BackgroundThe role of bacterial microbiota in the pathogenesis of nontuberculous mycobacterial pulmonary disease (NTM-PD) is unclear. We aimed to compare the bacterial microbiome of disease-invaded lesions and non-invaded lung tissue from NTM-PD patients.MethodsWe analyzed lung tissues from 23 NTM-PD patients who underwent surgical lung resection. Lung tissues were collected in pairs from each patient, with one sample from a disease-involved site and the other from a non-involved site. Lung tissue microbiome libraries were constructed using 16S rRNA gene sequences (V3–V4 regions).ResultsSixteen (70%) patients hadMycobacterium aviumcomplex (MAC)-PD, and the remaining seven (30%) hadMycobacterium abscessus-PD. Compared to non-involved sites, involved sites showed greater species richness (ACE, Chao1, and Jackknife analyses, all p = 0.001); greater diversity on the Shannon index (p = 0.007); and genus-level differences (Jensen-Shannon, PERMANOVA p = 0.001). Analysis of taxonomic biomarkers using linear discriminant analysis (LDA) effect sizes (LEfSe) demonstrated that several genera, includingLimnohabitans,Rahnella,Lachnospira,Flavobacterium,Megamonas,Gaiella,Subdoligranulum,Rheinheimera,Dorea,Collinsella, andPhascolarctobacterium, had significantly greater abundance in involved sites (LDA >3.00, p <0.05, and q <0.05). In contrast,Acinetobacterhad significantly greater abundance at non-involved sites (LDA = 4.27, p<0.001, and q = 0.002). Several genera were differentially distributed between lung tissues from MAC-PD (n = 16) andM.abscessus-PD (n = 7), and between nodular bronchiectatic form (n = 12) and fibrocavitary form (n = 11) patients. However, there was no genus with a significant q-value.ConclusionsWe identified differential microbial distributions between disease-invaded and normal lung tissues from NTM-PD patients, and microbial diversity was significantly higher in disease-invaded tissues.Trial registrationClinical Trial registration number:NCT00970801.