Epidemiological Research Of Colistin Resistance Escherichia Coli In Chickens In Partial Areas Of Jilin Province

Polymyxin is a polycationic antimicrobial peptide that is widely used to treat systemic infections caused by Gram-negative bacteria.It has been gradually replaced by new antibiotics for its low-toxic neurotoxicity and nephrotoxicity.It is only used in animal husbandry for the prevention and treatment of gastrointestinal diseases in livestock.In recent years,with the emergence of multi-drug resistant Gram-negative bacteria,especially the emergence of carbapenem-resistant Enterobacter,polymyxin has been re-used for clinical treatment.In the past,it was believed that the acquired resistance of bacteria to polymyxin was mainly due to the chromosomal encoding of the bacterial outer membrane component?lipopolysaccharide?,so there should have no transferable drug resistance mechanism.However,Chinese scientists reported the plasmid-mediated colistin resistance gene for the first time in 2015.After that,mcr-1 was detected in more than 40 countries around the world.The widely spread has made humans and animals really fall into the embarrassing situation of“no medicine available”.So far,we know very little about the epidemiological characteristics of mcr-1 pathogens.This study mainly collects and identifies E.coli carrying mcr-1 resistance gene from chicken in Jilin Province.Focusing on the epidemiological characteristics of mcr-1 positive E.coli,and providing reference for the prevention and cure of colistin-resistant infection in the future.From October 2017 to July 2018,406 strains of E.coli were isolated from 530 samples collected from 14 chicken farms in Jilin Province.22 strains of mcr-1 positive E.coli were detected by PCR,and the detection rate was 5.42%.There is no other subtypes were detected,it is indicating that mcr-1 is the major colistin resistance gene in Jilin Province.The minimum inhibitory concentration?MIC?of 22 strains of E.coli to polymyxin was 1-16?g/mL,of which8?g/mL was the highest rate?45.45%?,and the resistance level was medium.According to the statistics of collection location and sample type,we found the highest detection rate of mcr-1positive E.coli was in Yongji County?50.00%?,it is indicating that the situation of colistin resistance in this area is serious.273 strains of E.coli were isolated from the chicken swab samples,of which 19 strains were positive of mcr-1,the positive rate was 6.96%;12 strains of E.coli were isolated from air samples inside the chicken farm,of which 3 strains were positive of mcr-1,the positive rate was 25.00%;there no mcr-1 positive strain was detected in the soil and water samples around the chicken farm,indicating that the air might be the main route of transmission of mcr-1 positive E.coli.The mcr-1 genes of 22 positive strains were completely sequenced,and one mcr-1 positive strain?JZ10?was found to be mutated in its conserved region?860T?A?,result in the tyrosine?287Y?at position 287 is deleted of the encoded MCR-1protein,and 287Y is located in the core region of the catalytic domain of MCR-1.The deletion affects the secondary structure of the protein and involves chemical properties,which alters the Function and stability of D-glucose and Zn ion ligands in the catalytic domain,it may have an impact on MCR-1 resistance.Kirby-Bauer test was used to detect sensitivity of mcr-1 positive E.coli to 14antibiotics:ofloxacin,amikacin,ceftazidime,imipenem,florfenicol,chloramphenicol,gentamicin,amoxicillin,tetracycline,minocycline,fosfomycin,doxycycline,ciprofloxacin and cefotaxime.The results showed that 22 mcr-1 positive E.coli were highly resistant to chloramphenicol,amoxicillin,tetracycline and cefotaxime?resistance rate was 100.00%?,it showed different degrees of resistance to other antibiotics and only moderately resistant or sensitive to norcycline.It indicated that 22 positive E.coli belonged to multi-drug resistant.The drug resistance gene and the plasmid type on the mcr-1 positive E.coli plasmid were detected by PCR.Among the tested drug resistance genes,the detection rate of tetracycline resistance gene tetA was the highest?100%?,and the detection rate of broad-spectrum fluoroquinolone antibiotic resistance genes was also high,mainly oqxA,oqxB,qnrA;it is worth noting that 9 strains carrying the carbapenem resistance-related gene bla_CTX-M-9G,of which 2strains carrying bla_NDM at the same time.In this study,12 plasmid types were detected in 22mcr-1 positive E.coli,among which the positive rate of IncI2 and IncFIB was higher,the positive rate of IncK,IncY,IncI1,IncHI2 was slightly lower,the positive rate of IncX3?IncX4?IncFIA?IncN?IncA/C?IncB was the lowest.Five mcr-1 positive strains were successfully transferred,and the transconjugants were subjected to plasmid typing,and the mcr-1 gene was found to be located on the IncY,IncB,IncI2 type plasmid.In summary,we reached the following conclusions:?1?The colistin resistance gene in Jilin Province is mainly mcr-1,and the mcr-1 positive E.coli are moderately resistant to polymyxin.The detection rate of mcr-1 in chicken swab samples from Yongji County is relatively high,so the relevant local departments should strengthen the supervision of antibiotics used in livestock.?2?The multi-drug resistance of mcr-1 positive bacteria is serious,the types of plasmid are complex,and the rate of plasmid junction transfer is high.Efforts should be made to prevent the widespread spread of multi-drug resistant bacteria.?3?A new mcr-1 mutation was found?860T?A?,resulting in the deletion of the 287 tyrosine,it affects the function and stability of D-glucose and Zn ligands in the catalytic domain,and this mutation may lead to changes in colistin resistance induced by MCR-1 protein.