Phenotypic and genotypic assessment of antimicrobial and biocide resistance in enterobacteria from anaerobic digestion of dairy waste: implications for environmental and public health

Antimicrobial-resistant bacteria in agricultural waste represent a critical One Health concern. Anaerobic digestion of livestock waste is widely promoted; however, the fate of antimicrobial resistance and biocide tolerance in full-scale systems remains unclear, constraining safe digestate reuse. This study assessed phenotypic and genotypic resistance in Escherichia coli-like enterobacteria isolated from effluent and biofertilizer in a full-scale dairy anaerobic digestion system. A total of 173 isolates were screened for antimicrobial resistance genes (ARGs), antimicrobial susceptibility, multidrug resistance (MDR), and biocide inhibition-zone responses. No diarrheagenic virulence genes were detected. At least one screened ARG was detected in 91.0% of effluent and 83.3% of biofertilizer isolates. qacE∆1 was the most frequent marker and occurred significantly more often in effluent (70.8%) than biofertilizer isolates (44.0%; q = 0.00291). Phenotypic resistance to at least one antimicrobial was observed in 27.0% and 34.5% of effluent and biofertilizer isolates, respectively, while MDR occurred in 2.2% and 9.5% ( p = 0.0521). tet(a) carriage was strongly associated with tetracycline resistance (OR = 20.9; q < 0.001). H 2 O 2 inhibition zones were significantly smaller among biofertilizer isolates, whereas Lactic Acid (LA) yielded 6-mm measurements in all isolates and in the reference strain. These findings demonstrate that anaerobic digestion does not fully eliminate culturable resistant enterobacteria and underscore the need for integrated resistance monitoring in dairy-waste management.