Metagenomic Characterisation of Antibiotic Resistance in Anaerobic Digesters and Their Implications for Environmental Health

ABSTRACT Antimicrobial resistance (AMR) in environmental systems represents an emerging One Health challenge, with anaerobic digesters potentially serving as reservoirs of resistance‐associated genetic features. This study analysed four publicly available anaerobic digester shotgun metagenomic datasets (M2, M3, M12 and M17) retrieved from the NCBI Sequence Read Archive to characterise microbial functions, resistance‐associated annotations and antibiotic target loci. The datasets were generated using Illumina HiSeq 2000 sequencing and analysed through quality assessment, assembly, functional annotation and resistome profiling. A total of 1082 resistance‐associated annotations were identified, with the highest abundance detected in M2 (348), followed by M12 (276), M17 (259) and M3 (205). Detected annotations were associated with diverse antimicrobial categories, including aminoglycosides, quinolones, beta‐lactams, tetracyclines, glycopeptides, macrolides and sulfonamides. Several frequently detected loci, including rpoB , rpoC , gyrA , gyrB , EF‐Tu, EF‐G, Ddl and KasA, were interpreted as antibiotic target or housekeeping loci rather than confirmed acquired resistance genes. Exploratory co‐abundance analysis identified strong associations among functionally linked loci, including rpoB‐rpoC and gyrA‐gyrB , likely reflecting shared genomic occurrence or functional relationships. These findings highlight the resistome potential of anaerobic digesters while emphasising the need for host‐resolved metagenomics, mobile genetic element analysis and mutation‐level characterisation to clarify environmental AMR risks.