Characteristics Of Multidrug Resistance IncHI2 Plasmids Carrying OqxAB/Bla_CTX-M And Their Genetically Evolutionary Relationships

Plasmids are known to play a major role in bacterial evolution by allowing bacterial community to rapidly adapt to changing environments.Plasmid-mediated horizontal transfer of antibiotic resistance genes also plays a significant role in the spread of antibiotic resistance in bacteria,posing this problem increasingly serious.IncHI2 plasmid is one of the most prevalent plasmids involving in spread of resistance genes among Enterobacteriaceae,and has been found to harbor mosaic structures of multidrug resistance?MDR?regions and carry a large number of antibiotic resistance genes and metal tolerance genes.However,to date,it's still unclear of the prevalence and distribution of IncHI2 plasmids among Enterobacteriaceae,the molecular characteristics of IncHI2 plasmids,their genetically evolutionary relationships and the mechanisms involving in acquisition of MDR regions.This study is aimed to investigate the prevalence and distribution of IncHI2 plasmids among Enterobacteriaceae from food-producing animals and characterize the genotypic and phenotypic drug resistance among isolates harboring Inc HI2 plasmids,explore the molecular transmission mechanisms involved in spread of oqxAB/bla_CTX-M and the pco and sil operons mediated by MDR IncHI2 plasmids,elucidate the complete nucleotide sequences of IncHI2plasmids harboring oqxAB/bla_CTX-M and the mechanism involving in acquisition of MDR regions,reveal the genetically evolutionary relationships among IncHI2 plasmids.In this study,we comprehensively investigate the prevalence and distribution of multidrug resistance?MDR?IncHI2 plasmids among 2005 Enterobacteriaceae isolates from food-producing animals in South China.The results showed that a relatively low detection rate of IncHI2 plasmids?126/2005,6.28%?was found and positive isolates mostly belonged to Escherichia coli?E.coli??97.62%,123/126?.Among the 126 positive isolates,80.16%were resistance to more than 8 types of antimicrobial agents and the resistance rates to cefotaxime,gentamicin,ciprofloxacin and olaquindox were over 66%,most of these isolates exhibited a MDR phenotype.Among the 126 positive isolates,the floR gene was the most prevalent?83%?,followed by bla_CTX-M gene?73%?(including bla_CTX-M-9G?48%?and bla_CTX-M-1G?25%?),oqxAB?56%?,aac?6'?-Ib-cr?49%?and qnrS?44%?.This indicated that a relatively high prevalence of extended-spectrum-lactamases?ESBLs?genes(bla_CTX-M),plasmid-mediated quinolone resistance determinants?PMQRs??oqxAB,aac?6'?-Ib-cr and qnrS?and floR were present among E.coli isolates harboring IncHI2 plasmids.25 conjugative IncHI2 plasmids carrying bla_CTX-M/oqxAB were found from 739 E.coli isolates from disease food-producing animals,and bla_CTX-M and oqxAB were found to co-locate on the same IncHI2 plasmids in 13 transconjugants.In addition,aac-?6'?-Ib-cr,floR,fosA3 and rmtB were frequently co-transferred with bla_CTX-M/oqxAB in transconjugants harboring IncHI2 plasmids and bla_CTX-M/oqxAB-IncHI2 plasmids could confer MDR.The bla_CTX-M/oqxAB-IncHI2 plasmids are high molecular weight with sizes ranging from²60 kb to³80 kb,and mostly were fused plasmids and the most prevalent combination was IncHI2 in combination with IncFII?n=10?and followed by IncN?n=6?.Most of bla_CTX-M/oqxAB-IncHI2 plasmids were assigned to ST3?n=22?by pDLST,but a variety of plasmid patterns were observed by comparing the similarity of the 25 IncHI2 plasmids using XbaI-RFLP analysis.The diversity observed herein was probably due to the deletions or acquisition of a number of resistance genes by transposons,insertion sequences or fusion with other types of plasmids.Among the 25 bla_CTX-M/oqxAB-IncHI2 plasmids,20%were found to harbor the pco and sil operons,and they contributed to the increased MICs of Cu SO4 and AgNO3.The genetic context surrounding the two operons was well conserved except some variations within the pco operon.The³2 kb conserved region containing the two operons?tnsABCD-⁴.64-kb region-silESRCBAP-1.29-kb region-pcoEABCDRSE?identified in the IncHI2 plasmids was also found in chromosomes of different Enterobacteriaceae species.This may implicate mobilization of the sil and pco operons from plasmids into chromosomes or conversely,from the chromosomes into plasmids via Tn7-like-based transposition.Further,phylogenetic analysis of this structure showed that Tn7-like transposon might play an important role in cross-genus transfer of the sil and pco operons among Enterobacteriaceae of diverse origins in many countries.Four representative oqxAB/bla_CTX-M-IncHI2 plasmids were selected and subjected to high-throughput sequencing,and analysis of their complete nucleotide sequences showed that pZ13T,pP2-3T and pA74T were probably generated by fusion between IncHI2 and IncFII plasmids through IS26-mediated homologous recombination.In addition,of note,plasmid pP2-3T harbored two relatively large MDR regions with size of 15.6kb and 25.9kb,respectively,and one relatively large virulence region of 41.7kb.To clarify the phylogenetic relatedness among Inc HI2 plasmids,a phylogenetic tree was constructed based on the complete nucleotide sequences and core-genome of the IncHI2 plasmids,including 4 found in this study and 42 from the database GenBank.The results revealed two mainly distinct clusters,and IncHI2 plasmids belonging to the same ST type were usually closely related to each other,especially in two big groups which were mainly composed of ST3-IncHI2 and ST1-IncHI2 plasmids,respectively.However,IncHI2 plasmids belonging to the ST2 type were less related to each other than others belonging to the same ST type.There is a certain relation of the genetic evolution,ST types,and drug-resistant genes among IncHI2 plasmids.Overall,the highest number of the important resistance genes was found in two groups mainly composed of ST3-IncHI2 and ST1-IncHI2 plasmids,respectively;the oqxAB and fosA3 genes were prevalent exclusively in the former group,while the latter group contained various?-lactam resistance genes conferring reduced susceptibility to the third-generation cephalosporins.However,the number of the important resistance genes was relatively low in the groups composed of ST2-and ST4-IncHI2 plasmids.To our knowledge,this is the first description of the molecular transmission mechanisms involved in spread of pco and sil operons mediated by IncHI2 plasmids harboring oqxAB/bla_CTX-M,and the genetically evolutionary relationships of IncHI2 plasmids and the mechanism involving in acquisition of MDR regions by constructing phylogenetic trees based on the complete nucleotide sequences and core-genome of the IncHI2 plasmids and comparative genomic analysis.It would provide bases for assessment of fluoroquinolones and cephalosporins widely used in livestock and poultry resulting in accelerating the dissemination of resistant pathogenic bacteria from animals,and of heavy metal micronutrients using as animal feed additives in livestock and poultry resulting in facilitating the occurrence and persistence of MDR bacteria.Our study also would provide a scientific guidance in slowing down the development of resistance,and exploring novel strategies and approaches involved in controlling the antimicrobial resistance.