Three-Year Consecutive Field Application of Erythromycin Fermentation Residue Following Hydrothermal Treatment:Cumulative Effect on Soil Antibiotic Resistance Genes

Fermentation-based antibiotic production results in abundant nutrient-rich fermentation residue with high potential for recycling,but the high antibiotic residual concentration restricts its usefulness(e.g.,in land application as organic fertilizer).In this study,an industrial-scale hydrothermal facility for the treatment of erythromycin fermentation residue(EFR)was investigated,and the potential risk of the long-term soil application of treated EFR promoting environmental antibiotic resistance development was evaluated.The treatment effectively removed bacteria and their DNA,and an erythromycin removal ratio of up to approximately 98%was achieved.The treated EFR was utilized as organic fertilizer for consecutive field applications from 2018 to 2020,with dosages ranging from 3750 to 15000 kg·hm^(-2),resulting in subinhibitory levels of erythromycin(ranging from 0.83-76.00μg·kg^(-1))in soils.Metagenomic shotgun sequencing was then used to characterize the antibiotic resistance genes(ARGs),mobile genetic elements(MGEs),and bacterial community composition of the soils.The soil ARG abundance and diversity did not respond to the treated EFR application in the first year,but gradually changed in the second and third year of application.The highest fold change in relative abundance of macrolide-lincosamide-streptogramin(MLS)and total ARGs were 12.59 and 2.75 times,compared with the control(CK;without application),respectively.The soil MGEs and taxonomic composition showed similar temporal trends to those of the ARGs,and appeared to assist in driving increasing ARG proliferation,as revealed by correlation analysis and structural equation models(SEMs).The relative abundance of particular erm resistance genes(RNA methyltransferase genes)increased significantly in the third year of treated EFR application.The close association of erm with MGEs suggested that horizontal gene transfer played a critical role in the observed erm gene enrichment.Metagenomic binning results demonstrated that the proliferation of mac genecarrying hosts was responsible for the increased abundance of mac genes(efflux pump genes).This study shows that sub-inhibitory levels of erythromycin in soils had a cumulative effect on soil ARGs over time and emphasizes the importance of long-term monitoring for assessing the risk of soil amendment with treated industrial waste.

Field-based evidence for the enrichment of intrinsic antibiotic resistome stimulated by plant-derived fertilizer in agricultural soil

Animal manures have been demonstrated to enhance antibiotic resistance in agricultural soils.However,little is known about the effects of plant-derived fertilizer on soil antibiotic resistome.Herein,metagenomic sequencing was used to investigate the effects of a plantderived fertilizer processed from sugarcane and beet on soil antibiotic resistance genes(ARGs)in a soybean field along crop growth stages.ARG profiles in the soils amended by plant-derived fertilizer were compared with those in the soils amended by chicken manure.The abundance and diversity of total ARGs in the soils amended by plant-derived fertilizer were significantly(P<0.05)elevated at the sprout stage,to a level comparable to that in the manured soils.Whereas,unlike chicken manure mainly introducing manure-borne ARGs to soil,the plant-derived fertilizer was indicated to mainly enrich multidrug resistance genes in soil by nourishing indigenous bacteria.ARGs with abundances in amended soils significantly(P<0.05)higher than in unamended soils at the sprout stage of soybean were considered as enriched ARGs.Decrease in the abundance of the enriched ARGs was observed in both the amended soils from the sprout to the harvest.Network analysis further identified Proteobacteria and Bacteroidetes as the primary bacterial taxa involved in the temporal variation of the enriched ARGs in the soils amended by plant-derived fertilizer,while in manured soils were Firmicutes and Actinobacteria.As revealed by multivariate statistical analyses,variation of the enriched ARGs in the soils amended by plant-derived fertilizer was majorly attributed to the response of co-occurred bacteria to depleting nutrients,which was different from the failed establishment of manure-borne bacteria in the manured soils.Our study provided field-based evidence that plant-derived fertilizer stimulated the intrinsic antibiotic resistome,and proposed attention to the un-perceived risk since some clinically relevant ARGs originate and evolve from natural resistome.