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One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of <jats:italic>Legionella pneumophila<\/jats:italic> colonization of a <jats:italic>Pseudomonas fluorescens<\/jats:italic> biofilm, as information about the interactions between <jats:italic>Legionella<\/jats:italic> and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled <jats:italic>L. pneumophila<\/jats:italic> to tackle the following questions: (a) does the biofilm structure change upon <jats:italic>L. pneumophila<\/jats:italic> biofilm colonization?; (b) what happens to <jats:italic>L. pneumophila<\/jats:italic> within the biofilm over time and (c) where is <jats:italic>L. pneumophila<\/jats:italic> preferentially located within the biofilm? Results showed that <jats:italic>P. fluorescens<\/jats:italic> structure did not significantly change upon <jats:italic>L. pneumophila<\/jats:italic> colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled <jats:italic>L. pneumophila<\/jats:italic> showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old <jats:italic>P. fluorescens<\/jats:italic> biofilms, presumably entering in VBNC state by the end of the experiment. <jats:italic>L. pneumophila<\/jats:italic> was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced <jats:italic>Legionella<\/jats:italic> protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between <jats:italic>L. pneumophila<\/jats:italic> and biofilm structure that can provide, in the future, insights for public health <jats:italic>Legionella<\/jats:italic> management of water systems.<\/jats:p>","DOI":"10.1038\/s41598-024-67712-4","type":"journal-article","created":{"date-parts":[[2024,7,22]],"date-time":"2024-07-22T12:02:50Z","timestamp":1721649770000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Legionella colonization and 3D spatial location within a Pseudomonas biofilm"],"prefix":"10.1038","volume":"14","author":[{"given":"Ana Rosa","family":"Silva","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis F.","family":"Melo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"C. 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