Understanding associations between antimicrobial agents usage and antimicrobial resistance genes prevalence at the community level using wastewater-based epidemiology: A Spanish pilot study

Understanding the development and spread of antimicrobial resistance (AMR) is important for combating this global threat for public health. Wastewater-based epidemiology (WBE) is a complementary approach to current surveillance programs that minimizes some of the existing limitations. The aim of the present study is to explore WBE for monitoring antibiotics and antibiotic resistance genes (ARGs) in wastewater samples collected during 2021/2022 from the city of Castellon (Spain). Eighteen commonly prescribed antibiotics have been selected and measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), with triple quadrupole mass analysers. Moreover, qPCR for specific ARGs has been performed to obtain information of these genes in co-presence with antibiotics. All selected ARGs, along with a total of 11 antibiotics, were identified. The highest population-normalized daily loads were observed for the macrolide azithromycin, followed by the quinolones ciprofloxacin and levofloxacin. Subsequently, daily consumption estimates based on wastewater data were compared with prescription data of antibiotics. Statistical analyses were conducted to explore if there is correlation between antibiotics and ARGs. While no correlations were found between antibiotics and their corresponding ARGs, certain correlations (p < 0.05) were identified among non-corresponding ARGs. In addition, a strong positive correlation was found between the sum of all antibiotics and the intl1 gene. Moreover, population-normalized ARG loads significantly correlate with the 16S rRNA-normalized ARG loads, serving as an indicator for population size. Results provide a baseline for future work and a proof-of-concept emphasising the need for future work and long-term surveillance, and highlight the need of similar programs at a regional and global levels worldwide.