Research on physiology and ecology of pathogenic bacteri in viable but nonculturable state

在逆境条件下，一些细菌会进入一种生理状态，即细胞不能通过常规培养方法生长，但仍保持一定的代谢活性。在显微镜下检测到的天然细菌中，有相当一部分保持着代谢活性，但无法培养。本课题旨在阐明细菌“活但不可培养状态”（VNC）的生理基础及其生态学意义。首先，将大肠杆菌、霍乱弧菌、副溶血性弧菌等部分细菌置于应激状态，诱导其进入VNC状态。测定生物化学成分，如DNA、RNA和蛋白质含量，以及代谢活性，如呼吸活性或酯酶活性。对VNC状态下的收缩模型进行了综合分析。其次，研究了瞬时温度上移对低营养和低温胁迫下细菌细胞的影响。结果发现，在室温下暴露20分钟即可有效抑制活菌数的进一步下降。考察了影响这一过程的条件，并研究了这一现象的分子基础。第三，将分子技术与现代光学设备相结合，明确了传统培养方法无法检测到的天然细菌种群的分布和波动。此外，调查人员还努力提出和讨论VNC现象的问题。很明显，在过去两年中，日本的许多细菌学家、生物学家和科学家通过成员们的活动，第一次意识到了这个问题。

Under stressed conditions, some bacteria fall into a physiological state in which cells fail to grow by conventional culture method but still maintain certain metabolic activities. A considerable part of natural bacteria detected under the microscopy are mataintaining metabolic activity, but unable to be cultured. The purpose of the present project is to clarify the physiological basis and ecological implication of so called "viable but nonculturable state" (VNC) of bacteria The results are summarized as follows. First, some bacteria, such as Escherichia coli, Vibrio cholerae, V. parahaemolyticus were placed in the stressed condition and were induced to fall into the VNC state. Biochemical components such as contents of DNA, RNA and protein, and metabolica activities such as respiratory activity or esterase activity were measured. These parameters were analyzed together for contracting models of VNC state. Second, effect of transient temperature up-shift on bacterial cells stressed by low nutrient and low temperature were investigated. It was found that as short as 20 minutes exposure at room temperature was effective to suppress further decrease of viable counts. Conditions affecting this process were checked and molecular basis of this phenomenon has been investigated. Third, distribution and fluctuation of natural bacteiral population that are not detected by conventional culture methods were clarified by the combination of molecular techniques and modern optical devices. In addition, the investigators have made efforts to present and discuss on the issue of VNC phenomenon. It is quite clear that during the last two years, many bacteriologists, biologists and scientists in Japan have been aware of this issue for the first time through the activitis of the members.