Study On The Viable But Non-culturable State Of Vibrio Cincinnatiensis,V. Harveyi And V. Anguillarum

The viable but nonculturable state is a kind of special survival state when bacteria are under harsh surroundings conditions.The bacteria can not be detected with normal culture methods,but it is still viable and possesses metabolic activity. The aim of this study is to investigate the characteristics^- of Vibrio cincinnatiensis,V. anguillarum,V.harveyi in viable but nonculturable state including their morphological and virulence changes during they entry into viable but nonculturable state,resuscitated from the state.The physical characteristics and the virulence related gene expression of resuscitated cells of V.harveyi.were also investigated.Vibrio cincinnatiensis,V.anguillarum and V.harveyi cells were incubated in sterilized Seawater or artifial sea water microcosm at 4℃.Total cell counts,direct viable counts and cultural counts were determined by acridine orange direct count (AODC),direct viable count(DVC) and spread plate count(PC) on 2216E agar respectively.The results showed that large population of the V.cincinnatiensis became nonculturable after approximately 50 d cultured in sterilized seawater at 4℃. V.anguillarum kept viable for a long time,when this strain was incubated in the sterilized seawater microcosms or in the sterilized artificial seawater microcosms,the cultural counts declined at first and then remained fairly constant at a number of 10³-10⁴ CFU/ml.when it was incubated in the sterilized MMS microcosms or sterilized 0.5%- MMS microcosms,the cultural counts declined quickly at first,then rised to＞10⁴CFU/ml.The morphological changes of VBNC cells,normal and resuscitative cells were studied with epifluorescence microscope and transmission electron microscope.The VBNC cells changed from rod to coccoid and decreased in size compared to the normal cells.The periplasmic spaces increased greatly and nuclear region(electron clear area)was compressed the of the VBNC cells under TEM.Membrane curling was observed in some of the VBNC cells.The resuscitative cells showed similar morphology with the normal cells.Resuscitation of VBNC cells of Vibrio cincinnatiensis were achieved by temperature upshift in nutrition of yeast extract and peptone by addition of catalase or compound vitamin B.The VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately.The fish inoculated with the resuscitative cells died within 7 d,the LD₅₀ was 2.77×10⁵ CFU/L,which was close to the groups inoculated with the normal cells(3.87×10⁵ CFU/L).No death was observed in_the group inoculated with the VBNC cells,which suggested that the resuscitative cells might retain their pathogenicity.The sensitivity of the the resuscitative cells of V.harveyi to UV irradiation,heat, cold and osmotic stresses were determined.The sensitivity of the resuscitative Cells of Vibrio harvey to UV and high temperature(42℃) stresses did not changed obviously, sensitivity to osmotic(2.5M NaCl) decreased slightly,but the resistance to cold stress increased when compared with that of the normal cells.Reverse transcription-polymerase chain reaction(RT-PCR) showed that expression of Ser, rpoS genes were not detected in the VBNC cells but were detected in the resuscitative cells.The global transcription pattern of the VBNC cells and the resuscitative cells with that of normal cells V.harveyi were also studied.A total of 893 genes of VBNC cells were found to differ by compared with that of the normal cells.Among them 430 genes were up regulated and 263 genes were down regulated.In the resuscitative cells, 197 genes were up regulated and 779 genes were down regulated.