Vibrio chromoselect agar

Bacteria were isolated from gilthead sea bream (Sparus aurata L.) and shellfish (Penaeus indicus H. Milne-Edwards). These samples were obtained from a local market in Hail region-Saudi on 25 February 2022. Fish with red spots on their skin were targeted, as this is an indication of microbial infection. Upon arrival, gilthead sea bream and prawns were immediately washed using sterile seawater, gutted, headed, and shucked with a sterile knife. Twenty-five grams from prawn abdomen meat, and the intestines, gills, and muscle meat from S. aurata were enriched in 225 mL of alkaline peptone water supplemented with 1% NaCl [18 (link)]. The inoculated broth media was incubated overnight at 37 °C. After incubation, a loopful from each enrichment culture was steaked onto thiosulfate–citrate–bile salt–sucrose agar (TCBS) (Agar; Sigma Aldrich, Darmstadt, Germany) and onto Vibrio ChromoSelect agar (Sigma Aldrich, Germany), before incubating for 18 to 24 h at 37 °C.

Vibrio parahaemolyticus and Vibrio vulnificus were detected following the ISO 21872-1:2017 method. Mussels were washed, scrubbed, and opened aseptically. Flesh and intravalvar fluids were collected and homogenized for 120 s in a sterile bag using a Stomacher (IUL, S.A., Barcelona, Spain). Enrichment was carried out in two subsequent steps. First, 25 g of the homogenized sample was homogenized in 225 mL of alkaline saline peptone water (ASPW) and incubated at 37 °C for 6 h. Second, 1 mL was reinoculated into 10 mL of ASPW and incubated at 37 and 45 °C for 18 h. At the end of the incubation period, an inoculating loop of the culture broth was streaked onto thiosulfate citrate bile salt sucrose (TCBS, ISO Scharlau, Spain) and Vibrio ChromoSelect agar (Sigma-Aldrich, St. Louis, MO, USA). After incubation at 37 °C for 24 h, five typical colonies were subcultured on saline nutrient agar (3% NaCl) plates. Presumptive isolates were confirmed as V. parahaemolyticus by PCR amplification of the toxR and enteropathogenicity-associated genes (tdh and trh) or the vvh gene in the case of V. vulnificus.