Comparative Analysis of Concentration and Quantification Methods for Antibiotic Resistance Genes and Their Phage-Mediated Dissemination in Treated Wastewater and Biosolids

Antimicrobial resistance poses a growing threat to public health, and integrated surveillance strategies across environmental compartments such as treated wastewater and biosolids can substantially improve monitoring efforts. A key challenge is the diversity of available protocols, which complicates comparability for the concentration and detection of antibiotic resistance genes (ARGs), particularly in complex matrices. In this study, we compared two commonly used concentration methods-filtration-centrifugation (FC) and aluminum-based precipitation (AP)-and two detection techniques, quantitative PCR (qPCR) and droplet digital PCR (ddPCR), for the quantification of four clinically relevant ARGs: <i>tet(A)</i>, <i>bla_CTX-M group 1</i>, <i>qnr</i>B, and <i>catI</i>. Analyses were performed in both secondary treated wastewater and biosolid samples, including their purified bacteriophage-associated DNA fractions. Results showed that the AP method provided higher ARG concentrations than FC, particularly in wastewater samples. ddPCR demonstrated greater sensitivity than qPCR in wastewater, whereas in biosolids, both methods performed similarly, although ddPCR yielded weaker detection. Importantly, ARGs were detected in the phage fraction of both matrices, with ddPCR generally offering higher detection levels. These results provide comparative insights into established methodologies and highlight the value of selecting appropriate protocols based on matrix characteristics and surveillance objectives.