Distribution Characteristics And Transmission Mechanism Of Antibiotic Resistance Genes In Silage

Antibiotics resistance genes(ARGs)is found in various environmental media,which can lead to global public health and ecological and environmental security risks,and pose a huge threat to human health.Silage is a high-quality forage for herbivorous livestock,and it may be one of the potential sources of ARGs for animals.So far,studies on silage mainly focuses on the effects of different treatments(additives,temperature,dry matter(DM),and agronomic measure)on the changes of fermentation quality,nutrient composition,microbial interactions and succession,and the mutual transformation of metabolites,but little is known about the distribution and spread of ARGs.Therefore,in the present study,using metagenomics explained the response of different treatments(lactic acid bacteria,formic acid,DM,temperature,and growth years)to the distribution characteristics and transmission mechanism of ARGs in alfalfa and whole-plant corn silages.Meanwhile,the driving factors of ARGs transfer and spread in silage were also evaluated.The ARGs host and ARGs carried by clinical pathogens in silage were identified.The following study results are obtained:1.Study on the distribution characteristics and driving mechanism of antibiotic resistance genes in soil-fresh alfalfa-alfalfa silage system at different growth yearsWith increasing of alfalfa growth years,the ARGs diversity in the soil samples increased(P < 0.05),but did not affect the ARGs abundance.The diversity and abundance of ARGs in fresh alfalfa and alfalfa silage increased with the growing years of alfalfa(P < 0.05).The vancomycin and multidrug were the main resistance genes in soil and fresh alfalfa.The multidrug and bacitracin were the main resistance genes in alfalfa silage.The transposase was the most abundant mobile genetic elements(MGEs)type in each sample.Growth years did not alter the horizontal gene transfer potential of ARGs in soil and fresh alfalfa,but the horizontal gene transfer potential of ARGs in alfalfa silage decreased with increasing of growth years(P <0.05).The dominant bacteria in the soil and fresh alfalfa samples were Nocardioides.The dominant bacteria in the alfalfa silages were Enterobacter,Enterococcus and Bifidobacterium.Tracker source analysis showed that the longer the alfalfa grew,the more ARGs accumulated on the surface of fresh alfalfa,however,the source of most ARGs in alfalfa silage was obtained during the silage process.The main driver of ARGs variation in the soil-fresh alfalfa-alfalfa silage system is the bacterial community,and horizontal gene transfer in alfalfa silage might enhance the spread of ARGs.2.Effects of formic acid on the fate of antibiotic resistance genes and their potential hosts in high-moisture alfalfa silageMultidrug resistance genes were the most abundant ARGs type in high-moisture alfalfa silage.The natural silage process increased the ARGs enrichment(P < 0.05).However,after 5 days of ensiling,formic acid-treated alfalfa silage decreased the ARGs abundance.After 90 days of ensiling,formic acid-treated alfalfa silage increased the ARGs abundance(P < 0.05).Notably,some of pathogenic bacteria,such as Staphylococcus,Clostridium and Pseudomonas were annotated as potential hosts of ARGs in high-moisture alfalfa silage.Some clinical ARGs(acr B,mdt B,mdt C,and multidrug transporter)were also present in high-moisture alfalfa silage.Bacterial communities were the main drivers of ARGs distribution in the high-moisture alfalfa silage microecosystem.3.Study on biological reduction of antibiotic resistance genes in alfalfa silage inoculated with lactic acid bacteriaMultidrug and bacitracin resistance genes were the main ARGs type in alfalfa silage.The wilting process increased the ARGs enrichment in fresh alfalfa(P < 0.05).Naturally ensiling process increased the abundances of bacitracin,beta＿lactam and aminoglycoside resistance genes in alfalfa silage at 30% DM content(P < 0.05).Natural ensiling process increased the abundance of vancomycin resistance gene at40% DM content(P < 0.05).Interestingly,alfalfa silage inoculated with Lactiplantibacillus plantarum MTD/1 or Lactiplantibacillus buchneri 40788 decreased the total ARGs abundance as well as the abundances of multidrug,macrolides-lincoamide-streptomycin(MLS),vancomycin,aminoglycoside,and tetracycline resistance genes(P < 0.05).The main hosts of ARGs in alfalfa silage were harmful bacteria or pathogenic bacteria(Staphylococcus,Pseudomonas,Pantoea,and Erwinia).The clinical ARGs(Arl R,Emr B-Qac A,cpx R,and pen A)were found in alfalfa silage.In addition,the combined effects of bacterial communities,MGEs and fermentation quality were the main drivers of ARGs transfer and spread in alfalfa silage,in which bacterial communities were the key factor.4.Study on the distribution and transmission mechanism of antibiotic resistance genes in whole-plant corn silage inoculated with lactic acid bacteria at different dry matter contentsMacrolide and tetracycline were the main ARGs type in whole-plant corn silage and fresh whole-plant corn.Natural silage process increased the abundance of most ARGs in whole-plant corn(P < 0.05).Whole-plant corn before and after ensiling,the abundances of some ARGs types(peptide,aminoglycosides,glycopeptide,acridine,and lincosamide)at 40% DM content were higher than that at 30% DM content(P <0.05),but it had no effect on the total ARGs abundance.Although L.buchneri 40788 inoculant increased the distribution and transmission potential of ARGs in wholeplant corn silage(P < 0.05),L.plantarum MTD/1 inoculant had no significant effect on ARGs distribution.The dominant bacteria(Leuconostoc and Latilactobacillus)were the main ARGs hosts in whole-plant corn silage.Correlation analysis showed that the change of bacterial community succession caused by the change of DM content was the most important factor driving the ARGs distribution in whole-plant corn silage,while lactic acid bacteria inoculant and horizontal gene transfer driven by MGEs were not the main factors.In addition,high-risk ARGs such as tet W,mec A,vat E,mdt A,and mdt M were found in whole-plant corn silage.It was interesting that L.buchneri 40788 inoculant did not increase the abundance of high-risk ARGs in wholeplant corn silage.5.Study on the distribution and transmission mechanism of antibiotic resistance genes in whole-plant corn silage inoculated with lactic acid bacteria at different storage temperaturesThe natural silage process reduced the alpha diversity of ARGs in whole-plant corn silage at two storage temperatures(P < 0.05),but the abundances of total ARGs,glycopeptide,lincosamide,and oxazolidinone resistance genes were increased(P <0.05).Compared with 20℃ storage temperature,30℃ storage temperature decreased the ARGs diversity and total ARGs abundance as well as the abundances of fluoroquinolone,acridine and glycylcycline resistance genes in whole-plant corn silage(P < 0.05).Notably,L.buchneri 40788 inoculants increased the total ARGs abundance in whole-plant corn silage at both storage temperatures(P < 0.05),while L.plantarum MTD/1 inoculant had no effect on it.The main ARGs hosts in whole-plant corn silage were dominant bacteria(Leuconostoc and Lactobacillus).Integrase and insertion sequence were the main MGEs types in fresh whole-plant corn,while plasmid and transposase were the main MGEs types in whole-plant corn silage.Correlation analysis showed that bacterial community succession caused by the change of storage temperatures were the main driving factors affecting the variation in ARGs.Similarly,L.buchneri 40788 inoculant did not increase the high-risk ARGs abundances in whole-plant corn silage at two storage temperatures.It is the first time to elucidate the distribution characteristics and potential risks of ARGs in alfalfa silage and whole-plant corn silage using metagenomics.Meanwhile,the effects of lactic acid bacteria inoculant,DM content,formic acid and growth years on the distribution of ARGs in alfalfa silage,and effects of storage temperature,lactic acid bacteria inoculant and DM content on the distribution of ARGs in whole-plant corn silage were investigated.The results revealed the dominant resistance genes and host bacteria in whole-plant corn silage and alfalfa silage,and analyzed the contribution of physicochemical characteristics,fermentation quality,MGEs and bacterial communities to ARGs distribution and their correlation.At the same time,high-risk ARGs carried by clinical pathogens in silage were identified.The above results had important reference value for the biosafety control of ARGs in silage and herbivore animal production systems.