94 × 10-1 K27 + 1.27 × 10-1 K51 + 6.24 × 10-1 K54 + 11.1 K1179 + 9.06 × 10-1 Transformants     K744-T + <1 × 10-4 K2480-T + <1 × 10-4 To

test for the presence of the ß-lactamase gene, blaZ was amplified by PCR using a primer set K shown in Table 3. N315 and FDA209P cells were used as positive and negative references, respectively. As seen in Figure 2, the PCR products amplified from N315 cells showed a large distinct band with nucleotide numbers corresponding to about 170 bp, FK228 in vivo which was the expected PCR product. The PCR product was undetectable when the FDA209P DNA was used as a template. Similarly, PCR was carried out using the template DNA from Mu3, K101, K638, K670, K744 and K2480 cells and no detectable band was found (Figure 2). The results suggested that these BIVR strains did not have the ß-lactamase gene, which was fully consistent with the finding of undetectable ß-lactamase activity. In contrast, PCR experiments

using the DNA template from non-BIVR strains showed clear bands corresponding to the expected blaZ product. These results Cell Cycle inhibitor were again consistent with that of the ß-lactamase assay and with the above explanation (i); whether or not BIVR cells possessed the gene encoding ß-lactamase, but did not give the answer to the above question (ii); whether the expression of the ß-lactamase gene in BIVR could be suppressed. Therefore, the following experiments were conducted. Table 3 Primer sets used Code Nucleotide sequence A (F) 5’-GGTTGCTGATAAAAGTGGTCAA-3’ (R) 5’-CTCGAAAATAATAAAGGGAAAATCA-3’ B (F) 5’-AAGAAATCGGTGGAATCAAAAA-3’ (R) 5’-GTTCAGATTGGCCCTTAGGA-3’ C (F) 5’-TTGCCTATGCTTCGACTTCA-3’ (R) 5’-GCAGCAGGCGTTGAAGTATC-3’ D (F) 5’-TCAAACAGTTCACATGCCAAA-3’

(R) 5’-TTTTTGATTCCACCGATTTCTT-3’ E (F) 5’-GCCATTTTGACACCTTCTTTC-3’ (R) 5’-CGAAGCATAGGCAAATCTCTT-3’ F (F) 5’-TGAGGCTTCAATGACATATAGTGATAA-3’ (R) 5’-GTTCAGATTGGCCCTTAGGA-3’ else G (F) 5’-TGTTTAATAATAAAAACGGAGACACTT-3’ (R) 5’-TCAACTTATCATTTGGCTTATCACTT-3’ H (F) 5’-AAGAAATCGGTGGAATCAAAAA-3’ (R) 5’-TTTAAAGTCTTGCCGAAAGCA-3’ I (F) 5’-AAGAAATCGGTGGAATCAAAAA-3’ (R) 5’-TCGAAAATAATAAAGGGAAAATCA-3’ J (F) 5’-GCCATTTTGACACCTTCTTTC-3’ (R) 5’-AGCAGCAGGCGTTGAAGTAT -3’ K* (F) 5’-ACTTCAACACCTGCTGCTTTC-3’ (R) 5’-TGACCACTTTTATCAGCAACC-3’ * Primer K was from reference [19]. F and R denote the forward and reverse sequences, respectively. Codes correspond with that in Figure 3. Figure 2 Agarose gel electrophoretograms of the PCR product. Primer K was used for the PCR of blaZ and the conditions for the thermal cycler setting are given in the text. A fixed agarose concentration (2%) was used. The gel was stained with GelRed and visualised under UV light. Marker, LowRange 100 bp DNA markers; FDA209P, negative control; N315, positive control; the MRSA class and strain number are shown in the figure.