SPECIAL ISSUE Frontiers in Microbiology: Ensuring Food Safety and Quality in Sustainable Emerging Production Methods Sustainable Water Reuse in Food Production: Risks of extended-spectrum β-lactamase (ESBL)-Producing Escherichia coli and Antimicrobial Resistance Gene Release from Tertiary-Treated Reclaimed Water

Pilar Truchado¹Márcia Oliveira²Rebeca Cordero-García²manuel Abellán Soler³Amador Rancaño⁴Francisca García⁴Avelino Alvarez-Ordóñez²ANA ALLENDE^1*

• ¹Research Group on Microbiology and Quality of Fruit and Vegetables, Center for Edaphology and Applied Biology of Segura, Spanish National Research Council (CSIC), Espinardo, Murcia, Spain
• ²Department of Food Hygiene and Technology, University of León, León, Spain
• ³ESAMUR, Murcia, Spain
• ⁴ACCIONA AGUA, Madrid, Spain

This study evaluates the effectiveness of four tertiary wastewater treatment technologies—peracetic acid (PAA), PAA combined with low-intensity ultraviolet-C (PAA/UV Low), high-intensity UV-C (UV High), and ultrafiltration (UF)—in reducing extended-spectrum β-lactamase-producing Escherichia coli (ESBL-E. coli) and antimicrobial resistance genes (ARGs) in reclaimed water used for irrigation. The relative abundance of the genes, normalised to the 16S RNA gene present in the water samples, was then estimated to assess whether there is an amplification of these genes during the reuse process in the WWTP. The results indicate that while all treatments significantly reduced ESBL-E. coli (≥ 3 logs cfu/100 ml) and ARGs (≥ 1.5 logs gc/100 ml), complete elimination was not achieved in any wastewater treatment plant (WWTP). Among the treatments, UF demonstrated the highest removal efficiency (≈ 4 log gc ARG/100 ml), against ARGs, followed by UV High (≈ 3 log gc ARG/100 ml), whereas PAA and PAA/UV Low were less effective (≈ 2 log gc ARG/100 ml). The study also found that while absolute ARG levels were reduced, their relative abundance remained stable or showed minimal decline, suggesting a persistent environmental reservoir of resistance genes. The most frequently detected ARGs were associated with tetracyclines (tetW, tetA), quinolones (qnrB, qnrS), and sulfonamides (sul1, sul2). Moreover, multidrug-resistant (MDR) ESBL-E. coli isolates were present across all WWTPs, exhibiting resistance to β-lactams, quinolones, tetracyclines, and sulfonamides. Notably, low levels of resistance to last-resort antibiotics (tigecycline, colistin, and meropenem) were observed, highlighting potential public health concerns.These findings underscore the critical role of tertiary treatments in mitigating antimicrobial resistance (AMR) risks in water reuse systems. However, the persistence of ARGs in effluents suggests that current WWTP processes require further optimization.