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 colonization, by addressing three key questions: (1) How do low flow stagnation conditions affect biofilm response to colonization?, (2) How do biofilm structural variations mediate migration across the biofilm?, and (3) Can specific hydrodynamic conditions trigger entrance in a viable but nonculturable (VBNC) state? It was found that biofilms exhibit different responses to 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 spiking. Imposing stagnation after the spiking also seemed to accelerate migration towards the bottom of the biofilm. Water structures in the biofilm seem to be key to migration across the biofilm. Finally, shear conditions favoured the transition of to VBNC states (���94 %), despite the high viable cell counts (���8 log CFU/cm) found throughout the experiments. This research highlights the increased risk posed by biofilms and stagnation, emphasizing the importance of understanding the mechanisms that govern behaviour in diverse biofilm environments. These insights are crucial for developing more effective monitoring and prevention strategies in water systems.
