PAPER IN FOREFRONT Raman spectroscopy reveals distinct differences between two closely related bacterial strains, Mycobacterium indicus pranii and Mycobacterium intracellulare Taru Verma 1 & Santosh Podder 2,3 & Mansi Mehta 2 & Sarman Singh 4 & Amit Singh 2,5 & Siva Umapathy 1,6 & Dipankar Nandi 1,2,3 Received: 22 August 2019 /Revised: 24 September 2019 /Accepted: 7 October 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract 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 spectros- copy in differentiating two closely related mycobacterial strains. Keywords Mycobacterium indicus pranii . Mycobacterium intracellulare . Atomic force microscopy . Raman spectroscopy . Resonance Raman spectroscopy . Carotenoids . Mycolic acid Introduction Since the discovery of bacteria in the 1600s, several classifica- tion strategies have been developed for the identification of bacterial species to provide information related to their evolu- tion and phylogeny [1]. Bacterial identification can be per- formed using both genotypic and phenotypic methods. Genotypic techniques are based on sequencing an organism’ s genetic material, whereas phenotypic procedures classify bac- terial species on the basis of their metabolic features or chemical composition. A relatively easy and common procedure used to identify bacterial species is sequencing their 16S ribosomal genes [2, 3]. Despite being considered as the gold standard for identification of bacteria, the method fails for bacterial spe- cies exhibiting high sequence similarity in their 16S rRNA regions. Additionally, the assessment of bacterial diversity on the basis of a single gene can be misleading [4]. This is espe- cially true for bacteria belonging to the genus Mycobacterium which includes more than 170 species of bacteria [ 5]. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-019-02197-z) contains supplementary material, which is available to authorized users. * Siva Umapathy umapathy@iisc.ac.in * Dipankar Nandi nandi@iisc.ac.in 1 Centre for BioSystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India 2 Centre for Infectious Disease Research, Indian Institute of Science, Bangalore 560012, India 3 Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India 4 All India Institute of Medical Sciences, Bhopal 462020, India 5 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India 6 Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India Analytical and Bioanalytical Chemistry https://doi.org/10.1007/s00216-019-02197-z