Alcaligenes Faecalis MT02 Antagonizes Non-Tuberculosis Mycobacteria Through Hydroxylamine Secretion

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.