Transfer of antibiotic resistance genes in ESBL-producing Escherichia coli from raw milk and dairy farm environments under heavy metal stress.

This study was conducted to investigate the effects of heavy metals on the resistance levels exhibited by extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing Escherichia coli isolated from cattle farms and raw milk. The findings indicated that ESBL-producing E. coli strains exhibited significantly higher resistance to Cu, Zn, and Cd than non-ESBL-producing strains. Furthermore, ESBL-producing strains harbored various plasmid-mediated heavy metal resistance genes. Whole-genome sequencing (WGS) results showed that ESBL-producing strains carried plasmids encoding 148 heavy metal resistance genes. In addition, resistance to certain antibiotics (e.g., chloramphenicol) in E. coli exhibited a significant negative correlation with resistance to specific heavy metals, including Mn and Cd (p < 0.05). By establishing intrageneric (E. coli-E. coli) and intergeneric (E. coli-Klebsiella pneumoniae) conjugation models, the conjugation transfer frequency was measured at varying Cu2+ concentrations (0.5-100 μg/L). The results indicated that low-to-moderate Cu2+ concentrations (1-10 μg/L) significantly enhanced intrageneric conjugative transfer frequency in E. coli (up to approximately two-fold). This effect was reversed by the reactive oxygen species (ROS) scavenger thiourea, suggesting Cu2+ promotes transfer via oxidative stress induction. In contrast, K. pneumoniae conjugation remained insensitive to Cu2+ stress. Mechanistically, Cu2+ treatment increased the cell aggregation between donor and recipient bacteria, decreased the negative surface charge, improved the membrane permeability, and modulated multiple stress-related pathways (e.g., metal efflux, membrane metabolism, and the SOS response). This study revealed that Cu2+ drives the horizontal transmission of antibiotic resistance genes between environmental strains via ROS-dependent pathways, providing experimental evidence for understanding the coresistance mechanism between heavy metal and antibiotic resistance.