VBNC State Antibiotic Resistant Bacteria Induced By UV/Chlorine Disinfection And Its Resuscitation

Safe drinking water is a basic need for human health survival,but the widespread presence of antibiotic resistant bacteria(ARB)in the drinking water environment poses a new challenge to water safety and security.UV/chlorine technology as a new advanced oxidation process,but it does not completely inactivate ARB and has the potential to induce resistant bacteria into viable but nonculturable state(VBNC),posing a serious threat to the biosafety of drinking water.However,few studies have focused on the induction of VBNC ARB in the drinking water environment and the induction mechanism,and the recovery potential of VBNC ARB and the characteristics of the recovery are not clear.Compared with VBNC sensitive bacteria,VBNC ARB exists a greater health threat and may become an important link in promoting the spread of antibiotic resistance.Therefore,we investigated ARB with different resistance mechanisms(genetic mutation and carrying resistance plasmids),examined the law of UV combined with chlorine disinfection to induce ARB into VBNC state,elucidated its induction mechanism,and revealed the recovery potential and characteristics of VBNC ARB.The results will be an essential theoretical supplement to the study of VBNC bacteria in drinking water,and also provide a new scientific basis for the control of emerging biosafety hazards in drinking water.The main research findings are as follows.(1)Examination of the pattern of UV/chlorine disinfection induced entry of E.coli K12,rifampicin-resistant bacteria RIF(rpoB mutation)and plasmid-resistant bacteria RP4(containing RP4 plasmid)into the VBNC state.It was found that all three strains could be induced to enter VBNC,and ARB were more likely to enter the VBNC state than sensitive bacteria at higher disinfectant doses.The ability of ARB with different resistance mechanisms to enter the VBNC state also differed,with RP4 bacteria carrying the resistance plasmid becoming fully VBNC within a short time(20 s).Therefore,entering the VBNC state may be a preferred strategy for resistant bacteria,especially those carrying the resistance plasmid,to cope with harsh environments.(2)To investigate changes in the physiological characteristics,genetic features and expression levels of resistance-related genes in E.coli K12,RIF and RP4 after full entry into the VBNC state.The results showed that the cell membrane permeability of the three strains increased significantly after entering the VBNC state,and some bacterial bodies were wrinkled and their surfaces were concave inward.However,the ARB maintained their resistance gene integrity and bioactivity effectively.VBNC ARB still have the risk of promoting the spread of bacterial antibiotic resistance in the aqueous environment.(3)The expression of resistance genes associated with stress response regulation in VBNC bacteria was further analysed by RT-qPCR,which revealed differences in the gene regulation pathways of ARB entering the VBNC state by different resistance mechanisms.The expression of hipB and hipA,genes related to the toxins-antitoxins system(TA system),were significant regulatory mechanisms for the formation of VBNC mutant resistant bacteria.VBNC resistance plasmids carry resistant bacteria that rely more on the regulation of general stress response and functional resistance genes.(4)The recovery of VBNC state RIF and RP4 was tested under direct reversal culture conditions as well as under the addition of recovery-promoting factor pyruvate,bacterial culture supernatant.The results showed that the addition of LB broth,20 mM pyruvate,and culture supernatant could promote the recovery of VBNC status resistant bacteria,but the recovery needs of VBNC ARB with different resistance mechanisms were not identical.RIF was more effectively resuscitated under the condition of adding culture supernatant,and the highest recovery rate reached 26.7%,while RP4-resistant bacteria were more like to achieve resuscitated under pyruvate,with a recovery rate of 13.3%.The morphology of the resuscitated bacteria changed under various recovery conditions,with the surface of the bacteria recovered under LB broth being rough,the shape of some recovered bacteria still shortened under culture supernatant conditions,and the shape of the bacteria recovered under pyruvate conditions being similar to that of the original bacteria.