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Nano Lett. 2016 Nov 9;16(11):7113-7120. Epub 2016 Oct 14.

Near-Atomic Three-Dimensional Mapping for Site-Specific Chemistry of 'Superbugs'.

Author information

1
Department of Mechanical and Aerospace Engineering, Monash University , Clayton, Victoria 3800, Australia.
2
Institute for Frontier Materials, Deakin University , Geelong, Victoria 3216, Australia.
3
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University , Parkville, Victoria 3052, Australia.
4
Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University , Clayton, Victoria 3800, Australia.
5
ARC Centre of Excellence for Advanced Molecular Imaging, Monash University , Clayton, Victoria 3800, Australia.

Abstract

Emergence of multidrug resistant Gram-negative bacteria has caused a global health crisis and last-line class of antibiotics such as polymyxins are increasingly used. The chemical composition at the cell surface plays a key role in antibiotic resistance. Unlike imaging the cellular ultrastructure with well-developed electron microscopy, the acquisition of a high-resolution chemical map of the bacterial surface still remains a technological challenge. In this study, we developed an atom probe tomography (APT) analysis approach to acquire mass spectra in the pulsed-voltage mode and reconstructed the 3D chemical distribution of atoms and molecules in the subcellular domain at the near-atomic scale. Using focused ion beam (FIB) milling together with micromanipulation, site-specific samples were retrieved from a single cell of Acinetobacter baumannii prepared as needle-shaped tips with end radii less than 60 nm, followed by a nanoscale coating of silver in the order of 10 nm. The significantly elevated conductivity provided by the metallic coating enabled successful and routine field evaporation of the biological material, with all the benefits of pulsed-voltage APT. In parallel with conventional cryo-TEM imaging, our novel approach was applied to investigate polymyxin-susceptible and -resistant strains of A. baumannii after treatment of polymyxin B. Acquired atom probe mass spectra from the cell envelope revealed characteristic fragments of phosphocholine from the polymyxin-susceptible strain, but limited signals from this molecule were detected in the polymyxin-resistant strain. This study promises unprecedented capacity for 3D nanoscale imaging and chemical mapping of bacterial cells at the ultimate 3D spatial resolution using APT.

KEYWORDS:

Cell imaging; atom probe tomography; drug resistance; nanoscale chemical mapping; polymyxin

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