Targeted metagenomics reveals inferior resilience of farm soil resistome compared to soil microbiome after manure application

Authors: Goncalo Macedo, H. Pieter J. van Veelen, Lucia Hernandez-Leal, Peter van der Maas, Dick Heederik, Dik Mevius, Alex Bossers, Heike Schmitt. Abstract: Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the gene pool, such as the resistome, without the cost-prohibitive coverage depths, and can provide a valuable tool to study the fate of antimicrobial resistance (AMR) in the environment. Here, we used high-throughput sequencing and qPCR for 16S rRNA genes as well as targeted metagenomics (ResCap) to explore the fate of bacteria and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a strong increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families, increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also on a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.