| With the wide use of antibiotics,Enterobacteriaceae multi-drug resistant bacteria infection has become a serious challenge in medical treatment.Because polymyxin has a narrow antibacterial spectrum and is sensitive to other antibiotic-resistant strains,it is often used as the last drug for the treatment of multidrug resistant Enterobacteriaceae infections.However,since Chinese scholars have found that the mcr-1 gene encoding transferable colistin resistance gene and it can be transmitted through the food chain and the environment,human will face a very serious situation in the treatment of Enterobacteriaceae infection.During 2002 to June 2016,polymyxin has been used as a legal veterinary growth promoter or feed additive.In addition,in recent years,there have been more and more reports of new mobile colistin resistance(MCR)genes.It is of great significance to study the prevalence of MCR gene carried in E.coli from animal and human,analyze its epidemiological characteristics,and explore its molecular epidemiological laws,so as to effectively control the spread of drug-resistant genes,prevent and control drug-resistant strains,and ensure human health.In this study,3,663 strains of E.coli strains from 31 provinces(municipalities and autonomous regions)in the country were used(1,962 strains were preserved in the laboratory,and 1,701 strains were newly isolated and identified),including 3,049 strains of animal origin and 614 strains of human origin.Five kinds of MCR genes(mcr-1,mcr-2,mcr-3,mcr-4 and mcr-5)and calculate their respective positive rates,and systematically analyze the molecular epidemiology of the MCR gene through three aspects:host,age and region.At the same time,the minimum inhibitory concentration(MIC)test of 17 antibiotic drugs was carried out on the strains carrying the MCR gene to determine the multi-drug resistance of the MCR gene-positive strains.The results showed that among the 3,663 strains of E.coli,a total of 262 strains carried the MCR gene,and the positive rate was 7.2%(262/3663),of which 258 strains carried the mcr-1 gene with the positive rate of 7.0%(258/3663),5 strains carried mcr-3 gene with positive rate of was 0.1%(5/3663).There was only one strain carried both mcr-1 and mcr-3 genes,and no positive strains of mcr-2,mcr-4 and mcr-5 genes were detected.The molecular epidemiological characteristics of the MCR gene were further analyzed.From the host source,the MCR gene positive rate(11.5%)of E.coli from pigs was significantly higher than that of other hosts(8.0%from geese;7.5%from chicken).From the classification of the hosts livestock and poultry,the positive rate of MCR gene derived from livestock animals(9.9%)is higher than that of poultry(7.5%);the positive rate of animal source strains is significantly higher than that of human source strains(only 1.5%).From the chronological changes,the positive rate of mcr-1 gene has increased from 2009 to 2013,reaching the highest level in 2013(16.8%);remaining at around 16%from 2014 to 2015;dropping rapidly from 15.4%to 5.0%from 2016 to 2019.Here,the positive rate of mcr-1 gene showed a trend of rising firstly,then stable,and then rapidly decreasing,indicating that it is closely related to the national veterinary drug use policy.From the geographical distribution,the positive rate in the southeast region is generally larger than that in the northwest region.The positive rate of mcr-1(more than 15%)is the highest in central China,followed by northeast,east and south,north and southwest,and the positive rate in the northwest region is lowest.According to the MIC test results of 262 strains of E.coli with MCR gene on 17 drugs,all strains showed multi-drug resistance,of which the resistance to colistin ranged from 4?g/mL to>64 ?g/mL,MICso and MIC90 is 8 ?g/mL and 16 ?g/mL,respectively.The drug resistance rate of most other drugs is high(more than 50%).Only 3 drugs(meropenem,amikacin and nitrofurantoin)have drug resistance rates of below 30%,of which meropenem has the lowest resistance rate(2.3%).Among the drug-resistant strains,there were 6 strains that were simultaneously resistant to colistin and carbapenems,suggesting that there may be super-resistant strains.Based on the comprehensive epidemiological characteristics of the MCR gene,in order to further study the transmission mechanism of drug resistance genes,we carried out a genome-wide sequencing analysis of an E.coli strain C2624 which carrying both the mcr-1 gene and the mcr-3 gene.Conjugation experiment is also conducted to study the horizontal transmission ability of MCR genes.It was found that the mcr-3 gene can be transferred alone or with the mcr-1 gene to the recipient bacteria E.coli C600.The analysis of the junction frequency,resistance phenotype and transconjugants genotype showed that the junction frequency of the first type of transconjugants carrying the mcr-3 gene was 5×10-5/donor to 5×10-6/donor.The junction frequency of the second type of transconjugants carrying both mcr-1 and mcr-3 is 4×10-3/donor.Compared with the recipient strain C600,the two types of transconjugants carrying the mcr-3 gene and the mcr-1 and mcr-3 genes increased the MIC value of polymyxin by 64 times and 512 times,respectively.By gene prediction and annotation with RAST software and sequence comparision with ResFinder,BLAST and BRIG software,plasmid maps was drawn for genetic environment analysis.It is found that the structure of IncHI2 plasmid containing the mcr-1 gene is very stable,except for very few insertion sequence in the variable region and the transposon and related proteins,the skeleton structure is very stable,and the sequence coverage between different plasmids is quite high.The IncX1 type plasmid containing the mcr-3 gene is extremely unstable,with multiple variable regions.There might exist simultaneously of multiple drug resistance genes,insertion sequences and transposon proteins,and the genetic environment of each drug resistance gene is also very variable.The variable region and skeletion structure of IncXl plasmid(pYZUC2624.2)containing the mcr-3 gene was further compared and analyzed.It is speculated that the formation of mutations might be caused by the continuous horizontal transmission among the plasmids of several or more strains of bacteria.The study of drug resistance gene transmission mechanism provides a theoretical basis for the prevention and treatment of multi-drug resistant strains and super drug-resistant strains in clinical practice,and also provides a reference for the study of the mechanism of polymyxin resistance of other pathogenic bacteria. |
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