Factors Influencing The Development Of Viable But Non-Culturable State In Vibrio Cholerae And The Regulatory Mechanisms

Vibrio cholerae can enter into a viable but non-culturable(VBNC)state in order to survive in unfavourable environments.The VBNC state has been recognized for 30 years,but remains as a mystery to be resolved.To better understand this phenomenon,we designed and performed three parts of study as following.In the first part,we utilized propidium monoazide(PMA)combined with real-time PCR to quantitatively enumerate V.cholerae in the VBNC state.In this part,with an initial cell concentration of 1 x 108 CFU/ml,time and dose effect relationships of different PMA treatments were evaluated via quantitative real-time PCR using live cell suspensions,dead cell suspensions and VBNC cell suspensions of V.cholerae 01 El Tor strain C6706.The results suggested that a PMA treatment of 20μM PMA for 20 min was optimal under our conditions.This treatment maximized the suppression of the PCR signal from membrane-compromised dead cells but had little effect on the signal from membrane-intact live cells.In addition to the characteristics of PMA treatment itself,the initial concentration of the targeted bacteria showed a significant negative influence on the stability of PMA-PCR assay in this study.We developed a strategy that mimicked a 1×108 CFU/ml cell concentration with dead bacteria of a different bacterial species,the DNA of which cannot be amplified using the real time PCR primers.With this strategy,our optimal approach successfully overcame the impact of low cell density and generated stable and reliable results for counting viable cells of V.cholerae in the VBNC state.In the second part,we studied the roles of five physicochemical and microbiological factors or states,namely,different strains,growth phases,oxygen,temperature,and starvation,on the development of VBNC of V.cholerae in artificial sea water(ASW).Different strains of the organism,the growth phase,and oxygen levels affected the progress of VBNC development.It was found that the VBNC state was induced faster in V.cholerae serogroup 01 classical biotype strain 0395 than in 01 El Tor biotype strains C6706 and N16961.When cells in different growth phases were used for VBNC induction,stationary-phase cells lost their culturability more quickly than exponential-phase cells,while induction of a totally non-culturable state took longer to achieve for stationary-phase cells in all three strains,suggesting that heterogeneity of cells should be considered.Aeration strongly accelerated the loss of culturability.During the development of the VBNC state,the culturable cell count under aeration conditions was almost 106-fold lower than under oxygen-limited conditions for all three strains.The other two factors,temperature and nutrients-rich environment,may prevent the induction of VBNC cells.At 22℃ or 37℃ in ASW,most of the cells rapidly died and the culturable cell count reduced from about 108 CFU/mL to 106-105 CFU/mL.The total cell counts showed that cells that lost viability were decomposed,and the viable cell counts were the same as culturable cell counts,indicating that the cells did not reach the VBNC state.VBNC state development was blocked when ASW was supplied with Luria-Bertani broth(LB),but it was not affected in ASW with M9,suggesting that specific nutrients in LB may prevent the development of VBNC state.These results revealed that the five factors evaluated in this study had different roles during the progress of VBNC induction.Changing a single factor could influence and even block the development of the VBNC state.In the third part,we illustrate a confrontation adapted by V.cholerae 01 through quorum sensing(QS)system to against the development of VBNC state in ASW at 4℃.Firstly,the expression of HapR postponed the culturability lost during the VBNC induction.Deletions of C6706 in rpoN,luxO or qrrs genes,which leaded to up regulation of HapR,maintained their ability to form colony up to 50 days more than the deletion of hapR.Furthermore,the expression of HapR was employed by V.cholerae to confront the culturability lost during VBNC progress.During the induction,the hapR mRNA transcript was up regulated in the wildtype strain,resulted in 10 days delay of culturability lost compared with hapR deletion.When complemented with phapR,the hapR deletion regained hapR mRNA transcript and HapR protein together with the speed of culturability lost returned to the same level as the WT strain.Moreover,the ecological pressures acted on the evolution of quorum sensing and helped shaping particular quorum-sensing pattern to against the VBNC development.Some natural V.cholerae 01 isolates constitutively express high level of HapR protein.These strains exhibited more resistance to form non-culturable cells during VBNC induction.The existence of these strains gave evidences about the evolution of V.cholerae against these harsh environments.Our findings of these three parts provided useful data for and impetus for further studies into how the VBNC state is triggered and the regulatory mechanisms.And they also help us to understand the lives and activities of V.cholerae during VBNC development and how the environment stresses drive the evolution of this microorganism.