Prevalence Of Poultry-origin Mcr-1 Positive Escherichia Coli In Changsha, China And Screening Chinese Medicine Of Colistin Resistance Elimination

Although polymyxins are one of the last-line defense against difficult-to-treat MDR Gram negative pathogens,polymyxins resistant bacteria have been reported around the world,which indicate that the range of clinical selective drugs is shrinking in the future.mcr-1 was the first reported polymyxin resistance gene located on plasmid,and its discovery attracted worldwide attention on polymyxin resistance.Later,mcr-1 ～ mcr-10 were reported successively.Most reported mcr-1 gene had often been identified from pig,then after chickens,and few had been reported from duck and quail.Epidemic studies on mcr-1 gene positive bacteria from poultry in Changsha,Hunan Province have not been reported.In this study,10 poultry(chicken,duck,quail)farms in Liuyang and Changsha in Changsha were selected for sampling,investigated the molecular and epidemiological characteristics of polymyxin-resistant E.coli circulating in changsha area,hunan province,China.The mcr-1-positive E.coli were characterized phenotypically and subjected to whole genome sequencing(WGS).Using the WGS data we determined their sequence type,resistance gene content and genotypic environment of mcr-1.We aimed to explore the possibility of horizontal transmission of mcr-1-positive E.coli in local poultry farms,and to provide a significant basis for the assessment and prevention and control of bacterial resistance risk in Changsha area.On that,MCR protein was used as the target to preliminarily screen the traditional Chinese medicine extracts that could restore the bactericidal activity of polymyxin against MCR-1 positive E.coli through CADD,and further verified by in vitro drug sensitivity test,laying a foundation for the subsequent drug development of anti-drug resistant bacteria.1.In this study,366 polymyxin resistance isolates were collected from 659 samples,with a total isolation rate of 54.50 %.Result showed that the isolation rate is broiler farm>Quail farm > laying duck farms.A total of 17 strains of mcr-1 positive resistant E.coli were screened,and the positive rate of mcr-1 was 2.45%.The results showed that polymyxin resistance was common in poultry farms in Changsha area.2.Agar diffusion and broth dilution were used to test the minimal inhibitory concentration(MIC)of 17 strains of avian mcr-1 positive E.coli to 12 commonly used antimicrobials.The results showed that the MIC range of 17 avian mcr-1 positive E.coli to polymyxin E was 4 ～ > 128 ug / ml,and all resistant to sulfamethoxazole,florfenicol,tetracycline and ciprofloxacin.The resistance rates to tegacycline,piperacillin,tazobactam,cefotaxime,ceftiofur and gentamicin were 17.65%,42.11%,58.82%,70.59% and 1.18% of the 17 strains were sensitive to meropenem and amikacin.The results showed that the 17 strains of mcr-1-positive E.coli from poultry were multi-drug resistant.3.The whole genomes of 17 strains were sequenced in the second generation,and the sequencing results were analyzed.The results of Clermont system classification showed that17 strains of mcr-1 positive E.coli were mainly distributed in three groups A(58.8 %),B1(23.5 %)and D(17.6 %),and(MLST)analysis showed that there were 12 MLST types in 17 strains of positive bacteria,including ST93,ST3941,ST7153,ST1403,ST10338,ST69,ST156,ST1421,ST162,ST6321,and two new ST strains.Most of the mcr-1 contains typical plasmid conjugation transfer part and IV type secretory system,tellurium resistant protein.ISApl1 transfer element were not found.4.In this experiment,11 small molecules with high virtual screening score were selected for analysis.According to the virtual screening result,Astragalus polysaccharides and Fructus Aurantii extract were selected to continue the growth curve experiment and microchessboard experiment.The results showed that the extract of Fructus Aurantii had antibacterial effect on mcr-1-positive E.coli in vitro,and the combined action of astragalus polysaccharide and polymyxin E could effectively reduce the MIC of mcr-1-positive-E.coliConclusion: mcr-1-positive E.coli are common in three kinds of poultry farms in Changsha County and Liuyang County,which show multiple drug resistance.No ISApl1 insertion sequence was found in mcr-1 isolates.76.47% of the strains contained binding transfer elements in the upstream and downstream of mcr-1.Fructus Aurantii extract had antibacterial activity against mcr-1-positive E.coli in vitro.The combination of Astragalus polysaccharides and polymyxin E can effectively reduce the MIC of mcr-1 positive E.coli.