Evaluation of a hybrid capture-based sequencing panel for comprehensive detection of antibiotic resistance genes in wastewater

Introduction Antibiotic-resistant bacteria and antibiotic resistance genes (ARGs) in wastewater are of critical concern due to their potential to spread into natural ecosystems and pose risks to public health. This study evaluates a sequencing strategy based on a comprehensive multi-target enrichment probes panel for the detection of ARGs wastewater. Materials and Methods Eight influent and effluent wastewater samples were collected quarterly over a year from a central Italian wastewater treatment plant. ARG enrichment was performed using the QIAseq xHYB AMR panel (Qiagen) and enriched targets were sequenced with Illumina short reads technology. The QMI-AR was used for ARGs identification. Results The analysis of the 8 samples identified 743 (26.6%) out of 2,786 ARGs included in the panel, which are associated with 35 distinct antibiotic resistance classes. Out of 743 ARGs, 81 (11%) were exclusively detected in influents, 38 (5%) were found in effluents, while a total of 624 (84%) were detected in both influents and effluents. For each season, approximately 50% of the relative abundance (RA) was accounted for by a small group of dominant ARGs. In winter, the most abundant ARGs were tet(Q), conferring resistance to tetracycline, ant(3'')-Ia to aminoglycoside, blaOXA and cfxA6 to beta-lactams, erm(F) to macrolide and streptogramins. During spring and summer blaOXA, ant(3'')-Ia and aph(3”)-Ib (conferring resistance to 646 aminoglycoside), msr(E) (macrolide and streptogramins), arr (rifampin), predominated, while fall showed a similar profile with the additional presence of tetracycline-resistant genes. ARGs were similar across spring, summer, and fall, but winter showed a distinct pattern with more tetracycline resistance genes. The remaining ~ 50% of RA in each season is composed by a mean of 525 ARGs. ARGs were categorized by RA: major (>1%); minor (0.1%-1%); ultra-minor (<0.1%). Overall, among the major ARGs, only 2 exceeded 10% of relative abundance: ant(3'')- Ia at 13% and blaOXA at 11.6%; the others ranged between 6.8% and 1.2%. In addition, several minor ARGs conferring resistance to carbapenems, vancomycin, methicillin and colistin were detected. Discussion and Conclusions This study highlights the value of comprehensive multi-target sequencing approach, for a more accurate view of wastewater ARGs compared to methods targeting single genes or limited gene sets. The panel, originally designed for clinical applications, is in this study early applicated in an environmental context. Future optimization could involve targeted panels focused on key ARGs for improved cost-effectiveness and routine monitoring efficiency. Research supported by EU funding within the Next Generation EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, PE13 INF-ACT).