Abstract
Increasing antibiotic resistance in bacteria is a critical issue that often leads to infections or other morbidities. Mechanical properties of the bacterial cell wall, such as thickness or elastic modulus, may contribute to the ability of a bacterial cell to resist antibiotics. Techniques like atomic force microscopy (AFM) are used to quantify bacterial cell mechanical properties and image cell structures at nanoscale resolutions. An additional benefit of AFM is the ability to probe samples submerged in liquids, meaning that live bacteria can be imaged or evaluated in environments that more accurately simulate in vivo conditions as compared to other methods like electron microscopy.
However, because AFM measurements are highly sensitive to small perturbations in the deflection of the tip of a sensor probe brought into contact with the specimen, immobilization of bacteria prior to measurement is essential for accurate measurements. Traditional chemical fixatives crosslink the molecules within the bacterial cell wall, which prevent the bacteria from locomotion. While effective for imaging, chemical crosslinkers are known to affect the measured stiffness of eukaryotic cells and also may affect the measured stiffness of the bacterial cell wall. Alternative immobilization methods include Cell-Tak™, an adhesive derived from marine mussels that does not interact with the bacterial wall and filters with known pore sizes which entrap bacteria. Previous studies have examined the effect of these immobilization methods on successful imaging of bacteria but have not addressed differences in measured modulus. This study compares the effects of immobilization methods including chemical fixatives, mechanical entrapment in filters, and Cell-Tak™ on the stiffness of the bacterial cell wall as measured by force spectroscopy.
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References
Ding, Y., et al.: Are elastic moduli of biological cells depth dependent or not? Another explanation using a contact mechanics model with surface tension. Soft Matter. 14(36), 7534–7541 (2018)
Bailey, R.G., et al.: The interplay between cell wall mechanical properties and the cell cycle in staphylococcus aureus. Biophys. J. 107(11), 2538–2545 (2014)
Rojas, E.R., Huang, K.C.: Regulation of microbial growth by turgor pressure. Curr. Opin. Microbiol. 42, 62–70 (2018)
Wheeler, R., et al.: Bacterial cell enlargement requires control of cell wall stiffness mediated by peptidoglycan hydrolases. MBio. 6(4), 1–10 (2015)
Matheli-Guinlet, M., et al.: Bacterial cell mechanics beyond Peptidoglycan. Trends Microbiol. 28(9), 706–708 (2020)
Liu, L., et al.: Mechanical penetration of β-lactam-resistant Gram-negative bacteria by programmable nanowires. Sci. Adv. 6(27), 1–12 (2020)
Frieri, M., Kumar, K., Boutin, A.: Antibiotic resistance. J. Infect. Publ. Health. 10(4), 369–378 (2017)
Garcia-Bustos, J., Tomasz, A.: A biological price of antibiotic resistance: major changes in the peptidoglycan structure of penicillin-resistant pneumococci. Proc. Natl. Acad. Sci. U. S. A. 87(14), 5415–5419 (1990)
Neu, H.C.: The crisis in antibiotic resistance. Science. 257(5073), 1064–1073 (1992)
Longo, G., et al.: Antibiotic-induced modifications of the stiffness of bacterial membranes. J. Microbiol. Methods. 93(2), 80–84 (2013)
Gaboriaud, F., et al.: Surface structure and nanomechanical properties of Shewanella putrefaciens bacteria at two pH values (4 and 10) determined by atomic force microscopy. J. Bacteriol. 187(11), 3864–3868 (2005)
Sandin, J.N., et al.: Near simultaneous laser scanning confocal and atomic force microscopy (Conpokal) on live cells. J. Vis. Exp. 2020(162), 1–25 (2020)
Riethmller, C., et al.: Vacuolar structures can be identified by AFM elasticity mapping. Ultramicroscopy. 107(10–11), 895–901 (2007)
Chao, Y., Zhang, T.: Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy. Appl. Microbiol. Biotechnol. 92, 381–392 (2011)
Meyer, R.L., et al.: Immobilisation of living bacteria for AFM imaging under physiological conditions. Ultramicroscopy. 110(11), 1349–1357 (2010)
Cerf, A., et al.: Nanomechanical properties of dead or alive single-patterned bacteria. Langmuir. 25(10), 5731–5736 (2009)
Chen, Y., et al.: Bacterial cell surface deformation under external loading. MBio. 3(6) (2012)
Mularski, A., et al.: Atomic force microscopy reveals the mechanobiology of Lytic peptide action on bacteria. Langmuir. 31(22), 6164–6171 (2015)
Kopycinska-Mller, M., Geiss, R.H., Hurley, D.C.: Contact mechanics and tip shape in AFM-based nanomechanical measurements. Ultramicroscopy. 106(6), 466–474 (2006)
Ozkan, A.D., et al.: Atomic force microscopy for the investigation of molecular and cellular behavior. Micron. 89, 60–76 (2016)
Colville, K., et al.: Effects of poly(L-lysine) substrates on attached escherichia coli bacteria. Langmuir. 26(4), 2639–2644 (2010)
Acknowledgments
We gratefully acknowledge NIH Center of Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation (CPRI, P20GM130456) and NIH NIDCR funding (R03DE029547) for completion of these experiments. AFM was performed in the Light Microscopy Core at the University of Kentucky.
Funding This material is based upon work supported by the National Science Foundation CAREER Award grant number 2045853. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.
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Waldman, L.J., Grady, M.E. (2023). Do Immobilization Methods Affect Force Spectroscopy Measurements of Single Bacteria?. In: Amirkhizi, A., Furmanski, J., Franck, C., Kasza, K., Forster, A., Estrada, J. (eds) Challenges in Mechanics of Time-Dependent Materials & Mechanics of Biological Systems and Materials, Volume 2. SEM 2022. Conference Proceedings of the Society for Experimental Mechanics Series. Springer, Cham. https://doi.org/10.1007/978-3-031-17457-5_4
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