Epidemic Characteristics Of Lsa(E) Gene In Chicken And Cattle Farms,as Well As Analysis Of Its Abundance And Antibiotic Residue In Breeding Environment

Enterococci is an opportunistic pathogen widely distributed,which can bring a variety of diseases to humans and animals,and even endanger lives.With the extensive use of antibiotics,the drug resistance of enterococci is becoming more and more serious.It can be used as a repository of drug resistance genes,and it also contains a variety of transfer elements,which can promote the wide spread of drug resistance genes.Since its discovery,lsa(E)gene has been reported and widely distributed in a variety of pathogens from human,animal and environmental sources,which has caused serious harm to human health.It is necessary to study the prevalence of lsa(E)gene in animal breeding environment based on the concept of "One Health".Therefore,this paper intends to explore the transmission of lsa(E)gene in enterococci in farms,establish a fluorescence quantitative PCR(RT-qPCR)detection method of lsa(E)gene,and explore the relationship between the prevalence of lsa(E)gene in farms and the use of antibiotics.In this study,animal samples and environmental samples of feces,soil,bedding,sewage and solid surface were collected from 2 cattle farms and 4 chicken farms.Enterococci were isolated and identified.PCR detection,antimicrobial susceptibility test,sequencing techniques were used to analyze the drug resistance genes,SNP analysis and MLST typing to explore the epidemic rule of lsa(E)gene in enterococci.The RT-qPCR technique based on SYBR dye method was used to establish a method for rapid detection of lsa(E)gene from different samples(bacteria and various environmental samples),and the established RT-qPCR detection method was used to detect the relative abundance of lsa(E)gene in feces,soil,bedding and other environmental samples.Ultra high performance liquid chromatography-tandem mass spectrometry(UHPLCMS/MS)was used to determine the residues of tiamulin and valnemulin in environmental samples.To explore the relationship between the prevalence of lsa(E)gene in environmental samples of farms and the use of antibiotics.The isolation and identification of lsa(E)positive enterococci showed that the isolation rates of lsa(E)positive enterococci in cattle and chicken farms were 4% and29.5% respectively.The results of species identification showed that all lsa(E)positive enterococci in cattle farms were Enterococcus faecalis,while lsa(E)positive enterococci in chicken farms came from many species(Enterococcus faecalis,Enterococcus faecium,Enterococcus gallinarum,Enterococcus casseliflavus).In contrast,lsa(E)gene was more popular in enterococci in chicken farms.ST type and SNP analysis showed that lsa(E)positive enterococci were divided into 42 ST types,including 13 new ST type enterococci.ST16 enterococci were the main epidemic strains in chicken farms,and the genetic relationship of lsa(E)positive enterococci was complex,indicating that lsa(E)gene was widely spread in enterococci.The screening results of drug resistance genes showed that lsa(E)positive enterococci carried a large number of drug resistance genes,as many as 27,indicating that enterococci could integrate various drug resistance genes and was the repository of multiple drug resistance genes.Antimicrobial susceptibility test showed that the resistance rate of lsa(E)positive enterococci to 10 antibiotics was high.Compared with the screening results of drug resistance genes,it was found that the drug resistance phenotype was basically consistent with the carrying of drug resistance genes.Analysis of the genetic environment of lsa(E)genes showed that most of the lsa(E)genes were located in the drug resistance gene cluster formed by lnu(B)-lsa(E)-apt-ant1,and the flanking region of the drug resistance gene also harbored several IS insertion sequences,which were able to promote the spread of the drug resistance gene cluster.In this study,a RT-qPCR method for detection of lsa(E)gene was established.The results showed that the melting curve was unimodal,indicating that the specificity of the method was good.R2: 0.997 showed that the standard curve had a good linear relationship,a good amplification efficiency(101.56%),a wide detection range(Ct value: 15.22-34.27,gene copy number: 102-108 copies),and coefficients of variation between and within groups were less than 2%.It shows that the repeatability of the detection method is good.The established method for rapid detection of lsa(E)gene was used to detect the relative abundance of lsa(E)gene in various environmental samples collected.The results showed that the relative abundance of lsa(E)gene in cattle farm was9.99×10-4~5.96×100 and that in chicken farm was 1.66×100~7.25×103.In contrast,the relative abundance of lsa(E)gene in chicken farm environmental samples is higher,and lsa(E)gene is more widespread in chicken farms,which is mutually confirmed with the detection results of lsa(E)positive enterococci.UHPLC-MS/MS was used to determine the residues of tiamulin and valnemulin in environmental samples.The results showed that valnemulin was detected only in two environmental samples of cattle farm and chicken farm,and the residue of tiamulin was only detected in one environmental sample of cattle farm,and the residue was low,while it was detected in all environmental samples of chicken farm,the residue was 0.9529-13965 μg/kg.The relative abundance of lsa(E)gene and antibiotic residue analysis showed that there was a certain relationship between the relative abundance of lsa(E)gene and antibiotic residue(tiamulin)in environmental samples from chicken.To sum up,this study investigated the epidemic characteristics of lsa(E)gene in enterococci in cattle and chicken farms in Henan Province,and showed that the prevalence of the lsa(E)gene was more extensive in chicken farms,established a RT-qPCR detection method of lsa(E)gene in breeding environmental samples,and analyzed the relationship between lsa(E)gene abundance and antibiotic residues in cattle and chicken farms,showing that the relative abundance of the lsa(E)gene in the environmental samples of chicken farms had a relationship with tiamulin residues,which provided a theoretical basis for rational use of antibiotics and reducing the spread of drug resistance genes in livestock and poultry breeding.