Identification of mycobacteria by Raman spectroscopy

Abstract number: P1813

Willemse-Erix H.F.M., Buijtels P.C.A.M., Petit P.L.C., Endtz H.Ph., Puppels G.J., Verbrugh H.A., Maquelin K., van Belkum A., van Soolingen D.

Objectives: To analyse the capabilities of Raman spectroscopy for the identification of clinically relevant Mycobacterium species.

Methods: Strains were cultured in the MGIT system (Becton Dickinson Microbiology Systems, Cockeysville, Md., US) until indicated as positive by the system. The reproducibility of Raman spectroscopy was evaluated for killed/inactivated mycobacteria versus viable mycobacteria. 16S rRNA sequencing was used as a gold standard for the identification of M. tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). Sixty-three strains, belonging to 8 distinct species were analysed. Biomass from the MGIT system was suspended in 10 microliter of sterile distilled water, transferred onto a quartz slide and allowed to dry. Spectroscopic fingerprints were obtained using a dedicated Raman spectrometer, requiring approx. 1 min. per sample. Cluster analysis on these fingerprints was performed using the pair wise correlations as a distance measure in combination with Ward's cluster algorithm.

Results: The spectra of the inactivated bacteria showed minimal differences as compared to the spectra of viable mycobacteria. Therefore, identification of mycobacteria appears possible without biosafety level III precautions. The reproducibility of the Raman procedure was high. Correlation coefficients between repeated cultures of the same strain were significantly higher than the correlation coefficients between strains of different species. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7/7; 100%) as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains.

Conclusion: The spectra of the inactivated bacteria showed minimal differences as compared to the spectra of viable mycobacteria. Therefore, identification of mycobacteria appears possible without biosafety level III precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.