Occurrence of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus in the clam Ruditapes philippinarum (Adams & Reeve, 1850) from Emilia Romagna and Sardinia, Italy

Marine vibrios, Vibrio parahaemolyticus , V. vulnificus and V. cholerae are responsible of the majority of food-borne human infections by consumption of bivalve shellfish. The aim of the present study was to ascertain the occurrence of these bacteria, and their potential pathogenicity, in the Manila clam R. philippinarum from Emilia Romagna (ER) and Sardinia (SR) regions, Italy. Isolation was performed on CHROMagar TM vibrio with subculture on (thiosulfate-citrate-bile salts-sucrose) Agar and m-modified-cellobiose-polymyxin bcolistin (-CPC) Agar. Suspected strains were purified, biochemically characterized and genotyped by simplex polymerase chain reaction (PCR) for the specie-specific and pathogenic gene markers: V. parahaemolyticus ( tox RP, tdh and trh ); V. vulnificus ( vvhA , hsp , vcg C, vcg E, CPS operon allele 1, CPS operon allele 2, 16s-rRNA operon allele A, 16s-rRNA operon allele B; V. cholerae ( toxR C, hlya , tcpI , tcpA , ctxA , ctxB , stn/sto ). Moreover a multiplex PCR was applied to the SR bivalve shellfish, for the simultaneous detection of the three targets directly on homogenate samples, targeting the species-specific gene for V. cholerae ( toxR C), V. parahaemolyticus ( toxR P) and V. vulnificus ( vvhA ). As a result of phenotyping and genotyping of isolates, bivalve shellfish from ER resulted positive for V. parahaemolyticus (27.8%) and V. vulnificus (10.1%), but negative for V. cholerae . Shellfish from SR resulted positive for V. parahaemolyticus (30.3%), V. vulnificus (6.1%) and V. cholerae (3%). No significant differences emerged between the two areas (P>0.05).