5 kb EcoRI/SacII fragment containing stkP and flanking regions wi

5 kb EcoRI/SacII fragment containing stkP and flanking regions with cat cassette

inserted ApR, CmR [6] a: ApR, resistant to ampicillin; AtbS, Adriamycin molecular weight Susceptible to all tested antibiotics; CmR, resistant to chloramphenicol; EryR, resistant to erythromycin; NalR, resistant to nalixidic acid; PenR, non-susceptible to penicillin G; RifR, resistant to rifamycin; SmR, resistant to streptomycin; TetR, resistant to tetracycline. Table 2 Primers PU-H71 mw used for PCR amplification Primer Name Primer sequence Gene targeted Reference STKP-F 5′-AGGATGCCATATGATCCAAATCGGCAA-3′ stkP [6] STKP-R 5′-TTGATTATGAATTCGCTTTTAAGGAGTAGC-3′ stkP [6] STKP-F2 5′-GTAGGACAGAATTCAAGACAAGTCTACATACA-3′ stkP [6] pbp1aF 5′-CCAGCAACAGGTGAGAGTC-3′ pbp1A [12] pbp1aR 5′-GTAAACACAAGCCAAGACAC-3′

pbp1A [12] pbp1aF2 5′-GAACTTCAAGACAAGGCAGT-3′ pbp1A [12] pbp2bF 5′-CCGTCTTAATCCCGATACC-3′ penA [12] pbp2bR 5′-ATTTTTGGGTGACTTGTTGAG-3′ penA [12] pbp2xF 5′-GGAATTGGTGTCCCGTAAGC-3′ pbpX [12] pbp2xR 5′-CATCTGCTGGCCTGTAATTTG-3′ pbpX [12] Measurements of penicillin susceptibility The MIC of penicillin G for the strains constructed were determined in duplicate by E-test (AB Biodisk, Solma, Sweden) according to the manufacturer’s recommendations (incubation at 35°C in 5% CO2 for 18 to 24 H), and for clinical isolates by an agar dilution method with the testing conditions and susceptibility interpretation standards proposed by the Clinical and Laboratory acetylcholine Standards Institute (CLSI) [13]. Strains were considered penicillin susceptible for MIC values ≤ 0.06 μg ml-1, intermediate MIC for values of 0.1 – 1 μg ml-1, and highly Selleck TSA HDAC resistant for MIC values ≥ 1.6 μg ml-1. Strains were classified as non-susceptible for MIC values ≥ 0.1 μg ml-1, according to CLSI criteria. stkP genotyping by amplification and nucleotide sequencing The stkP gene of clinical strains was amplified by PCR using the primers listed

in Table 2 and a Qiagen multiplex PCR kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In brief, this routinely involved 40 cycles with an annealing temperature of 56°C for 1 minute. The PCR products were purified on ExoSAP-IT (USB, Cleveland, Ohio) and the nucleotide sequence was established (BigDye Cycle sequencing kit v1.1 from Applied Biosystems, Foster City, California). BioNumerics software v3.5 (Applied Maths, Sint-Martens-Latem, Belgium) was used for contig assemblages of the DNA sequences. Genetic diversity of the StkP kinase in 56 pneumococcal strains The amino acid sequences deduced from the 56 stkP genes were aligned using the CLUSTALW program built in the MEGA version 4 software package [14]. There were a total of 637 positions in the final dataset, of which 8 were parsimony informative. The evolutionary history was inferred using the Maximum Parsimony (MP) method [15].

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