Selective agents, microbial ecology, and horizontal gene transfer underly the fate of resistance genes during biological wastewater treatment

Abstract

Abstract Activated sludge is the centerpiece of biological wastewater treatment, as it facilitates removal of sewage-associated pollutants, fecal bacteria, and pathogens from wastewater through semi-controlled microbial ecology. Horizontal gene transfer (HGT) is believed to facilitate the spread of antibiotic resistance genes (ARGs) within sewage-associated microbiota, in part because of the presence of selective agents such as residual antibiotics in sewage. However, convincing evidence that selective agents promote resistance within activated sludge via HGT, or otherwise, has remained elusive. Here we used lab-scale sequencing batch reactors (SBRs) to simulate activated sludge treatment under controlled conditions. Replicate SBRs were fed field-collected municipal sewage augmented with either 0% or 10% hospital sewage to compare hypothetical low and high propensity conditions for ARG proliferation, respectively. Shotgun metagenomics, culture, pharmaceutical and personal care product screening, and bioinformatics were used to examine the impact of hospital sewage on SBR performance and the proliferation of ARGs. Hospital sewage had a modest impact on SBR performance and taxonomic composition determined via short read mapping. However, analysis of hospital- and municipal-sewage resistomes by the bioinformatic tool Kairos revealed microdiversity-level differences in ARG fate, including the disproportionate persistence of hospital-associated sulfonamide ARGs and municipal sewage derived macrolide ARGs. Correspondingly, it was found that levels of erythromycin and sulfamethoxazole sharply increased over the course of the experiment independent of hospital sewage. The relative abundances of metagenome assembled genomes bearing sulfonamide and macrolide ARGs showed drastically different trajectories in the presence of shifting antibiotic levels. In addition, in situ HGT events of the ARGs mphA and sul2 were detected in deeply sequenced samples. These included the potential transduction of mphA, which encodes an erythromycin phosphotransferase, between Dokdonella sp., Myxococcia, and Polyangia. Multiple potential pathways of transfer were noted for putative in situ HGT of sul2 between Bacteroidia, Gammaproteobacteria, and Alphaproteobacteria. These findings highlight complex ecological interactions enveloping the dissemination of ARGs in activated sludge and implicate roles for selective agents, microbial ecology, and HGT.