| The long-term use of antibiotics has led to the emergence and wide spread of drug-resistant strains.At present,bacterial resistance is becoming increasingly serious,which reduces the efficacy of antibacterial drugs and conventional combination drugs,restricts the development of aquaculture and threatens clinical anti-infection treatment.As the “last line of defense” drugs,tigecycline,colistin and linezolid for the treatment of multi-drug resistant bacterial infections are facing great challenges in clinical application due to the continuous emergence of new transferable drug resistance genes.Antibiotic resistance genes in livestock farms can be transmitted to crops through manure and sewage,and then to humans,posing a threat to human food safety and public health.In this study,fecal samples were collected from pig farm manure,sewage after dry and wet separation,and farmland soil irrigated by sewage.Metagenomic sequencing and screening of drug-resistant strains were used to compare the number and variation pattern of antibiotics resistance gene in pig farm environment,explore the transmission mechanism of drug-resistant genes,and evaluate the risk of transmission of drug-resistant genes,providing theoretical basis for clinical strain tracing and clinical antibacterial use.In the winter and summer of 2021,fecal samples from a manure tank,sewage after dry and wet separation,and farmland soil irrigated by sewage were collected from a pig farm in Henan Province.The metagenomic sequencing results showed that the number of antibiotic resistance genes(ARGs)decreased in the process of the transmission in the pig farm environment from feces to sewage to farmland.The clustering results of arithmetic mean method(UPGMA)and permutation multivariate analysis(ANOSIM)were relatively consistent,both showing that DS(winter sewage)and summer feces(XF),summer farmland(XT)and winter farmland(DT)samples were clustered,and the other samples were separated.The intergroup difference was greater than the intra-group difference.A total of 15 resistance mechanisms were detected from all samples,of which multi-drug resistance,macrolides-lincosamids-streptogramins(MLS)and tetracycline resistance mechanisms accounted for the highest proportion,with 32.44%~43.96%,10.79%~17.30% and 10.05%~12.87%,respectively.At the phylum level,Firmicutes,Proteobacteria,Bacteroidetes,Actinobacteria,Acidobacteria,Verrucomicrobia and Viscospheroid were the most common phyla in the six types of samples,among which Firmicutes was the most abundant.A total of 1173 ARGs subtypes were detected from all samples,with little difference in the number of ARGs subtypes in each sample.The most abundant ARGs subtypes were 983 in DT samples and the least were 883 in XF samples.There were 657 common ARGs types in the six types of samples.Among the 657 ARGs subtypes,the ABC transport system-related gene mac B was the most prevalent(7.24%).A total 19 strains of Gram-positive bacteria and 150 strains of Gram-negative bacteria were isolated from 162 environmental samples.Two of the 19 Gram-positive bacteria,ST506 E.faecalis 21W5 and ST2062 E.faecium 21W6,carried the optr A gene.The results of drug susceptibility test showed that 21W5 and 21W6 strains were resistant to 11 antibiotics except tigecycline,indicating that they were multi-drug resistant strains.S1-PFGE and Southern blot showed that the optr A genes of strains 21W5 and 21W6 were located on plasmids about 78 kb in size.Whole genome sequencing(WGS)and blast comparison results showed that there was 99.95% homology between plasmid p W5-2 in21W5 and plasmid p E211 in E.faecalis E211(No.MK784777.1),which covered 98% of the gene sequence.There was 98.46% homology between plasmid p W6-2 carrying optr A in 21W6 and plasmid p47-61(No.CP091102.1)in E.faecium p47,which covered 65% of the gene sequence.The optr A gene in both plasmids was located on the IS1216-fex A-optr A-erm(A)-IS1216 complex transposon mediated by IS1216,which was widely found in Enterococcus faecalis and Enterococcus faecium.A total of 150 strains of Gram-negative bacteria were tested to 14 antimicrobial agents of 8 classes.The results showed that the resistance rate of strains isolated from fecal samples was the most serious,followed by sewage and farmland soil,which was consistent with the results of metagenomic analysis.The strains isolated from fecal were the most resistant to ampicillin,with a resistance rate of 100%,and all strains were sensitive to amikacin.The resistance rates of strains isolated from sewage to doxycycline,tetracycline and florfenicol ranged from 68.25% to 82.54%,and all strains were sensitive to meropenem.The strains isolated from soil were most resistant to ampicillin,with a resistance rate of 80.00%,and were sensitive to cefquinome,meropenem,amikacin,and ciprofloxacin.PCR and sequencing results showed that 18 isolates carried tet(X4)gene and 10 isolates carried mcr-1 gene.Eighteen tet(X4)positive strains were divided into 15pulsed-field patterns by PFGE.Sixteen strains were successfully fused with E.coli C600,and Inc FIA,Inc W,Inc FIB and Inc P plasmid types were detected in transconjugant.PFGE results of the 10 mcr-1 carrying strains could be divided into 9 pulsed-field patterns.Nine of the 10 mcr-1 carrying strains were successfully transconjugated with E.coli C600,three plasmid types,Inc FIB,Inc Frep B,and Inc HI1,were identified in the transconjugants.In conclusion,the number of ARGs in pig farm environment decreased in the transmission process from feces to sewage to farmland,and the strains isolated from feces had the most severe drug resistance.The spread of IS1216-fex A-optr A-erm(A)-IS1216 gene fragments in 21W5 and 21W6 in E.faecalis and E.faecium may be related to the IS1216 mobile element.In Gram-negative bacteria,tet(X4)or mcr-1 carrying strains were distantly related to each other,and there was no obvious sign of clonal transmission,which was mainly caused by plasmid-mediated horizontal transmission.This study elucidated the transmission pattern of drug-resistant genes in pig farm environment,explored the resistance mechanism of “last line of defense” drugs,evaluated the possibility of transmission of drug-resistant genes from environment to humans,and provided theoretical basis for controlling the spread and evolution of drug-resistant strains in pig farm environment. |
PDF Full Text Request