Preliminary Study On The Mechanism Of Colistin Resistance Mediated By Mcr-9 Gene In Salmonella

Salmonella is an important zoonotic pathogen.Polymyxin is the "last line of defense" against multi-drug resistant Gram-negative bacterias infection in clinically.The expression of mcr-9 gene can be induced in the presence of colistin at subinhibitory concentrations,which increased the resistance of bacterias to colistin.However,the mechanism of drug resistance of mcr-9 gene is still unclearIn this study,porcine Salmonella preserved in laboratory was selected as the research object,and strains carrying mcr-9 gene were screened.The genetic environment and transferability of mcr-9 gene were analyzed,and the correlation between mcr-9 gene and the resistance of bacterias to colistin was verified.Using RNA-Seq and phenotypic analysis,the mechanism of colistin resistance mediated by mcr-9 gene under colistin stress environment was preliminarily explored,which provided certain data support for the study of the mechanism of Salmonella resistance.The main results are as follows:1.Identification and antimicrobial resistance of Salmonella carrying mcr-9 geneA total of 408 strains of porcine Salmonella isolates isolated and preserved in the laboratory were resuscitated and screened by PCR,2 strains(HB08035 and HB08370)carried mcr-9 gene.The minimum inhibitory concentration(MIC)of 8 antibiotics(ampicillin,meropenem,cefotaxime,colistin,gentamicin,tetracycline,chloramphenicol,ciprofloxacin)against 2 strains was determined by microbroth dilution method to evaluate their drug sensitivity.It was found that the strains were only resistant to cefotaxime and ampicillin.They were sensitive to other antibiotics such as colistin.2.Genetic environment analysis of Salmonella and its plasmids carrying mcr-9 geneGenome sequencing analysis showed that the sequence type of the two strains was ST34,and the relatives were very close,and they carried drug resistance genes such as lipopeptides,aminoglycosides and cephalosporins.All the plasmids carrying mcr-9 gene were Inc HI2/Inc HI2A-ST1 type plasmids.At the same time,the genetic environment of mcr-9 gene in Salmonella is very conservative.3.Study on transferability of the plasmids carrying mcr-9 gene and CTX-M-9 geneWith Salmonella HB08035 and HB08370 as donor bacterias,Escherichia coli NK5449 and Salmonella CVCC 542 as acceptor bacterias,no zygote was screened by cefotaxime resistance at 37 ℃ and 26 ℃ by filtration and electroconversion methods.The results showed that no conjugation transfer occurred in the plasmids in the strains.4.Functional analysis of mcr-9 geneThe full length of the mcr-9 gene was amplified from the DNA of HB08035 strain.The recombinant vector p HSG396-mcr-9 was constructed by double enzymes digestion method,and was introduced into HB08035 strain to obtain the recombinant Salmonella strain HB08035/p HSG396-mcr-9.The expression of mcr-9 gene in recombinant Salmonella strain was not up-regulated,and the resistance to colistin was not increased.Construction of recombinant Escherichia coli BL21(DE3)/p GEX-6P-1-mcr-9 was resistant to colistin(MIC=2 μg/m L)was higher than that of Escherichia coli carrying the empty vector(MIC<0.25 μg/m L),and the expression level of mcr-9 gene was significantly up-regulated(P < 0.001).5.Effect of colistin and disinfectant stress environment on Salmonella carrying mcr-9Colistin and seven disinfectants were used to stress the passage of Salmonella(HB08035)carrying mcr-9 gene.It was found that under the stress of colistin and sodium trichloroisocyanurate,the mcr-9 gene was significantly up-regulated(P < 0.001).Colistin showed more significant on stress(P < 0.05).Strains CL55(MIC=8 μg/m L)and CL510(MIC=16 μg/m L)were obtained after five and ten generations of stress with 1/2 MIC(1μg/m L)of colistin.Compared with WT,CL55 and CL510 exhibited cell elongation,decreased cell membrane permeability and enhanced hydroxyl radical scavenging ability(P < 0.001).Transcriptomic analysis showed that compared with WT,186 genes were up-regulated and 117 genes were down-regulated in CL510 strain,mainly concentrated in the regulatory pathways of lipopolysaccharide biosynthesis,CAMP resistance and two-component system.In this study,two Salmonella strains carrying the mcr-9 gene were screened,and the mcr-9 gene was located in the Inc HI2/Inc HI2 A plasmid,which was conserved.Under the stress of colistin and sodium trichloroisocyanurate,the mcr-9 gene of Salmonella bacteria was up-regulated,and the resistance to colistin was developed.The stress effect of colistin was more significant.High expression of mcr-9 gene can elongate the morphology of bacteria,decrease the permeability of cell membrane and enhance the ability of hydroxyl radical scavenging,thereby mediating the resistance to colistin.