Genomic Characterization and Taxonomic Classification of Mycobacterium shunyiense sp. nov., a Novel Nontuberculous Mycobacterial Species from a Human Respiratory Specimen.

Non-tuberculous mycobacteria (NTM) are increasingly recognized as opportunistic pathogens, yet accurate species identification remains challenging. In this study, two clinical isolates, 19-857T and 20-525, obtained from patients with suspected pulmonary tuberculosis in Beijing, were characterized using a polyphasic approach integrating phenotypic, biochemical, chemotaxonomic, and genomic analyses. Phylogenetic analysis based on 16S rRNA gene sequences placed both strains within the genus Mycobacterium, with highest similarity to M. peregrinum DSM 43271T and M. arcueilense DSM 46715T. However, whole-genome comparisons via average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) yielded values of 85.8-94.1% and 29.1-54.3%, respectively, against their closest relatives-well below the species delineation thresholds (95-96% for ANI; 70% for dDDH)-while the two isolates shared 98.8% ANI and 90.1% dDDH, confirming their co-assignment to a single novel species. Genomic analysis further revealed a repertoire of genes potentially associated with virulence, including glycopeptidolipid biosynthesis, mce operons, ESX secretion systems, and mycobactin synthesis, alongside antimicrobial resistance determinants against rifampicin, tetracyclines, and β-lactams. Mobile genetic element analysis also uncovered extensive insertion sequences and transposon-related elements with differential distribution between the two strains. Based on the combined evidence, we propose Mycobacterium shunyiense sp. nov., with strain 19-857T (=CGMCC 38906T) as the type strain. This discovery expands the understanding of NTM diversity and provides genomic reference data for accurate clinical identification.