“有活性但不可培养”状态抗生素抗性细菌的复苏及其潜在机理

Resuscitation of the VBNC state antibiotic-resistant bacteria and the related potential mechanisms

饮用水中“有活性但不可培养”(viable but nonculturable,VBNC)状态抗生素抗性细菌(antibiotic resistance bacteria,ARB)的流行极大地破坏了其水质安全性,严重危害人类健康.复苏是VBNC状态细菌的重要特征,但是目前饮用水环境中VBNC状态ARB的复苏及机理并不明确.本研究通过模拟饮用水管网生物膜复苏环境因子,考察了UV/Cl_(2)联合消毒诱导进入VBNC状态的携带多重耐药质粒(Rp4 plasmid,RP4)以及染色体突变(rpoB mutation,RIF)介导抗性的Escherichia coli K12菌株的复苏潜能和复苏规律.结果表明,通过移除消毒压力、添加可培养细胞上清液以及添加丙酮酸条件下,受试VBNC ARB的复苏比例可以达到3.3%~22.7%.并且,大多数复苏的细胞仍能保留原有抗生素抗性.通过长扩增子结合qPCR技术检测发现,即使是高消毒剂剂量诱导的VBNC RP4细菌中tetA和tnpA基因仍能够较好地维持其完整性,具有发生水平基因转移的风险.但高消毒剂剂量诱导的VBNC RIF则发生回复突变,抗性丢失.相较于染色体突变介导抗性的VBNC ARB,携带抗性质粒的VBNC细菌复苏后将具有更大的健康风险.总体而言,本研究证实了饮用水管网环境中存在VBNC ARB复苏的可能,且复苏细菌仍具有发生水平基因转移的潜能,能够促进细菌抗生素抗性在饮用水中的传播扩散.该研究结果不仅是对VBNC状态细菌,特别是VBNC状态ARB复苏研究的重要补充,同时,进一步加深了对VBNC ARB环境风险的认识,也为抑制或减缓饮用水消毒工艺所诱导的VBNC状态细菌的复苏提供了数据参考.

The prevalence of"viable but nonculturable"(VBNC)state antibiotic-resistant bacteria(ARB)have greatly damaged the quality of drinking water and seriously endangered human health.Resuscitation is an important feature of VBNC state bacteria,however,little data was available that explicitly characterized the recovery condition and mechanism of VBNC-state ARB.Here,we investigated the resuscitation potential of UV/Cl_(2)disinfection induced VBNC Escherichia coli K12 strain carrying multidrug-resistant plasmid(RP4)and that with chromosomal mutation(RIF)by simulating the resuscitation factors of drinking water distribution systems biofilm.The results showed that under the condition of removing disinfection pressure,adding cell-free supernatant and adding pyruvate,the resuscitation ratio of VBNC ARB could reach 3.3%~22.7%.Moreover,most of the resuscitated RP4 and RIF still retained the original antibiotic resistance.Through the detection of long amplicons combined with qPCR it was found that even the tetA and tnpA genes in VBNC RP4 induced by high disinfection dose could maintained their integrity,implying the potential for horizontal gene transfer(HGT).Nevertheless,reverse mutation was detected in the VBNC RIF induced by high disinfection dose,resulting in the loss of rifampicin resistance.Compared to VBNC ARB mediated by chromosomal mutations,VBNC bacteria carrying multidrug-resistant plasmids will have a greater health threat after resuscitation.Overall,this study confirmed the possibility of VBNC ARB resuscitation in the drinking water distribution systems,and the the resuscitated cells bacteria still have the potential of HGT which would facilitate the spread of bacterial antibiotic resistance in drinking water.The results of this study are not only an important supplement to the research of VBNC state bacteria,especially the resuscitation of VBNC ARB,but also further deepen the understanding of the environmental risks of VBNC ARB,and provide data reference for inhibiting or slowing down the resuscitation of VBNC state bacteria induced by drinking water disinfection processes.