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 Legionella. Stagnation periods, followed by sudden water flow, can promote biofilm detachment and the release of Legionella into the bulk water. Regardless of its importance, the simultaneous effects of shear stress on biofilm detachment and Legionella release into the bulk water remain poorly understood. This study investigates how shear stress affects biofilms containing Legionella pneumophila in terms of: a) biofilm detachment, b) release of L. pneumophila into the bulk phase, and c) shifting of L. pneumophila into the viable but nonculturable (VBNC) state. Pseudomonas fluorescens biofilms were formed in a Center for Disease Control (CDC) biofilm reactor at 125 RPM and spiked with L. pneumophila. 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, L. pneumophila viability, culturability, and spatial distribution were monitored. Results show that biofilms containing L. pneumophila maintained a similar basal thickness (12 μm) despite the detachment of the upper layers under different shear stresses. L. pneumophila, located at the bottom of the biofilm, remains surface-attached after biofilm detachment and seems to enhance the cohesiveness of these layers compared to P. fluorescens biofilms. On the contrary, when Legionella is not present, biofilm detachment increases with the increase of applied shear forces. All tested rotational velocities triggered L. pneumophila 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 Legionella control practices in water systems is discussed, highlighting the important insights that biofilms can provide in this context.