The Study On Epidemiology And Dissemination Mechanism Of Colistin Resistance Gene Mcr-1 In Escherichia Coli From Animal Origin

Antibiotic are the most important weapons for humans and animals to fight bacterial infections.However,bacterial resistance has become one of the biggest threats to global public health and food safety in the 21st century duo to the unreasonable use of antibiotic.In the past few decades,the development of new antibacterial drugs has been slow,and bacterial resistance has continued to emerge.Polymyxin is used as"last line of defense"for the treatment of multi-drug Gram-negative bacterial infections.The discovery of the movable colistin resistance gene,mcr-1,has caused worldwide attention to polymyxin resistance.Although there are reports on the prevalence of mcr-1 in E.coli in some areas of China,there is still a lack of understanding of the prevalence and transmission mechanism of mcr-1in China.This study aims to further elucidate the mechanism of mcr-1 transmission in E.coli from animal origin based on the investigation of the prevalence of mcr-1 in E.coli from northeastern regions of China.This study analyzed the strains isolated from the three northeastern provinces from June 2016 to September 2016.The prevalence of mcr-1 in E.coli from pigs,chickens and dairy cows showed that the mcr-1 carrying rate of chicken and pig-derived E.coli was significantly higher than that of E.coli of dairy cows origin.It showed that most of the mcr-1 positive isolates were resistant to many antibiotics,especially tetracycline,florfenicol and ciprofloxacin.The situation of multi-drug resistance in mcr-1-positive isolates was very serious.Secondly,two molecular typing methods were used to investigate the molecular prevalence of mcr-1 positive isolates in this study.The results showed that the PFGE profile and MLST typing of mcr-1-positive isolates exhibited significant diversity,and the genetic relationships among these strains were far.The location of mcr-1 in the isolates,the ability of conjunction transfer,the frequency of conjunction transfer and plasmid stability were analyzed.The results showed that the mcr-1 gene was distributed on both plasmids and chromosomes,mcr-1 genes of most isolates were located on plasmids with different sizes.The plasmid size ranged from³0kb to⁴30kb.Five different types of mcr-1 positive plasmids were identified by plasmid type identification.Among them,three types of plasmids,IncX4 type,IncI2 type and IncHI2 type,were dominant types.The conjunction transfer frequency ranged from 1.4 x 10^-66 and 8.8 x 10^-2.Plasmid stability tests indicated that five different types of plasmids were stably present in the recipient bacteria.The analysis of the draft genomes and the representative plasmids showed that the five representative plasmids of this study were highly homologous with the corresponding types of plasmids reported in other areas in China.It indicated that these plasmids had been prevalent in many regions of China.At the same time,the virulence-resistant hybrid plasmid carrying mcr-1 was firstly discovered by whole genome sequencing.Sequence analysis suggested that it might be fused by IncHI2-type resistant plasmid and IncF-type virulence plasmid by homologous recombination.In addition,by analysis that the genetic environment of mcr-1 gene,it was found that the Tn6330 transposon was the main mode that mediated the transmission of the mcr-1 gene between different types of plasmids and chromosomes.Two novel mcr-1 mutants were found by aligning the mcr-1 gene sequence of the isolates.Functional studies showed that the mutation did not alter the catalytic activity of the mcr-1 gene.The two positions of the mutation were not critical amino acid residues that determine its function.This study is aim to reveal the mechanism of mcr-1 transmission in E.coli from animal origin,and provide a scientific basis for risk assessment,prevention and control the transfer and spread of colistin-resistant bacteria and resistant gene.