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

在应激条件下，一些细菌进入一种生理状态，即细胞不能通过传统的培养方法生长，但仍保持一定的代谢活动。显微镜下检测到的相当一部分天然细菌仍保持代谢活性，但无法培养。本课题的目的是阐明细菌“可活但不可培养状态”(VNC)的生理基础和生态学意义。首先，将大肠杆菌、霍乱弧菌、副溶血性弧菌等置于应激状态，诱导其进入VNC状态。测定DNA、RNA、蛋白质等生化成分，呼吸活性、酯酶活性等代谢活性。对于VNC状态的收缩模型，这些参数被一起分析。其次，研究了瞬时升温对细菌细胞在低营养和低温胁迫下的影响。研究发现，在室温下暴露20分钟可以有效地抑制活菌数量的进一步减少。考察了影响该过程的条件，并对该现象的分子基础进行了探讨。第三，将分子技术与现代光学技术相结合，阐明了常规培养方法无法检测到的天然细菌种群的分布和消长。此外，调查人员还努力对VNC现象进行了介绍和讨论。很明显，在过去的两年里，日本的许多细菌学家、生物学家和科学家通过成员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.