Analysis of the Resuscitation-Availability of Viable-But-Nonculturable Cells of Vibrio parahaemolyticus upon Exposure to the Refrigerator Temperature

Abstract

Major pathogenic strains of Vibrio parahaemolyticus can enter into the viable-but-nonculturable (VBNC) state when subjected to environmental conditions commonly encountered during food processing. Especially, VBNC cells can be recovered to the culturable state reversibly by removing the causative stress, expressing higher levels of virulence factors. Therefore, the aim of this study was to determine if VBNC V. parahaemolyticus strains retain the resuscitation-availability upon eliminating the adverse condition, followed by the enrichment in developed resuscitation-facilitating buffers. Bacterial cells were shown to enter into the VBNC state in artificial sea water (ASW, pH 6) microcosms at 4{degrees}C within 70 days. VBNC cells were harvested, inoculated in formulated resuscitation-buffers, and then incubated at 25{degrees}C for several days. TSB (pH 8) supplemented with 3% NaCl (TSBA) exhibited the higher resuscitation-availability of VBNC cells. It was also shown that TSBA containing 10,000 U/mg/protein catalase, 2% sodium pyruvate, 20 mM MgSO4, 5 mM ethylenediaminetetraacetic acid (EDTA), and cell free supernatants extracted from the pure cultures of V. parahaemolyticus was more effective in resuscitating VBNC cells of V. parahaemolyticus, showing by 7.69-8.91 log10 CFU/ml.\n\nIMPORTANCEGenerally, higher concentrations ([≤]40%) of NaCl are used for preserving different sorts of food products from bacterial contaminations. However, it was shown from the present study that strains of V. parahaemolyticus were able to persist in maintaining the cellular viability, thereby entering into the VBNC state upon exposure to the refrigerator temperature for 80 days. Hence, the ability of VBNC V. parahaemolyticus to re-enter into the culturable state was examined, using various resuscitation buffers that were formulated in this study. VBNC cells re-gained the culturability successfully when transferred onto the resuscitation-buffer D, and then incubated at 25{degrees}C for several days. Resuscitation-facilitating agent D is consisting of antioxidizing agents, mineral, an emulsifier, and cell free supernatants from the actively growing cells of V. parahaemolyticus. It appeared that such a reversible conversion of VBNC cells to the culturable state would depend on multiple resuscitation-related channels.