Analytical and Bioanalytical Chemistry

, Volume 407, Issue 27, pp 8333–8341

Destruction-free procedure for the isolation of bacteria from sputum samples for Raman spectroscopic analysis

  • Sandra Kloß
  • Björn Lorenz
  • Stefan Dees
  • Ines Labugger
  • Petra Rösch
  • Jürgen Popp
Research Paper

DOI: 10.1007/s00216-015-8743-x

Cite this article as:
Kloß, S., Lorenz, B., Dees, S. et al. Anal Bioanal Chem (2015) 407: 8333. doi:10.1007/s00216-015-8743-x
Part of the following topical collections:
  1. Raman4Clinics

Abstract

Lower respiratory tract infections are the fourth leading cause of death worldwide. Here, a timely identification of the causing pathogens is crucial to the success of the treatment. Raman spectroscopy allows for quick identification of bacterial cells without the need for time-consuming cultivation steps, which is the current gold standard to detect pathogens. However, before Raman spectroscopy can be used to identify pathogens, they have to be isolated from the sample matrix, i.e., sputum in case of lower respiratory tract infections. In this study, we report an isolation protocol for single bacterial cells from sputum samples for Raman spectroscopic identification. Prior to the isolation, a liquefaction step using the proteolytic enzyme mixture Pronase E is required in order to deal with the high viscosity of sputum. The extraction of the bacteria was subsequently performed via different filtration and centrifugation steps, whereby isolation ratios between 46 and 57 % were achieved for sputa spiked with 6·107 to 6·104 CFU/mL of Staphylococcus aureus. The compatibility of such a liquefaction and isolation procedure towards a Raman spectroscopic classification was shown for five different model species, namely S. aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. A classification of single-cell Raman spectra of these five species with an accuracy of 98.5 % could be achieved on the basis of a principal component analysis (PCA) followed by a linear discriminant analysis (LDA). These classification results could be validated with an independent test dataset, where 97.4 % of all spectra were identified correctly.

Graphical Abstract

Development of an isolation protocol of bacterial cells out of sputum samples followed by Raman spectroscopic measurement and species identification using chemometrical models.

Keywords

Raman spectroscopy Single-cell pathogen identification Sputum Isolation 

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Sandra Kloß
    • 1
    • 2
  • Björn Lorenz
    • 1
    • 2
  • Stefan Dees
    • 2
    • 3
  • Ines Labugger
    • 2
    • 3
  • Petra Rösch
    • 1
    • 2
  • Jürgen Popp
    • 1
    • 2
    • 4
  1. 1.Institute of Physical Chemistry and Abbe Center of PhotonicsFriedrich Schiller University JenaJenaGermany
  2. 2.InfectoGnostics Forschungscampus JenaJenaGermany
  3. 3.Alere Technologies GmbHJenaGermany
  4. 4.Leibniz Institute of Photonic Technology e.V.JenaGermany

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