Tracking the distribution and persistence of clinically relevant antibiotic resistance genes in italian wastewater treatment plants

The increasing prevalence of antibiotic resistance across human, animal and environmental sectors poses a significant risk to public health. Wastewater treatment plants (WWTPs) act as reservoirs of a variety of contaminants, including Antibiotic Resistance Genes (ARGs). While WWTPs play a crucial role in protecting public health and environment by removing contaminants before the release into the effluent receiving water, they are not designed to completely remove ARGs. A better understanding of the presence and fate of ARGs in wastewater is essential to assess their potential impact on the environment and public health. This study examines the distribution of a selected panel of clinically relevant ARGs across Italy, assessing their presence in influent and effluent wastewater located in Northern, Central, and Southern regions. 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 represent resistance to multiple clinically relevant antibiotic classes, including beta-lactamases (blaOXA48, blaCTXM1 group, blaTEM, blaNDM), sulphonamides (sul1), tetracycline (tetA), vancomycin (vanA, vanB) and methicillin (mecA, mecC). Furthermore, Int1, a mobile genetic element, was quantified. The abundance of these ARGs was assessed by digital PCR both in absolute terms and normalised to 16S rRNA gene copies/L (g.c./L) to account for variations in bacterial load. Overall, all samples tested positive for one or more ARGs, with the exception of mecC, which was consistently undetected. The mean concentrations (g.c./L) of ARGs and Int1 in untreated wastewater, ranked by absolute abundance, were as follows: 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), and vanB (1.7 × 106). The mean concentrations in treated effluents showed reductions ranging from one to three log units. Following normalization to the 16S rRNA gene, 66 out 190 (35.5 %) measurements exhibited an increase in relative abundance of ARGs. Whit regard to the distribution and abundance across Italy, blaCTX-M-1 group and tetA were most prevalent in Southern Italy, while blaNDM was more abundant in Northern Italy. The other ARGs showed a more uniform distribution across the country. This study utilized an extensive panel of ARGs, covering multiple clinically relevant antibiotic classes, to assess their distribution and persistence in wastewater across different regions of Italy. The observed variations in ARGs distribution between influent and effluent samples, as well as across regions, suggest that multiple factors—such as antibiotic consumption patterns, population density, and treatment efficiency—may influence their prevalence. In line with previous findings, ARGs were not completely removed after treatment. This persistence in the environment raises concerns about their potential recirculation back to humans through water systems or other pathways. Further studies involving a larger dataset are necessary to confirm the geographical trends observed. Future research should focus on identifying key drivers of ARGs persistence, evaluating their impact on downstream ecosystems, and assessing the effectiveness of advanced treatment technologies.