Genomic-enabled classification of three <i>Mycobacteroides abscessus</i> subspecies and an effective subspecies-specific identification method

<i>Mycobacteroides abscessus</i> (Mab) is a clinically significant non-tuberculous mycobacterium. It comprises three distinct subspecies being considered to have different macrolide susceptibilities, transmission patterns, and treatment outcomes. In this study, systematic analysis was conducted on 2,006 Mab genomes deposited in the National Center for Biotechnology Information genome database, and the taxonomic classification of their subspecies was revised accordingly. The findings revealed that: (i) in terms of three distinct subspecies classification, the analysis based on core genes and average nucleotide identity (ANI) values was completely consistent; (ii) ANI was a reliable criterion for Mab species and subspecies classification, with defined thresholds of 95% ANI for species-level and 98% ANI for subspecies-level differentiations; and (iii) the integrity of the <i>erm</i>(41) gene or the similarity of the <i>rpoB</i> gene was an unreliable characteristic for Mab subspecies, and the assertions that subspecies <i>massiliense</i> lack inducible resistance to macrolides also cannot be sustained. Moreover, through a subspecies re-classification of genomes and pangenome analysis, Mab subspecies-specific genes were successfully identified, and a novel single-gene test with enhanced clinical applicability was developed. Additionally, the impact of reference genome selection on taxonomic classification highlighted the importance of adopting a standardized set of reference genomes in species/subspecies identification to significantly enhance the comparability across different studies.IMPORTANCE<i>Mycobacteroides abscessus</i> (Mab) is a clinically challenging non-tuberculous mycobacteria species. The accurate identification of subspecies is of utmost importance for clinical diagnosis and treatment, as well as for research on pathogenicity, drug resistance, and other related aspects. This study provided a clear average nucleotide identity threshold for Mab subspecies classification, as well as revised options of the three Mab subspecies, new and accurate Mab subspecies-special biomarker, and a detection technique with practical clinical application.