Molecular analysis of clinical isolates previously diagnosed as Mycobacterium intracellulare reveals incidental findings of "Mycobacterium indicus pranii" genotypes in human lung infection

Abstract

Background: Mycobacterium intracellulare is a major cause of Mycobacterium avium complex lung disease in many countries. Molecular studies have revealed several new Mycobacteria species that are closely related to M. intracellulare. The aim of this study was to re-identify and characterize clinical isolates from patients previously diagnosed with M. intracellulare lung disease at the molecular level.

Methods: Mycobacterial isolates from 77 patients, initially diagnosed with M. intracellulare lung disease were re-analyzed by multi-locus sequencing and pattern of insertion sequences.

Results: Among the 77 isolates, 74 (96 %) isolates were designated as M. intracellulare based on multigene sequence-based analysis. Interestingly, the three remaining strains (4 %) were re-identified as "Mycobacterium indicus pranii" according to distinct molecular phylogenetic positions in rpoB and hsp65 sequence-based typing. In hsp65 sequevar analysis, code 13 was found in the majority of cases and three unreported codes were identified. In 16S-23S rRNA internal transcribed spacer (ITS) sequevar analysis, all isolates of both species were classified within the Min-A ITS sequevar. Interestingly, four of the M. intracellulare isolates harbored IS1311, a M. avium-specific element. Two of three patients infected with "M. indicus pranii" had persistent positive sputum cultures after antibiotic therapy, indicating the clinical relevance of this study.

Conclusions: This analysis highlights the importance of precise identification of clinical isolates genetically close to Mycobacterium species, and suggests that greater attention should be paid to nontuberculous mycobacteria lung disease caused by "M. indicus pranii".

Fig. 1

A hsp65 sequence-based phylogenetic tree of 77 isolates including the M. intracellulare type, M. intracellulare clinical strains, “M. indicus pranii”, and other MAC species using the neighbor-joining method with Kimura’s two parameter distance correction model. Bootstrap analyses determined from 1000 replicates are indicated at the nodes. Bar, 0. 5 % difference in nucleotide sequence. GenBank accession numbers are given in parentheses

Fig. 2

The rpoB sequence-based phylogenetic tree of 77 isolates including the M. intracellulare type, M. intracellulare clinical strains, “M. indicus pranii”, and other MAC (sub-)species using the neighbor-joining method with Kimura’s two parameter distance correction model. Bootstrap analyses determined from 1000 replicates are indicated at the nodes. Bar, 0.5 % difference in nucleotide sequence. GenBank accession numbers are given in parentheses

Fig. 3

The phylogenetic tree based on concatenated hsp65 and rpoB sequences of 77 isolates including M. intracellulare type, M. intracellulare clinical strains, “M. indicus pranii”, and other MAC (sub-)species using the neighbor-joining method with Kimura’s two-parameter distance correction model. Bootstrap analyses determined from 1000 replicates are indicated at the nodes. Bar, 0.5 % difference in nucleotide sequence. GenBank accession numbers are shown in Figs. 1 and 2

Fig. 4

High-resolution computed tomography (HRCT) findings of “M. indicus pranii” lung disease. a A 27-year-old male with a prior history of pulmonary tuberculosis. Chest HRCT shows multiple bilateral large cavities in both upper lobes. The patient died after initiation of combination antibiotic therapy due to an accident. b A 72-year-old female. Chest HRCT shows severe bronchiectasis in the right middle lobe and lingular segment of the left upper lobe. Note a cavitary lesion in the right middle lobe, as well as multiple small nodules and tree-in-bud appearance in both lungs