16S ribosomal DNA sequence-based analysis of clinically significant gram-positive anaerobic cocci

Sequence analysis of the 16S rRNA gene represents a highly accurate and versatile method for bacterial classification and identification, even when the species in question is notoriously difficult to identify by phenotypic means. However, its use for identification based on public sequence databases is not without limitation due to the presence of ambiguous data in the databases. In this study, we evaluated the utility of 16S ribosomal DNA sequencing as a means of identifying clinically important gram-positive anaerobic cocci (GPAC) by sequencing 13 type strains of established GPAC species and 156 clinical isolates that had been studied only by phenotypic tests. Among the 13 type strains of GPAC species we tested, only 4 gave a "perfect" match with their corresponding sequences in GenBank, whereas the other 9 had lower sequence similarities (<98%). This indicates that data in the public database may be inaccurate at times. Based on the sequences of the 13 type strains obtained in this study, 84% (131 of 156) of the clinical isolates were accurately identified to species level, with the remaining 25 clinical strains revealing nine unique sequences that may represent eight novel species. This finding is in contrast to the phenotypic identification results, by which only 56% of isolates were correctly identified to species level.