Persistent Risks in the Effluents of Wastewater Treatment Plants: Mobile Genetic Elements and Viral-Mediated Dissemination of Pathogenic Antibiotic-Resistant Bacteria

Wastewater treatment plants (WWTPs) are recognized as reservoirs of pathogenic antibiotic-resistant bacteria (PARB), yet their genomic risk dynamics remain unclear. This study recovered PARB genomes from 102 influent and effluent metagenomes from six countries; their activity and risk potential were then experimentally validated with metatranscriptomics on samples from a Shanghai WWTP. A total of 44 PARB genomes were reconstructed, which carried both antibiotic resistance genes (ARGs) and virulence factor genes (VFGs), and they persisted in the effluent. Mobile genetic elements might mediate ARG transfer in 13 PARB genomes. Moreover, bacteriophages infecting PARB harbored and were transcribing ARGs/VFGs, and antiviral defense systems of PARB correlated with horizontal gene transfer (HGT). Evolutionary analyses indicated that influent PARB maintained high microdiversity via homologous recombination, while effluent populations underwent purifying selection, suggesting that wastewater treatment reduced the genetic diversity of PARB through purifying selection. However, the persistent accumulation of PARB as well as HGT might maintain the dissemination of ARGs. This study emphasized the necessity of selecting the PARB genomes for wastewater monitoring, thereby optimizing treatment strategies and mitigating the potential health risks posed by pathogenic bacteria.