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Comparison Of Two Transmission Electron Microscopy Methods To Visualize Drug-Induced Alterations Of Gram-Negative Bacterial Morphology

Hang Thi Nguyen, Lisa A. O’Donovan, Henrietta Venter, Cecilia C. Russell, Adam McCluskey, Stephen W. Page, Darren J. Trott, Abiodun D. Ogunniyi

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In this study, we optimized and compared different transmission electron microscopy (TEM) methods to visualize changes to Gram-negative bacterial morphology induced by treatment with a robenidine analogue (NCL195) and colistin combination. Aldehyde-fixed bacterial cells (untreated, treated with colistin or NCL195 + colistin) were prepared using conventional TEM methods and compared with ultrathin Tokuyasu cryo-sections. The results of this study indicate superiority of ultrathin cryo-sections in visualizing the membrane ultrastructure of Escherichia coli and Pseudomonas aeruginosa, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. We suggest that the use of ultrathin cryo-sectioning can be used to better visualize and understand drug interaction mechanisms on the bacterial cell membrane.