The Study On Epidemiology And Resistance Transmission Mechanisms Of Colistin Resistance Gene Mcr-1 In Salmonella Spp.

Background Colistin is used as“last line”of defense for the treatment of multi-drug resistant Gram-negative bacterial infections.However,the discovery and transmission of the mobile colistin resistance gene,mcr-1,threatened colistin efficacy in medicine,arising a worldwide attention.Plasmids with horizontal transfer capacity were the predominant location for mcr-1,and its global dissemination poses a great threat to public safety.At present,mcr-1 was mainly detected from Enterobacteriaceae,such as Escherichia coli,Klebsiella pneumoniae,etc.Only few studies reported other mcr-1-carrying bacteria species,such as Acinetobacter baumannii and Salmonella spp..Salmonella is a common zoonotic pathogen,which can cause infectious diarrhea,a severe foodborne transmission disease in humans.The increasing trend of prevalence of multidrug-resistant Salmonella challenged available therapeutic options.Previous studies reported the prevalence of mcr-1 in clinical infected Salmonella isolates in some areas of China,however,these studies lack the understanding of the prevalence and dissemination mechanisms of mcr-1 in different source,which is epidemic in different ecological niches.In this study,we screened Salmonella isolates from multi-source samples to investigate the prevalence of mcr-1 in Guangdong,China.In addition,we sought to detail the characteristics of mcr-1-positive Salmonella and reveal the mechanisms of antimicrobial resistance and transmission of these strains by bioinformatics analysis.Methods 1,701 samples were collected from pigs,pork and Salmonella infected patients from 2015 to 2017 in Guangdong,China.PCR and Sanger sequencing were used to screen and identify mcr-1-positive Salmonella.We performed antimicrobial susceptibility testing with 16 common antibiotics to all mcr-1-positive Salmonella using agar dilution methods.Conjugation and hybridization with S1-PFGE were used to determine transferability and location of mcr-1.In addition,we did whole genome sequencing?WGS?for all mcr-1-positive Salmonella isolates to evaluate the genomic characteristics,including strain characteristics,plasmids and transposons.Lastly,the population structure of strains was analyzed by combining the genome data of 68 mcr-positive Salmonella from published literatures worldwide.Results Nineteen mcr-1-positive Salmonella strains were identified from all samples?n=1,701?,in which the prevalence rates were 0.53%?5/936?,1.80%?3/167?and 1.84%?11/598?for pigs,pork and Salmonella infected patients.All mcr-1-positive Salmonella strains were multi-drug resistant and resistant to colistin and ampicillin.63.2%of these strains were resistant to cefotaxime and ciprofloxacin.More importantly,the mcr-1 genes were located on plasmids with?200-300 kb size and 12 mcr-1 genes can be transferred to E.coli J53 by conjugation.All transconjugants showed 16 to 32-fold MICs for colistin higher than the recipient E.coli J53.Seven transconjugants were resistant to both ampicillin and cefotaxime.Seventeen monophasic Salmonella Typhimurium strains belonged to ST34 and other two strains were assigned to ST5401 and ST6020,respectively.The antimicrobial resistance genes profile showed that 17,11 and 16 strains harbored the bla_TEM,bla_CTX-MTX-M and oqx genes,respectively.All strains carried SPI-1,SPI-3,SPI-4,SPI-5,SPI-13 and SPI-14.Identification of plasmid replicon revealed that all mcr-1-carrying plasmids were Inc HI2 type.In addition,we found that Tn6330?ISApl1-mcr-1-pap2-ISApl1?was the main way mediating the mcr-1 transmission.Among them,mcr-1 in 9 Salmonella strains were located on composite Tn6330,9 strains contained only the upstream ISApl1,and one contained no ISApl1.The analysis combined with 68 mcr-positive Salmonella from public database revealed that Salmonella Typhimurium ST34 was the main epidemic clone and 13 mcr-1-positive strains in this study were clustered together with the strains from Thailand and Denmark.Conclusions In this study,the prevalence of mcr-1 in Salmonella isolates was low in different sources in Guangdong,China.However,high resistance rate and multi-drug resistance of mcr-1-positive Salmonella,especially resistant to cefotaxime and ciprofloxacin,pose a serious threat to clinical treatment of Salmonella infection.mcr-1-carrying plasmids of Salmonella can be transferred to E.coli with or without other antibiotic resistance genes,which endanger public safety and human healthy.WGS confirmed that IncHI2 type plasmids and transposon Tn6330 plays an important role in the spread of the mcr-1 in Salmonella.The use of other antibiotics may supply a co-selection pressure on mcr-1-positive Salmonella strain carrying multiple antimicrobial resistance genes,promoting the spread of colistin resistant bacteria with other resistance genes.In addition,mcr-1-positive Salmonella isolates are widespread in the world.The strains in this study were more closely related to strains from Thailand and Denmark.Most of strains were Salmonella Typhimurium ST34,which implies that Salmonella Typhimurium ST34 is a new epidemic clone and has the risk of global transmisison.In conclusion,the prevalence of mcr-1-positive Salmonella in animals,foods,community and hospitals needs to be continuously monitored to prevent further spread of colistin resistance in animals and humans.