As the sizes of the homologous regions varied due to differences in the left- and right-flanking regions, it could be presumed that PVL phage acquired the region encoding lukS-PV, lukF-PV, and int H 89 purchase by non-site-specific illegitimate recombination events. The 12.4-kb

region after ant and before ter in φ7247PVL carried 17 ORFs (FP07–FP23) related to DNA replication/transcriptional regulation. Among these 17 ORFs, the functions of only three ORFs could be predicted. FP13 encodes a single-strand DNA-binding protein (ssb), FP15 encodes a protein related to DNA replication, and FP20 encodes dUTPase (dut). FP13 (ssb) is highly homologous (98.9% identity) only to that of φSLT. FP15 has 100% identity with φSLT and 80.5% identity with φ108PVL. FP20 (dut) has the highest identity (77.3%) with φSa2usa. The two PVL phages (φ7247PVL and φ5967PVL) identified in this study shared several characteristics in common with previously reported PVL phages: (1) the same integration site; (2) carriage of a 29-bp core sequence at both ends of the prophage; (3) the same structural organization; and (4) carriage of five (or six) genes that are highly homologous to those of extant PVL phages. However, the regions encoding genes for the structure module and DNA replication/transcriptional regulation in these two PVL phages differed greatly

from those of extant PVL phages. The genomes of 15 phages, the aforementioned six PVL phages and nine representative prophages, were compared by dot plot (data not shown). Dot plots showed that φ7247PVL belonged to group 3 Sfi21-like cos-site Siphoviridae. Tanespimycin Electron

microscopic observation of φ5967PVL indicated that the phage shared an isometric head similar to that of group 1 phage (Fig. S1). These data indicate that the phages identified in ST59 strains are distinct from previously reported PVL phages and should be regarded as a novel third type of PVL-carrying phage. In this study, we also demonstrated that ST59 MRSA strains isolated from Japan and Taiwan are lysogens of the same novel third type of PVL phage. PVL-positive phage particles were induced from 11 of 12 Taiwanese MRSA strains. The sequences of φ5967PVL, chosen as a representative of the inducible Taiwanese strains, and φ7247PVL from a Japanese strain, are identical DNA ligase except for one nucleotide, resulting in a difference of amino acid in ORFs, glutamic acid in FP32 of φ7247PVL and glycine in TP32 of φ5967PVL. All 13 MRSA strains carried the same type V(5C2&5) SCCmec. Moreover, their pulsed-field gel electrophoresis banding patterns were closely similar (data not shown). As PVL-positive ST59 MRSA strains have rarely been identified in Japan, whereas they are the predominant Taiwanese CA-MRSA (Chen et al., 2005, 2009; Takizawa et al., 2005; Ma et al., 2006), JCSC7247 may have originated from Taiwan. PVL-positive ST59 MSSA strains have also been isolated in Taiwan (Chen et al., 2009).