Genomic population structure, antimicrobial susceptibility, and clinical features of <i>Mycobacterium xenopi</i> isolates, Frankfurt, Germany, 1995–2020

<i>Mycobacterium xenopi</i> causes non-tuberculous mycobacterial pulmonary disease (NTM-PD) that is difficult to treat. However, data on the genomic population structure, antimicrobial susceptibility, and the clinical significance of this pathogen remain scarce. We analyzed 76 clinical <i>M. xenopi</i> isolates from 70 patients collected between 1995 and 2020 in Frankfurt am Main, Germany. All isolates underwent phenotypic drug susceptibility testing and whole-genome sequencing. Cluster analysis, including isolates from this study and all hitherto available high-quality <i>M. xenopi</i> genome data sets in the Sequence Read Archive (<i>n</i> = 11), was performed by core genome multilocus sequence typing. In our cohort, only 26.5% of patients met criteria for clinically relevant NTM-PD. Phylogenetic analysis identified three large hospital-associated clusters (≤10 allelic difference), each involving between 7 and 20 patients and persisting for over 18 years, suggesting prolonged transmission chains or a common environmental source. We also defined three major clades (≤50 allelic difference), two of which contained isolates from the United Kingdom. Clofazimine and guideline-recommended antimycobacterial agents showed good <i>in vitro</i> efficacy, except rifampicin, with 23.6% resistance. This study represents a major expansion of <i>M. xenopi</i> genomic resources and provides insights into the genomic population structure, phenotypic susceptibility, and clinical characteristics of <i>M. xenopi</i>. Guideline-recommended antimycobacterials show good <i>in vitro</i> activity, while clofazimine may be a valuable addition to <i>M. xenopi</i> therapy. The identified clusters underscore the need for further investigation into transmission dynamics and globally successful clones.IMPORTANCE<i>Mycobacterium xenopi</i> is an increasingly recognized opportunistic lung pathogen that is difficult to treat. Infections often occur in patients with pre-existing health conditions and can present substantial diagnostic and therapeutic challenges. A deeper understanding of its genetic diversity and resistance mechanisms is essential for optimal patient management and for clarifying potential transmission routes. By analyzing 76 whole-genome sequences together with detailed clinical information and phenotypic drug-susceptibility data, this study substantially expands the available genomic repertoire for <i>M. xenopi</i>. While clinical relevance was limited in our cohort, most guideline-recommended antimicrobial agents showed good efficacy <i>in vitro</i>. The detection of closely related strains might point toward a common environmental source of infection. These findings highlight the need for continued surveillance and provide a comprehensive foundation that supports more accurate monitoring, improved understanding of disease behavior, and future investigations into <i>M. xenopi</i> pathogenicity.