低温寡营养条件下副溶血弧菌形成活的非可培养状态及其复苏研究

STUDIES OF VIABLE BUT NONCULTURABLE VIBRIO PARAHAEMOLYTICU S AT LOW TEMPERATURE UNDER POOR NUTRITION CONDITIONS AND ITS RESUSCITATION

  • 摘要: 为研究在低温寡营养条件下副溶血弧菌(Vibrio parahaemolyticus)能否进入活的非可培养状态(VBNC),将浓度为1×1010CFU/mL的副溶血弧菌HW799在陈海水中4℃保存,每隔5天取样分别用吖啶橙染色荧光显微镜直接计数法(AODC)、活菌直接计数法(DVC)和涂布平板法(PC)测定细菌总数、活细菌数和可培养细菌数。在第30天时总细菌数基本不变,仍保持在109CFU/mL,活菌数为106CFU/mL,比总菌数低了约三个数量级,可培养细菌数为零,表明绝大部分副溶血弧菌HW799进入了VBNC状态;用扫描电镜、流式细胞仪对副溶血弧菌HW799活的非可培养状态、正常状态以及复苏后的细胞形态的研究表明进入VBNC状态后副溶血弧菌HW799形状变为球状,体积比正常状态明显变小,活细胞数也略有减少;采用在培养液中添加营养物质升温培养的方法,使VBNC状态的副溶血弧菌细胞在48h内复苏为可培养状态,复苏后的副溶血弧菌HW799与正常状态的细菌形态相似。

     

    Abstract: Vibrio parahaemolyticus, a halophilic Gram negative bacterium, causes acute gastroenteritis in humans.This bacteriumis a prevalent foodborne pathogen isolated from coastal water and from a variety of seafood, including fish.Most clinical isolatesare hemolytic on Wagatsuma agar (Kanagawa-positive,KP+) and produce amajorvirulence factor,thermostabledirect hemolysin(TDH).Seawater represents a nutrien-t limiting environment for V.parahaemolyticus.During its persistence in seawater, V.para-haemolyticus is exposed to a variety of environmental stresses, including fluctuations in temperature and cold stress.Many bacteriahave developed strategies for metamorphosis into more or less sophisticated survival forms in response to such harsh environmentalconditions.The formation of (Viable but nonculturable) VBNC cells of bacteria has been proposed as a survival strategy to ad-verse conditions.The VBNC cell is a metabolically one although cells are incapable of undergoing the sustained cellular divisionrequired to form a colony on regular agar media.This state has been demonstrated in other human and fish bacterial pathogens, in-cluding Vibrio vulnificus, Salmonella enteritidis, enterotoxigenic Escherichia coli, Helicobacter pylori, Campylobacter jejuni, Le-gionella pneumophila, Shigella dysenteriae and Aeromonas hydrophila.

     

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