CTM transformation medium was used to induce competence and for transformation, as described

previously [11]. The CSP concentration was 100 ng ml-1 and DNA concentration was 1 μg ml-1. The chromosomal source of DNA carrying mutated PBP alleles was the 9V derivative Spain23F-1 clone (strain URA1258) which carries the following mutations near or within the conserved motifs on the PBPs: Gln443Glu, Thr451Ala, Glu481Gly, Ser485Ala and Thr494Ala in PBP2B, Thr338Ala, Met343Thr, Ala346Ser, Ala347Ser, Leu364Phe, Ile371Thr, Arg384Gly, Leu546Val and Asn605Thr in PBP2X, and Thr371Ala, Glu388Asp, click here Pro432Thr, Asn546Gly, Thr574Asn, Ser575Thr, Gln576Gly, Phe577Tyr, Leu606Ile, Asn609Asp, Leu611Phe and Thr612Leu SB202190 price in PBP1A. Transformants were selected on plates containing 0.1 μg ml-1 and 0.5 μg ml-1 penicillin, and appropriate integration of PBP mutations was confirmed by nucleotide sequencing. Plates containing 2 μg ml-1 rifampicin and 10 μg ml-1 chloramphenicol were used to select rif-23

and Δstkp::cat transformants. All constructions were verified by PCR with the primers AZD3965 described in Table 2[6, 12]. Spontaneous mutation to penicillin in DNA free medium was < 10-9. Penicillin G was from Atral, Castanheira do Ribatejo, Portugal, and rifampicin was from Aventis Pharma. To assess StkP and PBPs conservation 50 strains were randomly selected among those isolated between 1994 and 2005 in various areas in Portugal; they included forty invasive isolates from blood and cerebrospinal fluid and ten colonizing isolates from the nasopharynx of asymptomatic carriers. Half of the isolates (n = 25) were non-susceptible to penicillin [minimal inhibitory concentration (MIC) > 0.1 μg ml-1]. These isolates were compared to the following reference strains: the highly for resistant serotype 9V strain URA1258, two susceptible and three non-susceptible strains provided by the ATCC and the unencapsulated strain R6 (Table 1). Table 1 Strains and plasmids used in the study Strain or plasmid Genotype or description Phenotypea Source or reference S. pneumoniae       R6 Non-capsulated

D39 derivative, susceptible reference strain; genome sequence available (R6) AtbS Laboratory stock ATCC BAA-334 Virulent reference strain, genome sequence available (TIGR4) AtbS ATCC ATCC 51916 Reference strain Tennessee 23F-4 PenR, EryR, ATCC ATCC 700670 Reference strain Spain 6B-2 PenR, CmR, TetR ATCC ATCC 700673 Reference strain Hungary 19A-6 PenR, EryR, CmR, TetR ATCC URA1258 Multiresistant strain closely related to Spain 23F-1 clone PenR, CmR, TetR [21] Cp1015 D39 derivate, str1; hexA SmR [31] Cp1016 D39 derivate, str1; hexA, rif23 RifR [31] Cp7000 Cp1015, stkP::cat SmR CmR This study Pen1 Cp1015, penA, and pbpX from URA1258 SmR PenR This study Pen2 Cp1015, penA, pbpX and pbp1A from URA1258 SmR PenR This study Pen1STK Cp1015Pen1, stkP::cat SmR PenR CmR This study Pen2STK Cp1015Pen2, stkP::cat SmR PenR CmR This study E.