QCM-D for non-destructive real-time assessment of Pseudomonas aeruginosa biofilm attachment to the substratum during biofilm growth.

Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to investigate initial adhesion and subsequent biofilm growth of wild-type Pseudomonas aeruginosa PAO1 and a pili-deficient (ΔpilA) mutant PAO1 strain. Clean, sterilized, silica-coated QCM-D crystals were pre-coated with lysogeny broth (LB), seeded with a PAO1 strain and allowed to grow for 20 h at 37 °C in fresh LB injected at 100 μL/min. QCM-D signals obtained for the wild-type PAO1 strain during the seeding period depict a large positive frequency shift that returns to baseline after ~20 min that is absent in the ΔpilA mutants, suggesting a dynamic pili-mediated attachment event for the wild-type PAO1 strain. During the subsequent growth period, significant and characteristic differences in the acquired QCM-D signals were observed between the wild-type and the ΔpilA mutant. Confocal laser scanning microscopy (CLSM) of the biofilm on the crystal surface showed that these differences could not be explained by differences in the extent of biofilm growth alone. When interpreted according to a coupled resonance model, the QCM-D observations suggest that pili are essential for coupling the developing biomass to the sensor surface. Total internal reflection fluorescence microscopy (TIRF) supports the hypothesis that the characteristic QCM-D signal is indicative of a dynamic attachment event, mediated by pili cell surface appendages pulling the wild-type PAO1 closer to the surface during the seeding period. We show that QCM-D offers direct, non-disruptive, in situ measurements of biofilm-substrate attachment. This technique has the potential to improve the current understanding of biofilm formation phenomena.