Analysis of the Resuscitation-Availability of Viable-But-Nonculturable Cells ofVibrio parahaemolyticusupon Exposure to the Refrigerator Temperature

Abstract

27Major pathogenic strains of Vibrio parahaemolyticus can enter into the viable-but-28 nonculturable (VBNC) state when subjected to environmental conditions commonly 29 encountered during food processing. Especially, VBNC cells can be recovered to the 30 culturable state reversibly by removing the causative stress, expressing higher levels of 31 virulence factors. Therefore, the aim of this study was to determine if VBNC V. 32 parahaemolyticus strains retain the resuscitation-availability upon eliminating the adverse 33 condition, followed by the enrichment in developed resuscitation-facilitating buffers. 34 Bacterial cells were shown to enter into the VBNC state in artificial sea water (ASW, pH 6) 35 microcosms at 4 o C within 70 days. VBNC cells were harvested, inoculated in formulated 36 resuscitation-buffers, and then incubated at 25 o C for several days. TSB (pH 8) supplemented 37 with 3% NaCl (TSB A ) exhibited the higher resuscitation-availability of VBNC cells. It was 38 also shown that TSB A containing 10,000 U/mg/protein catalase, 2% sodium pyruvate, 20 mM 39 MgSO 4 , 5 mM ethylenediaminetetraacetic acid (EDTA), and cell free supernatants extracted 40 from the pure cultures of V. parahaemolyticus was more effective in resuscitating VBNC cells 41 of V. parahaemolyticus, showing by 7.69-8.91 log 10 CFU/ml. 42 43 IMPORTANCE 44 45Generally, higher concentrations (≤40%) of NaCl are used for preserving different sorts of 46 food products from bacterial contaminations. However, it was shown from the present study 47 that strains of V. parahaemolyticus were able to persist in maintaining the cellular viability, 48 thereby entering into the VBNC state upon exposure to the refrigerator temperature for 80 49 days. Hence, the ability of VBNC V. parahaemolyticus to re-enter into the culturable state 50 was examined, using various resuscitation buffers that were formulated in this study. VBNC 51 cells re-gained the culturability successfully when transferred onto the resuscitation-buffer D, 52 and then incubated at 25 o C for several days. Resuscitation-facilitating agent D is consisting of 53 antioxidizing agents, mineral, an emulsifier, and cell free supernatants from the actively 54 growing cells of V. parahaemolyticus. It appeared that such a reversible conversion of VBNC 55 cells to the culturable state would depend on multiple resuscitation-related channels. 56 57 KEYWORDS cell free supernatant, pathogen, resuscitation, ROS-detoxifying, viable-but-58 nonculturable 59 60 62 63 Major food-borne pathogens, including Vibrio parahaemolyticus, Vibrio vulnificus, 64 Camphylobacter jejuni, Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, 65 and Shigella dysenteriae, are known to become the viable-but-nonculturable (VBNC) state 66 when challenged by various environmental stresses such as low temperatures (≤15 o C), 67 starvation, copper, and CO 2 (1-3). It should be noted that VBNC cells of these pathogens are 68incapable of producing their own colonies on culture media on which these organ...

Effect of sunlight and salinity on the survival of pathogenic and non‐pathogenic strains of Vibrio parahaemolyticus in water microcosms

“…Several methods and processes have been reported for the resuscitation of viable, but no culturable bacteria, such as the use of diluted or semi-solid media (Wai et al, 2000;Whitesides & Oliver, 1997), the addition of some components such as catalase, sodium pyruvate, or α-ketoglutarate or by adding fertilizers, such as decaying phytoplankton (Collin & Hernroth, 2020;Morishige et al, 2013;Yoon et al, 2018). However, the resuscitation results are not always successful and depend on the species, the cells physiological state, and the duration of time for which the cells remained into the VBNC state (Oliver, 2016).…”

Pseudomonas fluorescens Cells’ Recovery after Exposure to BAC and DBNPA Biocides

“…Many authors claim that the recovery of cells observed in most cases is due to the regrowth of some remaining culturable cells [ 17 , 18 ]. Other studies showed that cells in a dormancy state can also resuscitate [ 19 , 20 , 21 , 22 ] when conditions are favourable. Most probably, regrowth and resuscitation co-exist.…”

Transcriptome analysis and prediction of the metabolic state of stress-induced viable but non-culturable Bacillus subtilis cells

“…Importantly, cells that enter the VBNC state retain their metabolic activity and membrane integrity [3][4][5] , however they lost their ability to regrow immediately on a medium that typically supports proliferation [6][7][8][9][10] . Several studies demonstrated that VBNC cells can be resuscitated with appropriate environmental stimuli or permissive growth conditions [10][11][12][13][14][15][16] .…”