Drug Resistance Mechanism Of Colistin Resistance Gene In Foodborne Pathogens

In recent years,with the extensive use of antibiotics in livestock farming and clinical treatment,microbial resistance has been widely recognized in the food safety and public health fields worldwide.In particular,the emergence of carbapenem-resistant strains has directly led to the emergence of “no drug available” in the face of multi-drug resistant strains.In such a severe background,the peptide antibiotic colistin has begun to receive more and more attention.colistin has effective bactericidal and bacteriostatic effects against Gram-negative bacteria,especially for the frequent emergence of carbapenem multi-drug resistant strains.However,since 2016,mcr-1 and its homologous genes,which are caused by plasmid-mediated colistin resistance genes,have been reported continuously and made the use of colistin seriously threatened.Therefore,the study on the resistance of mcr-1 positive strains,the mechanism of mcr-1 and related family genes is of great significance for the prevention and control of colistin resistance.This paper studies from the following aspects: screening of mcr-1 positive resistant strains and analysis of drug resistance;development of a multiplex PCR detection assay for five mcr family genes,and verification of the specificity and sensitivity of the method;construction of mcr-5 gene-related engineering strains and study of their resistance mechanisms.(1)Screening of mcr-1 gene positive Escherichia coli and analysis of its drug resistance.Eight strains of colistin-resistant strains were isolated from 100 strains of chicken farms from the Faisalabad district of Pakistan.After PCR and chemical identification,8 strains of polymyxin-resistant strains were all mcr-1 Positive E.coli.The minimum inhibitory concentration(MIC)of colistin for 8 resistant strains was in the range of 2~8 ?g/mL,and the minimum bactericidal concentration(MBC)was in the range of 4~16 ?g/mL.Among the 8 resistant strains,the plasmid containing the mcr-1 gene can be divided into two types,and none of them contain the ISApl1 insertion element.Eight resistant strains belong to ST10,ST2847,ST155,ST361,ST6395 and two new multilocus sequence typing(MLST).The result of sequencing of the resistant strain PK105 genome revealed that the mcr-1 gene is located on a 60499 bp IncI2 type plasmid containing a series of vir and pil family genes involved in the conjugation.Matrix-assisted laser desorption tandem time-of-flight mass spectrometry(MALDI-TOF MS)analysis of lipid A of drug-resistant strain PK105 showed that it contained a 1791.122(m/z)peak and a 1920.136(m/z)Modified peak.Eight drug-resistant strains were resistant and intermediate-resistant to most of the 12 antibiotics.Eight drug-resistant strains were resistant and intermediate-resistant to most of the 12 antibiotics.(2)Establishment of a multiplex PCR assay for mcr family genes.We designed five pairs specific primers,and the PCR reaction procedures range from 219 to 908 bp in length for the mcr family genes mcr-1,mcr-2,mcr-3,mcr-4,and mcr-5 to construct a multiplex PCR assay for five mcr family genes.The specificity and sensitivity of the assay were verified by using a single plasmid containing the mcr genes and a multiplexed plasmids containing the mcr genes as templates.The detection of 156 wild-type strains as templates was verified the effectiveness of the detection method in practical application.(3)Study on drug resistance mechanism of colistin resistance gene mcr-5.The drug-resistant strain MG1655 and the protein expression strain BL21 of the polymyxin resistance gene mcr-5 were constructed as the test subjects.After bioinformatics prediction,12 key amino acid sites of phosphoethanolamine transferase MCR-5 were obtained,and the corresponding site mutant strains were constructed.The minimum inhibitory concentration of colistin,lipid A modification and western blot were detected.These 12 amino acid sites all play important roles in the enzyme activity of MCR-5.The extracellular and transmembrane regions of the phosphoethanolamine transferases MCR-1,MCR-2,and MCR-5 proteins were predicted to construct extracellular-transmembrane exchange strains of the MCR-1,MCR-2,and MCR-5.The drug resistance of extracellular-transmembrane exchange strains was detected.The extracellular-transmembrane exchange strain of MCR-1 and MCR-2 with amino acid sequence similarity of 81% retained the colistin resistance function.The amino acid sequence similarity between MCR-5 and MCR-1/MCR-2 was less than 35%,so the extracellular-transmembrane exchange strains lost their resistance function.In vitro expression,purification and enzyme activity assay of phosphoethanolamine transferase MCR-5 were carried out to verify the mechanism of phosphoethanolamine modification to produce colistin resistance.Using laser confocal microscopy and compound rescue tests,colistin can stimulate bacteria to produce reactive oxygen in the cells for sterilization.The presence of the drug resistance gene mcr-5 can reduce the production of reactive oxygen species caused by colistin,which makes the strain resistant to colistin.The study also verified the effect of mcr-5 gene expression on the growth of the strain,and compared the level of colistin resistance of MG1655 strains of five mcr family genes.