Vibrio

Samples from clinical cases and shellfish were obtained and analyzed as described previously (Fuenzalida et al., 2006 (link)). Briefly, samples of shellfish soft tissue were enriched for V. parahaemolyticus in three-tube serial dilutions in alkaline peptone water (APW). Tubes with bacterial growth were tested for tlh, tdh, and trh by multiplex PCR (mPCR) (Bej et al., 1999 (link)). Positive tlh enrichment tubes were plated on CHROMagar Vibrio (CHROMagar Microbiology, Paris, France), and bacterial colonies with the morphology and color expected for V. parahaemolyticus were purified. mPCR was performed again using approximately 10 ng of total bacterial DNA per reaction tube; strains positive for tdh and/or trh were discarded. DGREAs were performed as described previously (Fuenzalida et al., 2006 (link)). Information about strains used in this study is showed in Table 1.

The presence of three pathogenic Vibrio spp. in water samples was investigated using the most probable number polymerase chain reaction (MPN-PCR) method according to the modified ISO 8914 standard method (ISO 8914:1990) [16 (link)]. Briefly, 500 mL of each water sample were concentrated by filtration through a 0.45 μm pore filter (Sartorius, Goettingen, Germany) with a vacuum pump, after which the filters were placed in 225 mL of alkaline peptone water (APW; 1% (wt/vol) peptone, 1% (wt/vol) sodium chloride; pH 8.2). After incubation at 30 °C for 18 h, 1 mL aliquots of enrichment broths showing growth were analyzed by PCR to detect the presence of a species-specific toxR gene for V. parahaemolyticus and V. cholerae [17 (link)] and the vvhA gene for V. vulnificus [18 (link)].
Moreover, to obtain the isolates, 0.1 mL aliquots of enrichment broths were plated on thiosulfate–citrate–bile salts–sucrose (TCBS) (Oxoid, Hampshire, UK) and CHROMagar Vibrio (CHROMagar Microbiology, Paris, France) agar plates and were incubated for 18 h at 30 °C. Presumptive Vibrio spp. colonies were subcultured on TSA. A partial sequence of the 16S rRNA of Vibrio species was amplified by PCR and submitted for sequencing for bacterial identification [19 (link)]. Bacterial isolates were stored at −80 °C in LB broth containing 50% (v/v) glycerol.

A pre-enrichment was performed by diluting 25 g of each sample in 225 mL of alkaline peptone water (APW) and incubating at 37/41.5 °C overnight. At the end of the incubation period, an inoculating loop of the culture broth was seeded onto thiosulfate citrate bile salt agar (TCBS) (Microbiol, Cagliari, Italy) and CHROMagar Vibrio (CHROMagar™, Paris, France) plates. Plates were incubated for 24 h at 37 °C. After incubation, presumptive colonies from V. parahaemolyticus (green on TCBS Agar, mauve on CHROMagar), V. cholerae (yellow on TCBS Agar, turquoise on CHROMagar), and V. vulnificus (green on TCBS Agar, turquoise on CHROMagar) were streaked onto trypticase soy agar (TSA) supplemented with 3% NaCl, and their phenotypes were characterized by the following laboratory protocol: oxidase and catalase tests, Gram staining, sugar fermentation, and sensitivity to vibriostatic agent O12. Biochemical identification was performed following the API 20NE identification system (BioMerieux, Marcy l’Etoile, France). Also the strains identified as V. cholerae were processed by agglutination tests, using commercial polyvalent anti-O1 and anti-O139 antisera (Denka Seiken Co., Ltd., Tokyo, Japan). All isolates presumptively identified as V. parahaemolyticus, V. cholerae, and V. vulnificus were confirmed and further characterized by means of molecular tests.

A total of 120 raw seafood samples of five different food types (fish, n = 35; shellfish, n = 25; shrimp, n = 20; squid, n = 20; and crab, n = 20). All the samples were selected from markets by convenience sampling; they were subsequently stored in sterile bags on ice and were transferred to the laboratory within 2 h. Sample processing was conducted according to the ISO 21872-1: 2017 (Microbiology of the food chain – Horizontal Method for the determination of Vibrio spp.) protocol for the determination of Vibrio spp., with some modifications [26 ]. Briefly, 25 g of individual samples was pre-enriched in alkaline peptone water (Merck, Germany) at 37°C for 6 h. Then, the first enriched cultures were further inoculated into alkaline saline peptone water and were incubated at 37°C for 18 h. Subsequently, the overnight cultures were spread onto selective agar plates, thiosulfate citrate bile salts sucrose, and CHROMagar™ Vibrio (CHROMagar, Paris, France) for Vibrio spp., also the selective media for Aeromonas species (HiMedia, India) and the plates were incubated at 37°C for 18–24 h. The suspected colonies were subjected to conventional biochemical assays for the Vibrio and Aeromonas genera.

The collected samples were placed in sterile bags and transported in refrigerated trucks (12 °C) to the laboratory to be processed for analysis in less than one hour. Samples were removed from the bags and washed in running potable water. Dead shellfish, or those with broken shells, were discarded.
For the microbiological analysis of Vibrio, 25 g of bivalve samples obtained from 12 to 15 individuals (meat and liquor) were weighted, and 225 mL of alkaline saline peptone water (ASPW, OXOID, Hampshire, UK) were added. The matrix was homogenized for 2 min in a Stomacher (IUL, Barcelona, Spain), and directly incubated at 37 °C for 18 h. After incubation, a 3 mm loopful from the top 1 cm of each broth showing growth was streaked on different plates: thiosulfate-citrate-bile salt-sucrose (TCBS) agar (Oxoid, Hampshire, UK), and ChromAgar Vibrio (ChromAgar, Paris, France). The plates were incubated at 37 °C for 24 h. At least five typical sucrose-negative and positive colonies from each plate (TCBS), and mauve and white colonies from ChromAgar Vibrio were isolated and subjected to identification by biochemical tests on API 20E strips (bioMérieux, Marcy-l’Etoile, France) and other complementary biochemical assays. The isolated strains were stored at −80 °C for further analysis.

Foodborne strains, used in this work, were isolated from aquacultured fresh fish (D. labrax: sea bass, S. aurata: sea bream) and from shellfish (Parapenaeus longirostris: rose shrimp, Mytilus galloprovincialis: blue mussel). These samples were obtained from a local market (Souk El Blat, Tunisia) packed in cold boxes and transferred to the laboratory within 2 h for analyses [2 (link)]. Upon arrival, fish were immediately gutted, headed, washed and filleted. Mussels were washed, scrubbed free of dirt, shucked with a sterile knife. Twenty-five grams of meat and liquor of blue mussels muscle, and from each fish species were transferred aseptically and diluted in Stomacher packet each containing 225 mL of alkaline peptone water supplemented with 1% NaCl. The mixture was homogenized for 3 min using a laboratory blender Stomacher then incubated at 37 °C for 18–24 h. The enrichments (100 μL) were then streaked onto Thiosulfate-citrate-bile salt-sucrose agar (TCBS agar, Tulip Diagnostics. (P) LTD, Verna Industrial Estate, India), CHROMagar™ Vibrio and CHROMagar™ S. aureus media (CHROMagar Microbiology, Paris, France) and incubated for 18–24 and 37 °C.