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Streptococcus Pneumoniae and Streptococcus Pseudopneumoniae: Correct Dept Correspondance: Jens Jørgen Christensen Streptococcus pneumoniae and Streptococcus pseudopneumoniae: Correct Dept. of Clinical Microbiology Slagelse Hospital, 4200 Slagelse Denmark identification by a multiplex-PCR based on species specific 16S rRNA gene bp email: [email protected] differences. ECCMID2014 Rimtas Dargis1, Michael Kemp2, Jens Jørgen Christensen1 P 1448 Departments of Clinical Microbiology at 1Slagelse Hospital, Slagelse and 2Odense University Hospital, Odense, Denmark Table 1. Presence or absence of PCR products in different streptococcal species when tested with the two Objective: primer systems 623F/802R and 203F/623R The species Streptococcus pneumoniae, Streptococcus mitis, Bacterial species 623F/802R 203F/623R Streptococcus oralis, Streptococcus pseudopneumoniae, Streptococcus (S. mitis/S. oralis) (S. pneumoniae) infantis and the newly designated Streptococcus tigurinus are Streptococcus pneumoniae (n=18) 0 + Streptococcus mitis (n=11) + 0 phylogenetically very close. Especially, S. pneumoniae and S. mitis, Streptococcus oralis (n=3) + 0 hampering the exact identification of S. pneumoniae, when performing Streptococcus infantis (n=5) + 0 molecular examination on strains or specimens from normally sterile Streptococcus tigurinus (n=1) + 0 sites. Based on BLAST screened differences in 16S rRNA gene sequences Streptococcus pseudopneumoniae (n=7) 0 0 between S. pneumoniae and related species, a multiplex-PCR was Table 2. NCBI screened base differences (ref. 1 and own observations) at three base positions for different developed separating pneumococcal and S. pseudopneumoniae strains streptococcal species from the other closely related species. The method was applied on 16S rRNA gene base positions 203 630 860 S. pneumoniae C G T collection strains of the mentioned streptococcal species. S. mitis/oralis/infantis/tigurinus A C C S. pseudopneumoniae A G C Materials and methods: Conclusions: Strains: Fourty-five collection strains were examined, see Table 1. Multiplex-PCR: The multiplex-PCR tested for presence of three previous extensively NCBI- • The assay was useful in correctly identifying collection strains of S. database screened differences at positions 203, 623 and 802 (relative to the annotated 16S rRNA pneumoniae. gene sequence in the genome of strain R6 [accession no. AE007317.1], see Table 2) giving band • S. pseudopneumoniae strains were as well separated from the S. sizes of 470 bp for S. pneumoniae (203F/623R) and 235 bp for S. mitis/S. oralis (623F/802R). The multiplex-PCR was performed with touchdown. The annealing temperature of the reaction was pneumoniae strains as from other mitis group strains. decreased 1°C for every cycle from 70 °C to a “touchdown” at 60 °C at which temperature 20 • It seems that the multiplex-PCR definitively can confirm presence of S. cycles were carried out with the following PCR program: 95 °C in 15 sec, 60 °C in 30 sec, 72 °C in pneumoniae and separate presence of S. pseudopneumoniae from other 30 sec. Finally, 1 cycle at 72 °C in 7 minutes was carried out. closely related S. mitis group species. Results: Reference: Pneumococcal strains exhibited presence of the S. pneumoniae specific band (see Table 1). S. 1. Scholz CF, Poulsen K, Kilian M. 2012. Novel molecular method for identification of Streptococcus mitis, S. oralis, S. infantis, and S. tigurinus strains the S. mitis/S. oralis band. No bands were pneumoniae applicable to clinical microbiology and 16S rRNA sequence-based microbiome detected for the seven S. pseudopneumoniae strains. studies. J. Clin. Microbiol. 50(6):1968-73. .
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