Label-free optical vibrational spectroscopy to detect the metabolic state of M. tuberculosis cells at the site of disease

TL;DR

This study introduces a label-free optical spectroscopy method combining Raman and fluorescence techniques to identify and differentiate lipid-rich and lipid-poor M. tuberculosis cells in infected tissue, aiding understanding of relapse mechanisms.

Contribution

The paper presents a novel non-destructive spectroscopy approach capable of detecting phenotypically resistant M. tuberculosis cells directly in tissue samples.

Findings

Successfully differentiated dormant and non-dormant cells in tissue

Lipid-rich phenotype dominates in infected tissue

Correlated in-vitro and ex-vivo spectra of M. tuberculosis

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.