There is a growing concern about the risk of antibiotic resistance emergence and dissemination in the environment. Here, we evaluated the spatio-seasonal patterns of the impact of wastewater treatment plant (WWTP) effluents on antibiotic resistance in river sediments. To this purpose, sediment samples were collected in three river basins affected by WWTP effluents in wet (high-water period) and dry (low-water period) hydrological conditions at three locations: (i) upstream the WWTPs; (ii) WWTP effluent discharge points (effluent outfall); and (iii) downstream the WWTPs (500 m downriver from the effluent outfall). The absolute and relative abundances of 9 antibiotic resistance genes (ARGs), 3 mobile genetic element (MGE) genes, and 4 metal resistance genes (MRGs) were quantified in sediment samples, as well as a variety of physicochemical parameters, metal contents, and antibiotic concentrations in both sediment and water samples. In sediments, significantly higher relative abundances of most genes were observed in downstream vs. upstream sampling points. Seasonal changes (higher values in low-water vs. high-water period) were observed for both ARG absolute and relative abundances in sediment samples. Chemical data revealed the contribution of effluents from WWTPs as a source of antibiotic and metal contamination in river ecosystems. The observed positive correlations between ARG and MGE genes relative abundances point out to the role of horizontal gene transfer in antibiotic resistance dissemination. Monitoring plans that take into consideration spatio-temporal patterns must be implemented to properly assess the environmental fate of WWTP-related emerging contaminants in river ecosystems.

The concern on the fate and distribution of contaminants of emerging concern (CECs) is a burning topic due to their widespread occurrence and potential harmful effects. Particularly, antibiotics have received great attention due to their implications in antimicrobial resistance occurrence. The impact of wastewater treatment plants (WWTP) is remarkable, being one of the main pathways for the introduction of CECs into aquatic systems. The combination of novel analytical methodologies and risk assessment strategies is a promising tool to find out environmentally relevant compounds posing major concerns in freshwater ecosystems impacted by those wastewater effluents. Within this context, a multi-target approach was applied in three Spanish river basins affected by different WWTP treated effluents for spatio-temporal monitoring of their chemical status. Solid phase extraction followed by ultra-high-performance liquid chromatography were used for the quantification of a large panel of compounds (n = 270), including pharmaceuticals and other consumer products, pesticides and industrial chemicals. To this end, water samples were collected in four sampling campaigns at three locations in each basin: (i) upstream from the WWTPs; (ii) WWTP effluent discharge points (effluent outfall); and (iii) downstream from the WWTPs (500 m downriver from the effluent outfall). Likewise, 24-h composite effluent samples from each of the WWTPs were provided in all sampling periods. First the occurrence and distribution of these compounds were assessed. Diverse seasonal trends were observed depending on the group of emerging compounds, though COVID-19 outbreak affected variations of certain pharmaceuticals. Detection frequencies and concentrations in effluents generally exceeded those in river samples and concentrations measured upstream WWTPs were generally low or non-quantifiable. Finally, risks associated with maximum contamination levels were evaluated using two different approaches to account for antibiotic resistance selection as well. From all studied compounds, 89 evidenced environmental risk on at least one occasion in this study.

Antibiotic resistance (AR) and contamination are critical public and environmental health issues. In the last years, the environmental component of AR has acquired much interest due to its potential links with the human resistome. In particular, freshwater ecosystems are considered strategic sites for environmental AR surveillance, since they can act as both reservoirs and transmission routes for antibiotic-resistant bacteria and antibiotic resistance genes. Many studies are needed to deepen our understanding of AR evolution and dynamics in freshwater ecosystems and, specifically, on the existence of links between environmental and human AR. This calls for the design of robust and adaptive AR surveillance strategies and, concomitantly, the implementation of routine monitoring programs that effectively capture the environmental dimension of AR in freshwater ecosystems. Here, a roadmap for AR monitoring in freshwater ecosystems, framed around four essential questions (how? what? where? when?), is presented to guide researchers and decision-makers in designing and implementing effective environmental AR routine monitoring programs. It was concluded that, due to the complexity, heterogeneity, and dynamic nature of freshwater ecosystems, it seems foreseeable that environmental AR monitoring programs need to be carefully adjusted to the particular casuistry of each freshwater ecosystem, as well as to the specific interests of the corresponding program and the resources available. Still, much research is needed to properly assess and monitor the risks derived from the emergence and dissemination of AR determinants in freshwaters for both ecosystem and human health. By synthesizing current knowledge and methodologies, this review consolidates existing approaches and can serve as a guide for planning AR monitoring programs in freshwater ecosystems.

Wastewater treatment plants (WWTPs) disrupt freshwater ecosystems and are recognised as hotspots for antimicrobial resistance. This study assessed the effect of treated municipal wastewater discharge on both planktonic and sessile freshwater microbial communities. A monitoring network was established in a Spanish urban river basin during 2022 dry season, with sampling points upstream and downstream of a WWTP. Artificial glass-based substrates enabled biofilm colonisation, while river water and effluent samples were collected during biofilm harvesting. Chemical exposure was assessed by characterising nutrients and antimicrobial concentrations in water, while shotgun metagenomics revealed taxonomic composition and resistome pools in both matrices. River water exhibited greater genus richness and pathobiome diversity than biofilms. Wastewater discharge shaped microbial composition, leading to shifts in less dominant groups, with partial attenuation of this effect downstream. Resistome richness was higher in river water compared to biofilm, though the latter displayed greater total abundances and distinct resistance profiles suggesting their role as genetic reservoirs. While no consistent resistome trends were linked to sampling location, higher total resistance abundances occurred in river water at sites impacted by wastewaters. Background resistance levels found upstream likely persisted downstream due to pollutant-driven selection. Lower resistome richness in effluent water suggests downstream abundances may also result from microbiome shifts and environmental factors. Assembly-based analysis revealed resistance genetic contexts and indicators of gene dissemination.