Abstract
Tuberculosis relapse is a barrier to shorter treatment. It is thought that lipid rich cells, phenotypically resistant to antibiotics, may play a major role. Most studies investigating relapse use sputum samples although tissue bacteria may play an important role. We developed a non-destructive, label-free technique combining wavelength modulated Raman (WMR) spectroscopy and fluorescence detection (Nile Red staining) to interrogate Mycobacterium tuberculosis cell state. This approach could differentiate single “dormant” (lipid rich, LR) and “non-dormant” (lipid poor, LP) cells with high sensitivity and specificity. We applied this to experimentally infected guinea pig lung sections and were able to distinguish both cell types showing that the LR phenotype dominates in infected tissue. Both in-vitro and ex-vivo spectra correlated well, showing for the first time that Mycobacterium tuberculosis, likely to be phenotypically resistant to antibiotics, are present in large numbers in tissue. This is an important step in understanding the pathology of relapse supporting the idea that they may be caused by M. tuberculosis cells with lipid inclusions.
Highlights
Tuberculosis is established as the most important cause of death due to infectious disease, yet treatment has not improved in fifty years
In wavelength modulated Raman spectra (WMR spectra) zero-crossings are equivalent to peak positions in standard Raman spectra and the peak-to-valley corresponds to the peak intensity in standard Raman spectra
We showed that in-vitro lipid rich (LR) and lipid poor (LP) cells could be discriminated by WMR spectroscopy (sensitivity (84.0%) and specificity (80.2%)) (Fig. 4a and b)
Summary
Tuberculosis is established as the most important cause of death due to infectious disease, yet treatment has not improved in fifty years. Raman spectroscopy has been used previously as a means of identifying bacterial taxonomy at a single cell level in a range of species including mycobacteria[10, 11]. These studies, have only previously been performed on isolated cultured cells[12] and isolated cells from sputum[13]. Signal-to-noise ratio compared to other methods including standard Raman spectroscopy[16] Using this approach, we were able to detect LR M. tuberculosis cells in infected tissue with very high sensitivity and specificity. This is a major step towards understanding the pathogenesis of tuberculosis at the infection site and provides a paradigm for its application to a broad range of infectious diseases
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