| Carbapenems are important drugs used in veterinary and clinical medicine to treat infectious diseases caused by carbapenem-resistant strains.In recent years,with the extensive use of antibiotics,antibiotic-resistant strains have been steadily increasing.The problem of drug resistance is getting worse day by day.Therefore,the choice of anti-multi-drug resistant strains of the drug is limited.which makes colistin,the last line of defensing against antibacterial drugs,has to be reactivated.Unfortunately,a large number of reports about colistin resistance emerging in an endless stream.In particular,the appearance of the mcr-1 gene resistant to colistin has drawn even more waves in academia and has drawn wide attention from scholars both at home and abroad.Therefore,the study of mcr-1 transmission and drug resistance mechanism is particularly important.In this study,we analyzed the resistance of colistin in CTX-M chicken Escherichia coli in different years and the detection rate of mcr-1 gene,and explored the transmission of mcr-1 gene and its mechanism of drug resistance,Providing a theoretical basis for controlling the further spread of mcr-1 gene in the veterinary clinic.The result of drug sensitivity test showed that among the 13 CTX-M chicken isolates that were isolated and preserved in 2007,all of them were susceptible to colistin,and the drug resistance rate was 0,and the minimum inhibitory concentration（MIC）was less than 0.5.2012-2013 Six strains isolated and isolated from 66 CTX-M chickens were resistant to colistin,with a resistance rate of 9.1%.The results of PCR showed that these six strains all carried the mcr-1 gene,which showed different effects on colistin Degree of resistance.Among 83 strains of CTX-M chickens isolated from 2016,36 strains were resistant to colistin,with a resistance rate of 42.2%.The results of PCR showed that 35 strains carried mcr-1 gene with a detection rate of 42.1 %.Among the 41 Escherichia coli strains producing CTX-M gene and mcr-1 gene,there were 29 transconjugants of CTX-M gene transferred by plasmid conjugation test,the rate of conjugation was 70.7%.The frequency of CTX-M conjugation 1.23×10-5-9.11 ×10-2.There were 14 matrices that only transferred mcr-1 gene,with a rate of 34.15%.The frequency of mcr-1 gene binding ranged from 3.78×10-5 to 4.24×10-1.There were 14 zygotes of CTX-M gene and mcr-1 gene,with a rate of 34.15%.The strain GZM6 was cultured in LB broth containing cefotaxime（16μg/m L）for 100 generations to ensure that each generation of aseptic procedures was not contaminated and was designated as GZM6-100.GZM6 and GZM6-100 plasmids were extracted separately and the plasmids were found The results showed that CTX-M-9 existed and CTX-M-1 disappeared,indicating that the gene of junctional transfer was CTX-M-9,According to the drug susceptibility test,the MIC of GZM6-100 to cefotaxime（2560μg/m L）was 4 times higher than that of GZM6（640μg/m L）.In addition to the resistance to colistin and third generation cephalosporins,14 strains of Escherichia coli and their corresponding conjugates that produced CTX-M and mcr-1 genes were resistant to gentamycin,amikacin,florfenicol,Ciclofen,enrofloxacin and olaquindox also showed different drug resistance,and most strains showed multi-drug resistance.This shows that in addition to these two gene transfer,the other resistance genes have also been transferred.The analysis of gene subtypes showed that there were more CTX-M-9 subtypes and CTX-M-1 followed by CTX-M-9 gene.Plasmid incompatibility group results showed that most of the 14 donor bacteria had multiple plasmid types,and more FII plasmids were involved in conjugation and transfer.MLST results showed that 11 strains of Escherichia coli from a total of 11 strains of ST were obtained,indicating that most of the isolates were distantly related and none of the three types of strains had been submitted to the website for a new ST number.Through S1-PFGE and Southern blot hybridization results showed that most of the original strains contain multiple types of plasmids,one to two plasmids are obtained by conjugation test,and the mcr-1 gene is located on one of the plasmids with a size of about 33.3kb-310.1kb,mostly 78.2kb,FII plasmid,strain 26 is not color,we analysis mcr-1 gene may be located in the chromosome above.In conclusion,the mcr-1 gene carried by Escherichia coli of CTX-M isolated from different age has significant difference,increasing rapidly with age.The conjugation test showed that both the CTX-M and mcr-1 genes could be transferred by plasmid conjugation and the horizontal transmission dominated.In addition to the resistance to third generation cephalosporins and colistin,the donor bacteria and their corresponding transconjugates that simultaneously transferred both CTX-M and mcr-1 genes were resistant to gentamicin,amikacin,enrofloxacin,Superoxide,florfenicol,olaquindox and other drugs also showed a certain degree of resistance,showing multi-drug resistance,indicating that in the process of conjugal transfer,drug-resistant genes associated with these drugs also occurred at the same time.Hybridization of S1-PFGE and southern blot indicated that most of the mcr-1 gene was located on the plasmid,mediated by the plasmid and propagated horizontally,and the sizes of these plasmids were different. |
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