Biofilm and Wastewater Dynamics in an activated-sludge process in the UK: Insights into microbiome composition, metabolic activity, and antimicrobial resistance using 16S rRNA sequencing

• Findings highlight substantial shifts in the bacterial populations of both wastewater and biofilm communities during later treatment stages of wastewater treatment works. • Although the overall population of bacteria was reduced from influent to effluent, the predicted beta-lactamase production was significantly higher for biofilm and wastewater in the effluent compared to the influent. • Wastewater-treatment-works biofilms may be a potential source of environmental contamination with antimicrobial resistance genes. • This study underscores the need for future research into WWTW biofilms, as they could contribute to the persistence of antimicrobial-resistant bacteria in treated WW effluent, posing environmental and public health risks. This study explores the bacterial microbiome contributing to the resistome in wastewater and biofilm communities within a wastewater treatment works in Scotland, UK. By applying 16S rRNA sequencing at various stages of treatment, from influent to effluent, the bacterial composition could be established and then aligned with a tool for the prediction of metabolic pathways. This WWTW is shown to be working beyond its regulatory parameters, reducing bacteria from the influent and discharging decreased levels of bacteria in the effluent. The findings highlight substantial shifts in the bacterial communities of both wastewater and biofilm during later treatment stages, particularly post-oxidation ditch, suggesting that this process, with the addition of biofilms, actively shapes the WWTW resistome. The biofilms contained a high abundance of Zoogloea , a bacterial genus with known competency for horizontal gene transfer of antimicrobial resistance genes and Cyanobacteriota species which are known for their efficient exchanging and preservation of antimicrobial resistance genes in the environment. These features make biofilms potential reservoirs for dissemination of antimicrobial resistance into surrounding ecosystems. Moreover, predicted beta-lactamase enzyme levels, were higher in relative terms in the final effluent than in the influent in both wastewater and biofilms but this inference needs to be directly demonstrated by further experimentation. This points to the finding, that biofilms from these treatment works may be acting as a reservoir of AMR and underscores the need for further research into naturally occurring biofilms in wastewater management systems.