Utility of rpoB Gene Sequencing for Identification of Nontuberculous Mycobacteria in the Netherlands

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In the Netherlands, clinical isolation of nontuberculous mycobacteria (NTM) has increased over the past decade. Proper identification of isolates is important, as NTM species differ strongly in clinical relevance. Most of the currently applied identification methods cannot distinguish between all different Mycobacterium species and complexes within species. rpoB gene sequencing exhibits a promising level of discrimination among rapidly and slowly growing mycobacteria, including the Mycobacterium avium complex. In this study, we prospectively compared rpoB gene sequencing with our routine algorithm of reverse line blot identification combined with partial 16S rRNA gene sequencing of 455 NTM isolates. rpoB gene sequencing identified 403 isolates to species level as 45 different known species and identified 44 isolates to complex level, and eight isolates remained unidentifiable to species level. In contrast, our reference reverse line blot assay with adjunctive 16S rRNA gene sequencing identified 390 isolates to species level (30 distinct species) and identified 56 isolates to complex level, and nine isolates remained unidentified. The higher discriminatory power of rpoB gene sequencing results largely from the distinction of separate species within complexes and subspecies. Also, Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium interjectum were separated into multiple groupings with relatively low sequence similarity (98 to 94%), suggesting that these are complexes of closely related species. We conclude that rpoB gene sequencing is a more discriminative identification technique than the combination of reverse line blot and 16S rRNA gene sequencing and could introduce a major improvement in clinical care of NTM disease and the research on the epidemiology and clinical relevance of NTM.