Mycobacterium smegmatis (M.sm) is frequently used as an alternative model organism in Mycobacterium tuberculosis (M.tb) studies. While containing high sequence homology with M.tb, it is considered non-pathogenic in humans. As such it has been used to study M.tb and other infections in vivo and more recently been explored for potential therapeutic applications. A body of previous research has highlighted the potential of using genetically modified M.sm displaying rapid growth and unique immunostimulatory characteristics as an effective vaccine vector. Novel systems biology techniques can further serve to optimize these delivery constructs. In this article, we review recent advancements in vaccinomics tools that support the efficacy of a M.sm-based vaccine vector. Moreover, the integration of systems biology and molecular omics techniques in these pioneering studies heralds a potential accelerated pipeline for the development of next-generation recombinant vaccines against rapidly developing diseases.

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens that pose a continuous public health risk, and new control strategies are urgently needed. Alcaligenes faecalis is an environmental bacterium with broad antagonistic activity against various microorganisms, but the mechanism underlying its antagonism against NTM remains unclear. In this study, we isolated A. faecalis strain MT02 from mouse tail tissue and investigated its anti-NTM activity using inhibition zone assays, time–kill curves, hydroxylamine (HA) quantification, dialysis, recombinant extracellular serine protease (ESP) purification, RT-qPCR, and whole-genome sequencing. MT02 exhibited broad-spectrum antagonism against Mycobacterium smegmatis, M. abscessus, M. marinum, and M. avium. HA concentration was spatially correlated with the inhibition zone, and MT02 supernatant showed bactericidal effects similar to those of pure HA. Dialysis of the supernatant removed HA and markedly reduced its bactericidal activity, whereas purified ESP had no significant effect against M. smegmatis. HA production was carbon source-dependent, with fatty acids supporting the highest yield, and was also temperature-dependent: at 37 °C, HA production and dnfABC expression were significantly reduced compared with 30 °C. Whole-genome sequencing revealed in silico-predicted potential antibiotic resistance genes and virulence factors. Together, these results indicate that HA is a major effector molecule mediating the anti-NTM activity of A. faecalis MT02, and its production is regulated by both carbon source and temperature. This study provides new insights into the antagonistic mechanism of A. faecalis against NTM.