| The tetracycline resistance gene tet(M)encodes a ribosomal protection protein that usually located on conjugative transposons of chromosome,and and there are also a few conjugative plasmids.The transmission of tet(M)gene mediated by transposons and plasmids that were plays a crucial role in the expression,transmission and diffusion.Therefore,it is of great significance to explore the gene environment of tet(M).So far,the epidemiological characteristics and molecular transmission mechanism of tet(M)have been well-studied in Gram-positive bacteria,which is mostly linked to the transposons of Tn916,Tn5801 families.While the transmission mechanism of tet(M)gene is rarely reported in Gram-negative bacteria.In this study,the distribution characteristics of tet(M)in Escherichia coli and Salmonella isolated from swine and poultry were analyzed,the molecular characteristics of the transosons and plasmids of strains carrying tet(M)from different species and genera were compared,and the mechanism of action and specific transmission were further researched,so as to provide reference for prevention and control of drug resistance and research and development of new antibiotics in clinical practice.1.In this study,44 strains carring tet(M)were detected from 1134 strains isolated from pig and chicken in E.coli and Salmonella,with the separation rate was 3.9%.The MIC and resistant genes related to the phenotype of 44 strains were studied,which showed that all strains were resistant to sulfonamide,amoxicillin and terramycin,have higher resistance rates to doxycycline and flufenicol,while lowest resistance rates to amicacin,most of them were multiple antibiotics resistant strains.Besides tet(M),the detection rate were 95.5%,25%and 2.3%of other tetracyclines resistance genes tet(A),tet(B)and tet(D),respectively.The genes floR that mediated flufenicol resistance had the highest detection rate,followed by sul3,oqxB,sul1,blaTEM,oqxA,blaOXA-1,mcr-1 and so on,the lowest were rmtA and rmtB.2.All the strains haboring tet(M)were investigated by epidemiological analysis.The results of MLST showed that the 30 E.coli strains bearing tet(M)could be divided into 23 ST types,among which ST 10 was the epidemic strain.The 14 Salmonella strains haboring tet(M)could be divided into 6 ST types,of which 9 strains were all ST 34 type,indicating that ST 34 had a wide prevalence in Salmonella.The results of PFGE showed that 26 E.coli strains(83.3%)were typed successfully,and most of the strains have 80%similarity approximately,indicating that E.coli carrying tet(M)had genetic diversity.14 Salmonella strains carrying tet(M)were all successfully typed,and the similarity of 9 strains(64.3%)was 97.9%,while the similarity of the other 5 strains was low.3.Plasmids from strain S13 and three corresponding transconjugants were subjected to whole genome sequencing.Strain S13 contained four plasmids,including mcr-1-bearing pS13-1,blaCTX-M-55-carrying pS13-2,tet(M)-bearing pS13-3,and floR-carrying pS13-4.tet(M)was located on a novel composite transposons Tn6942(16,493 kb).Tn6942,includes integrons IntI1,tet(M),aadA1,cmlAl,aadA2 and dfrAl2,is flanked by two IS26 in the same direction.The IncFI:A-:Bplasmid pS13-3 harboring tet(M)could not be transferred.PS13-3 fused with IncN1-F33:A-:Bplasmid pS 13-2,which generated cointegrate plasmid pS13D,and then spread horizontal.In addition,pS13-2 could also be fused with IncX1 plasmid pS13-4 to generate the fusion plasmid pS13F.Above two intermolecular replicative mechanisms mediated by IS26 and the novel transposon Tn6952(TnAS3-IS26-?SEcp1-ramA-?IS26-?TnAS1),respectively.The fusion plasmid pS13D is highly stable in the host bacteria,and the formation and evolution of cointegrate plasmids could expand the resistance and host spectrum of fusion plasmids.4.To explore the genetic and biological features of the tet(M)-harboring plasmid pTS14 in Salmonella enterica strain S14 isolated from chicken.The 119-kb tet(M)-bearing IncF2:A1:B1 conjugative plasmid pTS14,contained a novel transposon Tn6709 with the genetic structure IS26-tnpA1-tnpA2-?orf13-LP-tet(M)-tnpX-?tnpR-IS26,and the resistance genes tet(B),tet(D),strAB,sul2,and blaTEM-1b.In addition,pTS14 was found to be highly stable in the recipient strain E.coli J53.The transconjugant TS14 exhibited a higher survival ratio than E.coli J53 under permanent starvation-induced stress.The tet(M)-bearing IncF2 epidemic plasmid lineage may accelerate the dissemination of tet(M)and other genes by coselection,which could constitute a potentially serious threat to clinical treatment regimens.5.The novel transposon Tn6539 harboring tet(M)were recovered from E.coli isolate ST162 from a duck.Tn6539 with the resistance module orf456-floR-lysR and strA-strB-sul2 were co-located on the MDR of the conjugable lncHI2 plasmid pTW4.Plasmid pTW4 remained stable in E.coli J53 for 7 days of passage in an antibiotic-free environment.The direct competition assays presented a fitness disadvantage of 6%each 10 generations for J53/pTW4 against its host E.coli J53.The survival percentage revealed that J53/pTW4 was significantly lower than the recipient strain J53 in the permanently starvation stress.The plasmid pTW4,a mobile multidrug resistance reservoir,may accelerate the dissemination of above resistance genes by coselection and constitute a potentially serious threat to clinical treatment regimens.6.A novel complex transposon Tn7124(IS26-ctp-lp-tet(M)-hp-IS406 tnp-IntI4-IS26)haboring tet(M)has been found in E.coli A2.The particial sequence of transposon Tn7124 on the IncR plasmid pTA2 was consistent with that of Tn6942.Plasmid pTA7 was consist of sul3,tet(M),qnrS1,bleO,oqxAB,floR,aadA1,cmlA1,aadA2 and tet(A)-tetR(A)and 22 insertion sequences.A fragment carrying the tet(M)gene(IS26-ctp-1p-tet(M)-IS406 tnp-ctp-aadA1-cmlAl-aadA2dfrA12-IntIl)was found in E.coli A7,which was similar to the transposons Tn6942 found in porcine Salmonella S13,and with the resistance module ISVsa3-VirD2-floR-lysR?tet(A)-tetR(A)both located on the IncR plasmid pTA7.The skeleton of IncR plasmid pTA7 was same to pTA2,only some differences in the MDR region.7.A novel complex transposon Tn7125 haboring tet(M)located on the IncF2:A6:B20 plasmid pTA13-1 has been found in E.coli A13 isolated from swine.Tn7125,Tn7124,Tn6942-like and Tn6942 all carring fragment IS26-ctp-lp-tet(M)-hp-IS406 tnp,while Tn 7125 also contains a resistance module qac-aadA1-cmlAl-aadA2-DUF1010-dfrA12 and ?ISVSa3-VirD-floR-lysR-ISVSa3.These results indicated that the transposons carrying tet(M)continuously integrates under the mediation of insertion sequence,which accelerates the transmission of tet(M)in E.coli isolates through the integration of other drug-resistant genes,and poses a potential serious threat to clinical treatment. |
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