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Download Hi-Res ImageDownload to MS-PowerPointCite This:Anal. Chem. 2022, 94, 2, 669-677
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Multi-Excitation Raman Spectroscopy for Label-Free, Strain-Level Characterization of Bacterial Pathogens in Artificial Sputum Media

  • Adam P. Lister
    Adam P. Lister
    School of Chemistry, Faculty of Engineering and Physical Sciences, University of Southampton, SO17 1BJ Southampton, United Kingdom
    National Biofilms Innovation Centre (NBIC) and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
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  • , 
  • Callum J. Highmore
    Callum J. Highmore
    School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, SO17 1BJ Southampton, United Kingdom
    Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
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  • , 
  • Niall Hanrahan
    Niall Hanrahan
    School of Chemistry, Faculty of Engineering and Physical Sciences, University of Southampton, SO17 1BJ Southampton, United Kingdom
    National Biofilms Innovation Centre (NBIC) and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
    More by Niall Hanrahan
  • , 
  • James Read
    James Read
    School of Chemistry, Faculty of Engineering and Physical Sciences, University of Southampton, SO17 1BJ Southampton, United Kingdom
    National Biofilms Innovation Centre (NBIC) and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
    More by James Read
  • , 
  • Alasdair P. S. Munro
    Alasdair P. S. Munro
    NIHR Southampton Clinical Research Facility and Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, UK
    Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
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  • , 
  • Samuel Tan
    Samuel Tan
    Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
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  • , 
  • Raymond N. Allan
    Raymond N. Allan
    School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, SO17 1BJ Southampton, United Kingdom
    NIHR Southampton Clinical Research Facility and Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, UK
    School of Pharmacy, Faculty of Health and Life Sciences, De Montfort University, Leicester LE1 9BH, UK
    More by Raymond N. Allan
  • , 
  • Saul N. Faust*
    Saul N. Faust
    NIHR Southampton Clinical Research Facility and Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, UK
    Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
    National Biofilms Innovation Centre (NBIC) and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
    *Email: [email protected]
    More by Saul N. Faust
  • , 
  • Jeremy S. Webb*
    Jeremy S. Webb
    School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, SO17 1BJ Southampton, United Kingdom
    National Biofilms Innovation Centre (NBIC) and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
    *Email: [email protected]
    More by Jeremy S. Webb
  • , and 
  • Sumeet Mahajan*
    Sumeet Mahajan
    School of Chemistry, Faculty of Engineering and Physical Sciences, University of Southampton, SO17 1BJ Southampton, United Kingdom
    National Biofilms Innovation Centre (NBIC) and Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom
    *Email: [email protected]
    More by Sumeet Mahajan
    Orcidhttps://orcid.org/0000-0001-8923-6666
Cite this: Anal. Chem. 2022, 94, 2, 669–677
Publication Date (Web):January 3, 2022
https://doi.org/10.1021/acs.analchem.1c02501
Copyright © 2022 American Chemical Society
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    Abstract

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    The current methods for diagnosis of acute and chronic infections are complex and skill-intensive. For complex clinical biofilm infections, it can take days from collecting and processing a patient’s sample to achieving a result. These aspects place a significant burden on healthcare providers, delay treatment, and can lead to adverse patient outcomes. We report the development and application of a novel multi-excitation Raman spectroscopy-based methodology for the label-free and non-invasive detection of microbial pathogens that can be used with unprocessed clinical samples directly and provide rapid data to inform diagnosis by a medical professional. The method relies on the differential excitation of non-resonant and resonant molecular components in bacterial cells to enhance the molecular finger-printing capability to obtain strain-level distinction in bacterial species. Here, we use this strategy to detect and characterize the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus as typical infectious agents associated with cystic fibrosis. Planktonic specimens were analyzed both in isolation and in artificial sputum media. The resonance Raman components, excited at different wavelengths, were characterized as carotenoids and porphyrins. By combining the more informative multi-excitation Raman spectra with multivariate analysis (support vector machine) the accuracy was found to be 99.75% for both species (across all strains), including 100% accuracy for drug-sensitive and drug-resistant S. aureus. The results demonstrate that our methodology based on multi-excitation Raman spectroscopy can underpin the development of a powerful platform for the rapid and reagentless detection of clinical pathogens to support diagnosis by a medical expert, in this case relevant to cystic fibrosis. Such a platform could provide translatable diagnostic solutions in a variety of disease areas and also be utilized for the rapid detection of anti-microbial resistance.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.1c02501.

    • Detailed classification methods (Figure S1). Raw Raman spectra at 532, 633, and 785 nm (Figure S2). Pre-processed Raman spectra (Figure S3). UV–visible absorbance spectra and Raman spectra (532 nm and 785 nm excitations) of β-carotene, xanthophyll, hemin, and protoporphyrin IX (Figure S4). Images of dried bacterial pellets of all P. aeruginosa and S. aureus strains used in the study (Figure S5). Peak assignments for major vibrational modes (Figure S6). Projections and classification accuracies for PCA of the 8 strains used in this study after polynomial background subtraction (Figure S7). Comparison of the classification of bacteria using the luminescent background versus Raman spectra (Figure S8). Classification accuracy for PCA–SVM of pure bacterial pellets (Figure S9). Sample preparation for bacterial suspensions in ASM (Figure S10). Raman spectra of blank ASM, and PA, and SA strains in ASM, taken at 532 nm and 785 nm excitations (Figure S11). Classification accuracy for SVM of bacteria suspended in ASM (Figure S12). Classification accuracy for PCA–SVM of bacteria suspended in ASM (Figure S13) (PDF)

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    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

    Cited By

    This article is cited by 5 publications.

    1. Muhammad Usman, Jia-Wei Tang, Fen Li, Jin-Xin Lai, Qing-Hua Liu, Wei Liu, Liang Wang. Recent advances in surface enhanced Raman spectroscopy for bacterial pathogen identifications. Journal of Advanced Research 2022, 46 https://doi.org/10.1016/j.jare.2022.11.010
    2. André Müller, Martin Maiwald, Bernd Sumpf. Compact, Watt-class 785 nm dual-wavelength master oscillator power amplifiers. Journal of Physics Communications 2022, 6 (12) , 125007. https://doi.org/10.1088/2399-6528/acac6e
    3. Aikaterini Pistiki, Stefan Monecke, Haodong Shen, Oleg Ryabchykov, Thomas W. Bocklitz, Petra Rösch, Ralf Ehricht, Jürgen Popp, . Comparison of Different Label-Free Raman Spectroscopy Approaches for the Discrimination of Clinical MRSA and MSSA Isolates. Microbiology Spectrum 2022, 10 (5) https://doi.org/10.1128/spectrum.00763-22
    4. Niall Hanrahan, Adam P. Lister, Callum J. Highmore, Leena Rajith, Ekaterina Avershina, Jawad Ali, Rafi Ahmad, Jeremy S. Webb, Sumeet Mahajan, , . Enhanced Raman techniques for infection diagnostics. 2022, 6. https://doi.org/10.1117/12.2635324
    5. Chenyang Zhang, Minjie Mou, Ying Zhou, Wei Zhang, Xichen Lian, Shuiyang Shi, Mingkun Lu, Huaicheng Sun, Fengcheng Li, Yunxia Wang, Zhenyu Zeng, Zhaorong Li, Bing Zhang, Yunqing Qiu, Feng Zhu, Jianqing Gao. Biological activities of drug inactive ingredients. Briefings in Bioinformatics 2022, 23 (5) https://doi.org/10.1093/bib/bbac160
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