Characterization Of Plasmid-Mediated Antibiotic Resistance Genes Contamination In Feed Of Hubei Chicken Farm

The long-term and extensive use of antibiotics causes selective pressure on microorganisms in the environment,constantly promoting the emergence of antibiotic resistant bacteria（ARB）,and accelerating the spread of antibiotic resistant genes（ARGs）.ARGs can migrate and transform in various environmental media through vertical transmission and horizontal transfer,and finally pose a threat to human health through the food chain.Most ARGs are encoded in plasmids,and plasmid mediated conjugation transfer is an important mechanism of ARGs transmission,and its transfer frequency is very high,which is the main cause of the widespread spread of antibiotic resistance.At present,a large number of ARGs have been detected in a variety of environmental media,and the pollution of ARGs in livestock and poultry breeding environment has become a topical issue.Poultry products are a major source of foodborne diseases.Animal feed is at the beginning of the food safety chain and is an essential part of livestock and poultry breeding.Animal feed is a potential vehicle for the transmission of ARB,and ARGs carried by ARB may be transferred to feed.However,the contamination characteristics of plasmid-mediated ARGs in animal feed and the transfer and transmission mechanism of ARB carrying ARGs in the microbial community remain unclear.In this study,common PCR was used to identify the types of plasmid-mediated ARGs in chicken feed,and then qPCR was used to detect the abundance of ARGs and characterize the distribution characteristics of plasmid-mediated ARGs in chicken feed.Microbial community structure diversity and distribution characteristics were analyzed with Illumina sequencing of 16S rRNA genes.The correlation between ARGs and microbial community was explored by procrustes analysis and redundancy analysis,and the potential host bacteria of ARGs was explored by network analysis.Culturable microorganisms were isolated from chicken feed and chicken feed ingredients and identified by 16S rRNA sequencing.Plasmid mediated antibiotic resistance genotypes in the isolated strains were detected by PCR,and then the susceptibility of the isolated strains to 11 antibiotics was detected by micro-broth dilution method to get the resistance phenotype.In vitro conjugation and transfer experiments were performed on strains carrying ARGs by filtration incubation method,and antibiotic susceptibility tests were conducted on conjugants and recipient bacteria to explore the transfer mechanism of ARGs in feed.The conjugants were continuously passaged in antibiotic-free conditions,and the stability of the plasmids in the strains was studied by PCR identification of ARGs in single colony on drug-containing plates.Common PCR and qPCR results showed thatβ-lactam,macrolide and chloramphenicol resistance genes,such as bla _TEM,fex A,sul1 and erm B,were the main ARGs types in chicken feed with high detection rates and abundance.Various high levels of ARGs were detected in trough feeds near the manure pond,suggesting that poultry manure may be an important source of ARGs in feed.16S rRNA high-throughput sequencing results showed that dominant bacteria groups in the chicken feed and chicken feed ingredients samples were generally similar at phylum and genus levels,and kinds of human pathogenic bacteria were detected in both of them,such as Staphylococcus,Pseudomonas and Klebsiella.There was a correlation between the microbial community and ARGs.Analysis showed that the common potential host of tet M,tet C and erm C were Bacteroides,and the enrichment and spread of host bacteria could affect the abundance of ARGs.Some of the isolated strains carrying ARGs could transfer the plasmid to Escherichia coli EC600 by conjugation,and could transfer between the same species and different species.The conjugants showed multi-drug resistance.The recipient bacteria carrying ARGs showed a fitness cost,and lost its plasmid.In summary,this study found that chicken feed is an important reservoir of ARGs.The bacterial community structure in chicken feed can affect the distribution and abundance changes of ARGs.The bacteria carrying ARGs can transfer their plasmids through conjugation transfer under in vitro conditions,suggesting that chicken feed can act as a vehicle for the transmission of ARB and ARGs,and may be a key risk control point for ARGs transfer.Therefore,it is necessary to strengthen the monitoring of ARB and ARGs contamination in animal feed and improve the biosafety of animal feed and animal food.