Raman spectroscopy reveals distinct differences between two closely related bacterial strains, Mycobacterium indicus pranii and Mycobacterium intracellulare.

A common technique used to differentiate bacterial species and to determine evolutionary relationships is sequencing their 16S ribosomal RNA genes. However, this method fails when organisms exhibit high similarity in these sequences. Two such strains that have identical 16S rRNA sequences are Mycobacterium indicus pranii (MIP) and Mycobacterium intracellulare. MIP is of significance as it is used as an adjuvant for protection against tuberculosis and leprosy; in addition, it shows potent anti-cancer activity. On the other hand, M. intracellulare is an opportunistic pathogen and causes severe respiratory infections in AIDS patients. It is important to differentiate these two bacterial species as they co-exist in immuno-compromised individuals. To unambiguously distinguish these two closely related bacterial strains, we employed Raman and resonance Raman spectroscopy in conjunction with multivariate statistical tools. Phenotypic profiling for these bacterial species was performed in a kinetic manner. Differences were observed in the mycolic acid profile and carotenoid pigments to show that MIP is biochemically distinct from M. intracellulare. Resonance Raman studies confirmed that carotenoids were produced by both MIP as well as M. intracellulare, though the latter produced higher amounts. Overall, this study demonstrates the potential of Raman spectroscopy in differentiating two closely related mycobacterial strains. Graphical abstract.

Misidentification of Mycobacterium leprae as Mycobacterium intracellulare by the COBAS AMPLICOR M. intracellulare test.

Commercially available nucleic acid probe- and amplification-based systems for detection and differentiation of mycobacteria are widely used in clinical microbiology laboratories. Here we report two cases of human leprosy in which the COBAS AMPLICOR Mycobacterium intracellulare test led to false- positive results. Correct identification of Mycobacterium leprae was possible only by amplification and comparative sequence analysis of the 16S rRNA gene.

Rapid and precise diagnosis of atypical mycobacterial infection by chemotaxonomical and immunological methods.

The species of 205 strains of acid fast bacteria isolated from swine and human mycobacteriosis were identified chemotaxonomically and numericaltaxonomically. The species of the isolates which were identified numericaltaxonomically as Mycobacterium avium intracellulare (MAI) complex were further classified by using both thin-layer chromatography of the antigenic glycopeptidolipids (GPL) from the bacteria and seroagglutination test devised by Schaefer. These MAI complex from swine fell into serotype 8 (45 strains), serotype 4 (32 strains), serotype 9 (9 strains) and untypable (9 strains), respectively. In contrast to swine, human isolates covered more wide ranges of serotypes such as serovar 7, 12, 16 besides serovar 4, 8 and 9. Furthermore, enzyme-linked immunosorbent assay (ELISA) which is based on the type specific glycolipid antigen and infected swine/human sera was applied to distinguish serological variants of the MAI complex. Of the fourteen cases in swine and five in human that had been typed by both the seroagglutination reaction and the thin layer chromatography (TLC) the thirteen in swine and two in human cases showed clear coincidence with the results of ELISA. The results demonstrated that enzyme-linked immunosorbent assay using infected sera was especially useful, and it was recommended from the sensitivity and rapidity as an adjunct to seroagglutination test and thin layer chromatography for the identification of serotypes of MAI complex.