Legionella affects biofilm structural response to detachment upon shear stress increase

Poor hydraulic management at water systems is associated with an increased risk of Legionnaires' disease caused by <i>Legionella</i>. Stagnation periods, followed by sudden water flow, can promote biofilm detachment and the release of <i>Legionella</i> into the bulk water. Regardless of its importance, the simultaneous effects of shear stress on biofilm detachment and <i>Legionella</i> release into the bulk water remain poorly understood. This study investigates how shear stress affects biofilms containing <i>Legionella pneumophila</i> in terms of: a) biofilm detachment, b) release of <i>L. pneumophila</i> into the bulk phase, and c) shifting of <i>L. pneumophila</i> into the viable but nonculturable (VBNC) state. <i>Pseudomonas fluorescens</i> biofilms were formed in a Center for Disease Control (CDC) biofilm reactor at 125 RPM and spiked with <i>L. pneumophila</i>. After 6 days, the system was set for 48 h to stagnation before flow was resumed at rotational velocities of 125, 225, and 400 RPM, corresponding to turbulent regimes with Reynolds numbers of 1552, 2794 and 4966, respectively. Biofilm properties, <i>L. pneumophila</i> viability, culturability, and spatial distribution were monitored. Results show that biofilms containing <i>L. pneumophila</i> maintained a similar basal thickness (12 μm) despite the detachment of the upper layers under different shear stresses. <i>L. pneumophila</i>, located at the bottom of the biofilm, remains surface-attached after biofilm detachment and seems to enhance the cohesiveness of these layers compared to <i>P. fluorescens</i> biofilms. On the contrary, when <i>Legionella</i> is not present, biofilm detachment increases with the increase of applied shear forces. All tested rotational velocities triggered <i>L. pneumophila</i> to enter the VBNC state in the bulk phase, while biofilm-associated VBNC cells were only observed at 400 RPM. Finally, the contribution of the present work to <i>Legionella</i> control practices in water systems is discussed, highlighting the important insights that biofilms can provide in this context.