Survey for Nonculturable form of Cholera Vibrios.

我们将霍乱弧菌TSI-4菌株在15ﾟC的M9盐或磷酸盐缓冲盐水中培养，建立了一种活的但不能培养的状态。25-35天后，细菌在营养琼脂平板上失去了菌落形成能力，细菌形态转变为球形。然而，细胞内的三磷酸腺苷浓度在整个实验期间保持在15ﾟC的恒定水平。当培养在45ﾟC加热1min时，活细胞就可以从这个阶段恢复过来。然而，在PBS中建立的非培养细胞并没有通过热休克处理而复苏。由于M9盐和PBS的不同之处在于M9盐中存在铵盐，而PBS中没有铵盐，因此我们进行了从PBS中添加NH4CL回收细菌的实验，并从该培养物热休克后在琼脂平板上获得了菌落。回收的细胞被证实与实验中最初使用的细菌相同。将该方法应用于孟加拉国环境水样中霍乱弧菌的检测。虽然水样中不含任何可培养的霍乱弧菌，也没有检测到ctx基因的聚合酶链式反应产物，但在45ﾟ℃的氨盐存在下，从一些样品中可以检测到ctx基因。在加热的水样中也有霍乱弧菌的菌落，经鉴定为霍乱弧菌的O1inaba血清群。

We have established a viable but nonculturable state of Vibrio cholerae strain TSI-4 by incubating in M9 salt or phosphate-buffered saline (PBS) at 15ﾟC.After 25-35 days the bacteria lost the colony forming ability on nutrient agar plate and the shape of the bacteria transferred to coccoid form. The ATP concentration in the cells was, however, kept at a constant level throughout experimental period at 15ﾟC.The recovery of viable cells from this stage was achieved when the culture was heated at 45ﾟC for one min. The nonculturable cells established in PBS,however, was not resuscitated by heat shock treatment. Since the difference of M9 salt and PBS was the presence of ammonium salt in M9 salt but not in PBS,we carried out the experiment to recover the bacteria from PBS supplemented with NH_4Cl and we had the colonies on agar plates after heat shock from this culture. The recovered cells were confirmed to be identical to the bacteria that originally used in this experiment. This reactivation method was applied to V.cholerae in environmental water samples conllected in Bangladesh. Though the water samples did not contain any cultivable V.cholerae and any detectable PCR product of ctx gene, from some samples that was incubated at 45ﾟC in the presence of ammonium salt we could detect ctx gene after amplification by PCR.We also had colonies of V.cholerae in such heated samples and identified them as O1 Inaba serogroup of V.cholerae.