Prevalence Characteristics And Molecular Transmission Mechanisms Of Colistin Resistance Mcr-1 Gene In Escherichia Coli And Salmonella Enterica

Colistin,a cationic antimicrobial peptide,is regarded as one of the last-resorts to treat clinical infections caused by MDR Gram-negatives bacteria.However,colistin resistance has been increasingly emerged recently,primarily mediated by plasmid-mediated colistin resistance gene mcr-1.Since then,worldwide spread and diversity of the mcr-1-carrying bacterial organisms were identified,posing an urgent threat to human and animal health.The aim of this study is to investigate the prevalence of mcr-1 gene in Enterobacteriaceae from livestock,migratory bird and flower,and explore the molecular transmission mechanisms,improving the regulation of colistin usage,and providing scientific basis for the preventing the spread of colistin resistant isolates and resistance genes.The prevalence of mcr-1 gene was investigated in Enterobacteriaceae from livestock,migratory bird and flower during 2015 and 2018.The result showed that the mcr-1 positive rates of the samples from livestock farm,migratory bird,and flower was 53.6%,4.4%and2.5%,respectively.In addition,five（1.8%）mcr-1-carrying Salmonella enterica isolates were identified from 276 nonduplicate Salmonella enterica.All the five mcr-1-positive Salmonella enterica exhibited multi-resistant phenotypes including streptomycin,gentamicin,florfenicol,nalidixic acid,tetracycline,trimethoprim-sulfamethox,in addition to colistin.The molecular dissemination characteristics of mcr-1 in Escherichia coli and Salmonella enterica were further assessed.The mcr-1 carrying E.coli displayed distinct genetic background,of which the ST48 and ST10 were most prevalent.Most of the mcr-1gene in E.coli were located on Inc X4 plasmids,followed by Inc HI2,Inc I2,Incp0111,and Inc P plasmids.In addition,chromosome encoded mcr-1 are more and more popular.Molecular typing shows that all of the mcr-1 positive Salmonella enterica belong to ST34Salmonella Typhimurium（S.Typhimurium）,and four of them had nearly identical PFGE type,although they were from different host species and diverse geographical locations.The mcr-1 gene was located on a transferable Inc I2 plasmid in the four genetically related strains.In the other one strain,mcr-1 was located on an Inc HI2 plasmid.Given that the positive rate of mcr-1 was high in Enterobacteriaceae and the Inc X4plasmids were the most popular mcr-1-carrying plasmid Inc-type among Enterobacteriaceae from livestock farm during 2015 to 2017,Enterobacteriaceae isolates from 2004 to 2013were subjected to a retrospective screening for Inc X4 to comparative analyze the Inc X4plasmids.Forty three of the 2470 isolates harbor Inc X4 plasmids,of which thirteen were found to be mcr-1 positive.The mcr-1 gene was located in Inc X4 plasmid in 11Enterobacteriaceae.Three representative complete Inc X4 plasmid sequences were obtained.Comparative genomics showed that the mcr-1-carrying Inc X4 plasmids exhibit remarkable similarity in the backbone,and the major distinction lies in the region containing mcr-1.The epidemic p CSZ4-like Inc X4 plasmid was also highly similarity with Inc X4 plasmid isolated during 2015 to 2017,and the mcr-1 harboring Inc X4 plasmids from Genbank database.This indicated that p CSZ4-like Inc X4 plasmids were widespread in Enterobacteriaceae.In addition,ISApl1 is located on downstream of mcr-1 gene in one Inc X4 plasmid,implying that the acquisition of mcr-1 on Inc X4 plasmid might be caused by the common ISApl1 mediated mechanism.In addition,one colistin-resistant Escherichia coli strain possessing chromosome encoded mcr-1 was further characterized.Whole genome sequencing revealed that mcr-1was in triplicate in the chromosome with another copy on a p HNSHP45-2-like Inc HI2plasmid in strain FS13Z2S.The mcr-1 cassettes in chromosome were bracketed by two copies of ISApl1,and the pap2 gene was truncated by an IS1294-like element.In plasmid p FS13Z2S,only single copy of ISApl1 was present upstream of the mcr-1 cassette.It implied that the triplication of chromosome copies of mcr-1 in FS13Z2S was due to intra-molecular transposition events via ISApl1.There was no significance difference on the colistin MIC level among the original strain FS13Z2S,the strain without p FS13Z2S and the conjugant harboring plasmid p FS13Z2S.All of these three strains are featured with PEA-addition to lipid A,without regard to the copy number variations of mcr-1.ISApl1 is presumably involved in the transposition of the mcr-1 cassette.To further explore the intermediates during the transposition of Tn6330,we engineered E.coli strains that carry an intact Tn6330 transposon or its deletion derivatives.PCR assays were performed to detect IR-IR junctions and possible circular intermediates.The transposition experiments demonstrated that the presence of an intact Tn6330 was essential for the successful transposition of mcr-1,although both Tn6330 and Tn6330-ΔIR could form circular intermediates.The insertion sequence junction structure was observed in all constructed plasmids but the ISApl1 dimer was only formed in one construct containing an intact Tn6330.The average frequency of mcr-1 transposition in an E.coli strain possessing an intact Tn6330 was～10^-6 per transformed cell.27 integration sites were identified among the Tn6330 transposition events.All the transposition sites were flanked by 2 bp target duplications and preferentially occurred in AT-rich regions,consistent with clinical isolates.In conclusion,the mcr-1 gene was identified in Enterobacteriaceae from livestock,migratory bird and flower.However,there was significant difference on mcr-1 positive rates.Additionally,the dissemination mechanisms were different among different bacteria species.Regardless of the year,source and species,the mcr-1 gene was transmitted through the common Inc X4,Inc HI2 and Inc I2 plasmids and ISApl1 mediated horizontal spread among Enterobacteriaceae.Moreover,this study experimentally proved that mcr-1 was spread mediated by intact Tn6330.