Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species

Abstract

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing approximately 440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1- to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.

FIG. 1

Phylogenetic tree of mycobacteria, including one representative strain for each species. Multiple sequence alignments were determined using the CLUSTAL method algorithm in the Megalign component of the Lasergene program (version 4.01). (A) Phylogenetic tree inferring relationships among rapid growers of the Mycobacterium genus. (B) Phylogenetic tree inferring relationships among slow growers of the Mycobacterium genus. The tree was rooted using Nocardia asteroides as the outgroup sequence. Sequences were determined in our laboratory unless indicated by a GenBank accession number.

FIG. 1

Phylogenetic tree of mycobacteria, including one representative strain for each species. Multiple sequence alignments were determined using the CLUSTAL method algorithm in the Megalign component of the Lasergene program (version 4.01). (A) Phylogenetic tree inferring relationships among rapid growers of the Mycobacterium genus. (B) Phylogenetic tree inferring relationships among slow growers of the Mycobacterium genus. The tree was rooted using Nocardia asteroides as the outgroup sequence. Sequences were determined in our laboratory unless indicated by a GenBank accession number.