Comparison of five genotypic techniques for identification of optochin-resistant pneumococcus-like isolates

Three PCR techniques (amplification of the psaA, ply, and lytA genes) and a commercial kit (AccuProbe [GenProbe, San Diego, Calif.], based on hybridization with the 16S rRNA gene), all four of which claimed to be specific for Streptococcus pneumoniae, were used to identify 49 alpha-hemolytic streptococcal isolates suspected of being pneumococci. The definite phenotypic identification of these organisms as S. pneumoniae was difficult when optochin susceptibility and the presence of a capsule were taken as markers. Furthermore, RsaI digestion of the amplified 16S rRNA gene was applied. All 49 strains were optochin resistant. Eleven of these were encapsulated and were identified as pneumococci by all tests. Twenty of the 38 unencapsulated strains were unambiguously identified as nonpneumococci by all tests. The identities of another 18 unencapsulated strains remained inconclusive due to highly variable reactions for all phenotypic and genotypic techniques applied. The AccuProbe test was positive for seven strains for which the results of the other tests were inconclusive. RsaI restriction of the amplified 16S rRNA gene confirmed the AccuProbe result for all strains, while the result of the psaA-specific PCR was in concordance with encapsulation for all strains. The results presented here indicate that identification problems continue to exist for some strains, despite the application of genotypic and phenotypic tests in combination. We found the psaA-specific PCR to be the genotypic technique best suited for the identification of genuine pneumococci and optochin-resistant pneumococci.