Abstract
Preventing legionellosis in water systems demands effective hydrodynamic management and biofilm mitigation. This study investigates the complex relationship between hydrodynamics (80 RPM and stagnation), biofilm mesoscale structure and Legionella pneumophila colonization, by addressing three key questions: (1) How do low flow vs stagnation conditions affect biofilm response to L. pneumophila colonization?, (2) How do biofilm structural variations mediate L. pneumophila migration across the biofilm?, and (3) Can specific hydrodynamic conditions trigger L. pneumophila entrance in a viable but nonculturable (VBNC) state? It was found that Pseudomonas fluorescens biofilms exhibit different responses to L. pneumophila based on the prevailing hydrodynamic conditions. While biofilm thickness and porosity decreased under shear (80 RPM), thickness tends to significantly increase when pre-established 80 RPM-grown biofilms are set to stagnation upon L. pneumophila spiking. Imposing stagnation after the spiking also seemed to accelerate Legionella migration towards the bottom of the biofilm. Water structures in the biofilm seem to be key to Legionella migration across the biofilm. Finally, shear conditions favoured the transition of L. pneumophila to VBNC states (∼94 %), despite the high viable cell counts (∼8 log(10) CFU/cm(2)) found throughout the experiments. This research highlights the increased risk posed by biofilms and stagnation, emphasizing the importance of understanding the mechanisms that govern Legionella behaviour in diverse biofilm environments. These insights are crucial for developing more effective monitoring and prevention strategies in water systems.