Initial denaturation of DNA at 95 °C for 10 min

was followed by 40 cycles of amplification (95 °C for 15 s and 60 °C for 45 s), ending with a dissociation phase at 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s, and 60 °C for 15 s. Primers were as follow: blaZ-F (ACGAAATCGGTGGAATCAAA) and blaZ-R (AGCAGCAGGCGTTGAAGTAT) for blaZ (product length, 115 bp); mecA1575 (AGGTTACGGACAAGGTGAAATACTG) and mecA1657 (TGTCTTTTAATAAGTGAGGTGCGTTAA) for mecA (product length, 106 bp); and 53D-F (CGACAAAAGGCATTCAACAA) buy AZD6738 and 53D-R (ACGTTCAAAAATCGCTTGCT) for the 4867-bp HindIII DNA fragment of bacteriophage φ53 cloned in the pUC18 vector (GenBank accession number, AF513856; product length, 139 bp) that is specific for all serogroup B phages (Doškař et al., 2000). The basis for calculating selleck inhibitor the unknown quantity of PCR product

is that the 10-fold difference in the amount of DNA in two samples will manifest in the difference in their quantification cycles with a value of 3.22 (Lee et al., 2006). By comparing quantities of the blaZ plasmid gene and mecA single-copy chromosome gene, the plasmid copy number (PCN) in the donor strain was determined, which is necessary to determine the number of transducing particles carrying the penicillinase plasmid in comparison with the standard consisting of genomic DNA. PCN was determined according to the equation PCN = [size of chromosomal DNA (bp) × amount of plasmid DNA (pg)]/[size of plasmid DNA (bp) × amount of genomic DNA (pg)] (Lee et al., 2006). To calculate the standard copy number (SCN), the following formula was used: SCN = (amount of DNA per reaction in ng)/(Mr/NA), where Mr = size of S. aureus USA300 genomic DNA × normalized weight of nucleotide base (650 Da),

and NA is the Avogadro constant. second The standard curves were repeatable, and amplification efficiency of PCR reactions ranged between 90% and 100%. Genotypic characteristics and plasmid content of clinical strains used as donors and/or recipients in transduction experiments are listed in Table 1. Ability to transduce plasmids from the USA300 strains was first confirmed using the φ80α phage. The prophageless, plasmidless, and restriction-deficient RN4220 strain was used as control recipient strain for the transductions. Transduction frequency in this system ranged from 3.9 × 10−6 CFU/PFU to 5.1 × 10−6 CFU/PFU for penicillinase plasmids and from 2.7 × 10−6 CFU/PFU to 3.4 × 10−6 CFU/PFU for tetracycline resistance plasmid pT181. Based upon successful transduction of penicillinase plasmids and the tetracycline resistance plasmid into RN4220, plasmids were transduced between the isolates from the USA300 clone by the φ80α phage and subsequently by the naturally occurring φJB prophage which in a number of our experiments demonstrated excellent transducing abilities (unpublished results).