Microbial Biofilms Dynamics and Functionality in an Urban <i>Mycobacterium</i> -Dominated Drinking Water Distribution System

Microbial communities in drinking water distribution systems (DWDS) develop primarily as biofilms on pipe surfaces. Despite their impact on water quality, infrastructure maintenance, and biosafety, biofilms are not routinely controlled. In this study, we investigated the bacterial community dynamics and functionality in an urban chlorinated DWDS, dominated by <i>Mycobacterium</i>, through a multiphasic approach which included 16S rRNA gene metabarcoding, metagenomics and microscopy. Our results showed that biofilm communities were more functionally diverse compared to those from water and that the biofilm maturity was positively correlated with the prevalence of potential <i>Mycobacterium</i> emerging pathogens and a broader distribution of antibiotic resistance genes (ARGs) within the microbial community. The reconstruction of metagenome-assembled genomes (MAGs) and the corresponding genomospecies allowed the identification of key microbial taxa involved in the biofilm matrix remodeling, with 22% of them strongly responsible for biofilm formation. A diverse and novel viral community was detected across the system, including new putative <i>Mycobacterium</i> phages that might act against mycolic acids and thus contribute to biofilm destabilization. Our findings enhance our understanding of DWDS microbial composition and biofilm formation dynamics, focusing on "who does what" and then providing a foundation for developing effective biofilm control strategies in water distribution systems.