title: Nanoscale focused ion beam tomography of single bacterial cells for assessment of antibiotic effects.
abstract: Antibiotic resistance is a major risk to human health, and to provide valuable insights into mechanisms of resistance, innovative methods are needed to examine the cellular responses to antibiotic treatment. Focused ion beam tomography is proposed to image and assess the detailed three-dimensional (3D) ultrastructure of single bacterial cells. By iteratively removing slices of thickness in the order of 10 nm, high magnification 2D images can be acquired by scanning electron microscopy at single-digit nanometer resolution. In this study, Klebsiella pneumoniae was treated with polymyxin B, and 3D models of both cell envelope and cytoplasm regions containing the nucleoid and ribosomes were reconstructed. The 3D volume containing the nucleoid and ribosomes was significantly smaller, and the cell length along the longitudinal axis was extended by 40% in the treated cells, implying stress responses to the drug treatment. More than a 200% increase in protrusions per unit surface area on the cell envelope was observed in the curvature analysis after treatment. Experiments by conventional transmission electron microscopy and atomic force microscopy were also performed, followed by comparison and discussions. In conclusion, the proposed 3D imaging method and associated analysis provide a unique tool for the assessment of antibiotic effects on multidrug-resistant bacteria at nanometer resolution.
authors.affiliation: 1 Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Boyin
authors.initials: B
authors.lastname: Liu
authors.affiliation: 2 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia.
authors.forename: Heidi H
authors.initials: HH
authors.lastname: Yu
authors.affiliation: 1 Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Tuck Wah
authors.initials: TW
authors.lastname: Ng
authors.affiliation: 3 Centre for Clinical Research, University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: David L
authors.initials: DL
authors.lastname: Paterson
authors.affiliation: 2 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia.
authors.forename: Tony
authors.initials: T
authors.lastname: Velkov
authors.affiliation: 2 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: 1 Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Jing
authors.initials: J
authors.lastname: Fu
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D021621:Imaging, Three-Dimensional / Q000379:methods; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D011112:Polymyxin B / Q000494:pharmacology; D014057:Tomography, X-Ray Computed / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents; D011112:Polymyxin B