Trajectory of Antibiotic Resistome Response to Antibiotics Gradients: A Comparative Study from Pharmaceutical and Associated Wastewater Treatment Plants to Receiving River

Pharmaceutical wastewater often contains significant levels of antibiotic residues, which continuously induce and promote antibiotic resistance during the sewage treatment process. However, the specific impact of antibiotics on the emergence of antibiotic resistance genes (ARGs), microbiomes, and mobile genetic elements (MGEs), as well as the dose-response relationship remain unclear. Herein, through metagenomic sequencing and analysis, we investigated the fate, transmission, and associated risk of ARGs over a ten-year period of exposure to a gradient of sulfonamide antibiotics at a pharmaceutical wastewater treatment plant (PWWTP), an associated wastewater treatment plant (WWTP), and the receiving river. Through abundance comparison and principal co-ordinates analysis (PCoA), our results revealed distinct ARG, microbiome, and MGE profiles across different antibiotic concentrations. Notably, there was a decreasing trend in the abundance of ARGs and MGEs as the antibiotic concentrations were attenuated (p < 0.05). Further partial least squares path modeling analysis, Procrustes analysis and network analysis indicated that variation in MGEs and microbiomes were the driving forces behind the distribution of ARGs. Based on these findings, we proposed an antibiotic-microbiome-MGE-ARG dissemination paradigm, in which integrons as key drivers were closely associated with prevalent ARGs such as sul1, sul2, and aadA. With a focus on human pathogenic bacteria and the associated health risks of ARGs, we conducted pathogen source analysis and calculated the antibiotic resistome risk index (ARRI). Our findings highlighted potential risks associated with the transition from PWWTP to WWTP, raising concerns regarding risk amplification due to the mixed treatment of antibiotic-laden industrial wastewater and domestic sewage. Overall, the results of our study provide valuable information for optimizing wastewater treatment practices to better manage antibiotic resistance.