| Objective Antibiotic resistant bacteria(ARB)is a serious threat to global public health.In order to control effectively the occurrence of antibiotic resistance bacteria and its propagation speed,it is imperative that we must discover the possible causes of the development and spread of ARB in the environment and clarify its mechanism.Bacteria can't be killed completely by the traditional water disinfection process,and some bacteria are left which have strong resistance,some bacteria still maintain in a healthy state among them,and some bacteria will form a special physiological state that can't be detected by traditional methods-sublethal injured bacteria.There has been no report on the association between the physiological state of bacteria,especially the injured state and the improvement in antibiotic resistance at present.The hypothesis that the injured bacteria state can improve the antibiotic resistance is made based on the above.The purpose of the study is to explore the effect of disinfection stress on bacteria resistance and to find out whether there is a correlation between the injured bacteria status and the increase of antibiotic resistance,trying to clarify its mechanism.Then we can lay the foundation for exploration of the mechanism that disinfection can promote the spread of ARGs in Water.Methods 1.Effects of sodium hypochlorite exposure and UV radiation on antibiotic resistance of bacteria:Taking Pseudomonas aeruginosa as the research object,after exposure to different doses of sodium hypochlorite or UV irradiation for different periods of time,the MIC of 13 representative antibiotics was determined by microbroth dilution method;at the same time,visible light method was used to detect the anti-oxidative enzyme activities of CAT enzyme,GSH-PX enzyme and SOD enzyme of bacteria;finally Spearman rank correlation was used to analyze the correlation between the concentration of injured bacteria and the antibiotic MIC value.2.The mechanism of bacteria increasing antibiotic resistance under disinfection stress: Extraction of bacterial RNA after exposure to sodium hypochlorite and UV radiation and transcriptomic sequencing was used,we have discovered key genes related to bacterial improvement of antibiotic resistance through differential expression analysis,SYBR Green fluorescence quantitative PCR method was used to verify the gene expression,finally we put forward the possible mechanism of bacteria under disinfection stress can increase antibiotic resistance.3.Investigation of antibiotic resistance of injured bacteria in drinking water: Injured bacteria in the tap water were isolated.Bacteria were identified based on 16 Sr RNA gene sequencing or biochemical identification kits,the MIC value of each isolate was determined by microbroth dilution method.Results: 1.The lethality of Pseudomonas aeruginosa at the dose of 4 mg/L sodium hypochlorite for 20 min was 50%(LD50),and the MIC of injured bacteria to AMP,CHL,and CAZ increased by 2,2 and 5.6 folds,respectively,and the MIC value is positively correlated with the concentration of injured bacteria under LD50.The content of CAT enzyme,GSH-PX enzyme and SOD enzyme was increased by 3.26,5.18,4.79 folds compared with the control group.The transcriptomic analysis showed that the most gene expression types was bacterial RND family drug efflux pump and the most obvious up-regulation genes was mex EF-opr N,the expression of mex E and opr P was up-regulated confirmed by q PCR.At the same time,the lethality rate of 2 mg/L and 8 mg/L sodium hypochlorite for 20 min was 20% and 90%,respectively,but no significant increase in MIC values was observed.2.After exposure to 11 m J/cm2 UV radiation,the MIC values of P.aeruginosa to TET,CIP,and PLB increased by 8.4,4.4,and 2 folds,respectively;the contents of CAT enzyme,GSH-PX enzyme and SOD enzyme were increased by 105.42,69.95,and 3.54 folds compared with the control group.Transcriptomic analysis showed that bacteria drug efflux pump family gene expression was significantly upregulated,the expression of mex C in RND family efflux pump was up-regulated confirmed by q PCR.3.From the 12 tap water samples,a total of 150 strains were isolated,involving 12 bacterial species,of which Pseudomonas is the largest,accounting for 38%.Compared with control bacteria,more than 90% of the isolates showed antibiotic resistance enhancement,among which the Pseudomonas aeruginosa's MIC values of AMP,CHL,CAZ,TET,CIP,and PB were all increased,and the MIC value of the CAZ has been increased by 128 times.The genus Bacillus might be resistant to five antibiotics.Conclusion: 1.P.aeruginosa at a semi-lethal dose of sodium hypochlorite significantly increased antibiotic resistance and was significantly associated with the concentration of injured bacteria.The mechanism is mainly related to the up-regulation of the expression of the mex EF-opr N efflux pump gene.2.Exposure to UV can increase antibiotic resistance of Pseudomonas aeruginosa,but there was no significant correlation with injured bacteria.The mechanism is mainly related to the up-regulation of gene expression in the mex C efflux pump family.3.The majority of isolates of injured bacteria in tap water show strong resistance to antibiotics,and Pseudomonas is the most abundant.The above research results show that both sodium hypochlorite and UV disinfection can cause bacterial damage,and the increased antibiotic resistance of injured bacteria can threaten the safety of drinking water. |
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