Identification of alpha-hemolytic streptococci by pyrosequencing the 16S rRNA gene and by use of VITEK 2

Alpha-hemolytic streptococci are very difficult to identify by phenotypic methods. In this study, a pyrosequencing method for the identification of streptococcal species based on two variable regions of the 16S rRNA gene is described. Almost all studied streptococcal species (n = 51) represented by their type strains could be differentiated except for some closely related species of the Streptococcus bovis or S. salivarius group. The pyrosequencing results of alpha-hemolytic streptococci isolated from blood (n = 99) or from the normal pharyngeal microbiota (n = 25) were compared to the results obtained by the VITEK 2 with GP card (bioWrieux, Marcy I'Etoile, France). As expected, the results of the two methods did not completely agree, but 93 (75.0%) of the isolates assigned to the same streptococcal group by both methods and 57 (46.0%) reached consistent results at the species level. However, 10 strains remained unidentified by VITEK 2, and 4 isolates could not be assigned to any streptococcal group by pyrosequencing. Identification of members of the S. mitis and S. sanguinis groups proved difficult for both methods. Furthermore, the pyrosequencing analysis revealed great sequence variation, since only 43 (32.3%) of the 133 isolates analyzed by pyrosequencing had sequences identical to a type strain. The variation was greatest in the pharyngeal isolates, slightly lower in the blood culture isolates, and nonexistent in invasive pneumococcal isolates (n = 17) that all had the S. pneumoniae type strain sequence. The resolution of the results obtained by the two methods is impeded by the lack of a proper gold standard.