| The emergence of mcr-1 gene,as the first reported plasmid-mediated colistin resistance gene,is of great global concern.Antibiotic resistance genes can be transmitted from animals into the environment through vertical or horizontal transfer.mcr-1 is prevalent in pig farms in China,however,its distribution in the environment of pig farm is still not clear.This study aimed to investigate the distribution of mcr-1 and the transmission mechanism of mcr-1 among various bacteria collected from the pig farm in Shanghai where the mcr-1 was first identified.A total of 388 samples were collected from pig faeces,the soil of pig house,sewage,the river and sedimen.The total DNA of all samples was purified and investigate the abundance of mcr-1 gene by real-time quantitative PCR.The results showed that mcr-1 was detected in all kinds of the environmental samples,and the abundance of the mcr-1 gene in soil was highest,followed by fecal,sewage showed the least abundance of mcr-1.The mcr-1 gene among soil in pigpen was significantly more abundant than that in the effluent of the pigpen,and the difference was over 10~4 times.The immobile character of the clay may be the reason for the largest enrichment of mcr-1.The abundance of mcr-1 in the soil of pigpen was greatly higher than that in the feces,the difference was between 100 and1000 times,and correlations between them were observed.Furthermore,the abundance of mcr-1 in the sediments of the river is largest,followed by middle and lower reaches which were higher than that in the upper reaches by 1000 times.A total of 458 colistin-resistant Enterobacteriaceae were obtained from the above 388 samples using MacConkey agar with2 mg/L colistin,and 136 strains were identified to carry mcr-1 by polymerase chain reaction,including 129 Escherichia coli,four Enterobacter sakazakii,and threeShiga bacteria strains,which were isolated from stool(n=69),anal swab(n=52),soil(n=4),manure pool(n=2)and river sediment(n=2).All mcr-1-positive isolates were tested for their MICs against 20 agents using the agar dilution method.The results showed that over90%of strains were resistant to tetracycline,florfenicol and cotrimoxazole with 98.5%(98/136),97.80%(133/136)and 94.11%(128/136),respectively.70%of strains were resistant to olaquindox,chloramphenicol and ampicillin,while 30.15%(41/136)strains showed resistanceto cefotaximer.All strains were multiple resistant.15 mcr-1 positive Escherichia coli isolates collected from soil(n=4),sedimen(n=2),feces-treatment pool(n=1)and faeces(n=8)were randomly selected for further investigation of transmission mechanism.The results of pulsed-field gel electrophoresis(PFGE)analysis showed that the mcr-1-positive isolates were clonally unrelated.To investigate the transferability of mcr-1,the above 15 Escherichia coli isolates,four E.sakazakii isolates and three Shigella isolates performed conjugation experiments,and 19 of them transferred mcr-1 to E.coli C600 successfully.All the transconjugants were subjected to S1-PFGE,Southern blotting and replicon typings.The results demonstrated that mcr-1was located on IncX4(~33.3 kb,n=3)and IncP(~54.7 kb)plasmids in four E.sakazakii isolates,and on IncX2(~54.7 kb)and IncFII(~138.9 kb)plasmids in two Shigella isolates.Among E.coli isolates,mcr-1 was located on IncX4(~33.3 kb,n=4),IncX2(~54.7 kb,n=3),IncI2(n=2),IncFII(~104.5 kb)and IncP(~54.7 kb)plasmids.In addition,mcr-1 was located on an~33.3 kb plasmid in the Shigella isolate which can not transfer mcr-1 to E.coli C600.Altogether,similar mcr-1-carrying plasmids detected in E.coli,E.sakazakii or Shigella isolates indicates mcr-1 transfer between different species and origins mediated by plasmid.The results showed that the mcr-1 gene is widely distributed in pig manure and in the environment and can diffuse from farmed animals into the environment through faecal and sewer discharges.The mcr-1 gene mainly located on plasmids with different sizes and replicon types,such as IncX4,IncP,and IncX2,and were detected in different speices and sources,suggesting that mcr-1 transmission in this pig farm was mediated by plasmid horizontal transfer. |
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