Effects of antibacterial agents and drugs monitored by atomic force microscopy

Originally invented for topographic imaging, atomic force microscopy (AFM) has evolved into a multifunctional biological toolkit, enabling to measure structural and functional details of cells and molecules. Its versatility and the large scope of information it can yield make it an invaluable tool in any biologically oriented laboratory, where researchers need to perform characterizations of living samples as well as single molecules in quasi-physiological conditions and with nanoscale resolution. In the last 20years, AFM has revolutionized the characterization of microbial cells by allowing a better understanding of their cell wall and of the mechanism of action of drugs and by becoming itself a powerful diagnostic tool to study bacteria. Indeed, AFM is much more than a high-resolution microscopy technique. It can reconstruct force maps that can be used to explore the nanomechanical properties of microorganisms and probe at the same time the morphological and mechanical modifications induced by external stimuli. Furthermore it can be used to map chemical species or specific receptors with nanometric resolution directly on the membranes of living organisms. In summary, AFM offers new capabilities and a more in-depth insight in the structure and mechanics of biological specimens with an unrivaled spatial and force resolution. Its application to the study of bacteria is extremely significant since it has already delivered important information on the metabolism of these small microorganisms and, through new and exciting technical developments, will shed more light on the real-time interaction of antimicrobial agents and bacteria. For further resources related to this article, please visit the . Conflict of interest: The authors have declared no conflicts of interest for this article.

Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

Sandor Kasas, Giovanni Longo, Petar Stupar

Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.

Royal Society of Chemistry2015

Resistance to disinfection agents: Adaptation of P. aeruginosa to sub inhibitory concentrations of quaternary ammonium compounds (QACs)

Florian Frédéric Vincent Breider, Leonardo Venturelli, Urs von Gunten, Margaux Océane Voumard

Quaternary ammonium compounds (QACs) are among the most used disinfection agents in consumer products and in industrial applications. Because of their poor biodegradabil ity, after use, they will persist in technical and natural aquatic systems at sub - inhibitory concentrations. Implications of QACs on the development of antimicrobial resistance was detected in several studies, however, the effect of a constant sub - inhibito ry concentration on the development of bacterial resistance remains unknown. The aim of this study was to determine if constant sub - inhibitory concentrations of benzalkonium chloride (BAC) and cetyltrimethylammonium chloride (CTMA) are able to promote a st able adaptation of populations of P. aeruginosa . Additionally, the study investigated cross - resistances between QACs and with antibiotics and nanomechanical properties of the membrane using atomic force microscopy (AFM) were explored. The exposure to sub - i nhibitory concentrations of QACs led to a maximal but reversible increase of the minimum inhibitory concentration (MIC) by a factor 1.45 for CTMA and a maximal irreversible increase of the MIC by a factor 1.9 for BAC. Cross - resistance was observed with the QACs but not with antibiotics: bacteria exposed to CTMA had an increased MIC for BAC of a factor 1.8 and bacteria exposed to BAC had an increased MIC for CTMA of a factor 1.75. Analyses of the membrane by AFM showed increase in the cell's roughness and st iffness after treatment with QACs. In populations of P. aeruginosa, constant sub - inhibitory concentrations of QACs were able to promote adaptations mechanisms, which were irreversible in the case of BAC. Furthermore, cross - resistance between the selected Q ACs were observed. Since low sub - inhibitory concentrations of QACs can be detected in natural and technical aquatic systems, this may lead to a dissemination of bacteria with higher QAC resistance or with QAC multiresistance, which can be a threat to the u rban water management.

2019

Time-lapse high-resolution microscopy to study the morphogenesis of microorganisms

Mélanie Thérèse Marie Hannebelle

Mycobacterium tuberculosis, the etiological agent for the tuberculosis disease, is a bacterial pathogen thought to infect about a quarter of the global human population. It is the first cause of death among infectious diseases, and is most prevalent in low-income populations. Much remains unknown about how these bacteria grow, divide, and manage to persist in the host even during month-long antibiotic treatments. Bacteria are typically at the edge of the spatial resolution that conventional optical microscopy techniques can offer, and so new tools are required to study bacterial substructures or surface morphologies with nanometer resolution. We developed a platform for automated optical microscopy and atomic force microscopy (AFM) in order to study the morphogenesis of mycobacteria. What is the growth dynamics of mycobacteria? Opposite models coexist in the literature for the pole growth dynamics of mycobacteria: the unipolar model and the bipolar model. We show that the growth dynamics of mycobacteria follows a new end take off NETO model. There is a surprising similarity between the NETO growth dynamics of mycobacteria and of fission yeast, which hints at shared mechanisms for pole growth in evolutionarily distant pole growing organisms. How do pole-growing organisms maintain their shape through insertion of new cell wall material at the tip? To explore further pole growth morphogenesis, we developed a method for imaging with AFM the pole of a growing rod-shaped microorganism. We observed the formation of a stiff fibril mesh on the pole of fission yeast cells, and showed that this mesh is stretched as the cell grows and insert new cell wall material at the tip. How do mycobacteria divide into two sibling cells? Another essential aspect of morphogenesis of rod-shaped cell is the division of a mother cell into two sibling cells. Using AFM, we measured the relative contributions of mechanical forces and molecular mechanisms driving the division process in mycobacteria. We showed that there is a concentration of mechanical stress at the future division site, using a combination of COMSOL finite element modeling and AFM measurements while controlling the cell turgor pressure. The accumulation of mechanical stress, in combination of enzymatic activity, ultimately leads to mechanical fracture and separation of the sibling bacteria. Conventional optical microscopy techniques tend to have low resolution and high speed, while conventional AFMs have comparatively high resolution and low speed. We designed and built a 3D-printed structured illumination (SIM) module, the openSIM, as an add-on for fluorescence microscopes. It provides structured illumination to obtain SIM images with higher resolution. With the aim of improving the imaging speed of AFM, we implemented photothermal actuation in a tip-scanning AFM head, and quantified the design constraints for high-speed photothermal off-resonance tapping imaging. AFM is most often used to scan and acquire high-resolution images of a sample. Its nanometer precision make it a unique tool for other applications. We combined an AFM head with a volcano-shaped probe for electrogram measurements, and demonstrated simultaneous recording of extracellular field potentials and contraction of the cell. This new tool opens the way for future studies exploring the intriguing links between mechanobiology and electrophysiology, with single-cell resolution.

EPFL2020

Time-lapse high-resolution microscopy to study the morphogenesis of microorganisms

Mélanie Thérèse Marie Hannebelle

EPFL2020

Resistance to disinfection agents: Adaptation of P. aeruginosa to sub inhibitory concentrations of quaternary ammonium compounds (QACs)

Florian Frédéric Vincent Breider, Leonardo Venturelli, Urs von Gunten, Margaux Océane Voumard

2019

Effects of antibacterial agents and drugs monitored by atomic force microscopy

Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

Resistance to disinfection agents: Adaptation of P. aeruginosa to sub­ inhibitory concentrations of quaternary ammonium compounds (QACs)

Time-lapse high-resolution microscopy to study the morphogenesis of microorganisms

Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

Time-lapse high-resolution microscopy to study the morphogenesis of microorganisms

Resistance to disinfection agents: Adaptation of P. aeruginosa to sub­ inhibitory concentrations of quaternary ammonium compounds (QACs)

Resistance to disinfection agents: Adaptation of P. aeruginosa to sub inhibitory concentrations of quaternary ammonium compounds (QACs)

Resistance to disinfection agents: Adaptation of P. aeruginosa to sub inhibitory concentrations of quaternary ammonium compounds (QACs)