| Carbapenems are considered as the last treatment options for life-threatening infections caused by multidrug-resistant?MDR?Gram-negative bacteria,but the increasing global resistance in Enterobacteriaceae to carbapenem poses a great challenge in clinical therapy.Carbapenems are forbidded to use in veterinary clinics,but domestic and foreign scholars have detected blaNDM in Escherichia coli?E.coli?from animal origin in recent years,posing a great threat to human health.In this study,molecular epidemiology of New Delhi metallo-?-lactamase?NDM?-producing Escherichia coli from a chicken farm in Anhui,revealed the prevalence and carrier characteristics of blaNDM in this chicken farm.Agar dilution method was used to test the susceptibility of 18 antibiotics such as ampicillin,isolated from 400 E.coli in this farm from 20132017.The results showed that no imipenem-resistant strains were detected from 2013 to 2014.A total of 18imipenem-resistant strains were detected from 2015 to 2017,and the drug resistance rates were 10.0%,10.5%,and 4.0%,respectively.The resistance rate of E.coli to tetracycline,ampicillin,cotrimoxazole,streptomycin,and florfenicol was higher than 90%.The results showed that the resistance rate of E.coli to tetracycline and ampicillin in the chicken farm was higher than 90%.High levels of resistance to streptomycin?81.4%?,doxycycline?84.6%?,ciprofloxacin?85.3%?,and cefotaxime?88.3%?.The rates of resistance to fosfomycin?68.8%?,neomycin?75.3%?,gentamicin?56.3%?,and cefquinoxine?71.1%?were also high.Ceftazidime?35.8%?,cefoxitin?19.8%?,amikacin?22.0%?,colistin?28.8%?,apramycin?43.8%?and imipenem?4.5%?drug resistance rates below 50%.The blaNDM was detected by PCR among 18 strains of imipenem-resistant E.coli.The results showed that all of the 18 strains carried blaNDM gene and only 2 strains were blaNDM-7,The others were bla NDM-5.At the same time,NDM-positive bacteria were detected for the carriage of mcr-1,fosA3,floR,rmtB,blaCTX-M-1G,blaCTX-M-9GTX-M-9G and oqxAB.The results showed that all strains carried oqxAB?100%?,the detection rates of mcr-1,fosA3 and floR were all 55.5%?n=10?,and the detection rates of blaCTX-M-1G and blaCTX-M-9GTX-M-9G were 22.2%?n=4?and 27.7%?n=5?respectively.The detection rate of rmtB was 38.9%?n=7?.Eighteen strains of NDM-positive bacteria were sequenced by PCR using multiple-site sequencing?MLST?.Eight ST-types were identified after sequencing and comparing sequences.ST5442?n=2?,ST10?n=3?and ST2935?n=1?in 2015,ST7507?n=6?are prevalent in 2016,then ST156?n=1?and ST93?n=1?.ST410?n=1?and ST5946?n=3?in2017.The frequency of NDM-positive bacteria in the conjugation and conjugation frequency experiments showed that bla NDM was located in the IncX3 plasmid and the conjugation frequency was 6.71×10-5 to 5.75×10-4.The transformation experiment was carried out to obtain blaNDMDM single-plasmid transformants,and several strains were randomly selected for stable experiment and competitive experiment.The blaNDM plasmid carried by IncX3 plasmid could be stably present in the recipient bacterium without antibiotics.The competitive result showed that the IncX3 plasmid had a fitness cost on its hosts.Whole genome sequencing results showed that the similarity of the reported sequence of the IncX3 plasmid was 99%.blaNDM is located in two different genetic environments,8of which are blaNDM-?ISAba125-IS5-?ISAba125-IS3000-?Tn2,which is identical to the human,animal,food,and environment-derived IncX3 plasmids carrying blaNDM.The 4strains were bla NDM-?ISAba125-IS5-?IS3000-?Tn2.Based on the PCR maping detection of blaNDM flanking sequences,6 strains were not sequenced,4 strains belong to the first structure and 2 strains were the second structure.In summary,the antibiotic resistance of E.coli in chickens from the farm in Anhui province was severe.The spread of clonal propagation and the horizontal propagation of the plasmid IncX3 mediated the prevalence of blaNDM in the chicken farm.The blaNDMDM positive bacteria also carry many important drug resistance genes such as oqxAB.E.coli,a source of food animals carrying blaNDM,may threaten human health,suggesting that the monitoring of carbapenem resistance of E.coli from food animals should be strengthened. |
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