR was performed to amplify part of the 16S rDNA and the PCR products were sequenced at McLab facilities. After 4 seasonal samplings, 58 strains of presumptive V. parahaemolyticus were isolated, where 36% were able to grow at 3%, 6% and 8% of NaCl; while, 64% of isolates were able to grow also at 10% of NaCl. Based on species-specific marker amplicon, and the absence of pathogenic genes signal, 90% of the isolates in the study are V. parahaemolyticus and none of them are potentially pathogenic. The PFGE analysis with NotI and SfiI allowed the discrimination of 52 isolates into 21 different strains. These strains were grouped into 18 types with >65% patterns similarity. Both, NotI and SfiI restriction patterns, revealed a similar discriminatory power. In-silico analysis of the bacteria isolated confirmed all isolates as Vibrio spp. In this study, do not detect the presence of pathogenicity markers (tdh, trh) in V. parahaemolyticus isolated from mollusk samples from the southwest coast of Puerto Rico. A comparison with isolates from patients with diarrhea associated to bivalve consumption will provide more information on potential foodborne disease associated to raw shellfish consumption from these waters. All restriction patterns were novel in comparison with the restriction patterns of the strains in the PulseNet USA V. parahaemolyticus database. This molecular study serves as a baseline to continue developing food safety studies of bivalves in the Island. Also, demonstrated not only the diversity of V. parahaemolyticus in the southwest coast of Puerto Rico, but the uniqueness and how they contrast to the isolates from outbreaks in USA.