High-risk E. coli clones in wastewater treatment plants of Barcelona
Antimicrobial resistance (AMR) is a major global health threat, and environmental reservoirs such as wastewater treatment plants (WWTPs) and drinking water treatment plants (DWTPs) can facilitate its persistence and spread despite their essential role in water safety. We investigated 152 antibiotic-resistant Escherichia coli strains collected across treatment stages from two WWTPs and one DWTP in Barcelona, Spain. Strains were characterized through antimicrobial susceptibility testing, whole-genome sequencing, multilocus sequence typing, biofilm formation assays, and the detection of antimicrobial resistance genes (ARGs), virulence factor genes (VFGs), biocide and heavy metal tolerance genes (HMTGs). Although bacterial loads decreased along treatment, AMR remained highly prevalent: 85.5% of strains were multidrug-resistant (MDR), 5.3% extensively drug-resistant, and 11.2% produced carbapenemases. Logistic regression analyses revealed significant associations among variables. Strains harboring integrons were 2.4 to 11.8 times more likely to carry ARGs to sulfonamide, aminoglycoside, phenicol, trimethoprim, mercury and quaternary ammonium compounds. Extended-spectrum beta-lactamase-producing strains carrying blaCTX-M genes were 3.0 to 20.3 times more likely to carry VFGs, while high-risk clones were 3.2 to 7.0 times more associated with VFGs. Phylogroup A strains were enriched in HMTGs, whereas phylogroup B2 carried more VFGs but fewer HMTGs.  MDR and high-risk E. coli clones persisted in reclaimed water, and one MDR strain was identified at the DWTP inlet. The broad distribution of virulence traits, including biofilm formation, siderophore production, and specific VFGs linked to diarrheagenic and extraintestinal pathogenic lineages, highlights the public health risks associated with environmental AMR reservoirs and the urgent need for improved surveillance and mitigation strategies.