In situ characterization of bacterial extracellular polymeric substances by AFM

Abstract

Although the production of extracellular polymeric substances (EPS) has long been recognized as an important factor in bacterial adhesion processes, little is known about the supramolecular organization of EPS adsorbed on solid surfaces. In this paper, atomic force microscopy (AFM) is used to probe, under aqueous conditions, the nanoscale morphology and molecular interactions of polystyrene substrata after adhesion of the Gram-negative bacterium Azospirillum brasilense under different experimental conditions. After cell adhesion under favourable conditions (24 h contact time at 30 °C), topographic images revealed that the substratum surface was covered by a continuous layer of adsorbed substances, ≈2 nm thick, from which supramolecular aggregates were protruding. These adsorbed substances, attributed to proteinaceous EPS, were found to cause a significant change of substratum solvation properties: adhesion force mapping performed over 5×5 μm areas with a silicon nitride probe showed adhesion forces of 0.8±0.2 nN ( n=1024) magnitude on bare substrata, while forces of only 0.2±0.2 nN magnitude were found after cell adhesion. Finally, when the cell adhesion test was performed under unfavourable conditions (2 h at 30 °C or 24 h at 4 °C), there was no/little indication of the presence of an adsorbed layer and of a change of substratum solvation properties. The correlation between the AFM data and the cell adhesion behaviour provides evidence for the involvement of proteinaceous EPS in the adhesion of A. brasilense to solid surfaces.