<b>Virus-host arms race driving antimicrobial resistance gene transfer in livestock manure anaerobic digestion</b>

Antimicrobial resistance (AMR) poses a critical global health challenge, with livestock manure acting as a significant environmental reservoir for antimicrobial resistance genes (ARGs). Anaerobic digestion (AD) is a pivotal process for mitigating ARG dissemination at the livestock-environment-human interface. This study aims to elucidate the global dynamics of ARGs in AD systems, focusing on virus-host interactions and arms race, to identify actionable strategies for AMR control. We analyzed 205 metagenomic (4.5 Tb) and 36 meta-transcriptomic (640 Gb) datasets, including 15 newly generated datasets, revealing that pig manure AD harbors the highest ARG abundance (0.709 ARGs/16S rRNA), while AD systems generally exhibit limited transcriptional activation of ARGs. We constructed a viral dataset for livestock manure AD (GVD_LMAD), comprising 59,316 DNA and 727 RNA viral operational taxonomic units (vOTUs). Virus-host interactions revealed 542 lytic infections of antimicrobial-resistant bacteria (ARB) compared to only 12 ARG transduction events, underscoring the predominant role of viral communities in reducing ARG abundance. Furthermore, an antiviral defense system catalogue (GADSC_LMAD), derived from 2,760 high-quality metagenome-assembled genomes (MAGs) containing 39,307 ADS genes, showed a higher ADS gene prevalence in ARB (7.8 ± 6.0 per ARB) compared to non-ARB (6.1 ± 4.6; p < 0.01), indicating an intensified virus-host arms race in AD that may shield ARB from phage lysis. The CRISPR-Cas immune network with expressed spacers, driven by the arms race, restricts horizontal gene transfer (HGT) of ARGs by targeting foreign sequences carrying them. Collectively, these findings highlight that viral community predominantly drive ARG reduction through phage lysis in AD, while the ADS-mediated arms race, despite protecting ARB, limits HGT. This study provides novel insights into virus-host dynamics as a key mechanism for controlling ARG dissemination in AD systems.