rpoB Gene Sequence-Based Identification of Aerobic Gram-Positive Cocci of the Genera Streptococcus , Enterococcus , Gemella , Abiotrophia , and Granulicatella

Abstract

ABSTRACT We developed a new molecular tool based on rpoB gene (encoding the beta subunit of RNA polymerase) sequencing to identify streptococci. We first sequenced the complete rpoB gene for Streptococcus anginosus , S. equinus , and Abiotrophia defectiva . Sequences were aligned with these of S. pyogenes , S. agalactiae , and S. pneumoniae available in GenBank. Using an in-house analysis program (SVARAP), we identified a 740-bp variable region surrounded by conserved, 20-bp zones and, by using these conserved zones as PCR primer targets, we amplified and sequenced this variable region in an additional 30 Streptococcus , Enterococcus , Gemella , Granulicatella , and Abiotrophia species. This region exhibited 71.2 to 99.3% interspecies homology. We therefore applied our identification system by PCR amplification and sequencing to a collection of 102 streptococci and 60 bacterial isolates belonging to other genera. Amplicons were obtained in streptococci and Bacillus cereus , and sequencing allowed us to make a correct identification of streptococci. Molecular signatures were determined for the discrimination of closely related species within the S. pneumoniae - S. oralis - S. mitis group and the S. agalactiae - S. difficile group. These signatures allowed us to design a S. pneumoniae -specific PCR and sequencing primer pair.