Resistance patterns of ST59 CA-MRSA strains isolated in Taiwan, compared to ST59 MRSA USA1000.

a<p>The number of strains tested was 24. Non-β-lactam agents: Ery, erythromycin; Cli, clindamycin; Kan, kanamycin; Stm, streptomycin; Chl, chloramphenicol; Tet, tetracycline. USA1000 is a type strain of PVL-positive ST59 MRSA.</p>b<p>Plasmid analysis was carried out for example strains.</p>c<p>Not determined.</p

Transfer of drug resistance in mating between ST59 CA-MRSA strain PM1and <i>S. aureus</i> RN2677.

a<p>Novobiocin was used for selection of RN2677.</p>b<p>MIC of ampicillin for transconjugants was lower than that for PM1 (1 µg/ml vs. 32 µg/ml).</p>c<p>No transconjugants were obtained.</p

Genetic structure of penicillinase plasmid pPM1 and its segregant.

<p>In A, pPM1 structure is shown. Inner circle indicates homologous regions to <i>S. aureus</i> plasmid SAP014A (GenBank accession number GQ900379), SAP063A (GenBank accession number GQ900418), and pSK156 (GenBank accession number GQ900448). The IS<i>431</i> region with the transposase gene (<i>tnp</i>) is shaded in light green. A Greek delta symbol indicates truncated coding sequence. In B, a segregant of pPM1 in MES<i>_tet</i> locus is shown. Tet^r, tetracycline-resistant; Tet^s, tetracycline-susceptible.</p

Comparison of IS<i>1216V</i>-containig mobile elements in MRSA and <i>E. faecium</i>.

<p>The structures of IS<i>1216V</i>-containing mobile elements: A, MES_PM1 determined in this study; B, Tn<i>1546</i> and Tn<i>5506</i> in <i>E. faecium</i>; C, vancomycin resistance transposon Tn<i>1546</i> in VRSA. The IS<i>1216V</i> region with the transposase gene (<i>tnp</i>) is shaded in blue. The <i>att</i> site is indicated by a vertical red line. The data for pVEF1 are from GenBank (GenBank accession number AM296544). The data for pHKK701 and Tn<i>1546</i> in VRSA-2 and VRSA-3 were obtained from previously described maps <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046987#pone.0046987-Lyon1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046987#pone.0046987-Heaton1" target="_blank">[32]</a>.</p

Genetic structure of MES_PM1 carrying four resistance genes, <i>ermB</i>, <i>aph(3′)-IIIa</i>, <i>aadE</i>, and <i>cat</i>.

<p>MES_PM1 was integrated into and disrupted the <i>sasK</i> gene, which encoded a putative cell-wall anchored surface protein with the LPXTG-motif. The intact <i>sasK</i> gene was present in PVL-positive ST59 CA-MRSA type strain USA1000. Three <i>att</i> sites are in a vertical red line. The region of IS<i>1216V</i> with the transposase gene (<i>tnp</i>) is shaded in blue. Homology regions with <i>E. faecalis</i> pLG2 plasmid (GenBank accession number HQ426665) and <i>S. aureus</i> SAP084A (GenBank accession number GQ900436) are shaded in yellow and purple, respectively. A Greek delta symbol indicates truncated coding sequence.</p

Primers used for PCR mapping of the MES_PM1 locus.

<p>Primers used for PCR mapping of the MES_PM1 locus.</p

Structure of νSAβ in PM1.

<p>The 15,551-bp νSAβ structure in PM1 is shown in the upper part. This sequence was compared to the data of ST8 CA-MRSA strain USA300 FPR3757 (GenBank accession number NC_007793) and ST5 HA-MRSA strain N315 (GenBank accession number BA000018). PM1 regions homologous to φSA3_USA (of USA300) and νSAβs (of USA300 and N315) are shaded in pink and yellow, respectively. A Greek delta symbol indicates truncated coding sequence.</p

The structure of MES_PM1 and its segregants in ST59 CA-MRSA strains.

<p>Structures of MES_PM1, MES_PM9, MES_PM8, and MES_PM18 (corresponding to IS<i>1216V</i>) are shown in A to D. Drug resistance (r): Ery/Cli, erythromycin/clindamycin; Kan, kanamycin; Stm, streptomycin; Chl, chloramphenicol. The IS<i>1216V</i> region with the transposase gene (<i>tnp</i>) is shaded in blue. The <i>att</i> site is indicated by a vertical red line. Arrows below the MES structures indicate PCR primers, which are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046987#s4" target="_blank">Materials and Methods</a>.</p

Genome comparisons of ST59 MRSA strain PM1 with two other <i>S. aureus</i> genomes.

<p>In A, two gray circles show the complete genomes of ST8 CA-MRSA strain USA300 FPR3757 (2,872,769 bp) and ST398 MRSA strain S0385 (2,872,582 bp); gaps in the genome circles indicate USA300 or S0385 sequences not present in PM1. The <i>sasK</i> gene with the MES_PM1 integration site (<i>att</i>) is shown outside the gray circles (on the USA1000 genome circle), since the USA300 and S0385 MRSA strains do not possess the <i>sasK</i> gene. The outermost (white) circle represents the alignment of PM1 contigs and provides PM1 genome information, including drug resistance traits, pathogenetic islands, bacteriophages, and genomic islands νSAα and νSAβ. Plasmid pPM1 is shown in the right bottom of the figure. Color region in the genome: orange, <i>orfX</i> with the <i>att</i> sequence for SCC<i>mec</i>; green, genetic elements with diversity; brown, genetic elements present in USA300 or S0385 (but not in PM1); pink, genetic elements present in PM1 (but not in USA300 or S0385). In B, relevant genotypes of PM1 is summarized. Drug resistance (r): Ery/Cli, erythromycin/clindamycin; Kan, kanamycin; Stm, streptomycin; Chl, chloramphenicol; Oxa, oxacillin; Tet, tetracycline. Virulence factors: SEB, staphylococcal enterotoxin B; SEK, staphylococcal enterotoxin K; SEQ, staphylococcal enterotoxin Q; PVL, Panton-Valentine leukocidin.</p

MES16K structure and phylogenetic tree analysis for the <i>ccrC</i> genes.

<p>The SCCmercury data for strain TW20 are from GenBank accession number FN433596. The GenBank accession number of MES16K is AB666466. Homologous regions are shaded. In A, for strain TW20, the <i>ccrC</i>-carrying unit was located within SCCmercury; the <i>ccrC</i> gene (marked in yellow) was split by the insertion of IS (IS200 family). For strain 16K, the <i>ccrC</i>-carrying unit was located within a mobile element structure MES16K, which was inserted into the <i>orf</i> (corresponding to SATW20_27600 of TW20) on the 16K genome, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029187#pone-0029187-g002" target="_blank">Fig. 2</a>; the <i>ccrC</i> gene (marked in yellow) was intact. The attachment sequence (<i>att</i>) of MES16K showed no homology to that (<i>att</i>) of the <i>orfX</i>, SCC<i>mec</i>III.1.1.1 or SCCmercury. Arrowheads PM1 and PM2 represent PCR targets for detection of SCCmercury of ST239/SCC<i>mec</i>III.1.1.1 MRSA and MES16K of the Russian variant (strain 16K) by multiplex PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029187#pone-0029187-g003" target="_blank">Fig. 3</a>). In B, the <i>ccrC</i> gene sequence of strain 16K and that of strain TW20 (expected intact <i>ccrC</i> gene sequence, obtained by excluding the inserted IS200-family sequence) were analyzed for phylogenetic diversity, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029187#pone.0029187-Higuchi2" target="_blank">[36]</a>. The <i>ccrC</i> gene of strain TW20 was a novel <i>ccrC1</i> allele (named <i>ccrC1</i> allele 10) and that of strain 16K was assigned as <i>ccrC1</i> allele 3, unambiguously demonstrating evolutionary diversity between them; moreover, the <i>ccrC1</i> gene of ST398 MRSA strain S0385 was also a novel allele (named <i>ccrC1</i> allele 11).</p