Effect of antibiotic use and composting on antibiotic resistance gene abundance and resistome risks of soils receiving manure-derived amendments

Manure-derived amendments are commonly applied to soil, raising questions about whether antibiotic use in livestock could influence the soil resistome (collective antibiotic resistance genes (ARGs)) and ultimately contribute to the spread of antibiotic resistance to humans during food production. Here, we examined the metagenomes of soils amended with raw or composted manure generated from dairy cows administered pirlimycin and cephapirin (antibiotic) or no antibiotics (control) relative to unamended soils. Initial amendment (Day 1) with manure or compost significantly increased the diversity (richness) of ARGs in soils (p < 0.01) and resulted in distinct abundances of individual ARG types. Notably, initial amendment with antibiotic-manure significantly increased the total ARG relative abundances (per 16S rRNA gene) in the soils (2.21×unamended soils, p < 0.001). After incubating 120 days, to simulate a wait period before crop harvest, 282 ARGs reduced 4.33- fold (median) up to 307-fold while 210 ARGs increased 2.89-fold (median) up to 76-fold in the antibioticmanure- amended soils, resulting in reduced total ARG relative abundances equivalent to those of the unamended soils. We further assembled the metagenomic data and calculated resistome risk scores, which was recently defined as a relative index comparing co-occurrence of sequences corresponding to ARGs, mobile genetic elements, and putative pathogens on the same scaffold. Initial amendment of manure significantly increased the soil resistome risk scores, especially when generated by cows administered antibiotics, while composting reduced the effects and resulted in soil resistomes more similar to the background. The risk scores of manure-amended soils reduced to levels comparable to the unamended soils after 120 days. Overall, this study provides an integrated, high-resolution examination of the effects of prior antibiotic use, composting, and a 120-day wait period on soil resistomes following manure-derived amendment, demonstrating that all three management practices have measurable effects and should be taken into consideration in the development of policy and practice for mitigating the spread of antibiotic resistance.