Asakura Hiroshi, Ishiwa Akiko, Arakawa Eiji, Makino Sou-ichi, Okada Yumiko, Yamamoto Shigeki, Igimi Shizunobu
Division of Biomedical Food Research, National Institute of Health Sciences, Kamiyoga 1-18-1, Tokyo 158-8501, Japan.
Environ Microbiol. 2007 Apr;9(4):869-79. doi: 10.1111/j.1462-2920.2006.01206.x.
Vibrio cholerae is an aetiological agent of cholera that inhabits marine and estuarine environments. It can survive harsh environments by entering the viable but non-culturable (VBNC) state, but the related changes in gene expression have not been described. Here, we experimentally induced the VBNC state in V. cholerae O1, by incubation in artificial seawater at 4 degrees C. Bacterial cells that were incubated for 70 days retained their membrane integrity and were pathogenic, colonizing the gut of iron-dextran-treated mice, even though they formed no colonies on tryptic soy agar (TSA) or TSA amended with pyruvate. We therefore used this stage of cells as the VBNC bacteria. We compared the global transcription pattern of the VBNC cells with that of stationary-phase cells grown in rich medium. A total of 100 genes were induced by more than fivefold in the VBNC state, and the modulated genes were mostly those responsible for cellular processes. Furthermore, real-time RT-PCR analysis verified the changes in the expression levels, showing that the VC0230 [iron(III) ABC transporter], VC1212 (polB), VC2132 (fliG) and VC2187 (flaC) mRNAs were increased in the non-culturable state. Thus, these genes may be suitable markers for the detection of VBNC V. cholerae. To our knowledge, this is the first report of a comprehensive transcriptome analysis of V. cholerae in the VBNC state. The significance of this gene expression profile compared with those of in vivo isolates and non-stressed bacteria (culturable in vitro) is its potential to provide information about the public health risk from dormant bacteria.
霍乱弧菌是霍乱的病原体，栖息于海洋和河口环境。它可通过进入活的但不可培养（VBNC）状态在恶劣环境中存活，但相关的基因表达变化尚未见报道。在此，我们通过在4℃的人工海水中孵育，实验性地诱导霍乱弧菌O1进入VBNC状态。孵育70天的细菌细胞保持其膜完整性且具有致病性，可在右旋糖酐铁处理的小鼠肠道中定殖，尽管它们在胰蛋白胨大豆琼脂（TSA）或添加丙酮酸的TSA上不形成菌落。因此，我们将此阶段的细胞用作VBNC细菌。我们将VBNC细胞的全局转录模式与在丰富培养基中生长的稳定期细胞的转录模式进行了比较。共有100个基因在VBNC状态下被诱导超过五倍，且调控的基因大多负责细胞过程。此外，实时RT-PCR分析验证了表达水平的变化，表明VC0230[铁（III）ABC转运蛋白]、VC1212（polB）、VC2132（fliG）和VC2187（flaC）的mRNA在不可培养状态下增加。因此，这些基因可能是检测VBNC霍乱弧菌的合适标志物。据我们所知，这是关于VBNC状态下霍乱弧菌全面转录组分析的首次报道。与体内分离株和非应激细菌（体外可培养）的基因表达谱相比，这种基因表达谱的意义在于它有可能提供有关休眠细菌公共卫生风险的信息。
