Drug resistance is a challenge that can be addressed using nanotechnology. We focused on the resistance of the bacteria Pseudomonas aeruginosa and investigated, using Atomic Force Microscopy (AFM), the behavior of a reference strain and of a multidrug resistant clinical strain, submitted to two antibiotics and to an innovative antibacterial drug (CX1). We measured the morphology, surface roughness and elasticity of the bacteria under physiological conditions and exposed to the antibacterial molecules. To go further in the molecules action mechanism, we explored the bacterial cell wall nanoscale organization using functionalized AFM tips. We have demonstrated that affected cells have a molecularly disorganized cell wall; surprisingly long molecules being pulled off from the cell wall by a lectin probe. Finally, we have elucidated the mechanism of action of CX1: it destroys the outer membrane of the bacteria as demonstrated by the results on artificial phospholipidic membranes and on the resistant strain.

Immobilization of live micro-organisms on solid substrates is an important prerequisite for atomic force microscopy (AFM) bio-experiments. The method employed must immobilize the cells firmly enough to enable them to withstand the lateral friction forces exerted by the tip during scanning but without denaturing the cell interface. In this work, a generic method for the assembly of living cells on specific areas of substrates is proposed. It consists in assembling the living cells within the patterns of microstructured, functionalized poly-dimethylsiloxane (PDMS) stamps using convective/capillary deposition. This versatile approach is validated by applying it to two systems of foremost importance in biotechnology and medicine: Saccharomyces cerevisiae yeasts and Aspergillus fumigatus fungal spores. We show that this method allows multiplexing AFM nanomechanical measurements by force spectroscopy on S. cerevisiae yeasts and high-resolution AFM imaging of germinated Aspergillus conidia in buffer medium. These two examples clearly demonstrate the immense potential of micro-organism assembly on functionalized, microstructured PDMS stamps by convective/capillary deposition for performing rigorous AFM bio-experiments on living cells.

Immobilization of living micro-organisms on predefined areas of substrates is a prerequisite for their characterizations by atomic force microscopy (AFM) in culture media. It remains challenging since micro-organisms should not be denatured but attached strongly enough to be scanned with an AFM tip, in a liquid phase. In this work, a novel approach is proposed to electrostatically assemble biological objects of interest on 2 nm thick polyethylenimine (PEI) patterns fabricated by nanoxerography. This nanoxerography process involves electrostatic trapping of PEI chains on negatively charged patterns written on electret thin films by AFM or electrical microcontact printing. The capability of this approach is demonstrated using a common biological system, Pseudomonas aeruginosa bacteria. These negatively charged bacteria are selectively assembled on large scale arrays of PEI patterns. In contrast to other PEI continuous films commonly used for cell anchoring, these ultrathin PEI patterns strongly attached on the surface do not cause any denaturation of the assembled Pseudomonas aeruginosa bacteria. AFM characterizations of large populations of individual living bacteria in culture media can thus be easily performed through this approach, providing the opportunity to perform representative statistical data analysis. Interestingly, this process may be extended to any negatively charged micro-organism in solution.