Rapid identification of mycobacteria by Raman spectroscopy

Abstract

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.

FIG. 1.

Raman spectra of M. kansasii after inactivation with formalin and heating in comparison to the procedure without inactivation. Shaded areas indicate spectral region in which significant effects of formalin inactivation can be observed. a.u., arbitrary units.

FIG. 2.

Dendrogram of HCA of Raman spectra of the isolates used in the pilot study. Numbers refer to isolates of the collection of the national tuberculosis reference laboratory at the National Institute for Public Health and the Environment.

FIG. 3.

Average intrastrain and interspecies similarities between spectra obtained from six native and heat-killed Mycobacterium species. The error bars show the 95% confidence intervals.

FIG. 4.

Representative Raman spectra from the eight Mycobacterium species used in the identification study. a.u., arbitrary units.

FIG. 5.

Dendrogram resulting from hierarchical cluster analysis of Raman spectra of the isolates used in the identification.