Recovery and detection of Vibrio vulnificus during cold storage

Different cultural techniques and molecular methods for the detection of Vibrio vulnificus during cold storage in a model broth system were compared. Two strains of V. vulnificus were grown to stationary phase and inoculated (10(6) CFU/mL) into tryptic soy broth with 2% sodium chloride (TSBN2) or artificial seawater (ASW), both pre-chilled to 5 degrees C. These were stored for 10 days, with sub-sampling conducted at time 0 and every 2 days thereafter. Each subsample was plated, by both pour and spread plate techniques, onto tryptic soy agar 2% sodium chloride (TSAN2) with or without catalase (400 or 600 U) or sodium pyruvate (80 or 160 mg) supplementation. Nucleic acids were extracted from subsamples and subjected to PCR and RT-PCR with hemolysin as the target. Higher recoveries of V. vulnificus were obtained with spread plating compared to pour plating (P<0.05). The addition of sodium pyruvate (80 mg) or catalase (400 U) significantly increased cell recovery (P<0.05). PCR amplification signals were stronger than RT-PCR signals at each timepoint, and results were generally consistent between TSAN2 and ASW for each strain. These results will aid in the design of optimum methods to recover and/or detect V. vulnificus cells subjected to sublethal stress that might be encountered in food processing and storage.