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.

Antimycobacterial potential of the juniper berry essential oil in tap water.

Mycobacterium avium complex-related diseases are often associated with poorly maintained hot water systems. This calls for the development of new control strategies. The aim of this study was to investigate the activity of essential oils (EOs) from the Mediterranean plants, common juniper, immortelle, sage, lavandin, laurel, and white cedar against Mycobacterium avium ssp. avium, Mycobacterium intracellulare, and Mycobacterium gordonae in culturing broth and freshwater as their most common habitat. To do that, we developed a new method of water microdilution to determine their minimal effective concentrations (MEC). The most active EO was the one from the common juniper with the MEC of 1.6 mg mL-1. Gas chromatography / mass spectrometry the juniper EO identified monoterpenes (70.54 %) and sesquiterpenes (25.9 %) as dominant component groups. The main monoterpene hydrocarbons were α-pinene, sabinene, and ß-pinene. The juniper EO significantly reduced the cell viability of M. intracellulare and M. gordonae at MEC, and of M. avium at 2xMEC. Microscopic analysis confirmed its inhibitory effect by revealing significant morphological changes in the cell membrane and cytoplasm of all three bacteria. The mode of action of the juniper EO on the cell membrane was confirmed by a marked leakage of intracellular material. Juniper EO has a great practical potential as a complementary or alternative water disinfectant in hot water systems such as baths, swimming pools, spa pools, hot tubs, or even foot baths/whirlpools.

Emergence of mmpT5 Variants during Bedaquiline Treatment of Mycobacterium intracellulare Lung Disease.

Bedaquiline (BDQ), a diarylquinoline antibiotic that targets ATP synthase, is effective for the treatment of Mycobacterium tuberculosis infections that no longer respond to conventional drugs. While investigating the off-label use of BDQ as salvage therapy, seven of 13 patients with Mycobacterium intracellulare lung disease had an initial microbiological response and then relapsed. Whole-genome comparison of pretreatment and relapse isolates of M. intracellulare uncovered mutations in a previously uncharacterized locus, mmpT5 Preliminary analysis suggested similarities between mmpT5 and the mmpR5 locus, which is associated with low-level BDQ resistance in M. tuberculosis Both genes encode transcriptional regulators and are adjacent to orthologs of the mmpS5-mmpL5 drug efflux operon. However, MmpT5 belongs to the TetR superfamily, whereas MmpR5 is a MarR family protein. Targeted sequencing uncovered nonsynonymous mmpT5 mutations in isolates from all seven relapse cases, including two pretreatment isolates. In contrast, only two relapse patient isolates had nonsynonymous changes in ATP synthase subunit c (atpE), the primary target of BDQ. Susceptibility testing indicated that mmpT5 mutations are associated with modest 2- to 8-fold increases in MICs for BDQ and clofazimine, whereas one atpE mutant exhibited a 50-fold increase in MIC for BDQ. Bedaquiline shows potential for the treatment of M. intracellulare lung disease, but optimization of treatment regimens is required to prevent the emergence of mmpT5 variants and microbiological relapse.