Dynamics of drinking water biofilm formation associated with Legionella spp. colonization

Drinking water biofilms can harbor pathogenic species of the genus Legionella. Understanding biofilm formation dynamics and how Legionella spp. proliferates in complex multi-species biofilms are essential to develop strategies to control their presence in building plumbing systems. Here we analyzed biofilm formation and Legionella spp. colonization on new plumbing material exposed to non-chlorinated municipal drinking water at 37 °C during eight weeks. Biofilm formation was characterized by an initial increase in intact cell concentrations up to 9.5x105 cells/cm2, followed by a steady decrease after five weeks. We identified Comamonas, Caulobacter, Schlegella, Blastomonas and Methyloversatilis as pioneer genera in the initial biofilm formation process. Importantly, L. pneumophila was the dominant Legionella species and rapidly colonized the biofilms, with culturable cell concentrations peaking at 3.1x104 MPN/cm2 after four weeks already. Moreover, different Legionella species co-occurred and had distinct dynamics of biofilm colonization. Vermamoeba vermiformis, a known Legionella spp. host, was the dominant protist identified with 18S rRNA gene amplicon sequencing over the whole analysis period. Together our results highlight that biofilm formation upon introduction of new building plumbing material is a highly dynamic process and that under the appropriate conditions, pathogenic Legionella species can be part of the earliest colonizers.