Phage predation mitigates the spread of antibiotic resistance in anaerobic digestion under shortened solid retention...

Optimizing anaerobic digestion (AD) via shortening solid retention time (SRT) enhances methane recovery, yet the mechanistic impact of SRT reduction on antimicrobial resistance (AMR) dissemination remains underexplored. Herein, we employed metagenomics to investigate how reduced SRTs (from 60 to 5 days) regulated the dynamics of antibiotic resistance genes (ARGs) mediated by pathogenic hosts, plasmids, and phages in mesophilic and thermophilic AD systems. Shortened SRTs elevated ARG abundance by 5.9-388% under mesophilic conditions, driven by the SRT-elicited niche expansion of antibiotic-resistant bacteria (ARB) and the persistent dominance of ESKAPE pathogen Enterobacter hormaechei, the latter intrinsically harbored and transmitted high-risk ARGs (aadA, sul1, and qacEdelta1) via multi-resistant plasmids. Notably, plasmid-mediated and cross-phylum transmission substantially enhanced ARG mobility. Contrastingly, thermophilic conditions eliminated ARGs by 17.0-57.1% under shortened SRTs, driven by thermophilic ARB niche differentiation. Crucially, both homology search and phage-host prediction indicated the lack of ARGs matching between phages and hosts under reduced SRTs, denoting a negligible contribution of transduction to horizontal ARG transfer. The dominance of lytic phages (85.3%), intensified lytic phage-host interactions, and heightened abundance of lytic phages lysing ARB collectively imposed potent phage top-down control over ARG hosts, with the lytic phage predation on ARB being validated by laboratory assays. We also identified 9 high-risk digestate ARG biomarkers (ANT(6)-Ia, aadA, ermA, mel, qacEdelta1, sul1, tet44, tetM, tetQ) by integrating criteria of prevalence, gene mobility, clinical relevance, and host pathogenicity to inform monitoring. Overall, this study underlined the significance of phage predation in mitigating ARG propagation under shortened SRTs, informing the development of novel AMR control strategies in AD practices.