| Zdroj: |
Zeng, G, Ogaki, R, Regina, V R, Müller, T & Meyer, R L 2014, ' Probing living bacterial adhesion by single cell force spectroscopy using atomic force microscopy ' SPM 2014, Toronto, Canada, 02/09/2014-06/09/2014, . |
| Popis: |
Bacteria initiate attachment to the surfaces with the aid of different extracellular polymers. To quantitatively study how these polymers mediate bacterial adhesion and possibly their interactions, it is essential to go down to single cell level, with in mind that cell-to-cell variation should be considered.We have therefore developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion with atomic force microscopy (AFM).[1] A single-cell probe was readily made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated with a commercial cell adhesive CellTakTM. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, mica). Attachment to the cantilever was stable during the 2 h of AFM force measurements, and viability was confirmed by Live/Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time of contact. The single-cell probe offers control of the cell immobilization, thus holds advantages over the commonly used multi-cell probes where random immobilization is obtained by submerging the cantilever in a bacterial suspension. The reported 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.An alternative way to study the adhesion of single bacterial cells is to measure the adhesion between immobilized bacterial cells and coated AFM cantilevers. This strategy was used to investigate the adhesive properties of novel high density poly(ethylene glycol) (PEG) coatings on titanium. We investigate the ability of a high density poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) coating to resist bacterial adhesion and biofilm formation from three clinically relevant bacteria: Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermis. The high density PLL-g-PEG coatings were about eight times as thick as the conventional PLL-g-PEG coatings. Adhesion forces toward high density PLL-g-PEG coatings were low (P. aeruginosa) or close to zero (S. aureus and S. epidermidis) compared to bare titanium surface. However, no decrease in adhesion force was observed for S. epidermidis toward conventional PLL-g-PEG coatings, whereas significantly lower adhesion forces were observed for S. aureus and P. aeruginosa.The adhesion force patterns were reflected by the colonization of bacteria after 48 h incubation of the coatings in bacterial cultures. The high density PLL-g-PEG coatings completely resisted bacterial colonization, whereas conventional coatings couldn’t resist colonization by S. epidermidis. The unique ability of S. epidermidis to colonize conventional PLL-g-PEG coatings was investigated by looking into the composition of S. epidermidis biofilm. The strain-dependent susceptibility to bacterial colonization on conventional PLL-g-PEG illustrates how bacterial diversity challenges development of “universal” antifouling coatings, and AFM single-cell force spectroscopy was proven to be a powerful tool to provide insights into the molecular mechanisms of adhesion by different bacterial strains. Bacteria initiate attachment to the surfaces with the aid of different extracellular polymers. To quantitatively study how these polymers mediate bacterial adhesion and possibly their interactions, it is essential to go down to single cell level, with in mind that cell-to-cell variation should be considered.We have therefore developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion with atomic force microscopy (AFM).[1] A single-cell probe was readily made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated with a commercial cell adhesive CellTakTM. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, mica). Attachment to the cantilever was stable during the 2 h of AFM force measurements, and viability was confirmed by Live/Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time of contact. The single-cell probe offers control of the cell immobilization, thus holds advantages over the commonly used multi-cell probes where random immobilization is obtained by submerging the cantilever in a bacterial suspension. The reported 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.An alternative way to study the adhesion of single bacterial cells is to measure the adhesion between immobilized bacterial cells and coated AFM cantilevers. This strategy was used to investigate the adhesive properties of novel high density poly(ethylene glycol) (PEG) coatings on titanium. We investigate the ability of a high density poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) coating to resist bacterial adhesion and biofilm formation from three clinically relevant bacteria: Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermis. The high density PLL-g-PEG coatings were about eight times as thick as the conventional PLL-g-PEG coatings. Adhesion forces toward high density PLL-g-PEG coatings were low (P. aeruginosa) or close to zero (S. aureus and S. epidermidis) compared to bare titanium surface. However, no decrease in adhesion force was observed for S. epidermidis toward conventional PLL-g-PEG coatings, whereas significantly lower adhesion forces were observed for S. aureus and P. aeruginosa.The adhesion force patterns were reflected by the colonization of bacteria after 48 h incubation of the coatings in bacterial cultures. The high density PLL-g-PEG coatings completely resisted bacterial colonization, whereas conventional coatings couldn’t resist colonization by S. epidermidis. The unique ability of S. epidermidis to colonize conventional PLL-g-PEG coatings was investigated by looking into the composition of S. epidermidis biofilm. The strain-dependent susceptibility to bacterial colonization on conventional PLL-g-PEG illustrates how bacterial diversity challenges development of “universal” antifouling coatings, and AFM single-cell force spectroscopy was proven to be a powerful tool to provide insights into the molecular mechanisms of adhesion by different bacterial strains. |
