Drug Resistance Characteristics And Typing Of Escherichia Coli Carrying Mcr-1 From Chicken

Antibacterial drugs are important weapons for humans and animals to resist infections caused by bacteria and viruses,and provide a guarantee for the health of humans and animals and reduce losses in the breeding industry.The unreasonable and irregular use of antibacterial drugs leads to the emergence of a large number of resistant bacteria,which directly or indirectly threatens human health.Therefore,bacterial resistance has become a public health problem that all countries have continued to pay attention to.As an effective method to treat multidrug-resistant bacteria resistant to carbapenems,the discovery of mcr-1,a transferable and mutable colistin resistance gene,limits the use of colistin.Poultry industry is a pillar industry in China.The safety of food animals,especially chicken,is the focus of attention.The purpose of this study is to detect colistin resistant strains and mcr-1 gene,and to further study the multi-drug resistance of Escherichia coli carrying mcr-1 from chicken gene and the transmission mechanism of mcr-1.In this study,182 strains of Escherichia coli from chicken were isolated from liver samples samples and feces samples of dead chickens in seven provinces of Henan,Hubei,Jiangxi,Shaanxi,Hunan,Shanxi and Anhui,and genomic were the test subjects.Detection of resistance of Escherichia coli from chicken to colistin using micro broth dilution method.The results of the drug susceptibility test showed that 49 strains of Escherichia coli from chicken were detected to be resistant to colistin,and the drug resistance rate was 26.92%(49/182).By PCR,33 strains were found to carry the mcr-1 gene.Among 182 strains of E.coli from chicken,the positive rate of mcr-1 was 18.13%(33/182).At the same time,the minimum inhibitory concentrations of 7 common antibacterial drugs against mcr-1 positive E.coli from chicken were tested.The results showed that 33 mcr-1 positive strains were resistant to different types of antibacterial drugs and the resistance rates to ceftiofur,amoxicillin,enrofloxacin,and doxycycline were 100%.The resistance rates to amikacin and gentamicin were 33.33%and 63.64%,respectively.Then,the drug resistance genes in bacteria were detected by PCR.The results showed that 33 strains carrying mcr-1 genes all carried blaTEM,with the detection rate of 100.00%,blaCTX-M-1G detection rate of 48.48%,tet(A)detection rate of 69.70%,and oqxA and oqxB detection rates of 39.40%.Meanwhile,genes such as blaCTX-M-9G,blaOXA-1,blaoOXA-10,rmtB and tet(M)were also detected,with the detection rates of 27.27%,24.24%,3.03%,18.18%and 3.03%,respectively.The results of XbaI-PFGE cluster analysis showed that the similarity of genotype was 70.8%?100%,and 33 strains were divided into 21 types.32 mcr-1 positive Escherichia coli strains could be divided into 22 ST types,and one strain did not match ST type.The dominant ST types were ST69,ST93,ST10 and ST2847.The comparison between MLST typing and PFGE typing showed that most of the typing results were consistent.In order to study whether mcr-1 can be transferred,the conjugation experiment was carried out,and 19 conjugations were obtained,with the conjugation rate of 57.58%(19/33).19 conjugations were resistant to colistin,and 57.89%(11/19)of them were resistant to gentamicin.The results of enzyme digestion and cloning showed that although there were base mutations and amino acid mutations,they did not affect the function of mcr-1 gene.Whole-genome sequencing analysis showed that mcr-1 was located on the horizontal transferable IncI2 plasmid with the size of?60 kb.The genetic environment of mcr-1 was nikeA-nikeB-mcr-1-pap2,and this plasmid did not carry other drug-resistant genes.Most of the resistant genes carried by the sequenced strains were located on chromosomes,including genes mediating drug resistance to macrolides,?-lactams,tetracyclines,quinolones,aminoglycosides,etc.The main resistant mechanisms were antibiotic efflux,antibiotic inactivation,and changes in target sites of antibiotics.After sequence alignment,the fragment containing blaTEM gene carried by the strain had 100%homology with P7 phage,and this fragment might be derived from P7.This study explored the transmission mechanism of colistin resistance gene mcr-1 and mcr-1 in Escherichia coli from chicken,and provided laboratory basis for slowing the spread of colistin resistance gene mcr-1.