| Vibrio parahaemolyticus is a kind of gram-negative bacterium,mainly exist in seafood and coast environment.It can cause diarrhea and vomiting by eating contaminated and undercooked food.It has been proved to enter viable but non culturable(VBNC)state,which cannot be detected by culture method but maintain metabolic activity in this state.It is a survival strategy when bacteria suffer from stress conditions and resuscitation occurs when they are changed to suitable conditions.Therefore,it is of great importance to do some research on it.In this thesis,different conditions were used to induce VBNC cells and the whole processes were monitored by counting the cells numbers.Variousresuscitate medium were prepared to comparing the effect of induction for resuscitation.The relation between biofilm and VBNC state was discussed.Real-time polymerase chain reaction combined with propidium monoazide(PMA-qPCR)was established to quantitative detect VBNC cells of V parahaemolyticus.Analyzation was performed to study physiological characteristic of cells in VBNC state.Finally,transcriptome andproteome were used to analyze the differential expressions of gene and protein.This thesis provided meaningful theoretical method and data for the detection and mechanism research of V.parahaemolyticus in VBNC state.V.parahaemolyticus was induced by different temperatures,salinity and concentration of chloromycetin.Among different inducing conditions,seven of them could entirely induced cells into VBNC state within 60 d.The minimum and maximum density of VBNC cells were 5.75×10~2 CFU/mL and 1.70×10~5 CFU/mL respectively.The induction efficiency of garlic extract and cinnamon extract were greater than chloromycetin.The VBNC cells induced by different conditions were sensitive to different resuscitate medium.The VBNC cells induced by 4?could be resuscitated by the most medium while the medium added 1%Tween 80 could resuscitate all the VBNC samples expect the one induced by 2MIC garlic extract.Cell-free supernatant(CFS)was confirmed to resuscitate VBNC cells with a good result,but heat-treated would impact its ability of resuscitation.Biofilm was detected by crystal violet staining method.It was found that,during induction the group which cannot be induced in to VBNC state were more likely forming biofilm.And compared with cells in biofilm,planktonic cells were more easily to be induced into VBNC state.The primers of PMA-qPCR were designed based on the tlh gene of V.parahaemolyticus.Results showed that the optimal PMA treatment condition was 5min-PMA treatment and 15 min-light exposure.The detection range was1.2×10~2 to1.2×10~9CFU/mL This method can significantly distinguish viable from dead cells and quantitatively detect VBNC state of V.parahaemolyticus rapidly.Viability of the cells was confirmed by Live/Dead detection kit under fluorescent microscope.In the respiration test,staining with C TC/DAPI couldclearly distinguish viable cells from dead cells under CLSM by fluorescent formazan.Flow cytometry was used to divide respiratory active and non-active cells into different groups rapidly.Transcriptome andproteome were used to analyze the differential expressions of gene and protein between VBNC cells,resuscitated cells and cells in logarithmic phase.There were 1309 differential expressed genes and 546 differential expressed proteins among these samples.In the comparison between cells in VBNC state and logarithmic phase,differential expressed genes were related to metabolism,metal ion transfer,locomotion,stimulus response,signaling,antioxtive and transport.Differential expressed proteins wereparticipated in ribosomesynthesis,amino acid metabolism,carbon fixation,fatty acid synthesis and metabolism.In the comparison between cells in resuscitation and logarithmic phase,differential expressed genes were related to ribosome synthesis,translation,fatty acid synthesis and metabolism,HIF-1 signal channel.Differential expressed proteins wereparticipated in ABC transporter(transmembrane transport). |
PDF Full Text Request