| Disinfectant has been widely used in medical treatment,food,industry,agriculture,cosmetics and other industries since its discovery.Quaternary ammonium compounds(QACs)disinfectant with no corrosion,without excitant,relatively stable and low toxicity and is widely used in food and environment disinfection,is currently one of the most commonly used disinfectants,its antibacterial effect is through the realization of positive interact with the cell membrane,through the interaction with cell membranes,membrane integrity of damage and leakage of cell contents.Every year,a small number of QACs are directly discharged into the environment after use,and the active ingredients in the formula far exceed the minimum inhibitory concentration(MIC)of the target microorganism.After discharge,the concentration of QACs will be decreased after dilution and biodegradation in the environment,resulting in the exposure of microorganisms to the sub-inhibitory concentration of QACs.Recent studies have shown that exposure to concentrations of repressed bacteria may promote the evolution of antimicrobial resistance,and may also lead to cross-resistance and multiple resistance to other antimicrobial agents such as antibiotics..Objective:By studying the expression of Acr AB-Tol C efflux pump regulatory genes marA,mar R,sox S and rob,to understand the role of Escherichia coli efflux pump in Escherichia coli resistance to quaternary ammonium compounds and the molecular mechanism of cross-resistance with fluoroquinolone,so as to provide ideas for the follow-up study of other mechanisms of cross-resistance of bacteria to disinfectants and antibiotics.Materials and methods:1.Six Escherichia coli stains were induced with benzalkonium bromide in vitro.Sensitivity of six resistant strains and their parent strains to benzalkonium bromide were tested by agar dilution method.2.Six resistant strains and their parent strains to benzalkonium bromide were tested by agar dilution method after efflux pump inhibitor Carbonyl cyanide3-chlorophenylhydrazone(CCCP)were added.3.The Acr AB-Tol C efflux pump regulatory genes mar R,marA,sox S and rob were amplified by PCR.4.Quantitative real-time PCR were used to detect the transcriptional level of mar R、marA、sox S、rob of both induced and their parent strains.5.The Acr AB-Tol C efflux pump regulatory genes mar R,marA,sox S and rob were sequenced after amplified by PCR.Results:1.The resistance level of 5 inducible strains to benzalkonium bromide was 4-8times higher than that of their parent strains,one strain had no change in sensitivity2.The minimum inhibitory concentrations(MIC)of benzalkonium bromide in 11 strains decreased by 2-256 times after addition of CCCP.3.The results of disinfectant resistance gene test showed that the mar R、marA、sox S、rob were commonly presently(93.75%-100%).4.The transcriptional level of marA gene in 4 induced strains was 17-33 times higher than that in their parent strains and mar R was 1-10 times higher than that in the parent strains in 5 induced strains(P<0.05).5.We found a tyrosine deletions at codon 142 of mar R in 10 strains,a mutation causing methionine to valine substitution at codon 1 and a serine deletions at codon145 of marA in 11 strains,a mutation causing serine to alanine substitution at codon12 of sox S in 10 strains.Conclusion:1.The increased transcriptional level of regulatory gene marA of Acr AB-Tol C efflux pump may be related to the resistance of quaternary ammonium compounds in Escherichia coli.2.More attention should be paid to the potential effects of the widespread use of quaternary ammonium compounds on Escherichia coli resistance and their resistance to fluoroquinolones. |
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