Integrated metagenomic and 16S rRNA analysis reveals temporal associations between resistance genes and microbial communities during dairy manure composting

Abstract Dairy manure composting is widely applied to stabilize organic waste and reduce environmental pollution, yet the behavior of resistance determinants during this process remains insufficiently resolved. In this study, shotgun metagenomic sequencing was used to characterize temporal changes in antibiotic resistance genes (ARGs), metal resistance genes (MRGs), biocide resistance genes (BRGs), mobile genetic elements (MGEs), and microbial community composition during dairy manure composting. Rather than inferring direct mechanistic causation, our analyses focused on identifying statistically supported trends, associations, and co-occurrence patterns across composting stages. We observed a rapid decline in the relative abundance of ARGs compared with MRGs and BRGs during the thermophilic phase, coinciding with increasing temperature, while specific genes such as sul2 persisted throughout the process. Shifts in microbial community composition, particularly changes in the relative dominance of Actinobacteria and Proteobacteria , were significantly associated with variations in resistome profiles. Correlation and network analyses further revealed strong associations among ARGs, MRGs, BRGs, and MGEs, suggesting potential co-selection and horizontal gene transfer linkages without implying direct causal mechanisms. In addition, several opportunistic bacterial genera showed positive associations with aminoglycoside- and macrolide–lincosamide–streptogramin-type ARGs, indicating possible dissemination risks following compost application. Overall, this study provides an integrated, association-based overview of resistome and microbial community dynamics during dairy manure composting and highlights the importance of considering multiple resistance determinants when evaluating composting as a manure management strategy.