| Polymyxin is the"last line of defense"for the treatment of multidrug-resistant Gram-negative bacteria,especially Carbapenem-resistance Enterobacteriaceae infections.For decades,polymyxin has been widely used in the prevention and treatment of animal infectious diseases on all continents and has been added to feed as a growth promoter in some Asian countries such as China,Japan,India and Vietnam.The discovery and prevalence of the plasmid-mediated polymyxin resistance gene mcr-1 has accelerated the horizontal transfer and spread of the polymyxin resistance gene.The widespread existence of polymyxin-resistant strains has brought great emergency to the farmed animals health and the food production safety.In this investigation,by clarifying the relevant transmission route of mcr-1 gene in wild-type foodborne Enterobacteriaceae bacteria not only in vitro but in vivo.Explore the influence of factors such as different temperatures and different media on mcr-1 gene transfer ability,as well as the changes in the environmental characteristics of mcr-1 gene during conjugation transfer.Analyze the transfer mechanism of mcr-1 gene from the genetic level.To provide a theoretical basis for seeking to slow down or block the rapid and widespread spread of mcr-1 gene,and reference for food animal breeding and food safety control.(1)We retrospectively investigated the prevalence of mcr-1-positive Salmonella and Escherichia coli isolated from retail food products,food supply chain,and clinical samples across China in the last decade(2006–2017).Twelve(0.67%)mcr-1-positive strains were identified from 1,789 non-duplicated isolates,including 1,283 Salmonella and 506 E.coli.Eleven(21.7‰)mcr-1-positive E.coli strains were identified from the isolates collected during 2006–2017,which was significantly more prevalent than mcr-1-positive Salmonella in this period(0.78‰,1/1,283;P<0.05).(2)In order to verify that the mcr-1 gene can transfer horizontally between bacteria and mediate low levels of polymyxin resistance,nine mcr-1-positive isolates resistant to polymyxin B were screened as donors(designated as D1–9).To explore the influence of factors such as different temperatures and different media on the horizontal transfer ability of mcr-1 gene,chicken was selected as a substrate to carry out a conjugative transfer experiment.And to simulate the horizontal transfer of mcr-1 gene in animal intestines after ingestion mcr-1 positive bacteria contaminated food,an animal model was constructed with Kunming mice for conjugative transfer experiments.In filter-medium conjugation transfer,mcr-1-carrying plasmids in donors were successfully transferred to recipients at frequencies of 1.75×10-5to 4.6×10-2 per recipient cell,which was significantly higher than frequencies of conjugation transfer on chicken 1.32×10-6 to 3.85×10-4.mcr-1-carrying plasmids in donors were successfully transferred to recipients on chicken even at 4?.In the mice gut,mcr-1 can not only spread to the recipient bacteria provided by intragastric administration,but also conjugated with intestinal bacteria.The negative effect of mice intestinal flora was showed by the horizontal transfer of mcr-1 gene to mice gut bacteria.In the same conjugative transfer environment,replicon type of plasmids was the most decisive factor that effect the frequencies.The peak number of transconjugants in group D6-E.coli C600with Inc HI2 type plasmid was 1.43×102 CFU/g feces,which was significantly higher than the peak number of transconjugants in group D7-E.coli C600 0.3×102 CFU/g feces.(3)A total of 3 types of plasmid replicators encoding the mcr-1 gene were detected,Inc X4(D1 and D7),Inc I2(D2,D4 and D5)and Inc HI2(D6 and D9),respectively.Inc X4-type plasmids carried mcr-1 with the insertion sequence IS26 upstream.The mcr-1 gene cassette IS26-mcr-1-pap2 did not change during the horizontal transfer.The mcr-1 gene cassette ISApl1-mcr-1-pap2 existed in Inc I2-type plasmids carried by D2 and D4.The insertion sequence ISApl1 existed in D2 was lost during filter-medium conjugation transfer and the ISApl1 existed in D4 was also lost during conjugation transfer on chicken in 25?.Inc I2-type plasmids in D5 carried the mcr-1 gene cassette ISEcp1-bla CTX-M-55-mcr-1-pap2.The mcr-1 gene cassette ISApl1-mcr-1-pap2 in Inc HI2 type plasmid was stable,which was an unreported mcr-1 genetic environment.The mcr-1 gene cassette ISApl1-mcr-1-pap2 in Inc HI2 type plasmid was stable.Inc HI2 was very active in the process of bacterial reproduction and evolution and it was easy to interact with other plasmids or gene cassettes to form a fusion plasmid.(4)Three mcr-1-bearing fusion plasmids were identified in the transconjugants.Sequence analysis showed that p D1(R1)1?mcr-1F in the transconjugant D1(R1)1 was an Inc HI2 plasmid formed upon the fusion of two plasmids in D1 and one plasmid in R1;the gene cassettes IS26–oqx A–oqx B in p D1?MDR and IS26–dfr A12–aad A2 and IS26–mcr-1 in p D1?mcr-1 were horizontally transferred to p R1,and the novel fusion plasmid shared the same backbone as p R1.p D2(R5)2?mcr-1F in the transconjugant D2(R5)2 was an Inc HI2plasmid formed upon fusion between two plasmids that originally existed in D2;the insertion of the polymyxin resistance-associated gene cassette ISApl1–mcr-1–pap2,carried by p D2?mcr-1,into p D2?ami led to the formation of a novel fusion plasmid,which was subsequently transferred to R5.p D6(EC)3?mcr-1 in the transconjugant D6(EC)3 was identified to be a novel Inc FIB–Inc HI2 hybrid plasmid,which was formed upon fusion between p D6?mcr-1 and p D6?tet(M)in D6 through interaction with a 38-bp homologous region.The three fusion plasmids carried mcr-1 and antibiotic resistance genes.Plasmid fusion occurred at frequencies of 1.48×10-3 to 3.94×10-3 per recipient cell.The fusion plasmids presented high stability and could transfer their antibiotic resistance-encoding gene cassettes including mcr-1 to E.coli C600 and J53 at frequencies of 1.73×10-3 to 2.32×10-4 per recipient cell.Plasmid fusion events occur frequently and the mechanism was complex.Fusion plasmids can carry out stable inheritance and have strong conjugative transfer capabilities. |
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