Environmental high-risk efflux pumps mediate concurrent enhancement of resistance and virulence in reclaimed water from urban wastewater treatment plants

Abstract

With the growing demand for urban water, reclaimed water is a key solution for mitigating shortages. However, wastewater treatment plants (WWTPs) can act as reservoirs for antibiotic resistance genes (ARGs) and virulence factors (VFs), posing environmental and public health risks. This study systematically investigated the distribution and co-selection mechanisms of ARGs and VFs across four treatment stages in five WWTPs in Nanjing over two years, with a focus on high-risk efflux pump-related ARGs. A total of 902 persistent ARGs and 1086 persistent VFs were identified, including 12 characteristic ARGs, 12 characteristic VFs, and 20 high-risk priority ARGs. Network analysis and linear regression revealed a significant correlation between efflux pump ARGs and VFs, particularly those linked to outer membrane proteins, pili, and iron ion transport. Their coexistence suggests potential selective advantages during wastewater treatment. Laboratory-simulated aquatic environment experiments, RT-qPCR, and virulence protein assays further confirmed the co-selection between resistance and virulence. Results demonstrated that environmental stress, including antibiotics, heavy metals, and disinfection, induces efflux pump overexpression, enhancing bacterial pathogenicity. These findings highlight the importance of monitoring high-risk efflux pump ARGs in reclaimed water safety. Targeted surveillance and advanced treatment strategies are crucial for controlling antibiotic resistance and ensuring safe water reuse.