P1448 Poster Session V Molecular Diagnosis of Respiratory Tract

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P1448 Poster Session V Molecular diagnosis of respiratory tract bacterial infections STREPTOCOCCUS PNEUMONIAE AND STREPTOCOCCUS PSEUDOPNEUMONIAE: CORRECT IDENTIFICATION BY A MULTIPLEX-PCR BASED ON SPECIES SPECIFIC 16S RDNA BP DIFFERENCES. R. Dargis1, M. Kemp2, J.J. Christensen1 1Department of Clinical Microbiology, Slagelse Hospital, Slagelse, Denmark ; 2Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark Objective: The species Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis , Streptococcus pseudopneumoniae, Streptococcus infantis and the newly designated Streptococcus tigurinus are phylogenetically very close, especially S. pneumoniae and S. mitis, hampering the exact identification of S. pneumoniae, when performing molecular examination on strains or on specimens from normally sterile sites. Based on BLAST screened differences in 16S rRNA gene sequences between S. pneumoniae and related species, a multiplex PCR test separating pneumococcal and S. pseudopneumoniae strains from the other closely related species was created and applied on collection strains of the mentioned streptococcal species. Materials: Strains: Fourty-four collection strains were examined. Strains belonged to 1) S. pneumoniae (n = 18) 2) S. mitis: (n = 11) 3) S. oralis (n = 3) 4) S. pseudopneumoniae (n = 6) 5) S. infantis (n = 5) and 6) S. tigurinus (n = 1). Multiplex-PCR: The Multiplex-PCR tested for presence of three previous extensively BLAST screened differences at positions 203, 623 and 802 ((relative to the annotated 16SrRNA gene sequence in the genome of strain R6 [accession no. AE007317.1] in the 16S rRNA gene; respectively base pair 'C', 'G' and 'T' for S. pneumoniae and 'A', 'C' and 'C' for S. mitis/S. oralis giving band sizes of 470 bp for S. pneumoniae (203F/623R) and 235 bp for S. mitis/S. oralis (623F/802R). The multiplex was performed with touchdown. The annealing temperature of the reaction was decreased 1ºC for every cycle from 70ºC to a 'touchdown' at 60ºC at which temperature 20 cycles were carried out with the following PCR programme: 95ºC in 15 sec, 60ºC in 30 sec, 72ºC in 30 sec. Finally 1 cycle at 72ºC in 7 minutes was carried out. Results: Pneumococcal strains exhibited presence of the S. pneumoniae specific band (see Table). S. mitis, S. oralis, S. infantis, and S. tigurinus strains the S. mitis/S. oralis band and no bands were detected for the six S. pseudopneumoniae strains. Bacterial species 623F/802R (S. mitis/S. oralis) 203F/623R (S. pneumoniae) Streptococcus pneumoniae (n=18) 0 + Streptococcus mitis (n=11) + 0 Streptococcus oralis (n=3) + 0 Streptococcus infantis (n=5) + 0 Streptococcus tigurinus (n=1) + 0 Streptococcus pseudopneumoniae (n=6) 0 0 Conclusion: The BLAST screened species specific bp differences in the 16S rRNA gene were usefull in correctly identifying collection strains of S. pneumoniae. Interestingly, the six S. pseudopneumoniae strains were as well separated from the S. pneumoniae strains as from the other strains belonging to other species of the mitis group. Thus, it seems that the multiplex PCR definitively can confirm presence of S. pneumoniae and separate presence of S. pseudopneumoniae from other closely related S. mitis group species..

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Supporting Information
Supporting Information Wade et al. 10.1073/pnas.1423754112 Materials and Methods alone (soluble) or after a 20-min incubation at 37 °C with 1-pal- Primary Sequence Alignment and Cluster Analysis. Sequence align- mitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol liposomes ment of the CDC primary structures was performed using ClustalW (20 μL) to allow assembly of the oligomer. For all experiments, (1), and the phylogenetic trees were derived from a maximum emission scans of the unlabeled soluble or liposome-bound toxin parsimony analysis of the ClustalW-derived alignments, using were measured and subtracted from the experimental data to Mega6geneanalysissoftware(2). eliminate any changes in fluorescence not resulting from the NBD probe. An excitation wavelength of 480 nm and 4 nm bandpass Steady-State Fluorometry. Steady-state experiments were performed were used. Resolution was set to 1 nm with 0.1 s integration. on a Fluorolog-3 Spectrofluorometer with the FluorEssence soft- β4β5 disengagement was measured as previously described (3) ware. The polarity of the environment surrounding Cys-substituted for PFOV322C or PFOV322C:Y181A, where a 1:1 molar ratio of PFOE183C or PFOK336C (176 pmol total: 25% NBD labeled, 75% NBD-labeled to unlabeled protein was used to minimize NBD unlabeled) was determined by performing an NBD emission scan self-quenching in the oligomeric complex. Settings for measuring (500–600 nm) of proteins either in buffer (2.5 mL final volume) NBD emission were set as described above. 1. Larkin MA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21): 3. Ramachandran R, Tweten RK, Johnson AE (2004) Membrane-dependent conforma- 2947–2948.
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