Dynamics of within-host genomic relatedness and emergence of resistance in serial Mycobacterium avium complex isolates during treatment

Mycobacterium avium complex infections require prolonged multidrug therapy and may develop antimicrobial resistance. We applied whole-genome sequencing (WGS) to assess within-patient genomic relatedness and resistance-associated mutations emerging during therapy. WGS was performed on cultured populations of 30 serial isolates (16 M. avium and 14 Mycobacterium intracellulare ) from 13 patients with repeated recovery of the same species. Genomic relatedness was classified by species/subspecies, sequence type (ST), and fastBAPS (FB) clustering. Resistance-associated mutations were assessed in isolates showing susceptible-to-resistant conversion or an increase of at least two dilutions in the minimum inhibitory concentration (MIC). Among 13 patients, 10 (76.9%) showed concordant genomic patterns, 2 (15.4%) showed discordant genomic patterns with genetically distinct cultured populations, and 1 (7.7%) showed both concordant and discordant relationships across intervals. Sixteen M. avium cultured populations belonged to four STs (ST112 and ST118, 75.0%) and four FB clusters. Fourteen M. intracellulare cultured populations belonged to four STs, predominantly ST1109 (71.4%). While most cultured populations of the same ST matched the same FB, M. avium ST112 and ST118 and M. intracellulare ST1109 each occurred in two FB clusters. Conversely, three STs (ST112, ST118, and ST1112) of M. avium belonged to the same FB. Among three cases with emergent clarithromycin resistance, rrl mutations (A2058C and A2059G) were identified in two. In one case, an embC-embA intergenic variant showed stepwise increases in variant allele frequency, accompanied by increasing ethambutol MICs. WGS provided two methods for analyzing within-patient genomic relatedness (ST assignment and FB clustering), which were mutually complementary and enabled longitudinal detection of resistance-associated mutations during treatment. IMPORTANCE Mycobacterium avium complex pulmonary disease often requires prolonged multidrug therapy, during which serial culture-positive isolates may differ in within-patient genomic relatedness and antimicrobial resistance. Distinguishing closely related from genetically distinct isolates and identifying emerging resistance are clinically important but cannot be reliably achieved using conventional species identification or sequence typing alone. In this study, whole-genome sequencing (WGS) of serial M. avium complex isolates enabled high-resolution assessment of within-patient genomic relatedness. WGS also identified emerging resistance-associated mutations, including acquired rrl mutations associated with high-level clarithromycin resistance during macrolide-containing therapy and an embC-embA intergenic variant accompanying increasing ethambutol minimum inhibitory concentrations during ethambutol-containing treatment. These findings support the potential clinical utility of WGS for characterizing genomic relationships among recurrent M. avium complex isolates and detecting emergence of resistance-associated mutations during therapy.