Limited data exist on longitudinal changes in the sputum bacterial microbiome during treatment in nontuberculous mycobacterial pulmonary disease (NTM-PD) patients. We prospectively collected serial sputum samples from 14 NTM-PD patients during treatment, at the start (n = 14) and at 1 (n = 10), 3 (n = 10), 6 (n = 12), and 12 (n = 7) months. The bacterial microbiome changes were analyzed using 16S rRNA sequences (V3-V4 regions). Subgroup analysis included culture conversion (n = 9) and treatment refractory (n = 5) groups. In all patients, sputum alpha-diversity (ACE, Chao1, and Jackknife) significantly decreased during antibiotic treatment at 1, 3, 6, and 12 months compared to treatment initiation levels. Within the culture conversion group, genus/species-level beta-diversity showed differences at 1, 3, 6, and 12 months compared to treatment initiation (all p < 0.05). However, in the refractory group, there were no differences in beta-diversity at the genus/species levels in the sputum at any time point. In the linear discriminant analysis (LDA) effect sizes (LEfSe) analysis, the culture conversion group exhibited decreasing taxa at various levels (phylum/genus/species), but no significant increase in taxa was observed. LEfSe analysis of the refractory patient group revealed multiple taxa decreased during treatment. However, proportions of Veillonella dispar (LDA = 4.78), Fusobacterium periodonticum (LDA = 4.35), and Pseudomonas aeruginosa (LDA = 2.92) increased as the treatment period progressed in the refractory group. Sputum microbiota diversity decreases during NTM-PD treatment. In the culture conversion group, most taxa decrease, while some increase in the refractory group. These findings suggest that a distinct respiratory microbial community may exist in refractory NTM-PD patients compared to responsive antibiotic-treated patients.
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