Molecular Characterization And Mechanism Of Dissemination Of Bla_CTX-M Genes In Multidrug Resistant Escherichia Coil From Dog

In October 2017,120 dogs fecal swabs samples were collected from three pet hospitals in Jinshui District,Zhengzhou City,Henan Province.A total of 106 E.coli isolates were identified.All isolates were examined for susceptibility to antimicrobial agents by the microdilution broth method following the Clinical and Laboratory Standards Institute?CLSI?recommendations.The following antimicrobials were tested: ampicillin,cefotaxime,ceftiofur,ceftazidime,cefoxitin,amikacin,gentamicin,doxycycline,flufenicol,enrofloxacin,profloxacin and colistin.The isolates showed high resistance rates to ampicillin,cefotaxime and cefotaxime with 39.62%,followed by ceftazidime,amikacin,gentamicin,doxycycline,florfenicol,enrofloxacin and ciprofloxacin with 20-35%,but relatively low resistance rates to cefoxitin and colistin with 3.8% and 4.7%,respectively.Forty-four cefotaxime resistance isolates harbored CTX-M genes,three of four had cefoxitin resistance producing bla CMY-2,the other resistant determinants rmt B,flor,mcr-1 was also indentified by PCR.The various of sub-type of CTX were sequenced:CTX-M-3?n=1?,CTX-M-14?n=10?,CTX-M-15?n=6?,CTX-M-24?n=1?,CTX-M-27?n=1?,CTX-M-55?n=16?,CTX-M-65?n=7?,CTX-M-123?n=3?.Twenty-six transconjugants were selected by conjugation experiments using the CTX producing isolates as the donor.Various plasmid incompatibility groups were identified by PCR and sequencing.The predominant group was Inc FIB and followed by Inc FII and Inc I1.Phylogenetic analysis showed that 22,8 and 14 isolates belonged to group A,B2 and D,respectively.Multilocus sequence typing?MLST?showed that CTX-M producing isolates divided into 29 types,5,4 and 3 of them belonged to ST10,ST457 and ST155,respectively.Others were one or two strains except four were not identified.PFGE was successfully conducted for 42 E.coli isolates,and divided into 9 clusters,designated A,B,C,D,E,F,G,H and I.The DNA profiles of the remaining 21 strains were all less than 85% similar,and were divided into 21 PT.A bla_CTX-M-123 producing isolate,HB37,co-harbored rmt B,co-carried Inc FII and Inc I1 plasmids,but only bla_CTX-M-123 producing Inc I1 plasmid transferd to corresponding transconjugant.The F33:A-:B-plasmid carrying rmt B was successfully obtained by electroporation.S1-PFGE and Southern blot hybridization indicated,HB37 carried three plasmids with different size,except Inc I1 and F33:A-:B-,the thirdly plasmid did not carry any resistance gene desighed p HB37-3.The chromosome contained two MRRS: One was bla_CTX-M-123 region,a complete ISEcp1 was on the upstream,the fragment inserted into the genome produced two repeat sequence?GAGTA?.The second was an incomplete Tn1721,tet?A?in integrons?tni A-tni B-sui1-qac E ? 1-aad A1-dfr A1-inti1?and followed by mercury-resistant clusters?mer R-mer T-mer P-mer C-mer A-mer D-mer E?,this region was located between the two IS1 Rs produced a repeat 9 bp sequence?CGCCGATAA?.This resistance region is most likely formed due to the insert of transposon which contribute to the dissemination of resistance genes.The genetic environment of bla_CTX-M-123 on the plasmid and the chromosome was consistent,presumably ISEcp1 facilitate the transfer of CTX.To sum up,CTX-M-producing E.coli have increased in prevalence and diversity in pets.Three bla_CTX-M-123 strains of E.coli were also present and the bla_CTX-M-123 were detected simultaneously in HB37 plasmids and chromosomes for the first time.The genetic environment of bla_CTX-M-123 on the plasmid and the chromosome was consistent,presumably ISEcp1 facilitate the transfer of CTX.The results of PFGE and MLST were generally consistent and indicated that horizontal gene transfer and clonal spread were responsible for the dissemination of the CTX gene.The rapid spread of multi-drug resistant strains of E.coli from pets poses a great threat to both humans and pets.