Study On The Transmission Mechanism Of Bla_CTX-M-55 Gene Among Salmonella Isolates From Food-producing Animals In Guangdong Province

Production of extended-spectrumβ-lactamases（ESBLs）is the principal mechanism of resistance to oxyimino-cephalosporins in gram-negative bacteria.The unreasonable use of antibiotics accelerated the formation and spread of CTX-M-type extended-spectrumβ-lactamases（ESBLs）.In recent years,CTX-M-55 has become an emerging type of ESBLs detected in humans,animals and the environment in Asia.In the present study,we investigated the prevalence of CTX-M-55 among Salmonella isolates from food-producing animals in different regions of Guangdong Province,and further explored the transmission mechanism of bla _CTX-M-55.A total of 176 Salmonella isolates from food-producing animals during 2015 to 2017stored in our laboratory were used in this study.The presence of bla _CTX-M-55 resistance genes were screened by PCR.The results showed that 27（15.4%）Salmonella isolates carried bla _CTX-M genes,the most common subtype was CTX-M-55（12）,followed by CTX-M-27（8）,CTX-M-64（3）,CTX-M-14（2）and CTX-M-65（2）.The susceptibility of bla _CTX-M-55-positive isolates to 20 kinds of antimicrobial agents was performed by the ager dilution method.The results showed all of these isolates were resistant to cefotaxime at high levels（MIC,128～256μg/m L）,10 of 12 strains showed resistance to ciprofloxacin（MIC,4～64μg/m L）and presented multidrug resistant phenotypes.The presence of both others ESBLs genes(bla _TEM、bla_SHV、bla_OXA)and fluoroquinolone resistance gene PMQR（qnr A,qnr B,qnr C,qnr D,qnr S,aac（6’）-Ib-cr,qep A,and oqx AB）、PMQR（gyr A,gyr B,par C,and par E）.The results showed that gyr A:S83F D87G+par C:T57S S80R double mutations occurred in all ciprofloxacin-resistant strains,and they all carried two PMQR genes.Serotypes were determined among using slide agglutination with hyperimmune sera.12 bla _CTX-M-55Salmonella strains were identified,the predominant serovars were S.enterica serovar Enteritidis Indiana（7）.S.enterica serovar Typhimurium（2）,S.enterica serovar Chester（2）.Pulsed-field gel electrophoresis（PFGE）and multi locus sequence typing（MLST）were applied to analyze the genetic relatedness of 12 bla _CTX-M-55-positive isolates.Twelve Salmonella strains were divided into five different profiles according to 85%similarity.The similarity of the same serotype in the same area was higher,suggesting clonal transmission.ST typing of Salmonella was ST17,ST34,ST343,ST321 and ST27,and one strain was not successfully typed.The horizontal transmission of bla _CTX-M-55 were analyzed by conjugation or transformation experiment,and the characteristics of bla _CTX-M-55-harboring plasmids were performed by plasmid replicon typing,S1-PFGE,southern blotting and genome sequencing.Only 2 of 12 Salmonella strains producing CTX-M-55 were conjugated.bla _CTX-M-55 was located on Inc HI 2 plasmid of 280 kb and 250 kb,respectively.The bla _CTX-M-55 gene of the other 10 strains without conjugation were all located on chromosomes.Interestingly,the bla _CTX-M-55 was located in a strain with 250 kb plasmid,and the resistance gene was also detected on chromosomes.Genome sequencing and plasmid sequencing showed that bla _CTX-M-55 gene located on chromosome and plasmid had the same genetic environment:ISEcp1-bla _CTX-M-55-orf477 transposon unit.The fitness of bla _CTX-M-55 plasmids were assayed by growth curve,plasmid stability test and competitive test.Results show that the growth of conjugate and recipient were same,and bla _CTX-M-55 gene were stable alone in the host.But Inc HI2 plasmid（～280 kb）had a disadvantage competition when cultured with the recipient.A strain of a drug-resistant gene located on a chromosome has a competitive advantage in a competition assay by conjugating a transconjugant that is transferred to an Inc HI2 type plasmid（～250 kb）.Both horizontal transfer of the resistance genes and clonal spread of resistant strains accelerated the dissemination of bla _CTX-M-55 among Salmonella isolates.In most of the bla _CTX-M-55 genes of salmonella are located on chromosomes.ISEcp1-bla _CTX-M-55-orf477transposon unit promotes the transfer of bla _CTX-M-55 gene between different bacteria,and also mediates the transfer between plasmids and chromosomes of Salmonella.