Study On Vibrio Parahaemolyticus In Viable But Non-Culturable State

In recent years, because of recurring problems of food contamination about food safety, food quality and safety issue has become the focus of the whole society’s attention. In a series of food contamination events, more than 80% have been caused by microorganism pathogenic bacteria. Staphylococcus bacteria, salmonellas and clostridium botulinum are common bacterial food poisonings. And that vibrio cholera, vibrio parahaemolyticus, escherichia coli O157:H7, listeria monocytogenes, etc, are pathogenic bacterium. But at present the monitoring on pathogenicity of the pathogen are all in the state of viable bacteria researching. Based on public health safety considerations, further research of bacteria in the state of VBNC will be imminence in China.This article was about Vibrio parahaemolyticus inducing in the state of VBNC by low temperature and the control of nutritional condition, also analyzed these two conditions. Besides the Vibrio parahaemolyticus in the state of VBNC had been resuscitated, indicators of resuscitative strains had been detected, and a preliminary study of resuscitative strains had been conducted. Then, applying PCR and RT-PCR detected the virulent genes and specific housekeeping genes of vibrio parahaemolyticus in the VBNC and recovery state. Finally, making use of the difference of results could build the way of rapidly detecting Vibrio parahaemolyticus in the state of VBNC in environment and food. The certain basis could be provided by the formation of this approach for the future research of bacteria in the state of VBNC and food safety control.This research has done from the following aspects:1. The induction of Vibrio parahaemolyticus in the state of VBNC and its resuscitative research This article was about Vibrio parahaemolyticus BJ1.1997 and Vibrio parahaemolyticus BJ1.1616 which were respectively induced in oligotrophic (Artificial seawater, Seawater) and eutrophic conditions (TSB-3% NaCl) at low temperatures(4℃). The results showed that both of them could induce in the VBNC state; the low temperature was the effectiveness factor, but correlated little with nutrients conditions; different virulent genes (V.pBJ6-tdh, V.pBJ7-trh) were not a significant predictor for the time(about 35 days) of inducing in VBNC state. Besides, rising column temperature and other programs resuscitated Vibrio parahaemolyticus in the VBNC state. The results indicated that V.pA6, V.pA7 and V.pH6 could be resuscitated by adding nutrient substance(25℃)and rising column temperature; V.pA7 could be resuscitated successfully by adding Tween-20; the others could not make Vibrio parahaemolyticus in the VBNC state recover to culturable state. The above conclusions provided references for research on Vibrio parahaemolyticus in the VBNC state and resuscitative bacterium.2. Pilot study on the characteristics of resuscitative bacterium in the VBNC stateIrritability, growth curves, animal toxicologic, physiological and biochemical indexes and virulence genes of resuscitated strains V.pA6F、V.pH6F、V.pA7F were detected. Cold shock showed that after resuscitation than the standard strains of bacteria survival rate is slightly higher, while the high temperature, ultraviolet radiation and tolerance of high osmotic pressure compared with the normal survival rate of decline of Vibrio, while the experiment of growth curves proved that the resuscitative bacteria stains’activity had declined, comparing with normal bacteria stains. Meanwhile, physiological and biochemical indexes and virulence genes of resuscitated strains’expression was basically the same as normal strains. But the urease reaction and virulence gene for trh of V.pA7F had a negative reaction, and it suggested maybe there was a positive correlation between urease and virulence gene, its mechanism still needed further study. Finally virulence verifying on strains in the VBNC state and resuscitative strains had been done by using mice. The results reveled that all the strains in the VBNC state and resuscitative strains V.pA7F were non-pathogenic on mice; however, V.pA6F and V.pH6F were pathogenic on mice.3. Application of Reverse Transcription-PCR to detect Vibrio parahaemolyticus in Viable but Non-Culturable State From applying PCR and RT-PCR to detect the virulent genes tlh, trh, tdh, and specific housekeeping genes rpoS of vibrio parahaemolyticus in the state of VBNC and recovery, the results showed, the virulent genes tlh,trh,tdh of strains in VBNC state could not be expressed by PCR and RT-PCR. The specific housekeeping genes rpoS of strains in the state of VBNC could not be expressed by PCR, but it could be expressed successfully by RT-PCR. Besides the virulent genes tlh, trh, tdh, and specific housekeeping genes rpoS of strains in recovery state could be expressed. The difference of this result played a leading role in detecting the pathogenicity dangers to vibrio parahaemolyticus and its potential pathogen or survival condition in the state of VBNC in the environment and food.