Healthcare- and Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) and Fatal Pneumonia with Pediatric Deaths in Krasnoyarsk, Siberian Russia: Unique MRSA's Multiple Virulence Factors, Genome, and Stepwise Evolution

Methicillin-resistant Staphylococcus aureus (MRSA) is a common multidrug-resistant (MDR) pathogen. We herein discussed MRSA and its infections in Krasnoyarsk, Siberian Russia between 2007 and 2011. The incidence of MRSA in 3,662 subjects was 22.0% and 2.9% for healthcare- and community-associated MRSA (HA- and CA-MRSA), respectively. The 15-day mortality rates for MRSA hospital- and community-acquired pneumonia (HAP and CAP) were 6.5% and 50%, respectively. MRSA CAP cases included pediatric deaths; of the MRSA pneumonia episodes available, ≥27.3% were associated with bacteremia. Most cases of HA-MRSA examined exhibited ST239/spa3(t037)/SCCmecIII.1.1.2 (designated as ST239Kras), while all CA-MRSA cases examined were ST8/spa1(t008)/SCCmecIV.3.1.1(IVc) (designated as ST8Kras). ST239Kras and ST8Kras strongly expressed cytolytic peptide (phenol-soluble modulin α, PSMα; and δ-hemolysin, Hld) genes, similar to CA-MRSA. ST239Kras pneumonia may have been attributed to a unique set of multiple virulence factors (MVFs): toxic shock syndrome toxin-1 (TSST-1), elevated PSMα/Hld expression, α-hemolysin, the staphylococcal enterotoxin SEK/SEQ, the immune evasion factor SCIN/SAK, and collagen adhesin. Regarding ST8Kras, SEA was included in MVFs, some of which were common to ST239Kras. The ST239Kras (strain OC3) genome contained: a completely unique phage, φSa7-like (W), with no att repetition; S. aureus pathogenicity island SaPI2R, the first TSST-1 gene-positive (tst +) SaPI in the ST239 lineage; and a super copy of IS256 (≥22 copies/genome). ST239Kras carried the Brazilian SCCmecIII.1.1.2 and United Kingdom-type tst. ST239Kras and ST8Kras were MDR, with the same levofloxacin resistance mutations; small, but transmissible chloramphenicol resistance plasmids spread widely enough to not be ignored. These results suggest that novel MDR and MVF+ HA- and CA-MRSA (ST239Kras and ST8Kras) emerged in Siberian Russia (Krasnoyarsk) associated with fatal pneumonia, and also with ST239Kras, a new (Siberian Russian) clade of the ST239 lineage, which was created through stepwise evolution during its potential transmission route of Brazil-Europe-Russia/Krasnoyarsk, thereby selective advantages from unique MVFs and the MDR.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) has been a major multidrug-resistant (MDR) pathogen since the early 1960s [1], with recent threats, such as intensive care unit (ICU)-associated bacteremia in London [2], serious invasive infections in the United States (US) [3], and global antimicrobial resistance in common infections, being alerted by the World Health Organization (WHO) [4].
In Russia, dominant MRSA are ST239/SCCmecIII and PVL-negative (PVL -) ST8/SCCme-cIV [49]. Although we previously reported PVL-positive (PVL + ) ST30 CA-MRSA [50], tst + ST239 HA-MRSA [51], and a whole genome structure [52], information on MRSA in Russia is still limited at the molecular level, especially in Siberian Russia, which is located between the European and Far Eastern regions. In the present study, we focused on episodes (and mortality rates) of MRSA hospital-acquired pneumonia (HAP) and community-acquired pneumonia (CAP) with pediatric deaths in Krasnoyarsk, Siberian Russia, as has been reported with initial fatal pediatric MRSA CAP episodes in the US North areas [5]. We discussed possible MRSA multiple virulence factors (MVFs), implicated in fatal cases of MRSA HAP and CAP. We then demonstrated their unique genomic structures and evolution of representative fatal-pneumonia-associated MRSA.

Ethics statement
The Ethics Review Boards of Krasnoyarsk State Medical University (Ethics Review Board No28/2010), Krasnoyarsk, Russia; Far Eastern Federal University School of Biomedicine, Vladivostok, Russia; National Taiwan University College of Medicine, Taipei, Taiwan; Niigata University School of Medicine, Niigata, Japan; and International Medical Education and Research Center, Niigata, Japan, specifically approved this study. Written informed consent was obtained from patients, if necessary.

Patients and bacterial strains
A total of 3,662 subjects were examined in Krasnoyarsk between 2007 and 2011. S. aureus specimens including MRSA were isolated in four hospitals in Krasnoyarsk, and all bacterial strains were isolated from different individuals. The data obtained are summarized in Table 1. The follow-up period used to determine the mortality for pneumonia was 15 days in this study; and 15-day mortality rates were compared between MRSA HAP and MRSA CAP cases. HA-MRSA was defined as MRSA isolated from inpatients 48 h after hospitalization while CA-MRSA was defined as MRSA isolated from outpatients who had no history of hospitalization within at least the past year and presented with no other established risk factors for MRSA infections [3].
Russian MRSA strains also included ten strains from inpatients (age, 1-41 years) with burn wound infections and respiratory tract infections in Vladivostok in 2012 and 2013; and nine strains from patients with burn and wound infections, osteomyelitis, respiratory tract infections, and blood stream infections in Moscow and Saint-Petersburg (European Russia) and in Kurgan (Ural Federal Region, Russia) in 2011 and 2012.

TSST-1 and SEA assays
The amounts of TSST-1 and SEA in the supernatants of bacterial cultures at 2.0 X l0 9 CFU/ml were examined using a TST-RPLA kit (Denka Seiken) and SET-RPLA kit (Denka Seiken), respectively, according to the instructions of the manufacturer.

Plasmid analysis
The plasmid DNA of MRSA was prepared using a Plasmid Midi Kit (QIAGEN Sciences, Tokyo) or according to the method by Kado and Liu [57] with a modification to the lysostaphin treatment. Plasmid DNA was analyzed by agarose (0.6-1.0%) gel electrophoresis. The Tn554 circular intermediate was detected by PCR (PCR product size, 772 bp), as previously described [52].

Conjugative transfer
Donor strains were mated with S. aureus RN2677, a recipient strain, which is resistant to rifampicin (Rif r ) and novobiocin and carries no plasmids, on tryptic soy agar (Difco, Sparks, MD, USA), with or without membrane filters [52].

Susceptibility testing
Susceptibility testing of bacterial strains was performed using the agar dilution method with Mueller-Hinton agar [58]. Inducible clindamycin resistance (Cli r ) was tested, as above, by using agar plates containing erythromycin (Em) at 0.1 to 1 μg/ml.

Genome analysis
The ST239 MRSA OC3 genome was analyzed by pyrosequencing using a genome sequencer FLX system with the assembler software GS De Novo Assembler version 2.6 (Roche Diagnostics, Branford, CT, USA

Entire sequencing of mobile genetic elements, phages, and plasmids
The gaps between contigs were filled by PCR and sequencing. We also assembled contigs using an LA PCR in vitro cloning kit (Takara Bio, Shiga, Japan) according to the manufacturer's instructions. In brief, after digestion with suitable restriction enzymes and ligation with the corresponding cassette adapters, amplification was performed with cassette primers and targetspecific primers.

Phylogenetic and homology analyses
Multiple alignments were performed up to 1,000 times using default settings with ClustalW software (version 2.1) and a phylogenetic tree analysis was performed using TreeViewX software (version 0.

mRNA expression assay
The mRNA expression levels of the cytolytic peptide (PSMα and Hld) genes (psmα and hld) and 16S rRNA genes were examined by an RT-PCR assay [56,59]. psmα and hld expression levels were normalized to 16S rRNA expression levels. The mRNA expression levels of the transcriptional regulator genes (sarA, sarR, mgrA, saeR, saeS, sarX, rot, and srrAB) were also examined.

Statistical analysis
Data were evaluated by Fisher's exact test for MRSA incidence and by an analysis of variance with repeated measurements for the mRNA expression assay. The level of significance was defined as a P value of <0.05. Regarding 15-day mortality rate estimates, 95% confidence intervals (95% CIs) were included.

Regarding healthy carriers
Among MRSA in Table 1, thirty-one isolates were subjected to molecular characterization ( Table 2). When pneumonia was targeted between 2007 and 2009, eight MRSA were characterized, all of which were from fatal or severe cases only; MRSA was not available from other non-fatal pneumonia cases. When pneumonia, osteomyelitis, SSTIs, and colitis were mainly targeted in 2010 and 2011, MRSA from fatal cases were initially selected and characterized: Healthy carrier (hospital worker) c Healthy carrier (student) d two from MRSA HAP and nosocomial SSTI/sepsis. Eighteen isolates were randomly selected from non-fatal cases: two out of 19 pneumonia isolates, five out of 19 osteomyelitis isolates, nine out of 32 MRSA SSTI isolates, one out of four colitis isolates, and one isolate from peritonitis. All three MRSA isolates from healthy carriers were included in the molecular analysis.
In Table 2, at least three out of the 11 MRSA pneumonia episodes (pneumonia/sepsis) were associated with bacteremia with the incidence being !27.3%; at least two out of seven HAP episodes (>2/7) and one out of four CAP episodes (>1/4), or two out of six ST239 MRSA cases (>2/6) and one out of five ST8 MRSA cases (>1/5) were associated with bacteremia. Although two ST239 and ST8 HAP-related bacteremia cases in 2007 were fatal, one ST239 HAP-related bacteremia case in 2011 was not.

Molecular characteristics of MRSA from Krasnoyarsk
The molecular data of the 31 MRSA isolates are summarized in Table 3 and Fig 1. MRSA strains were classified into three groups, A to C (Table 3).
The strains in group A2 were sea + (Table 3). Their PFGE patterns were divergent from group A1 (Fig 1). Group A2 was a common ST239 HA-MRSA in Russia, whereas group A2 showed a slightly narrower MDR spectrum (Table 3). Group A (A1 and A2) exhibited a high level of resistance to imipenem and oxacillin (Table 3), which is consistent with the characteristics of HA-MRSA [31].
Four ST8 Kras strains (OC8, OC22, OC23, and OC59) caused fatal CAP. One ST8 Kras strain (OC11) caused fatal HAP, suggesting that ST8 Kras even spread in hospitals. The ST8 Kras strains associated with fatal pneumonia were all sea + . Two ST8 Kras strains were isolated from carriers; OC217 was from a student while OC52 was from a hospital worker.
All plasmids were transferred to S. aureus RN2677 (recipient) in the bacterial mixed culture at frequencies ranging from 10 -5 to 10 -7 (S1D Fig). Tn554, carrying the ermA, spc genes, of ST239 Kras strains was also transferred to RN2677 (S1D Comparative genomics of ST239 Kras (strain OC3) The OC3 genome was estimated to be at least 2.93-Mb in size, with a 2,908-bp pCp r (pOC3). A total of 2.91-Mb (approximately 99.3% of the determined genome sequences) was mapped on the TW20 genome (Fig 2).
However, the 9-bp right-side sequence of φSa7-like (W) was divergent; there was no φSa7 att on the right side (Fig 4). Moreover, the insertion site (huNaDC-1 gene) of φSa7-like (W) was divergent from that of φSa7, which was generally inserted into the intercistronic region between the isdB and rpmF genes (Fig 4, the figure on the lower right side). φSa7-like (W) only showed 66% overall homology to φSa7 (NM2).
In the tst gene sequence comparison (Fig 5D-a), seven clusters were detected: i) cluster consisting of tst from Russia (OC3), UK, USA, South Korea, and Argentine; ii) tst cluster from USA (RN3984); iii) tst cluster from USA (pRN6101); iv) cluster consisting of tst from USA (including pRN6100), Japan (ST5/SCCmecII HA-MRSA, NY/J clone; and ST8/SCCmecIVl CA-MRSA, ST8 CA-MRSA/J clone), and Kenya (camel strain); and v-vii) three tst clusters from Ireland, Switzerland, and France (cow/bovine and ovine strains). The analysis at the deduced amino acid sequence levels produced very similar results (Fig 5D-b).
Russian (OC3) and UK TSST-1 precursors shared the same amino acid sequence with the same one amino acid replacement in the signal peptide region (S-11L; S!L at position -11), when compared with purified TSST-1 protein (MN8) or the precursor protein, deduced from the first USA tst gene (pRN6100) (Fig 5E). Regarding the tst genes from clinical isolates, amino acid replacements in the mature toxin (TSST-1) region were very rare, in contrast to the tst genes from animal isolates with distinct host specificity (Fig 5E). We were unable to determine why the tst gene from camel (Kenya) had the clinical type sequence.  Analysis of the tst + SaPI (SaPI2R) structure and tst nucleotide and deduced amino acid sequences of the ST239 Kras strain OC3. In A, the integration site (att) and att sequences of SaPI2R of the ST239 Kras strain OC3 are shown. In B, the SaPI2R structure was compared with those of tst -SaPI (ATCC25923) and tst + SaPI2 (RN3984). Homologous regions between SaPI structures are shaded with color. Genes: tst, toxic shock syndrome toxin-1 gene; eta, S. hyicus exfoliatin A gene; ter, terminase gene (which cleaves multimeric DNA); rep, replication initiator gene; int, the integrase gene. In C, the nucleotide sequences of tst + SaPIs and tst -SaPI (ATCC25923) were analyzed for phylogenetic diversity. In D-a, the nucleotide sequences of the tst genes were analyzed for phylogenetic diversity. In this figure, each GenBank record year is also shown. In D-b, the deduced amino acid sequences of the tst gene products were analyzed for phylogenetic diversity. The origin (reported source) of each isolate is indicated by the color of the isolate name: red, Russia; yellow, United Kingdom (UK); blue, United States (USA); dark red, Korea; light blue, Argentine; purple, Japan; green, those for animal isolates. In C and D, the scale bar represents substitutions per single-nucleotide polymorphism site. In E, the representative tst gene sequences were compared with the reference sequences (of pRN6100). Arrows indicate the positions of the nucleotide and amino acid changes for the representative tst genes. At the bottom of the figure (green), different amino acids from the amino acid sequences of purified TSST-1 (MN8; GenBank accession number EFH95768) and the deduced amino acid sequence of the tst gene (pRN6100) are indicated in red letters.

SaPI1 carrying sek and seq
doi:10.1371/journal.pone.0128017.g005 ϕSa3 carrying immune evasion genes φSa3 (OC3) was 43,681 bp in size (with 13-bp att at both ends) and was inserted into the hlb gene. φSa3 (OC3) showed 76% homology to that of strain CN1, sharing the same att and same integration site (S4 Fig). φSa3 (OC3) had the immune evasion cluster (IEC) on the left-end side, with the immune evasion genes sak (for staphylokinase, SAK) and scn (for staphylococcal complement inhibitor, SCIN), but lacked chp (for chemotaxis inhibitory protein of S. aureus, CHIPS) present in CN1 (S4 Fig). The IEC region of TW20 and ST8 Kras strain OC8 showed 99.3% homology and carried sea, in addition to sak, and scn (S4 Fig). Other relevant genetic structures Tn4001 (OC3), flanked by two IS256, was 6,483 bp in size and inserted into the noncoding region, located downstream of the ThiJ/PfpI family protein gene (Fig 2); in TW20, Tn4001 was present within φSPβ-like.
The pSK41-related resistance structure, flanked by two IS431, was 4,039 bp in size and carried the two drug resistance genes ble and aadD (Fig 2); its location on the genome currently remains unknown.
SaPI (fhuD) was 15,756 bp in size and inserted into the noncoding region, located downstream of the SsrA-binding protein gene. It showed only 69% overall homology to fhuD + SaPIm4 from the NY/J clone (Mu50). The 10.2-kb left-side half exhibited high homology (95%) to fhuD -SaPIj50 from Japanese ST8/SCCmecIVl CA-MRSA, suggesting that SaPI (fhuD) is a new mosaic SaPI (S5 Fig). φSa5 (OC3), with 10-bp att at both ends, was 44,424 bp in size and inserted into the hypothetical gene (for protein AGY89988.1), located downstream of the ThiJ/PfpI family protein gene. It was the most similar to φSa5 (XN108), albeit with only 63% homology (S6 Fig); φSa5 (OC3) is a new mosaic phage.
Elevated mRNA expression of cytolytic peptide genes in ST239 Kras ST239 Kras , including strain OC3, expressed the psmα and hld genes at high levels, similar to CA-MRSA USA300 and Russian CA-MRSA (RS08 and ST8 Kras ), but significantly higher than HA-MRSA, ST5/SCCmecII (the NY/J clone) and other ST239/SCCmecIII, including reference strains HU25 and ANS46 (P <0.05), as shown in Fig 6A. Regarding the expression levels of transcriptional regulatory genes (Fig 6B), although no significant difference was observed among the ST239 strains for sarA, ST239 Kras , including OC3, showed higher levels of expression for sarR, mgrA, saeR, saeS, sarX, rot, and srrAB (P <0.05), compared with the other ST239 strains, including reference strains HU25 and ASN46.
However, regarding MRSA types, the most prevalent HA-MRSA was ST239 Kras , a novel regional variant of the ST239 lineage (S2 Table, [52]. ST239 Kras is highly-virulent HA-MRSA with fatal HAP cases (with bacteremia) being reported. The ages of patients with fatal HAP were consistent with previous HA-MRSA data [34]. The genetic divergence in PFGE patterns strongly suggested that ST239 Kras infections persisted and spread among patients and carriers (hospital workers) at least since 2007.
Regarding the ST8 lineage, a single unique MDR MRSA clone (ST8 Kras ) with very similar PFGE patterns had persisted and spread. The spa type (spa1-t008) was the same as previous Russian ST8 [49], but divergent from that (spa826; t, unknown) of Vladivostok ST8 [52]. ST8 Kras was Lvx r with the same mutations as HA-MRSA ST239 Kras . ST8 Kras was a successful CA-MRSA, with not only fatal CAP, but also fatal HAP cases (with bacteremia). The fatal CAP cases included one infant and one young child death, consistent with previous CA-MRSA infections [34]. ST8 Kras carriers were also identified, suggesting its potential to be become widespread.
In the present study, we also focused onto the MVFs of MRSA. The hyper virulence of CA-MRSA USA300 has been attributed to MVFs, such as PVL, ACME-related factors, α-hemolysin (Hla), the elevated production of PSMs, and SEK (sek2) and SEQ (seq2) [37,68].
The acquisition of sek and seq with synonymous substitutions (sek2, seq2) may partly explain the hyper virulence of USA300 [37]. ST239 Kras (OC3) shared the same (unique) SEK and SEQ sequences with TW20, which were distinct from those of USA300.
ST8 Kras possessed a distinct set of MVFs, which included SEA, the strong expression of PSMα/Hld, SAK/SCIN, and Hla. Of these factors, SEA is associated with the severity of infections (sepsis and shock) [100] and promotes bacterial survival in vivo [101]. ST8 Kras has attracted attention because of its high mortality rate for MRSA CAP, including pediatric deaths. The whole genome of ST8 Kras is now being investigated to further characterize ST8 Kras ' MVFs.
Moreover, ST239 Kras carried the tst gene on SaPI [42,118], for the first time in the ST239 lineage. The same tst gene was present in the United Kingdom before the isolation of ST239 Kras , suggesting the potential salvage of tst in Europe. ST239 Kras also carried a completely unique, domestic phage, φSa7-like (W). Phages [19,20,44,[119][120][121] are a possible tool for S. aureus diversification, and classified according to the integrase gene types [120]. φSa7-like (W) was classified as integrase type 7 (Sa7int); however, φSa7-like (W) had no repeats of the terminal att sequence, similar to Tn554 [52,[122][123][124], and had a unique insertion site distinct from φSa7. In addition, ST239 Kras exhibited the characteristics of CA-MRSA, i.e., the strong expression of the cytolytic peptide gene (as described above).
Regarding ST239 MRSA transmission, the Brazilian clone spread to Portugal [9,46], Central Europe (Germany, Poland, and Czech Republic), Northern Europe (Finland) [17], and Eastern Europe/West Asia (Georgia) [125]. Krasnoyarsk has had a historically close relationship to the European region (St. Petersburg and Moscow). The (Southeast) Asian clade, including London strain TW20, which was likely transmitted from Southeast Asia [2,20], carried characteristic φSPβ-like (S2 Table) [19,20,[126][127][128], while ST239 Kras lacked φSPβ-like. Based on these findings and our results, we herein proposed a new Russian clade (representative strain, OC3) in the ST239/SCCmecIII lineage, and also speculated that ST239 Kras originated in the Brazilian clone, with the possible transmission route of Brazil-Europe (West-Central-North/East)-Russia (European-Siberian) (S9 Fig). Further genome-level analysis is needed for the understanding of evolution.
The plasmid distribution in Krasnoyarsk was unique. Many MRSA only carried pCp r and often carried two pCp r species, in contrast to some other country's cases with no pCp r [19,23,28,29,71,129,130], or Vladivostok's cases with multiple plasmids [52]. In Russia, inexpensive Cp is commonly administered to patients without a doctor's prescription as an ointment for skin injuries or burns, as a tablet for gastroenteritis, and as an eye lotion, providing MRSA with strong pressure to carry a pCp r . A small (2.9-kb) pCp r must be transferred, even in nature, possibly through the rolling circle (RC) manner of replication [41,131,132], similar to the replication that occurs during the conjugation of large Tra + plasmids [1,41,[132][133][134]. pEM r [135] and Tn554, with a circular intermediate [52,136], may follow pCp r -like transfer.
In conclusion, we identified novel regional variants of the ST239 and ST8 lineages (ST239 Kras and ST8 Kras ), in Siberian Russia (Krasnoyarsk), in which international research had never previously focused on MRSA and its invasive infections. ST239 Kras and ST8 Kras were MDR and had clonally (albeit with divergence) and widely spread, with fatal cases of HAP and CAP with bacteremia. The 15-day mortality rate for MRSA CAP was significantly higher than that for MRSA HAP, and fatal cases of ST8 Kras CAP included infant and young child deaths. According to the recent accumulation of information showing that successful MRSA, associated with large epidemics, has a unique set of MVFs, we speculated that fatal cases of ST239 Kras HAP were caused by the unique combination of TSST-1, the strong expression of PSMα/Hld, Hla, SEK/SEQ, SAK/SCIN, and Cna, while fatal cases of ST8 Kras CAP were attributed to the combination of SEA, the strong expression of PSMα/Hld, Hla, and SAK/SCIN. ST239 Kras carried a completely unique phage and mobile DNA, and exhibited unique virulence phenotypes; therefore, ST239 Kras represented a new (Siberian Russian) clade of the ST239 lineage, which was created through regional stepwise evolution during its possible Brazil-Europe-Russia transmission. Small resistance plasmids spread widely enough to not be ignored and in a unique manner among MRSA.
Supporting Information S1 Fig. Plasmid analysis (A to C) and plasmid transfer in a mixed bacterial culture (D) of MRSA from Krasnoyarsk, in comparison with the ST239 MRSA strain 16K from Vladivostok. In A; RN, RN2677 (recipient). Covalently closed circular (CCC) plasmid DNA, isolated from MRSA and transconjugants (RN2677 carrying plasmids), was electrophoresed in 1% agarose. Plasmid sizes were determined using reference plasmids with known molecular sizes. Plasmids (color): Cp r (yellow), chloramphenicol resistance plasmid; Em r (blue), erythromycin resistance plasmid; Gm r (red), gentamicin resistance plasmid. Regarding plasmids marked with Ã , the entire plasmid sequence was determined. In B-a, CCC plasmid DNA was electrophoresed in 0.6% agarose. RN, RN2677. In B-b, CCC plasmid DNA was digested with EcoRI, and the digests were electrophoresed in 0.5% agarose. Marker 1, 2.5 kb DNA Ladder; marker 2, λ-HindIII digest. In C; RN, RN2677. The Tn554 circular intermediate was detected by PCR; the ST239 Kras strain OC3 (lane 2) and Em r transconjugant (Em r RN2677, lane 3) produced positive results (carried the Tn554 circular intermediate), while RN2677 (lane 4) had no such structure. In D, bacterial mating between MRSA (plasmid-donor) and RN2677 (recipient) was performed by filter mating and non-filter mating methods. Nov, novobiocin; Cp, chloramphenicol; Em, erythromycin; Gm, gentamicin; Cli, clindamycin; Spc, spectinomycin; Amp, ampicillin; Cd, cadmium; EtBr, ethidium bromide; Acr, acriflavin. Transfer frequency, plasmidpositive (drug-resistant) transconjugants/donor. (TIFF) S2 Fig. Structure analysis of small plasmids specifying for chloramphenicol resistance (A-a to c) and erythromycin/clindamycin resistance (B). Plasmid sequence data were from the GenBank accession numbers described. Homologous regions are shaded in each comparison. Genes: cap, chloramphenicol resistance; rep, replication initiator protein; pre, pre protein; rlx, RLX protein; repL, replication initiator protein L. Em/Cli r , constitutive resistance to erythromycin and clindamycin; Em/Cli ind , inducible resistance to erythromycin and clindamycin (due to the presence of the leader peptide sequence in the promoter region upstream of ermC). (TIFF) S3 Fig. Analysis of the sek + seq + SaPI1 structure of the ST239 Kras strain OC3. In A, SaPI1 (OC3) showed the highest homology to SaPI1 (TW20). SaPI1 (OC3) was also compared with SaPI5 (USA300). Homologous regions between the SaPI structures are shaded with color. ear, penicillin-binding protein fragment. In B, the deduced amino acid sequences of the sek and seq genes (of OC3, TW20, and USA300) were compared with those of COL. Arrows indicate the positions of the amino acid changes. Different amino acids from the amino acid sequences of COL are indicated in red letters. (TIFF) S4 Fig. Structure of φSa3 of the ST239 Kras strain OC3. φSa3 (OC3) exhibited the highest homology to φSa3 (CN1). The left-side immune evasion cluster (IEC) region was also compared with those of φSa3 (TW20) and ST8 Kras strain OC8. Homologous regions are shaded in each comparison. Genes in IEC: scn, staphylococcal complement inhibitor (SCIN) gene; chp, chemotaxis inhibitory protein of S. aureus (CHIPS) gene; sak, staphylokinase (SAK) gene; sea, staphylococcal enterotoxin A (ETA) gene. The IEC region, carrying scn and sak, of OC3 (a region from attL to sak) was 3,541 bp in size, and showed 99% homology to the corresponding region of TW20. The IEC region, carrying scn, sak, and sea, of OC8 (a region from attL to sea) was 6,022 bp in size, and showed 99.3% homology to the corresponding region of TW20.