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《Acta Hydrobiologica Sinica》 2008-02
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STUDIES OF VIABLE BUT NONCULTURABLE VIBRIO PARAHAEMOLYTICUS AT LOW TEMPERATURE UNDER POOR NUTRITION CONDITIONS AND ITS RESUSCITATION

DU Meng,CHEN Ji-Xiang,SUN Feng-Rong,LUAN Xiao-Yan,WANG Dan,LI Yun and ZHANG Xiao-Hua(College of Marine Life Sciences,Ocean University of China,Qingdao 266003)  
Vibrio parahaemolyticus,a halophilic Gram negative bacterium,causes acute gastroenteritis in humans.This bacterium is a prevalent foodborne pathogen isolated from coastal water and from a variety of seafood,including fish.Most clinical isolates are hemolytic on Wagatsuma agar(Kanagawa-positive,KP+) and produce a major virulence factor,thermostable direct hemolysin(TDH).Seawater represents a nutrient-limiting environment for V.parahaemolyticus.During its persistence in seawater,V.parahaemolyticus is exposed to a variety of environmental stresses,including fluctuations in temperature and cold stress.Many bacteria have developed strategies for metamorphosis into more or less sophisticated survival forms in response to such harsh environmental conditions.The formation of(Viable but nonculturable) VBNC cells of bacteria has been proposed as a survival strategy to adverse conditions.The VBNC cell is a metabolically one although cells are incapable of undergoing the sustained cellular division required to form a colony on regular agar media.This state has been demonstrated in other human and fish bacterial pathogens,including Vibrio vulnificus,Salmonella enteritidis,enterotoxigenic Escherichia coli,Helicobacter pylori,Campylobacter jejuni,Legionella pneumophila,Shigella dysenteriae and Aeromonas hydrophila.It has been demonstrated that VBNC cells are active in metabolism and can nevertheless be able to recover from this dormant state,which becoming metabolically active and fully culturable.Although the loss caused by V.parahaemolyticus occurs over a long period of time in the year,V.parahaemolyticus is focusing on high temperature months,and is seldom isolated when the temperature of the water is low.A suggestion had been made that V.parahaemolyticus can survive for long period and may become nonculturable in the aquatic environment.In order to investigate the viable but nonculturable state of Vibrio parahaemolyticus at low temperature under poor nutrition conditions,V.parahaemolyticus HW799 was cultured in sterilized seawater microcosms at 4℃.Total cell counts remained constant throughout the 30d period,while plate counts declined to undetectable levels(0.1CFU/mL) within 30d.On the other hand,the viable cells declined to ca.106 CFU/mL and remained fairly constant at this level.These results indicated that a large population of cells existed in the VBNC state.The morphological changes of VBNC cells,normal cells and resuscitative cells of V.parahaemolyticus were studied with scanning electron microscope and flow cytometry.The results showed that compared to normal cells,the VBNC cells gradually changed in cell shape from curved to coccoid and decreased in size when they entered into the VBNC state.Another important characteristic of VBNC cells is their ability to be resuscitated in vitro and in a natural estuarine environment as a consequence of environmental changes.The experimental chick embryos injected with the VBNC cells remained alive and no microorganisms were reisolated from the injected chick embryos during the experimental time.The viable but nonculturable cells could be resuscitated with temperature upshift in the presence of nutrition in 48h.The resuscitative cells showed no differences in morphology with the normal cells.But when the cells were subjected to a temperature upshift in the absence of nutrition,no culturable population was observed on the medium.
【Fund】: 国家自然科学基金项目(30371108);; 上海市科技兴农项目(沪农科攻字(2005)第4-2号)资助
【CateGory Index】: S941
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