Use of Flow Cytometry for Quantitative Analysis of Metabolism of Viable but Non-culturable (VBNC) Salmonella.
Use of Flow Cytometry for Quantitative Analysis of Metabolism of Viable but Non-culturable (VBNC) Salmonella.
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DOI：
10.1248/bpb.b15-00005
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发表时间：
2015-09
影响因子：
2
通讯作者：
Y. Morishige;K. Fujimori;F. Amano
中科院分区：
文献类型：
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作者：
Y. Morishige;K. Fujimori;F. Amano
建立了一种简单、快速的活的但不可培养的沙门氏菌代谢活性分析方法。肠道沙门氏菌（SE）的环境分离株，生长到对数期，在Luria-Bertani（LB）培养基中与1-10 mM H2 O2孵育期间迅速失去其可培养性。为了评估细菌的活力，我们测量了3种不同的代谢活性：通过5-氰基-2，3-二甲苯基-四唑氯化物（CTC）还原的呼吸活性，用2-[N-（7-硝基苯-2-氧杂-1，3-二唑-4-基）氨基]-2-脱氧葡萄糖（2-NBDG）评估的葡萄糖摄取，以及通过5-乙炔基-2 '-脱氧尿苷（EdU）掺入评估的DNA合成活性。这些活动进行了分析，共聚焦激光扫描显微镜和流式细胞术，连同LB-琼脂平板上的菌落形成试验。结果表明，H_2O_2处理的SE细胞中的一些处于VBNC状态，并且H_2O_2诱导的每种代谢活性的降低程度根据活性而变化。也就是说，葡萄糖摄取活性没有明显变化，保持在最高水平;而呼吸活性小于葡萄糖摄取活性，DNA合成活性是其中最低的。这些结果表明，VBNC状态的特征可能是不同的代谢活动，这些活动的变化和对应于压力的种类和强度，威胁着细菌在不利环境中的生存。
A simple and rapid assay method for analysis of the metabolic activity of viable but non-culturable (VBNC) Salmonella was established. An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost its culturability during incubation with 1-10 mM H2O2 in Luria-Bertani (LB) medium. To assess the viability of the bacteria, we measured 3 different metabolic activities: Respiratory activity by 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) reduction, glucose uptake assessed with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and DNA synthesis activity evaluated by 5-ethynyl-2'-deoxyuridine (EdU) incorporation. These activities were analyzed by both confocal laser-scanning microscopy and flow cytometry, together with colony-formation assays on LB-agar plates. The results showed that some of the H2O2-treated SE cells were in the VBNC state and that the extent of H2O2-induced decrease in each metabolic activity varied according to the activity. That is, glucose-uptake activity was not markedly changed, being kept at the highest level; whereas the respiratory activity was less than that of the glucose-uptake, and DNA synthesis activity was the lowest among them. These results suggest that the VBNC state might be characterized by different metabolic activities that vary and correspond to the kind and strength of the stress, threatening bacterial survival in an adverse environment.