Synthesis and evaluation of lipophilic fluorescent probes for the labelling of Listeria and their impact on biofilm formation

Imaging bacterial biofilms using confocal fluorescence microscopy is used to study their structures, but its wider application is constrained by the limited availability of effective labelling tools. Small chemical fluorescent probes offer a versatile alternative to heterologous expression of fusion or reporter proteins, but data on their effects on biofilm formation are lacking. In this study, we synthesized a series of new lipophilic fluorescent probes based on Nile blue, Nile red and coumarin scaffold. We investigated them for the labelling of Listeria biofilms and determined their effects on the growth and biofilm biomass formation. The Nile red probe SP-AM 7 and the coumarin probe PAG 31 inhibited biofilm development and showed a strong bactericidal effect. The Nile blue probe PAG 19 had the least effect on the tested parameters, but labelled slowly, while the fast-labelling Nile red probe SP-AM 8 promoted biofilm formation. Both are suitable for use during biofilm growth, resulting in less variation in biomass-related measurements than probes added prior to imaging. In the 3D imaging-based measurements for selected probes, we found no difference in the total biomass formed compared to the control dye, but a redistribution of biomass in the 3D layers was observed. Other probes were found to be slow to label, leave traces of unused probes or interfere with attachment to the surface. Our results show that fluorescent probe labelling should be evaluated from chemical, physical and biological points of view to understand their reliability and credibility.