Abstract

We investigated the fate of Escherichia coli in natural waters, addressing survival, viable but non-culturable (VBNC) state, changes in phenotype and genomic diversification under laboratory microcosm environment. Five E. coli isolates (three marine, one clinical and one laboratory strain) were inoculated in microcosms of sterile distilled water, as well as, water collected from river, pond, sea, and estuary. A viable count was measured every week for up to 33 weeks. Strains which undergone VBNC state in microcosms were resuscitated. The revived isolates were tested to determine changes in antibiotic susceptibility, thermotolerance and genetic fingerprinting. We found extended persistence of five E. coli strains in water microcosms at room temperature for durations ranging from four to 33 weeks. Even in distilled water, some isolates persisted up to seven weeks; proving E. coli have excellent tenacity to survive. At VBNC state in microcosms, bacteria were small coccoid in shape and formed biofilms in most cases. Resuscitated laboratory strains showed changes in antibiotic susceptibility and thermotolerance compared to the original counterpart. However, the revived marine isolates showed very little to no difference in those tests. Resuscitation media could not revived the pathogenic isolates. The REP- and BOX-PCR genomic fingerprinting also showed some genetic diversification among resuscitated strains during the extended survival and VBNC state. The overall observation supports for the E. coli to survive in natural waters and to enter into VBNC state. It ultimately proves that their presence in water does not necessarily indicate faecal contamination but still beyond acceptance.

中文翻译：

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大肠杆菌在水微观研究中的存活及其对指示剂的使用的重新思考

摘要

我们研究了大肠杆菌在天然水域中的命运，探讨了生存，生存但不可培养的状态（VBNC），表型的变化以及实验室微观环境下基因组多样化的问题。五个大肠杆菌分离物（三个海岸，一个临床和实验室一个菌株）中的无菌蒸馏水缩影接种，以及，从河流，池塘，海，河口收集的水。每周测量一次可行的计数，最多持续33周。复苏在缩微中经历VBNC状态的菌株。测试了恢复的分离物以确定抗生素敏感性，耐热性和基因指纹图谱的变化。我们发现了五种大肠杆菌的持久性在室温下在水的微观世界中产生毒株，持续时间为4到33周。即使在蒸馏水中，某些分离株也能持续长达七个星期。证明大肠埃希菌具有出色的生存韧性。在微观世界中，在VBNC状态下，细菌的形状呈小球状，并形成了生物膜。复苏的实验室菌株与原始菌株相比，显示出抗生素敏感性和耐热性的变化。但是，恢复的海洋分离物在那些测试中显示出很小的差异或没有差异。复苏培养基无法使病原分离株复活。REP-和BOX-PCR基因组指纹图谱还显示了在延长的生存期和VBNC状态下复苏菌株之间的一些遗传多样性。总体观察支持大肠杆菌在天然水域中生存并进入VBNC状态。最终证明，它们在水中的存在并不一定表明粪便受到污染，但仍然无法接受。