| In recent years,with the rapid development of bacterial drug resistance,multidrug resistant bacteria have been frequently reported and became one of the world public health safety problems.Plasmid-mediated multidrug resistant genes widely spread among bacteria from different sources,posing a potential threat to human and animal health.The food-producing animals have been normally regarded as the reservoir of antibiotic resistant bacteria,and help the antibiotic resistance genes transfer to human via the food chain.In order to explore the development of important drug resistance genes and plasmids in bacteria isolated from domestic food-producing animals,and the impact on the prevalence as well as distribution of antibiotic resistance genes after the implementation of antibiotic use policies.This research investigated the epidemiological characteristics of Inc HI2,Inc F?,Inc I2,Inc I1 and Inc X4 plasmids and the bla CTX-M,fos A3,rmt B and mcr-1 genes in E.coli isolated from samples from a broiler farm in Anhui Province from 2013 to 2018,thus to provide theoretical basis for slowing down the development of drug resistant bacteria and instruction for antibiotics as therapeutic drugs.The 552 strains of E.coli isolated from samples from a broiler farm in Anhui from2013 to 2018 were tested by using PCR technique for the detection of Inc HI2,Inc F?,Inc I2,Inc I1 and Inc X4 plasmids and bla CTX-M-1G,bla CTX-M-9G,fos A3,rmt B and mcr-1 genes.Results showed that the detection rates of the Inc HI2,Inc F?,Inc I2,Inc I1 and Inc X4plasmids were 32.61%(n=180),26.63%(n=147),26.45%(n=146),6.25%(n=36)and2.54%(n=14),respectively.From 2013 to 2018,the detection rates of Inc HI2 fluctuated between 20.48%and 46.67%;the detection rates of Inc F?increased from 8.70%to43.85%.However,the detection rates of Inc I2 decreased from 47.25%to 19.23%.The detection rates of Inc I1 and Inc X4 plasmids were quite high in 2015 and 2016,while these two plasmids were less found in other years,which the detection rates were lower than 10%and 5%,respectively.The detection rates of bla CTX-M,fos A3,rmt B and mcr-1 genes were78.8%(n=435),54.17%(n=299),20.29%(n=112)and 35.68%(n=197),respectively.During the 6 years,the detection rate of bla CTX-Mfluctuated between 70%and 94%,the detection rate of fos A3 gene decreased from 83.52%to 21.54%,the detection rate of rmt B gradually increased from 6.59%to 31.54%,the detection rate of mcr-1 gene showed a downward trend,especially from 2017 to 2018.The flanking of mcr-1 gene was detected by PCR to analyze its genetic environment and determine the plasmid type in which the mcr-1 gene was located.A total of four different genetic environments were found,the insertion sequence ISApl1 was detected in the upstream of mcr-1 gene in 87 strains of all,while ISApl1 was found in the upstream and downstream of mcr-1 gene in 23 strains.However,intact ISApl1 was detected in the upstream but being incomplete in the downstream of mcr-1 in 5 strains,and ISApl1couldn't be found in 114 strains neither in the upstream nor the downstream of the mcr-1gene.Among 197 mcr-1-positive bacteria,192 mcr-1 genes were located on Inc HI2,Inc I2and Inc X4 plasmids.From 2017 to 2018,mcr-1-Inc X4 had been gradually disappeared,while mcr-1-Inc I2 showed a decreasing trend and mcr-1-Inc HI2 is rising.72 strains,of different years,plasmid types and genetic environments,were selected from 197 strains of mcr-1 positive E.coli for conjugation and transformation experiments,and 61 transconjugants were obtained.Inc HI2,Inc I2 and Inc X4 plasmids were dectected in18,38 and 5 conjugants/transformants,respectively.The conjugants and transformants were subjected to S1-PFGE experiments and results showed that all transconjugants contained single plasmids,and the size of Inc HI2 plasmid was 210.0-310.0 kb,the size of Inc I2plasmid was 54.7-78.2 kb,and the size of Inc X4 plasmid was approximately 33.3 kb.Agar dilution method was used to detect the sensitivity of 61 conjugants/transformants to 15 antibacterial drugs,microdilution method was used to detect the sensitivity of colistin.Results showed that the colistin MIC value of all conjugants/transformants was between 2and 16?g/m L.The resistance rates to AMP and CTX were higher than 40%;the resistance rates to FOS and FFC were between 20%and 30%;the resistance rates to CAZ,GEN and NEO were all between 10%and 20%;the resistance rates to DOX,TET and SXT were below 10%.61 conjugants/transformants were sensitive to FOX,AMI,IPM and CIP.PCR was applied to detect the presence of other drug resistance genes in the conjugants/transformants.One Inc X4 conjugant carried bla CTX-M-14;10 Inc I2 plasmids carried bla CTX-M-64(n=3),bla CTX-M-55(n=6)and bla CTX-M-132(n=1);the other Inc X4 and Inc I2conjugants/transformants did not carry other resistance genes.Among all Inc HI2 conjuants,17 of them carried bla CTX-M-14,fos A3 and flo R;4 of them carried tet A;3 of them carried oqx AB as well as 1 conjugant carried bla CTX-M-15,indicating that mcr-1 in the Inc HI2plasmid often co-propagated with other drug resistance genes,mediating a variety of antibiotic resistance.In conclusion,the multi-drug resistance genes and plasmids in the E.coli of the broiler farm are widely spread,and the mcr-1 gene is mainly transmitted in the farm through the Inc HI2,Inc I2,Inc X4 plasmids.Moreover,mcr-1 gene carrying Inc HI2 plasmids usually carries a plurality of drug resistance genes at the same time.It is worth noting that significant changes have taken place in the distribution and transmission characteristics of the mcr-1 gene from 2017 to 2018,which may be related to the prohibition on colistin use as feed additive.Therefore,we should regulate the use of drugs in farms and strengthen the monitoring of bacterial resistance to ensure the health of humans and the sustainable development of animal husbandry. |
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