Comparative genomic analysis of Mycobacterium kansasii reveals population structure, virulence-associated gene profiles, and pangenome dynamics

Mycobacterium kansasii is a clinically important nontuberculous mycobacterial pathogen; however, its genomic diversity remains underrepresented in Southeast Asia. We analyzed 200 confirmed M. kansasii genomes, comprising six clinical isolates from northeastern Thailand, 193 public genomes, and one reference genome, to contextualize the Thai isolates and characterize conserved and variable genomic features. Genome-based species reassessment, multilocus sequence typing, core-SNP phylogeny, in silico virulence-associated gene screening, pangenome analysis, Heaps' law modeling, and core-gene network enrichment analyses were performed. Five Thai isolates belonged to ST38 and clustered within a geographically widespread lineage predominant. Screening identified broadly conserved virulence-associated homologs related to cell wall biosynthesis, intracellular survival, and ESX secretion. Pangenome sensitivity analyses used Roary at 90%, 95%, and 98% BLASTp identity and Panaroo in strict cleaning mode for the full collection and a 172-genome ST38 subset. Roary identified 21,905-26,230 clusters in the full collection, whereas Panaroo identified 9956, including 4798 core and 4251 cloud clusters. Panaroo total and cloud counts were 57.39% and 75.56% lower, respectively, than Roary at 95% identity, indicating substantial method dependence. All eight Heaps' law decay estimates were below 1 (α = 0.394-0.495) and supported an open pangenome under the tested analytical frameworks. Core-gene network enrichment predominantly reflected conserved ribosomal and translation-related functions. These findings expand the genomic representation of M. kansasii from Thailand and places these isolates within the currently available global genomic context. Larger, geographically representative, phenotype-linked clinical and environmental collections are required to clarify population structure and the biological significance of variable genomic features.