Differentiation of Streptococcus pneumoniae from Streptococcus mitisStreptococcus oralis by recA-gene-based PCR

Abstract number: P1850

Zbinden A., Köhler N., Bloemberg G.V.

Objectives: 16S rRNA gene analysis is the gold standard for molecular identification of bacteria. However, the discriminatory power of the 16S rRNA-gene is too low to differentiate S. pneumoniae from S. mitis and S. oralis. We propose the recA-gene as additional, more discriminatory target for proper identification to species level.

Methods: A recA-PCR was developed and investigated with a preliminary number of strains. Type strains of S. pneumoniae, S. mitis and S. oralis as well as 18 strains of S. pneumoniae identified by conventional phenotypic methods including colony morphology, susceptibility to optochin and bile solubility, were analysed. With a specific set of recA-primers, a 313-bp fragment of the recA-gene was amplified and sequenced. The recA-sequences were aligned for the search of positions that distinguished those closely related species (molecular signatures). 20 strains formerly identified as belonging to the group S. pneumoniae/mitis/oralis by 16S rRNA-gene sequencing were tested using the recA-PCR for species identification.

Results: Partial recA-gene sequence similarity between the type strains was 95.2% for S. pneumoniae and S. mitis, 91.4% for S. pneumoniae and S. oralis and 91.7% for S. oralis and S. mitis. The recA-sequences of all 18 isolates phenotypically identified as S. pneumoniae showed >99.7% sequence homology to recA-sequences of S. pneumoniae. Within the amplified recA-fragment of the type strains and the 18 S. pneumoniae isolates, we found 7 signature nucleotides specific for S. pneumoniae. Those nucleotides were found in all the S. pneumoniae strains and differed from those of the type strains of S. mitis and S. oralis in every single position.

The recA-sequences of the 20 strains grouped to S. pneumoniae/mitis/oralis revealed <95.9% sequence homology to recA-sequences of S. pneumoniae. Additionally, differentiation from S. pneumoniae according to the 7 specific nucleotide positions was determined for all 20 isolates, the identification of which resulted in S. mitis or oralis.

Conclusion: Our investigations present a more discriminatory molecular tool than the 16S rRNA-gene for proper identification of pneumococci. A recA-gene based PCR and analysis of 7 signature positions within the amplified fragment allowed an accurate differentiation of S. pneumoniae from S. mitis/S. oralis.