Molecular Mechanism Of Environmental Stress On The Growth Of Drug Resistant Escherichia Coli

Bacterial resistance has become an important public safety problem threatening the global human and animal health.Reducing the use of antibiotics(reducing resistance)is the most important strategy proposed by WHO to reduce bacterial resistance.The theory is based on the fact that drug-resistant bacteria increase the cost of adaptation in drug-free environment,decreasing their competitiveness for survival and thus gradually being replaced by sensitive bacteria,allowing regional bacteria to recover their susceptibility to drugs.In this study,multiple drug resistant E.coli QL15and standard E.coli ATCC25922 from pigs were selected as the research objects.Growth curve and competitive test were used to explore the growth status and adaptability of resistant E.coli under different environmental stress factors,and the effects of environmental stress factors on the morphological structure and drug resistance genes of drug-resistant E.coli were investigated,To reveal the effect of environmental stress factors on the survival competitiveness of drug-resistant E.coli and its molecular mechanism,we detected the changes of efflux pump and outer membrane proteins by re sequencing analysis and fluorescence quantitative PCR,and provided theoretical support for exploring the theory of"reducing resistance"to accelerate the recovery of antimicrobial susceptibility of multi drug resistant strains.The results are as follows:(1)Escherichia coli(QL15)isolated from large pig farms is a high level of multi-drug resistance bacteria,which could resist 14 kinds of antibiotics in 7categories.The resistance level to enrofloxacin and spectinomycin is the highest,which is 32 times of the standard minimum inhibit concentration,while florfenicol and sulfaisoxazole are the lowest(about 1 time).(2)QL15 contained 15 drug resistance related genes,including aac(3)-IV?cmlA?ompC?ompF?acrA?tolC?emrB?emrD?tetA?bla_TEM?acrB?sugE?mdtL?sul2 and sul3.There were 8 efflux pump genes and 3 outer membrane protein related resistance genes.Therefore,the multidrug resistance of QL15 is closely related to the efflux pump and outer membrane protein mechanism.As biofilm negative strains,QL15 and ATCC25922 they could not produce biofilm.(3)The results of growth curve showed that under independent culture conditions,the growth of QL15 in 0.1%C,0.5%C,1%C&0.1%N,0.05%N,0.01%N,pH5,pH6,pH8,20?and 42?were better than that of the sensitive strain ATCC25922,while the growth in 3.5%NaCl,6%NaCl,pH9,1/4 LB,1/8 LB and1/16 LB was slightly worse.Under mixed culture conditions,the relative adaptability of QL15 in 0.1%C,0.5%C,1%C&0.1%N,0.05%N,0.01%N,pH5,pH6,pH8,20?,42?,3.5%NaCl and 6%NaCl was higher than that of sensitive bacteria and also higher than the independent culture results,but it was inferior in pH9,1/4 LB,1/8LB and 1/16 LB.The surface morphology of QL15 under 6%NaCl did not change much,indicating that it is more salt tolerant.(4)Using ATCC25922 as the reference genome,mutations in various functional protein components of QL15 were measured.Among these,The mutation sites of biofilm related genes such as cell wall and cell membrane were the most.The mutation may have a positive impact on the adaptation of drug-resistant bacteria to a wider range of environmental stresses.The expression of QL15 resistance gene was different under different environmental stress factors.The expression of emrB?acrB?tolC and ompC was were mostly down-regulated,while the expression of ompF was up-regulated under salt stress,acid-base stress,total nutrition stress as well as high and low temperature stresses.Therefore,the high expression of ompF gene may be beneficial to the growth of bacteria under the above stress conditions.Conclusion:under drug-free pressure conditions,QL15 was more adaptive than sensitive bacteria under carbon,nitrogen,acid-base,high and low temperature and salt stresses except under the condition of pH9 and LB dilution.The results were contrary to the traditional theory that drug-resistant bacteria are less adaptable than sensitive bacteria in drug-free environment.QL15 has a variety of efflux pump and outer membrane pore protein related resistance genes,and most of the InDel sites were biofilm related functional genes.Its multiple drug resistance of QL15 is mainly mediated by a variety of efflux pumps and outer membrane pore proteins.The high expression of ompF gene under different environmental factors may be responsible for improving the survival competitiveness of the bacteria.Therefore,whether the drug-resistant bacteria with efflux pump and outer membrane porin mediated resistance mechanism could recovery their sensitivity to antibiotics through"reducing resistance"needs to be further studied.