Atomic Force Microsocopy: Key Unconventional Approach for Bacterial Nanotubes Characterization In Vivo.

Bacterial communication is essential; understanding its mechanisms could lead to better control of bacterial growth, which is a key factor in important bacteria-related issues like the emerging problem of antimicrobial resistance (AMR) or energy production using MFC (Microbial Fuel Cell). We know that bacteria exchange metabolites through the extracellular environment and communicate primarily via these secreted extracellular factors, yet it remained unclear whether bacteria can also use cell-cell connections to directly exchange nutrients, as evidenced in plant or mammal cells. Recent studies opened up great prospects by introducing some startling new discoveries on bacterial communication. Indeed, a previously uncharacterized type of bacterial communication mediated by nanotubes, small filaments connecting bacteria, that bridge neighboring cells was recently identified, making evidence of the existence of microstructures connecting bacterial cells together and allowing the transfer of molecules. Exploring the formation and the functioning of this new mechanism of bacterial communication in biofilms could lead for example, to better control of the major global problem of AMR. Thus, current studies have highlighted the crucial function of establishing tight connections between bacterial cells without answering a number of unresolved questions, especially those about their submicrometric biophysical characteristics. In this study, we provide new evidence of the existence of these nanotubes, using an unconventional approach in the analysis of biological samples based on scanning probe microscopy (SPM), precisely atomic force microscopy (AFM) in liquid, using Quantitative imaging mode, to investigate locally the properties of these intercellular bridges. This is, to our knowledge, the first time bacterial nanotubes are characterized by AFM in liquid on living bacteria. A protocol we developed enabled us to visualize these intercellular links on native bacteria without any immobilization. This challenging technique, unconventional for characterizing biological materials, enabled real-time nanomechanical mapping of these structures. By analyzing approach curves, the Young modulus was calculated, revealing new insights into their nanomechanical properties. Their lower Young's modulus (compared to that of the main body of the bacteria) suggest flexibility, which underscores an essential role in facilitating intercellular communication and material transfer.