Resuscitation and characterization of VNC vibrio cholerae

我们构建了一种不同条件下的不可培养（活菌但不可培养：VNC）霍乱弧菌，以阐明病原菌作为传染源在环境中的生存方式，并分析其生理特性。此外，我们还尝试建立一种能使VNC霍乱弧菌在琼脂培养基上持续复苏的方法。将霍乱弧菌悬浮于M9盐中，低温保存约1个月后，细菌进入VNC状态，发现进入VNC状态的条件不仅与霍乱弧菌菌株不同，而且与细菌周围的各种条件也不同。特别是，霍乱弧菌的细胞浓度和细胞生长阶段都影响VNC的时间。低细胞浓度和中对数期的细菌迅速进入VNC状态。霍乱弧菌在含有灰尘或生物等杂质的水中比在清洁水中存活的时间更长。这再次证实了霍乱弧菌依附于环境中的浮游生物并能够长期存活的事实。我们证实，VNC霍乱弧菌是复苏形成菌落在铵离子存在的热处理。该复苏方法对环境中的霍乱弧菌是有效的，在孟加拉国达卡附近采集的水样中可以复苏VNC霍乱弧菌。我们认为这种复苏方法对霍乱弧菌以外的细菌是有用的。然而，从VNC复苏的速度并不是恒定的，我们必须摸索更复杂的复苏方法。从我们获得的数据来看，VNC状态很难被认为是“生存的主动机制”，而VNC状态似乎是由于细胞成分或功能的各种损伤积累而导致的“细胞死亡”过程的初始阶段。目前，我们正在研究这种修复机制是否可能是由热处理等刺激诱导的，从而修复了VNC霍乱弧菌的损伤。少

We constructed a nonculturable (viable but nonculturable : VNC) form of Vibrio cholerae under various conditions to clarify mode of life of pathogenic bacteria as infectious sources in the environment and analized their physiological characterizations. Furthermore, we tried to establish the method that could constantly resuscitate the VNC V.cholerae to grow on agar medium.When V.cholerae was suspended into M9 salt and was kept at low temperature, the bacteria entered into a VNC state after about 1 month incubation, It was found that the terms for entering into the VNC were different from not only strains of V.cholerae but also various conditions surrounding the bacteria. Especially, both cell concentrations of V.cholerae and stages of cell growth affected the time for VNC.The bacteria in low cell concentration and middle log phase have quickly entered into the VNC state. V.cholerae showed longer survival in the water containing many impurities such as dust or living things rather than … More in clean water. This indicated reconfirmation of the fact that V.cholerae attached to the planktons in the environment and was able to survive for long time.We confirmed that the VNC V.cholerae was resuscitate to form colonies by heat treatment in the presence of ammonium ions. This resuscitation method was effective to V.cholerae in the environment, since we could resuscitate VNC V.cholerae in the water sample collected near Dhaka in Bangladesh. We think that the resuscitation method will be useful to the bacteria other than V.cholerae. The rate of resuscitation from VNC, however, was not constant and we have to grope for the more sophisticated method for resuscitation.From our data obtained, it is difficult to consider the VNC state "active mechanism for Survival", rather the VNC state seems to be the initial stage of process to "cell death" caused by the accumulation of various damages to cell components or functions. We are now investigate the possibility that the repair mechanism is induced by the stimuli such as heat treatments and repair the damages in the VNC V.cholerae. Less