Preliminary Study On The Viable But Nonculturble State Of Vibrio Parahaemolyticus Vp L4-90 Strain

Since the original 1982 paper (Xu et al., 1982), more and more papers have been appearing which describe various aspects of the phenomenon most commonly referred to as the"viable but nonculturable (VBNC) state". A great many pathogens, as well as nonpathogens, are now known to enter this dormancy state, and its significance in medicine, bioremediation, the use of bacteria as fecal indicators, and indeed in most microbiological studies where culturability is employed as the (often sole) indicator of viability, is becoming increasingly evident.Bacteria in the VBNC state fail to grow on the routine bacteriological media on which they would normally grow and develop into colonies, but are alive and capable of renewed metabolic activity. Cells in the VBNC state typically demonstrate very low levels of metabolic activity, but on resuscitation are again culturable. The bacteria described to enter this condition, and the concerns this physiological state raises in public health and bioremediation.V. parahaemolyticus is a gram-negative halophilic bacterium that is indigenous to coastal marine waters throughout the world. As a common cause of acute gastroenteritis and the source of some cases of septicemia, it is an organism of public health concern. The stresses of low nutrient and low temperature have been shown to be the main causes of the induction of VBNC stages in some pathogenic bacteria.in this study,we used V. parahaemolyticus VP L4-90 as the Experiment material to research the viable but nonculturable state of bacteria. V. parahaemolyticus VP L4-90 was incubated in The stresses of low nutrient and low temperature to induce the VBNC state.During the inducement ,total cell counts was determined by AODC ,viable cell counts determined by the DVC method and Culturable cell counts determined by PC. Finally,we verified that V. parahaemolyticus VP L4-90 can enter into VBNC state.After V. parahaemolyticus VP L4-90 entering into VBNC state, We studied the change of its morphology and nucleic acid content.Then we optimized the factors that affect the entry of V. parahaemolyticus into VBNC state. These factors include temperature, bacterial growth phase and bacterial concentration. According to our results ,we know that it is easier for V. parahaemolyticus to enter into the VBNC state at low temperature. V. parahaemolyticus also can enter into the VBNC state at high temperature condition but it is unsteadily. Bacterial concentration would affect the time of entering into the VBNC state of V. parahaemolyticus.In this study,we explore the recovery of culturability of VBNC state V. parahaemolyticus after temperature upshift and to determine whether regrowth or resuscitation occurred. V. parahaemolyticus Vp L4-90 strain was rendered VBNC state by exposure to artificial seawater (ASW) at 4℃. Aliquots of the ASW suspension of cells (0.1, 1 and 10 ml) were subjected to increased temperatures of 25℃and 37℃. The results suggest that a portion of VBNC cells is able to undergo cell division. It is concluded that a portion of VBNC cells of V. parahaemolyticus subjected to cold temperatures remain viable. After temperature upshift, regrowth of those cells, rather than resuscitation of all bacteria of the initial inoculum, appears to be responsible for recovery of culturability of VBNC cells of V. parahaemolyticus. Nutrient in filtrates of VBNC cells is hypothesized to allow growth of the temperature-responsive cells, with cell division occurring.We optimized the EMA procedure to set up a novel method(EMA-PCR) for detecting viable V. parahaemolyticus. Ethidium bromide monoazide (EMA) treatment selectively allow conventional PCR amplification of target DNA from viable but not dead V. parahaemolyticus cells. The specific primers that amplify a sequence of tlh gene were used for the PCR. After treating by EMA stain ,real time PCR was used to quantity the viable bacteria in the bacterial samples.Based on the results,we found that definitely can enter into the VBNC state at high temperature(46℃). Farther,we utilized the EMA-PCR method to detect bacterial samples cultured at high temperature to vertify the entry of V. parahaemolyticus into VBNC state. During the treatment at high temperature there would be much dead cells in the sample, Although there also be much VBNC state cells. It would be a new consideration for food safety and bacterial detection.