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Nanoscale Focused Ion Beam Tomography of Single Bacterial Cells for Assessment of Antibiotic Effects

Published online by Cambridge University Press:  04 March 2014

Boyin Liu ,
Heidi H. Yu ,
Tuck Wah Ng ,
David L. Paterson ,
Tony Velkov ,
Jian Li  and
Jing Fu
Boyin Liu
Affiliation:
Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC 3800, Australia
Heidi H. Yu
Affiliation:
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia
Tuck Wah Ng
Affiliation:
Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC 3800, Australia
David L. Paterson
Affiliation:
Centre for Clinical Research, University of Queensland, Brisbane, QLD 4072, Australia
Tony Velkov
Affiliation:
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia
Jian Li*
Affiliation:
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia
Jing Fu*
Affiliation:
Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC 3800, Australia
*
*Corresponding author. jing.fu@monash.edu; jian.li@monash.edu
*Corresponding author. jing.fu@monash.edu; jian.li@monash.edu
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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.

Keywords

focused ion beamelectron microscopy3D tomographycurvature analysisantibiotic resistance
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 2 , April 2014 , pp. 537 - 547
Copyright
© Microscopy Society of America 2014 

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