Mapping clinically significant ARGs in Italian wastewater: distribution, persistence and seasonal dynamics

Introduction Antibiotic resistance is a growing threat across human, animal and environmental sectors. Wastewater treatment plants (WWTPs) are reservoirs of antibiotic resistance genes (ARGs), which are not fully eliminated before water is discharged into receiving bodies. This study examines the distribution of clinically relevant ARGs across influent and effluent wastewater in Italy. Materials and Methods A total of 38 wastewater samples (19 influents and 19 effluents) were collected during 2024 from WWTPs located in Piedmont (North), Lazio (Central), and Sicily (South). The selected ARGs confer resistance to multiple clinically relevant antibiotic classes, including beta-lactamases (blaOXA48, blaCTXM1 group, blaTEM, blaKPC, blaNDM), sulphonamides (sul1), tetracycline (tetA), vancomycin (vanA, vanB) and methicillin (mecA, mecC). The integrase gene Int1, a mobile genetic element, was also quantified. ARGs abundance was measured by digital PCR, both in absolute terms and normalised to 16S rRNA gene copies/L (g.c./L), to account for differences in bacterial load. Results All samples tested positive for one or more ARGs, except for mecC, which was consistently undetected. The mean concentrations (g.c./L) in untreated wastewater, ranked by absolute abundance, were: Int1 (5.0 × 109), sul1 (3.4 x 109), tetA (7.6 × 108), blaTEM (5.0 × 108), blaNDM (2.6 × 108), vanA (2.6 × 108), mecA (1.9 × 108), blaCTX-M-1 group (1.0 × 107), blaOXA-48, (7.3 × 106), blaKPC (3.2 × 106) and vanB (1.7 × 106). Mean concentrations in treated effluents showed reductions from one to two log units. After normalization to the 16S rRNA gene, 90 out 228 (~ 40%) measurements exhibited an increase in relative abundance of ARGs in effluents (e.g . 644 blaOXA-48 and blaTEM). Geographycally, blaCTX-M-1 group and tetA were most prevalent in Southern Italy, while blaNDM was more abundant in the North. Other ARGs showed similar distribution pattern. Most ARGs showed minimal seasonal variation, with some exceptions: tetA, Int1 and blaCTX-M-1 group increased in spring while blaNDM and vanA increased in spring/summer. Discussion and Conclusions Regional and sample-level differences in ARG distribution likely reflect multiple factors, including antibiotic consumption patterns and population density. Consistent with previous findings, ARGs persisted after treatment, raising concerns about their potential recirculation to humans through water systems or other pathways. Future efforts should focus on identifying the drivers of ARG persistence and developing strategies to mitigate their environmental dissemination. Research supported by EU funding within the Next Generation EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, PE13 INFACT).