Deep sequencing reveals subpopulation dynamics associated with treatment failure in a rare non-tuberculous mycobacterial infection

Low-frequency genetic variants may drive the evolution of non-tuberculous mycobacterial populations, contributing to treatment failures during antibiotic therapy.

Summary and findings

This study investigates the genetic heterogeneity of a rare non-tuberculous mycobacterial infection over a 29-month period. Whole genome sequencing identified Mycobacterium sp. SMC-2 and revealed 444 unique SNPs and 26 indels in 12 longitudinal isolates. The emergence of drug resistance was associated with low-frequency mutations in antibiotic resistance genes.

Abstract

<h4>Background</h4> Nontuberculous mycobacteria (NTM) are an increasingly common group of pathogens that remain challenging to diagnose and treat effectively. The lack of standardization of NTM management, from identification to antibiotic resistance prediction, results in imperfect correlations between treatment and outcomes. This study characterizes the genetic heterogeneity of a previously uncharacterized NTM during a 29-month bacteremia with acquired drug resistance. <h4>Results</h4> In contrast to the initial diagnostic result identifying M. nebraskense , a rare NTM causing disease in humans, whole genome sequencing (WGS) identified Mycobacterium sp. SMC-2 , a species with only one publicly available genome. High-resolution analysis of variants revealed 444 unique SNPs and 26 indels in 12 longitudinal isolates, with the highest number of low-frequency mutations between 3-5% frequency. Seven candidate drug-resistance mutations across five evolutionary trajectories showed frequency shifts that correlated with changes in minimum inhibitory concentrations to the corresponding antibiotics. These included a 23S rRNA clarithromycin-resistance SNP detected at 7% frequency when phenotypic resistance emerged, suggesting that low-frequency variants drive subpopulation evolution. Acquisition of drug resistance during therapy was associated with several low-frequency mutations in genes associated with resistance to antibiotics, including clarithromycin and quinolones, in other NTM species. <h4>Conclusion</h4> This study highlights the importance of low-frequency variants as drivers of intra-patient bacterial population diversity, allowing subpopulations to adapt to antibiotic pressure and ultimately contributing to treatment failure. Additionally, it underscores their potential implications for the development of molecular diagnostic tests for NTM resistance prediction.

Limitations

Not reported in abstract.

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