Mechanically Induced Bacterial Death Imaged in Real Time: A Simultaneous Nanoindentation and Fluorescence Microscopy Study

Mechano-bactericidal nanomaterials rely on their mechanical or physical interactions with bacteria and are promising antimicrobial strategies that overcome bacterial resistance. However, the real effect of mechanical versus chemical action on their activity is under debate. In this paper, we quantify the forces necessary to produce critical damage to the bacterial cell wall by performing simultaneous nanoindentation and fluorescence imaging of single bacterial cells. Our experimental setup allows puncturing the cell wall of an immobilized bacterium with the tip of an atomic force microscope (AFM) and following in real time the increase in the fluorescence signal from a cell membrane integrity marker. We correlate the forces exerted by the AFM tip with the fluorescence dynamics for tens of cells, and we find that forces above 20 nN are necessary to exert critical damage. Moreover, a similar experiment is performed in which bacterial viability is assessed through physiological activity, in order to gain a more complete view of the effect of mechanical forces on bacteria. Our results contribute to the quantitative understanding of the interaction between bacteria and nanomaterials.This work was supported by the Spanish Ministerio de Ciencia, Innovacion y Universidades (MAT2015-66605-P, PGC2018-094802-B-I00, and SEV-2016-0686), and the Comunidad Autonoma de Madrid (PEJD-2016/IND-2774, S2017/BMD- ́3867). We thank Prof. Piet A. J. de Boer (Case Western Reserve University) for the gift of plasmid pDR122 (GFPMinD) and Prof. Pedro J. de Pablo (Universidad Autonoma de Madrid) for insightful discussions, and Dr. Pawel Hermanowicz for assistance with AtomicJ and the implementation of version 2.1.2