PCR products were purified using the UltraClean™ PCR Clean-up Kit (MoBio) according to the manufacturer’s instructions. 16S rRNA
gene nucleotide sequences were determined using ABI Prism® BigDye™ dye-terminator chemistry (Applied Biosystems) and an automated ABI Prism® 3700 Genetic Analyzer (Applied Biosystems). Sequences were aligned to known sequences in the GenBank database using blast (Altschul et al., 1990). To identify possible chimeras within the 16S sequences, all sequences were analyzed using the RDP program check_chimera. The sequences obtained in this study were deposited in the GenBank database Epacadostat chemical structure under accession numbers GQ332269–GQ332300. The effectiveness, of each biochemical method and for a group of tests, was evaluated based on sensitivity and specificity. One hundred percent sensitivity was sought in order to eliminate
false negatives. Sensitivity and specificity were calculated as follows: sensitivity=[(number of isolates positive as determined by biochemical tests and PCR)/(total number of isolates positive as determined by PCR)] × 100; specificity=[(number of isolates negative as determined by biochemical tests and PCR)/(total number of isolates negative as determined by PCR)] × 100 (Choopun et al., 2002). The environmental conditions in both sampling sites are well described in Celussi & Cataletto (2007): seawater temperature ranged from 6.4 to 25.3 °C following a typical selleck seasonal
progression, while the salinity showed a different trend: in C1, it ranged between 37.0 and 38.2 p.s.u., remaining fairly constant throughout the year, while in D2, we detected strong variations underlined by a wide annual range between 25.5 and 37.7 p.s.u. D2 is, in enough fact, located more close to the Isonzo River mouth and the season-dependent amount of freshwater inputs is reflected in strong variations in salinity. Out of the 269 sucrose-negative isolates subjected to the screening phase, only 171 were confirmed as Vibrio spp. and then analyzed to verify their identity as V. parahaemolyticus. Twenty-three strains died during the analyses; 35 strains showed an arginine dihydrolase-positive reaction that is inconsistent with a V. parahaemolyticus typical response. One hundred and thirteen strains selected as presumptive V. parahaemolyticus were tested using API systems, and even among these, three strains yielded K/K in the KIA test, 32 strains were sensitive to 10 μg Vibriostat O/129 and 40 did not grow in 8% NaCl. API systems characterized only 19 strains as V. parahaemolyticus (Table 1); the urease production was recorded only for one strain (#PVP408). PCR amplification and sequencing of the 16S rRNA gene and the detection of toxR, tlh, tdh and trh genes were carried out on 32 strains (19 presumptively identified as V.