| Since the discovery of transferable colistin resistance gene for the first time in China,the mcr-1 gene has been detected in more than 40 countries and regions around the world.The mcr-1 gene can be spread between different strains of bacteria through plasmids or transposon.This is not only a great threat to public health,but also a big challenge to clinical anti-infective therapy.At present,the strains carrying the mcr-1 gene detected from many different areas and included a lot of different species,but the sample sources mainly from animals,raw meats,environment and clinical,the number of mcr-1 positive strains isolated from clinical are relatively rare.Most of the reported strains carrying mcr-1 gene are Escherichia coli and Klebsiella pneumonia isolated from nosocomial infections or detected from environmental.However,there is only a few reports of the important intestinal pathogens carrying mcr-1 gene such as E.coli,Salmonella and Shigella.These Enterobacteriaceae could cause gut infection and lead to acute diarrhea.In addition,the spread characteristics of mcr-1 gene in intestinal pathogenic bacteria and the phylogenetic relationship of mcr-1 positive bacteria are still not clear,the related studies are therefore urgently needed.Research Objective In order to revealing the characteristic of distribution and spread of mcr-1 gene in intestinal pathogens we screened some important intestinal pathogens such as E.coli,Salmonella and Shigella collected from samples of patients with diarrhea in some local hospitals.We analysis of the resistance characteristics of pathogenic bacteria carrying mcr-1 to provide a basis for the scientific treatment of colistin resistant bacteria.We analysis of the genetic characteristics of mcr-1 positive E.coli,Salmonella and Shigella isolated from human,in order to effectively monitor and control the spread of colistinresistant gene mcr-1 in human popμlations and provides early warning for outbreaks.Research Methods We collected clinical samples of patients with diarrhea and the patients’ information based on the military infectious disease pathogen monitoring platform.The collected strains from diarrhea specimen were isolated and cultured in accordance with the “National Clinical Laboratory Operation Regulations”.Antimicrobial susceptibility of mcr-1 positive bacteria was analyzed by gradient broth microdilution.Molecular Typing of enteric pathogens carrying mcr-1 was analyzed using PFGE technology.The Type and localization of plasmids were determined by S1-PFGE and Southern-blot techniques.The spread characteristics of mcr-1 positive plasmids were detected through plasmid conjugate transfer experiment.PCR detection and whole genome sequencing techniques were used to study the characteristics of strains.Research Resμlts 1.Based on the military infectious disease pathogen monitoring platform,we collected 16950 strains from different places in China from 2003 to 2015.There are 3821 strains of Shigella,1024 strains of E.coli and 12105 strains of Salmonella.76 mcr-1 positive strains were identified by PCR and Sanger sequencing including 7 Shigella,28 Salmonella and 41 Escherichia coli.Among the mcr-1 positive strains,47.36% were atypical enteropathogenic Escherichia coli(a EPEC)strains and 32.89% were Salmonella typhimurium.2.Among all patients infected with mcr-1 positive bacteria,only 12.86%(9/70)of the human sourced samples were isolated from adults,77.41%(54/70)were isolated from children under 4 years of age and the average age of children was only 1.4 years.3.Through antibiotic susceptibility analysis of mcr-1 positive bacteria,the three antibiotics with the highest resistance rates were colistin 100.00%(76/76),78.95%(60/76)tetracycline,and 69.74%(53/76)ampicillin.The three antibiotics with the highest sensitivity were 90.79%(69/76)ciprofloxacin,80.26%(61/76)amoxicillin and clavμlanic acid,and 84.21% of sμlfisoxazole(64/76).4.Through analyzing the resistance spectrum of mcr-1 positive bacteria,it was found that lots of strains have multidrug resistance.There were 12(15.79%)strains have the resistance to the third generation of cephalosporins and quinolones.69(90.79%)strains were resistant to three or more kinds of antibiotics.Only 2 strains were found have single antibiotic resistance spectrum of colistin.There were differences in antibiotic resistance profiles among mcr-1 positive E.coli,Salmonella and Shigella.Of these,42.86% of Salmonella strains were resistant to the third generation cephalosporins.5.Four types of plasmid backbone were detected from the mcr-1 positive Enterobacteriaceae,including Inc X4,Inc I2,Inc HI2 and Inc F.26 were Inc X4(36.6%),24 were Inc I(31.6%),19 were Inc H(25%),and 1 were Inc F(2.4%).6.Among the E.coli strains,the most popular type of the mcr-1 plasmid was Inc X4(19/41,46.3%).The the most popular type of the mcr-1 plasmid in Salmonella strains was Inc H(13/28,46.0%).Only Inc I plasmid type was detected in Shigella.7.PFGE results of 41 mcr-1 positive E.coli strains showed that theie genomes had heterogeneity,and 3 highly homologous prevalent strains were detected in 28 strains of Salmonella.8.Through analyzing of SNPs phylogenetic tree,it was found that some Salmonella typhimurium strains had closed genetic relationship,indicating that it may be the same clonal cluster.Research Conclusion 1.It’s the first time to find Shigella sonnei,Salmonella Enteritidis and Salmonella wandsworth stains which isolated from human carried the mcr-1 gene in China.Among mcr-1 positive strains,the atypical enteropathogenic E.coli(a EPEC)and Salmonella typhimurium are the dominant types.Both of these two kinds of strains can cause nosocomial infection and fulminant of food poisoning.It is a great threat to public health.2.Among the mcr-1 positive bacteria isolated in Shanghai,the positive rate in E.coli(4.0%)was significantly higher than that in Salmonella and Shigella.And mcr-1 positive strains were also detected in asymptomatic populations 3.The infection caused of mcr-1 positive enteropathogenic bacteria has a high morbidity in children under 4 years old.Public health safety knowledge should be popularized in schools and households,and personal awareness of protection should be strengthened.Therefore,the threats to children’s health brought by food and water pollution could be reduced.4.The phenomenon of mμltidrug resistance of mcr-1 positive intestinal pathogens were very common.Multi-drug resistance and pan-drug-resistant strains carrying mcr-1 become a serious problem in clinical treatment 5.There is a significant difference in the resistance to the same drug among different mcr-1 positive intestinal pathogenic bacteria.The number of third-generation cephalosporin resistant mcr-1 positive Salmonella is relatively more than E.coli.However,E.coli strains showed higher levels of resistance to tetracyclines and sulfadiazine.Four strains of Shigella spp.showed pan-drug resistance.They carried azithromycin and third-generation cephalosporin resistance simultaneously.6.The heterogeneity feature of a EPEC E.coli is obvious.region,A clonal population of mcr-1 positive Salmonella isolated from 2012 to 2015 was found in the Shanghai.Among them,5 strains had a high homology of more than 95%,and they all had the resistance to the third generation cephalosporins.7.Through analyzing the mcr-1 plasmid in the intestinal pathogenic bacteria,the type of mcr-1 plasmid carried by enteric pathogenic bacteria is complex and diverse,Main of the plasmids were Inc X4,Inc I and Inc H plasmid types.The Inc I type plasmid were widely distributed in the enteric bacteria detected in this study.Among them,the type of mcr-1 plasmid carried by E.coli was more complex.Two kinds of main types of plasmids including Inc X4 and Inc I were found in E.coli strains.In Salmonella strains,the predominent type was Inc H.A new mcr-1 gene variant mcr-1.9 was identified in a mcr-1 positive E.coli by plasmid sequencing.8.Through analyzing of SNPs phylogenetic tree,it was found that some Salmonella typhimurium strains had closed genetic relationship,indicating that it may be the same clonal cluster.However,they showed a significant difference in the type of mcr-1 plasmid,the insertion sequence,and the carrying of other drug resistance genes.The difference probably due to the horizontal transfer.Through the gene horizontal transfer,plasmids which carried different drug resistance genes influenced each other and then get the resistance genes they need.Significance and Innovation 1.So far,there has been no large-scale investigation and analysis of the distribution of the mcr-1 gene in human-originated intestinal pathogens.We have screened the samples collected from patients with diarrhea in some hospitals and then revealed the characteristic of distribution of mcr-1 gene in E.coli,Salmonella,and Shigella spp.Through this work,we provide a reliable theoretical basis for controlling the spread of colistin resistance.2.Through the screening of the collected strains,we studied the distribution,spread relationship,antibiotics resistance,genetic polymorphism and transfer mechanism of mcr-1 positive human-originated intestinal pathogens in some regions which provided a scientific basis to the assessment of the hazards of mcr-1 positive intestinal pathogens.3.Through genomic sequencing analysis,we studied the transfer characteristics of the mcr-1 plasmid in intestinal pathogens and the structural features of the mcr-1 plasmids carried between different species which provided the basis for preventing and controlling the spread of mcr-1 gene. |
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