0 ± 0.18 8 3281 4.0 ± 0.22 25 2687 1.6 ± 0.22 9 2932 2.8 ± 0.19 26 2688 7.6 ± 0.07 10 3543 14 ± 1.21 27 2689 9.8 ± 0.28 11 3573 12 ± 0.20 28 2690 3.1 ± 0.16 12 V432 7.0 ± 0.25 29 2701 5.0 ± 1.12 13 V637

7.6 ± 0.30 30 2702 2.8 ± 0.23 14 V666 5.2 ± 0.11 31 2165 4.0 ± 0.13 15 V700 check details 8.0 ± 0.21 32 3624 1.0 ± 0.19 16 V723 1.3 ± 0.34 33 3878 2.2 ± 0.20 17 V694 4.0 ± 0.22 34 3890 6.4 ± 0.08 Strain 1, genome strain. Strains 2 to 34 were randomly selected clinical MRSA isolates that were all tst-positive and assigned to agr class 2 by PCR. Amounts of TSST-1 varied among strains and ranged from 0.8 to 14 μg/ml. A comparison of the nucleotide sequences from the 9 strains with the corresponding sequence of the agr class 2 reference strain S. aureus SA502A (GenBank accession no., AF001782), revealed no relevant changes in the agrD and agrB regions, whereas 4 strains had allelic variations in the coding region of agrC, which is the receptor for two component regulatory systems. Strain 3 had a point mutation at nucleotide position 28 of the coding region that replaced phenylalanine with isoleucine. Strain 10 also had a point mutation at nucleotide position 651 of the coding region that replaced glutamine with

histidine. Strain 8 had a 9-nucleotide deletion (nt 495 to 504 of the agrC coding sequence) that resulted in the deletion of leucine, lysine and isoleucine. Strain 2 had a nucleotide insertion that caused a frame-shift mutation, which in turn generated numerous stop codons. Although both strains 10 and 2 produced large amounts of TSST-1, Mdm2 inhibitor the agr locus did not consistently

vary in any way from that of the other strains (Table 2). We also sequenced the promoter regions Pembrolizumab of the tst gene, sar (staphylococcal accessory regulator) and the entire region of sigma factor B of these 9 strains. The sar is another positive regulatory locus for TSST-1 production that is required for maximal agr expression and sigma factor B is an important factor that feeds into the global regulatory network governing the expression of accessory genes [2, 8–10]. No relevant nucleotide changes were evident in the sequences of both promoter regions of the tst gene and sar as well as the entire sigma factor B region (Table 2). Table 2 Summary of nucleotide changes and predicted outcomes of mutations in the agr locus. Strain number Amount of TSST-1 produced (μg/ml) Changes in agrC region nucleotide sequence Predicted outcome tst promoter sarA sigB 1 3.5 NC NC NC NC 2 14 T(321) insertion Frameshift→Truncated AgrC NC NC NC 3 5 T 281A phe→ile NC NC NC 7 2 NC NC NC NC 8 4 Δ495~504 Deletion of leu-lys-ile NC NC NC 9 2.8 NC NC NC NC 10 14 G651T glu→his NC NC NC 11 12 NC NC NC NC 16 1.3 NC NC NC NC Data are from DNA sequencing of agr loci, tst promoter region, sarA and sigB from 9 strains.