pneumoniae culture from normally sterile body fluid (blood/cerebrospinal fluid). The IMPACT surveillance study has research ethics board approval at each participating centre to obtain demographic, clinical and microbiologic information on all cases without the requirement for written informed consent. S. pneumoniae strains were VRT752271 verified and serotyped as part of

IMPACT’s routine surveillance protocol. The investigation described here was undertaken using IMPACT’s 19A invasive strains, collected with ethical approval between 1991 and 2009. Strains were grown overnight at 5% CO2 on Columbia Blood Agar (prepared according to manufacturer’s instructions, Becton see more Dickinson and Company, Difco™, Sparks, Maryland, USA) plates with Optochin Disk (used according to manufacturer’s instructions, Sigma-Aldrich, Oakville, Ontario, Canada) susceptibility and the presence alpha hemolysis used for species PX-478 verification. Genomic DNA was then isolated with the QIAamp DNA Mini Kit (used according to manufacturer instructions, Qiagen, Toronto, Ontario, Canada). Sequencing methodology Each of the seven typing alleles was evaluated with both the standard (Table 1) and alternative (Table 2) MLST primers. PCR solutions were prepared for each primer set consisting of: 11 μl sterile

distilled water, 2.5 μl of 10× reaction buffer (5 ml 1 M KCL, 5 ml 1 M (NH4)2SO4, 5 ml 2 M Tris–HCl pH 8.8, 5 ml 200 mM MgSO4, 5 ml 10% Triton X-100, water to 50 ml), 2.5 μl of 2 mM dNTPs, 2.5 μl of each primer at 5 μM, 1 unit pfu enzyme (Thermo Scientific, Ottawa, Ontario, Canada) and 2 μl of genomic DNA template at 50 – 300 ng/μl. All PCRs were performed in a BioRad (Mississauga, Ontario, Canada) Thermocycler with annealing temperatures specific to each primer set (Table 1 and 2). Amplification was verified by visualizing gene products with gel electrophoresis on a 1% ethidium bromide agarose gel with a voltage of 110 V for 25 minutes. Verified PCR products were purified with the E.Z.N.A Cycle Pure Kit (used according to

manufacturer’s instructions OMEGA Biotek, Norcross, Georgia, USA). Purified products were subsequently verified via spectrophotometry (used according to manufacturer’s instructions NanoDrop 1000 Spectrophotometer, until Thermo Scientific, Ottawa, Ontario, Canada). Purified samples with a concentration of greater than 3 ng/μl, and 260 nm/280 nm absorbance values between 1.0 and 2.0 were accepted to send for sequencing. Sequencing was carried out at both Macrogen Corporation, Rockville USA, and the University of Calgary, Calgary Canada, DNA Core Services facility. Assessing sequence coverage The sequencing results were manually inspected for quality with the open source program 4Peaks, and sequence coverage was inspected by using the Multiple Sequence Alignment by Fast Fourier Transform (MAFFT) program, available through the European Bioinformatics Server [27].