Genomic insights into non-tuberculous mycobacteria isolated from patients in a Chinese hospital

Non-tuberculous mycobacteria (NTM) are increasingly recognized as important opportunistic pathogens, yet the genomic basis of their antimicrobial resistance and virulence in clinical settings remains incompletely understood. We performed whole-genome sequencing on 28 clinical isolates of NTM collected from Chinese patients (primarily bronchoalveolar lavage fluid) between 2022 and 2025, combined with broth microdilution susceptibility testing and comprehensive bioinformatics analysis. The collection comprised diverse species, including Mycobacterium intracellulare , Mycobacterium chimaera , Mycobacterium marseillense , Mycobacterium abscessus , Mycobacterium fortuitum , Mycobacterium lentiflavum , Mycobacterium kansasii , and Mycobacterium colombiense . Phenotypic testing revealed extensive multidrug resistance: 100% of isolates were resistant to rifampicin, erythromycin, imipenem, and doxycycline; 82.1% were resistant to ciprofloxacin; 89.3% were resistant to linezolid; and only amikacin retained substantial activity (50% susceptible). Resistome profiling using the Comprehensive Antibiotic Resistance Database/Resistance Gene Identifier identified heterogeneous distributions of efflux pumps (tap), ribosomal methylases [Erm(39)], β-lactamases (blaF), and other determinants, consistent with both intrinsic and accessory-genome-mediated resistance. Virulence factor annotation against the Virulence Factor Database revealed 531 unique factors, with hierarchical clustering separating isolates into two major clades: (i) rapid-growing species ( M. abscessus and M. fortuitum ) enriched in secretion systems (ESX/T7SS), polyketide synthases, and lipid biosynthesis genes, and (ii) slow-growing Mycobacterium avium complex species showing lower overall virulence gene abundance. Pangenome analysis indicated a relatively closed pangenome structure, with 1,026 core genes and substantial shell/cloud compartments contributing to strain-specific adaptations. These findings provide genomic insights into the multidrug-resistant and virulence-heterogeneous nature of contemporary Chinese clinical isolates of NTM. IMPORTANCE In this study, we sequenced 28 clinical non-tuberculous mycobacterial strains, performed comprehensive drug susceptibility testing, and analyzed virulence factors to elucidate genomic determinants of resistance and pathogenicity, as well as evolutionary characteristics. These findings aim to bridge existing knowledge gaps, informing improved diagnostic and therapeutic strategies for non-tuberculous mycobacterial infections.