Isolation And Identification Of Avian-derived Tet(X4)-positive Escherichia Coli And Characterization Of Molecular Transmission Of Tet(X4) In Sichuan Province

Escherichia coli（E.coli）can carry a variety of antibiotic resistance genes（ARGs）,which can be transferred with mobile genetic elements.Tigecycline is one of the last resort treatments for complex infections caused by Multidrug-Resistant（MDR）bacteria,and the tet（X4）gene mediates high levels of bacterial resistance to tigecycline.In this study,1286 manure samples of avian origin from Sichuan Province were isolated and identified by tet（X4）positive E.coli and the molecular transmission characteristics of tet（X4）gene were studied.The results were as follows:1.Isolation and identification of tet（X4）-positive Escherichia coli and analysis of antibiotic resistance21 strains of tet（X4）-positive E.coli were isolated from 1286 samples,and 9 kinds of ARGs were detected in 6 categories,including tet（X4）,bla _OXA,bla _SHV,bla _TEM,qnr S,tet A,add A1,flo R and sul3.21 strains of tet（X4）-positive E.coli were tested for antimicrobial susceptibility test to 9 kinds of antibiotics by broth microdilution method.The results showed that all strains were MDR bacteria.2.Characterization of molecular transmission of tet（X4）geneThe 21 tet（X4）-positive E.coli could transfer the tigecycline resistance into the recipient E.coli J53 by conjugation transfer,and the MIC value of tigecycline in the recipient bacteria was increased 32-64 flods.The plasmid maps of all donor bacteria and transconjugants were obtained by S1-PFGE,PCR and sequencing techniques were used to detect the presence of tet（X4）gene in transconjugants,and it was concluded that tet（X4）gene was located in transferable plasmid.The conjugation frequency of 21 tet（X4）-positive E.coli was 5.69×10^-6-1.02×10^-4 at 37℃and 5.69×10^-6-2.14×10^-4 at 42℃.The stability of tet（X4）-positive plasmids was determined by passing through the transconjugants.The results showed that the stability of the plasmids passed under tigecycline selection pressure was higher than that of the plasmids passed without tigecycline selection pressure.Recombinant strain PMD19-tet（X4）/DH5αexpressing tet（X4）gene was obtained by molecular cloning,and the adaptation of tet（X4）-positive plasmids was studied.Based on the results of growth curve,biofilm formation ability and in vitro competition experiment,the acquisition of tet（X4）-positive plasmids imposes a certain fitness cost to the strain.3.Genetic diversity and whole-genome sequencing of tet（X4）-positive E.coliAccording to MLST typing and phylogenetic typing,the 21 tet（X4）-positive E.coli could be divided into 6 ST types:ST206,ST761,ST155,ST1638,ST542 and ST767.There are three phylogenetic groups:A,B1,C.Two tet（X4）positive E.coli strains L6-21 and MY1-8 were selected for whole genome sequencing,and the tet（X4）gene in L6-21 and MY1-8 was mapped to IncX1 plasmid.The core genetic structure of the tet（X4）gene in L6-21 was rdmc-tet（X4）-ISVsa3,and the core genetic structure of the tet（X4）gene in MY1-8 was IS26-rdmc-tet（X4）-ISVsa3.In summary,a total of 21 tet（X4）-positive E.coli were isolated and identified in this study,and tet（X4）gene was located on the efficient and stable transmission plasmid.The whole genome sequencing further confirmed that tet（X4）could be located on the IncX1 plasmid,and there were insertion sequences around tet（X4）that could mediate its transfer.