Background : Antibiotic resistance, which is mediated by environmental reservoirs, poses a threat to human and animal health. Aquatic biofilms impacted by treated wastewater (WW) are known environmental reservoirs for antibiotic resistance, however the specific influence of biotic factors and abiotic factors from WW on the abundance of antibiotic resistance genes (ARGs) within aquatic biofilms remains unclear. Additionally, experimental evidence is limited as to whether genes with low sequence similarity to reference ARGs actually encode for functional ARGs, particularly within understudied and complex aquatic microbial communities. Results : To disentangle the effects of abiotic and biotic factors on ARG abundances, natural biofilms were previously grown in flume systems with different proportions of stream water and either ultrafiltered or nonultrafiltered WW. In this study, we conducted deep shotgun metagenomic sequencing of 75 biofilm, stream, and WW samples from these flume systems and compared the taxonomic and functional microbiome and resistome composition. Statistical analysis revealed an alignment of the resistome and microbiome composition and a significant association with experimental treatment. Several ARG classes exhibited an increase in metagenomic abundances in biofilms grown with increasing percentages of nonultrafiltered WW. In contrast, sulfonamide and BEL family beta-lactamase ARGs showed greater abundances in biofilms grown in ultrafiltered WW compared to nonultrafiltered WW. Overall, our results pointed toward the dominance of biotic factors over abiotic factors in determining ARG abundances in WW-impacted stream biofilms and suggested gene family-specific mechanisms for ARGs which exhibited divergent abundance patterns. To investigate one of these specific ARG families experimentally, we biochemically characterized a new beta-lactamase from the Planctomycetota (Phycisphaeraceae). This beta-lactamase displayed activity in the cleavage of cephalosporin analog despite sharing low sequence identity with known ARGs. Conclusions : This discovery of a functional planctomycete beta-lactamase ARG is noteworthy, not only because it was the first beta-lactamase to be biochemically characterized from this phylum, but also because it was not detected by standard homology-based ARG tools. In summary, this study conducted metagenomic analysis on the relative importance of biotic and abiotic factors in the context of WW discharge and their impact on both known and new ARG abundances in aquatic biofilms.

Disentangling abiotic and biotic effects of treated wastewater on stream biofilm resistomes enables the discovery of a new planctomycete beta-lactamase