| Colistin,a cationic polypeptide antibiotic,has been recently as a last-line treatment option for multidrug-resistant Gram-negative bacteria.Colistin is widely used in veterinary medicine for the treatment of gastrointestinal infections caused by Enterobacteriaceae and used as a feed additive for growth promotion in China.In 2016,Liu et al.identified the first mobile colistin resistance gene,mcr-1,in Enterobacteriaceae Escherichia coli and Klebsiella pneumoniae).Shortly afterwards,mcr-1 and its slightly altered gene variants were identified in various Enterobacteriaceae of different origins in over 40 countries across five continents.Several other mcr genes(mcr-2 to mcr-8)were subsequently identified in Enterobacteriaceae.Antimicrobial resistance against colistin has emerged worldwide threatening the efficacy of one of the last-resort antimicrobials used for the treatment of multi-drug resistant Gram-negative bacteria.In this study,quantitative real-time PCRs(qPCR)for the detection of 8 mcr genes were established.The 1,552 pig swabs from 9 provinces,3,343 poultry swabs from 24 provinces and 134 human swabs were investigated using the specific mcr-qPCR.At the same time,the persistence influence of mcr-1 was evaluated after two years of implemented the withdrawal of colistin as a food additive for growth promotion in food animal.Finally,this study further analyzed the characteristics of antimicrobial resistance,the molecular epidemiological characteristics of mcr-1 carrying E.coli strains from different animal resources and the molecular transmission mechanism of mcr-1 in E.coli strains.1.Establishment of mobile colistin resistance genes(mcr-1-mcr-8)quantitative real-timePCR(qPCR)The specificity of the primers for the mcr-1 to mcr-8 PCRs was verified by BLASTN and DNA sequencing of the amplicons obtained using synthesized plasmids containing portions of the target mcr-1 to mcr-8 that were cloned into the SacI site.Our specific mcr-qPCR detected the positive-control plasmids containing their target gene sequences with a detection limit of one gene copy per reaction.Each qPCR amplified the plasmids containing its own mcr gene but did not amplify plasmids containing the other mcr genes.2.Molecular detection of mobile colistin resistance genes(mcr-1-mcr-8)from pigs inChinaAntimicrobial resistance in bacteria is an important issue related to the health of both human and animals.Colistin use in food animals and particularly in pig production has been singled out as responsible for the emergence of colistin resistance.In this study,PCRs were used to detect mcr genes(mcr-1,mcr-2,mcr-3,mcr-4,mcr-5,mcr-6,mcr-7 and mcr-8)in 213 anal and 1,339 nasal swabs from pigs(n=1,454)in nine provinces of China.The different mcr prevalences in pigs were 79.2%(mcr-1)?56.3%(mcr-2)?18.7%(mcr-3)?42.7%(mcr-4)?32.9%(mcr-5)?58.9%(mcr-6)?1.6%(mcr-7)and 4.5%(mcr-8).The prevalences of the mcr(mcr-1,mcr-2,mcr-3,mcr-4 and mcr-5)were significantly higher in nasal swabs than in the anal swabs of pigs(n=98).In total,five pigs were positive for seven mcr genes.Phylogenetic studies identified 31 new mcr-2 variants,2 new mcr-3 variants and 16 new mcr-7 variants.This study further confirms the presence of the different mcr genes(mcr-1-mcr-8)in pigs,and indicates these genes are prevalent and widespread from pigs.3.Molecular detection of mobile colistin resistance genes(mcr-1-mcr-8)from poultry inChinaIn this study,PCRs were used to detect mcr genes(mcr-1,mcr-2,mcr-3,mcr-4,mcr-5,mcr-6,mcr-7 and mcr-8)in 1,696 cloacal and 1,647 oropharyngeal samples from poultry(n=1,836)at live-bird markets in 24 provinces.The different mcr prevalences in poultry were 273%(mcr-1)?2.8%(mcr-2),3.8%(mcr-3)?11.5%(mcr-4)?5.6%(mcr-5)?5.5%(mcr-6)?0.03%(mcr-7)and 0.2%(mcr-8).The prevalences of the mcr(mcr-1,mcr-2,mcr-3 and mcr-5)were significantly higher in oropharyngeal swabs than in the cloacal swabs in avian(n=1,507).In total,one oropharyngeal swab from chicken was positive for five mcr(mcr-1,mcr-2,mcr-3,mcr-4 and mcr-5)genes and two cloacal swabs from goose was positive for 4 mcr(mcr-1,mcr-3,mcr-4,mcr-5)genes.Phylogenetic studies identified 5 new mcr-2 variants and 5 new mcr-3 variants.This study further confirms the presence of the different mcr(mcr-1-mcr-8)in poultry and indicates these genes are prevalent and widespread in poultry in China.4.Molecular detection of mobile colistin resistance genes(mcr-1-mcr-8)in humanvaginal swabsBased on the molecular epidemiology from pigs and poultry,we found the mobile colistin resistance genes are prevalent and widespread in food-producing animals in China.Although there is close relationship between humans and animals,few data are available on the prevalence of wcr-genes other than mcr-1 in human samples.In this study,the presence of five currently described colistin resistance genes(mcr-1 to mcr-5)in 134 vaginal swabs of women undergoing infertility evaluation was reported.Most samples were found to be positive for the rrmcr-4(12.7%),followed by two for the mcr-2(1.5%),two for the mcr-3(1.5%),one for the mcr-1(0.7%),and one for the mcr-5(0.7%).Phylogenetic comparison demonstrated identical(mcr-1,mcr-2,mcr-3,mcr-4 and mcr-5)nucleotide sequences of human samples and those of animal origins from the same city,suggesting the potential transmission of mcr genes from animals to humans.This is the first detection of mcr-2,mcr-4 and mcr-5 genes in human samples.5.Detection of mobile colistin resistance gene 1(mcr-1)after removal of colistin from feedadditiveThe efficiency of colistin as the last resort antimicrobial is now compromised by the presence of an increasing number of mobile colistin resistance genes.Since 1 April 2017,the Chinese government has implemented the withdrawal of colistin as a feed additive for growth promotion in food animal.In this study,PCRs were used to detect mcr-1 in 203 anal swabs collected in March,2019 from pigs in Jiangsu and Zhejiang provinces.The mcr-1 prevalence was not significant difrerences between 2019 and 2016.The average copy numbers of mcr-1 positive samples in 2016(9,750 copies/reaction)was higher than that in 2019(3,353 copies/reaction).The mimic of mcr-1 transformation in vitro demonstrated that the resistance gene is maintained at an unchanged level through 60 generations in the absence of colistin,and that transfer occurs even in the absence of selective pressure by colistin.Our data indicate high prevalence of the mcr-1 in pigs do not appear to be readily lost after withdrawal of colistin as a food additive.6.Antimicrobial resistance in mcr-1-positive Escherichia,coliAntimicrobial resistance is a global health concern in both human and veterinary medicine where antimicrobial agents have been used widely for treating bacterial diseases.The use and misuse of antimicrobial agents has lea to the development of resistance which is threatening their effectiveness in the treatment and prevention of bacterial infections.The aim of this study was to investigate the differences antimicrobial resistance in Escherichia colt isolated from different types of animals in Jiangsu province.The antimicrobial susceptibility testing and disc diffusion method were used to determine susceptibility to colistin and other 16 antimicrobial agents in 28 clinical isolates collected between 2016 and 2017,respectively.Overall,all of the isolates showed low susceptibility of colistin(4-8?g/ml)and resistant to at least one drug with 96.4%being resistance to at least three different classes of antimicrobials.The isolates from different animals were most commonly resistant to ampicillin(100%,27/28),cefotaxime(82.1%,23/28),ceftriaxone(82.1%,23/28),sulfamethoxazole(78.6%,22/28)and chloramphenicol(67.9%,19/28)and high sensitive to amoxicillin/clavulanic acid(100%,28/28),ertapenem(100%,28/28),amikacin(100%,28/28),nitrofurantoin(100%,28/28).The resistant to streptomycin and naproxinic acid in isolates from food-producing animals were significantly higher than that in isolates from elks,and only the isolates from food-producing animals were resistant to ciprofloxacin.MDR was most common in isolates from two different types of animals.The isolates from food-producing animals and elks showed resistance to nine and eleven different classes of antimicrobials,respectively.Our finding showed the characteristics of antimicrobial resistance in mcr-1 E.coli isolates from Jiangsu province.It provided a guideline for control the antimicrobial resistance development and clinical drug selection of mcr-1 E.coli.7.Transmission mechanism of mcr-1 positive Escherichia coliIn this study,the molecular epidemiological characteristics of 28 mcr-1 positive E.coli from different sources(food-producing and non-food-producing)were analyzed by multi-locus sequence typing(MLST)and pulsed-field gel electrophoresis(PFGE).The MLST result showed a total of 17 sequence types(STs)were obtained from 28 E.coli strains.The PFGE showed most of the E.coli strains had the similarity between 70%and 90%.At the same time,there were strains with 100%homtology in both different sources and indicated that they had the possibility of monoclonal transmission in the same area.Conjugation assay showed a total of 19 of 28 E.coli strains could be successfully transferred and the colistin resistance for the trans-coniugant increasing two-fold to four-fold compared with the untransformed control.The S1-PFGE result showed that 26 of wcr-1-positive E.coli strains contained 1 to 4 plasmids,and most strains had a plasmid about 60kb in size.The result of antimicrobial resistance showed other antimicrobial resistances were co-transferred to the recipient strain in this study.In summary,this study further analyzed the molecular epidemiological characteristics of mcr-1 E.coli strains from different sources and the molecular transmission mechanism of mcr-1 in E.coli strains.In conclusion,this study established specific mcr-qPCR and applied this molecular detection method to investigate the prevalence of mcr genes from food-producing animals(pigs and poultry)in China,and in human swabs from Yangzhou,respectively.Our study showed mcr.genes are prevalent and widespread in China.Moreover,the high prevalence of the mcr-1 in pigs do not appear to be readily lost after withdrawal of cohstin as a food additive.In addition,this study further analyzed the characteristics of antimicrobial resistance,the molecular epidemiological characteristics of mcr-1 carrying E.coli strains from different animal resources and the molecular transmission mechanism of mcr-1 in E.coli strains.The result showed mcr-1 can be transmitted either by monoclonal method or by plasmid-mediated method.This study reveals the distribution of mcr gene in food-producing animals in China and provides the guideline for the clinical antibiotic selection in veterinarians and lays the foundation for effectively monitoring and controlling of the prevalence of colistin-resistant genes in animals. |
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