| Poultry and livestock are the most important reservoirs for pathogenic Escherichia coli and the using of antimicrobials in animal farming is considered the most important factor for promoting the emergence,selection and dissemination of antimicrobial-resistant microorganisms.Many countries established surveillance and investigation system to reduce antimicrobial resistance.This surveillance include both phenotypic and genotypic procedures to describe and improve our understanding of bacterial evolution,outbreaks,and transmission of antimicrobial resistance.Antimicrobial resistance against colistin has emerged worldwide threatening the efficacy of one of the last-resort antimicrobials used for the treatment of multidrug-resistant Enterobacteriaceae infection in humans.1.Antimicrobial resistance in clinical Escherichia coli isolates from poultry and livestockThe disc diffusion method was used to determine susceptibility to 18 antimicrobial agents in 862 clinical isolates collected from chickens,ducks,pigs,and cows between 2004 and 2012 in China.Overall,94%of the isolates showed resistance to at least one drug,and 83%being resistance to at least three different classes of antimicrobials.The isolates from the different species were most commonly resistant to tetracycline,nalidixic acid,sulfamethoxazole,trimethoprim/sulfamethoxazole and ampicillin,and showed increasing resistance to amikacin,aztreonam,ceftazidime,cefotaxime,chloramphenicol,ciprofloxacin as time going on.They were least resistant to amoxicillin/clavulanic acid(3.4%)and ertapenem(0.2%).Multi-drug resistance(MDR)was most common in isolates from ducks(44/44,100%),followed by chickens(568/644,88.2%),pigs(93/113,82.3%)and cows(13/61,21.3%).2.Whole genome sequence of selected Escherichia coli isolatesNineteen clinical E.coli isolates collected from chicken,pig,cow and duck,were selected for the whole genomic sequencing based on their phenotyping background of antimicrobial resistance.Genomic DNA was purified from all isolates and sequenced as paired-end reads on the Illumina platform.The web servers Centre of Genomic Epidemiology was used to analyze the whole genome sequence and identify antibiotic resistance genes and virulence factors.Overall,the 78.9%of isolates carried 29 different resistance genes conferring resistance to 12 classes of antimicrobials.All the 19 E.coli stains carried more than one resistance gene.Only one isolate(5.2%)carried mcr-1 gene in full-length copy of a colistin resistance gene.Thirteen types of plasmid replicons in 15/19(78.9%)isolates were recognized.3.Epidemiology of mcr-1 gene in ChinaTo investigate the presence of the colistin resistance gene(mcr-1)in China,we developed two qPCRs(mcr-1 qPCR,total-bacteria qPCR)to determine directly the prevalence of the mcr-1 and the total bacteria in samples.The mcr-1 was positive with poultry samples from 21 of the 25 provinces.The highest prevalence was nasal and anal swabs from pigs(81.2%;1,128/1,389)followed by fecal swabs from dogs(35.0%;14/40),oro-pharyngeal and cloacal swabs from poultry(27.1%;899/3,318),vaginal swabs from women(0.7%,1/134),and bovine fecal samples(0%;0/64).The ratios of the mcr-1 and the total-bacteria copy numbers were 31.6%with fecal swabs from dogs,1.7%with cloacal and 0.4%with oro-pharyngeal swabs from poultry,0.1%with nasal and anal swabs from pigs,and 0.0008%with vaginal swabs from women.The mcr-1 levels did not differ significantly between nasal and anal swabs in pigs,and between oro-pharyngeal and cloacal swabs in poultry,showing that mcr-1 positive bacteria are present in multiple ecosystems.The ratio of the copy numbers from the mcr-1 and total-bacteria qPCRs suggested there were high numbers of the mcr-1 in affected bacteria.4.The role of flies in transmission of mcr genesQuantitative PCRs we developed showed that 34.1%of Musca domestica(86/252)and 51.1%of Protophormia terraenovae(23/45)were positive for the mcr-1,1.2%of M.domestica(3/252)and 2.2%of P.terraenovae(1/45)were positive for the mcr-2,and 5.2%of M.domestica(13/252)and 44.4%of P.terraenovae(20/45)were positive for the mcr-3.Overall,4.8%(9/189)of the bacteria isolated from the flies were positive for the mcr-1(Escherichia coli:8.3%,4/48;Enterobacter cloacae:12.5%,1/8;Providencia alcalifaciens:11.8%,2/17;Providencia stuartii,4.9%,2/41)while none were positive for the mcr-2 and mcr-31 Four mcr-1 positive isolates(two P.stuartii and two P.alcalifaciens)from blow flies trapped near the dumpster had a MIC above 4 mg colistin/mL.This is the first report of mcr-1 carriage in Providencia spp.,and the first detection of mcr-2 and mcr-3 after their initial identification in Belgium and China,respectively.5.Identification and characterization of mcr-mediated colistin resistance in pathogenic Escherichia coliWe investigated the presence of colistin resistance genes(mcr-1,mcr-2,mcr-3)in pathogenic Escherichia coli strains isolated from poultry and livestock collected between 2004 and 2012 in China.Furthermore,we studied the maintenance and transfer of the mcr-1 gene in E.coli after serial passages.Overall,2.7%(17/624)of the E.coli isolates were positive for the mcr-1 while none were positive for the mcr-2 and mcr-3.The prevalences of mcr-1 were similar in E.coli isolates from chickens(3.2%;13/404),pigs(0.9%;1/113),and ducks(6.8%;3/44)and was absent in isolates from cattle(0/63).The mcr-1 was maintained in the E.coli after six passages(equivalent to 60 generations).In vitro transfer of mcr-1 was evident even without colistin selection.Our data indicate the presence mcr-1 in pathogenic E.coli from food producing animals in China,and suggest that high numbers of the mcr-1 positive bacteria in poultry and livestock does not appear to be readily lost after withdrawal of colistin as a food additive.In summary clinical E.coli isolates from poultry and livestock are commonly resistant to multiple antibiotics by carrying number of resistance genes.Our study shows the high prevalence of the mcr-1 gene in wide-spectrum animals from China.Moreover,this study suggests flies might contribute significantly to the dissemination of bacteria carrying these colistin resistance genes into a large variety of ecological niches.Further studies will perform to explore the roles of flies playing in the spread of colistin resistance genes in the future.Our data indicated the presence of mcr-1 gene in pathogenic E.coli from food producing animals in China,and suggested that high numbers of the mcr-1 positive bacteria in poultry and livestock does not appear to be readily lost after withdrawal of colistin as a food additive. |
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