ANTIBIOTIC RESISTANCE GENES IN WATER MATRICES: QUANTITATIVE ANALYSIS AND NEXT-GENERATION SEQUENCING

Antibiotic resistance is a growing threat across human, animal and environmental sectors. Wastewater Treatment Plants (WWTPs) act as important reservoirs of antibiotic resistance genes (ARGs), which may persist throughout treatment and be released into receiving water bodies. As part of an ongoing doctoral project, this study investigates the distribution, persistence and seasonal dynamics of 12 clinically relevant ARGs, along with bacterial community composition, in influent and effluent wastewater collected across Italy. To date, 46 wastewater samples (23 raw influents and 23 treated effluents) have been collected and analyzed from six WWTPs located in Northern, Central and Southern Italy. Twelve ARGs conferring resistance to clinically relevant antibiotic classes (blaOXA-48, blaCTX-M-1group, blaTEM, blaKPC, and blaNDM; sul1; tetA; vanA, vanB; mecA, and mecC), together with the integrase gene Int1, were quantified by digital PCR, as both absolute concentrations and values normalised to 16S rRNA gene copies. Full-length 16S rRNA sequencing was employed to characterize the bacterial community composition and explore potential associations between ARGs occurrence and microbiome composition. Most ARGs were consistently detected across samples, with the exception of mecC, which was never detected. Influent wastewater was dominated by Int1, sul1 and tetA, followed by clinically relevant β-lactamase and glycopeptide resistance genes. Treated effluents generally showed reductions of one to two log units, although ~ 40% of normalized measurements (102/276) exhibited an increase of ARGs in effluents, particularly for blaOXA-48 and blaTEM. Distinct geographic patterns were observed, with Int1, blaCTX-M-1 group and tetA more abundant in Southern Italy and blaNDM more prevalent in the North. Seasonal variability was generally limited, although selected ARGs displayed WWTP-specific increases during spring or summer. Full-length 16S rRNA gene sequencing revealed distinct bacterial community compositions across WWTPs, including the presence of several species belonging to the World Health Organization Priority Pathogens List, a classification that identifies pathogens of greatest concern due to their clinical relevance and limited treatment options. Overall, these results show that clinically relevant ARGs and priority bacterial pathogens can persist through wastewater treatment, with clear geographic and plant-specific patterns. This highlights the value of wastewater surveillance as a complementary tool to clinical data for tracking antimicrobial resistance at the community level and identifying potential hotspots of environmental dissemination.