Association of Antibiotic Resistance Gene Migration with Heavy Metals in Large-Scale Dairy Farms Using Recycled Manure Solids Bedding

Antibiotic resistance genes (ARGs) are prevalent in livestock farms, with manure serving as a major reservoir. Their distribution and transmission are associated with husbandry practices. To evaluate the presence and abundance of ARGs along the feces-bedding-milk-calves chain, particularly under the practice of recycled manure solids for bedding (RMSB), we sampled from 5 critical stages: fecal slurry before anaerobic digestion, solids after solid-liquid separation, bedding derived from dried solids, unpasteurized mixed milk before feeding calves, and calf feces in 4 large-scale dairy farms (>1,000 lactating cows each). We analyzed ARGs and mobile genetic elements (MGEs) via PCR, characterized bacterial communities using 16sRNA sequencing, measured the concentrations of heavy metals and nutrients (total carbon and nitrogen), and evaluated the correlations between ARGs abundance and these factors using partial least squares path modeling analysis and redundancy analysis. A total of 27 ARGs and 10 MEGs subtypes were detected. Among these, 8 ARGs were found to be prevalent across all groups: blaTEM and cfxA (β-lactamase resistance), dfrA1 (sulfonamide resistance), strA and strB (aminoglycoside resistance), tetH, tet40, and tetQ (tetracycline resistance) besides 7 MGEs. Notably, higher ARG level in calf feces underscores urgent intervention needs. We also found that heavy metals, especially Zn and Pb, were positively correlated with ARG abundance. Most ARGs exhibited co-occurrence and significant correlation with microorganisms (including Enterococcus and Bacteroides), and MGEs (such as IS26, intI1, and Tp614). These findings suggested that the presence of continuous ARGs along the chain may be associated with RMSB, but this association requires further verification. Consequently, improved manure and bedding management, optimized dietary Zn levels, and enhanced removal or inactivation of heavy metals during bedding treatment could substantially lower the ecological and public health risks associated with ARG dissemination.