Bacteria initiate attachment to surfaces with the aid of different extracellular proteins and polymeric adhesins. To quantitatively analyse the cell-cell and cell-surface interactions provided by bacterial adhesins, it is essential to go down to single cell level where cell-to-cell variation can be considered. We have developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion by force spectroscopy using atomic force microscopy (AFM). A single-cell probe was readily made by picking up a bacterial cell from a glass surface by approaching a tipless AFM cantilever coated with the commercial cell adhesive CellTakTM. We applied the method to study adhesion of living cells to abiotic surfaces at the single-cell level.
Immobilisation of single bacterial cells to the cantilever was stable for several hours, and viability was confirmed by Live/Dead staining and fluorescence microscopy. This method for preparing a single-cell AFM probe offers control of the cell immobilization, and therefore holds advantages over the commonly used approach where multiple cells are immobilized at random positions by submerging the cantilever in a bacterial suspension. The method provides a general platform for investigating single cell interactions of bacteria with different surfaces and other cells by AFM force spectroscopy, thus improving our understanding of the mechanisms of bacterial attachment.
To explore the influence of biological and physicochemical parameters on the adhesion force, we explored the bond formation and adhesive strength of four different bacterial strains towards three abiotic substrates with variable hydrophobicity and surface roughness. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time of contact. Staphylococcus xylosus DSM 20266 and Staphylococcus epidermidis DSM 20044 showed much higher adhesion forces than Pseudomonas fluorescens AH1, but bond strengthening by P. aeruginosa (2 s) was faster than for the staphylococci (10 s) . Escherichia coli DSM 429, which was the only strain unable to form biofilm, showed almost no adhesion to any surface. The differences between staphylococci and P. fluorescens in adhesion pattern reflects their differences in the composition of extracellular adhesins. Both adhesion force and rupture length were significantly smaller on mica compared to glass. Staphylococci adhere stronger on fresh glass than on hydrophilic glass, while the weaker adhesion by P. fluorescens was similar on both types of glass. These results confirmed the importance of surface hydrophobicity in bacterial adhesion.
This study has demonstrated that single-cell force spectroscopy allows quantitative measurement of the adhesion of different bacteria toward surfaces of different physicochemical properties. We expect that this method can be combined with bacterial mutation or enzymatic treatment to knock off specific surface components, thus providing more insights into the contribution of various surfaces components in bacterial initial attachment.