| Pathogenic Escherichia coli often causes diarrhea,sepsis and other clinical symptoms,and is still one of the main intestinal pathogens affecting human and animal health.?-lactam antibiotics are the oldest and most widely used class of antibiotics,among which ampicillin resistance rate increases year by year.Antimicrobial agents are transmitted to the environment through animal excreta,which not only pollute the environment,but also do great harm to human health and the sustainable development of aquaculture.The resistance of bacteria to antibiotics is mainly by encoding?-lactamase,changing the target protein in cell wall,reducing the permeability of outer membrane,and improving the expression of drug excretion pump in the efflux system.Multi-drug-resistant Escherichia coli seriously confine human health,especially bacteria resistant to?-lactam antibiotics,but the mechanism for drug resistance is still unknown.We conducted a study to elucidate the mechanism and law of drug resistance.ObjectTo understand the mechanism of resistance of Escherichia coli to beta lactam antibiotics by inducing drug resistance of sensitive bacteria in laboratory.Methods1.Clinical sensitive E.coli strain was induced into resistance strain by 1/2minimum inhibitive concentration(MIC)induced trails of ampicillin.The drug resistance spectrum was measured by modified K-B susceptibility test.2.Whole genome sequencing analysis was used to analyze primary sensitive strain,and resequencing was used to analyze induced strains.3.RT-PCR was performed to verify the differential expression of non-SNPs genes in sensitive and drug-resistant strains.4.Protein structure encoded by the gene containing SNP was analyzed by bioinformatics.Results1.In the experiment,we successfully induced two strains of Escherichia coli.After induction,the MIC value reached 256?g/mL,which was 32 and 64 times higher than that of the sensitive strain,respectively.Compared with the susceptible strains,the resistance spectrum of Escherichia coli to antibiotics was changed.2.The regression equation of MIC value(y)and induction time(x)of E.coli 181was y=1.1447 ln~x-0.8036.Regression analysis was performed on the parts with MIC value less than or equal to 32?g/mL,and the regression equation was y=0.0442x+1.5752.The fitting effect of the equation was evaluated,R~2=0.7806,P<0.05.The fitting equation of the MIC value(y)and induction time(x)of E.coli 15743was y=1.0435ln~x-0.7316.The regression equation for the part with MIC value less than or equal to 32?g/mL was y=0.0358x+1.2812.The fitting effect of the equation was evaluated,R~2=0.991,P<0.05.3.E.coli 181 contained 4,498 genes,and drug-resistant strains E.coli 181-32 and181-256 contained 1 and 7 non-synonymous SNPs,which were distributed in frdD and unknown genes.E.coli 15743 contains 4,408 genes,and E.coli 15743-32 and15743-256 contain 4 and 8 non-synonymous SNPs,which were distributed in frdD,ftsI,acrB,OmpD,marR,VgrG and envZ genes.4.The functional classification of non-snps in COG was related to energy production and conversion,origin of cell wall,transport and metabolism of inorganic ions,transcription,and signal transduction mechanism.5.The protein secondary structure encoded by these genes was mainly composed of alpha helix and random coil.6.The tertiary structure of the protein encoded by acrB and envZ genes were altered before and after resistance.Conclusions1.With the induction,MIC of 32?g/mL was a key point for the bacteria to be resistant to ampicillin.2.FrdD?ftsI?acrB?OmpD?marR?VgrG and envZ genes were associated with ampicillin resistance.3.Changes in non-synonymous SNPs of Escherichia coli were related to the resistance of ampicillin.These researches will help to improve the molecular mechanism of tolerance of large intestine to?-lactam bacteria and provide research foundation for the prevention and control of multi-drug resistant bacteria and the target of new antibiotics. |
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