Article Figures & Data
- FIG 1
Phylogeny based on single-copy core orthologs (n = 1,822). Phylogeny showing the relationship of VRSA genomes to other completely sequenced S. aureus genomes. The date (month/day/year) and geographic location of the VRSA strain and multilocus sequence type (MLST) are indicated in the expanded section. MI, Michigan; PA, Pennsylvania; NY, New York; DE, Delaware; MD, Maryland; JP, Japan; NIR, Northern Ireland. Bootstrap values are indicated at each node. Scale bars correspond to number of changes per site.
- FIG 2
Haplotype network of Tn1546 sequences. Numbering of the nucleotide changes refers to the position in sequence in comparison to the prototypical Tn1546 (GenBank accession no. M97297).
- FIG 3
Plasmid sequences in VRSA. Heat map showing the extent of occurrence of known enterococcal (graded green shades) and staphylococcal plasmids (graded yellow to red) (based on detail in Table S5 in the supplemental material). Plasmids that are definitely present in the strains are indicated by squares outlined with a thick black border. The plasmid on which Tn1546 resides is indicated by a white v, and superscript letters a through g denote unique insertion sites. pLW043* is the pSK41 homolog into which Tn1546 inserted. pWZ909⊥ is the prototype Inc18 enterococcal plasmid involved in vancomycin resistance transfer. pN315 and pUSA300 were shown in one column, as these plasmids are nearly identical.
- FIG 4
Restriction systems and dprA in VRSA. (A) (1) Alignment of inferred primary translation products of genes encoding the Sau1 restriction nuclease subunit (HsdR) compared to the CC5 strain originating in Japan, S. aureus N315. Polymorphisms are highlighted. Periods in blocks of sequence denote stretches that are identical and not shown. Numbers above the sequences represent the amino acid position in the prototype, from which distances represented by dots can be discerned. (2) Alignment of inferred primary translation products of genes encoding the modification (HsdM) and specificity (HsdS) subunits in the νSaα island. The locations of restriction-modification subunits relative to other key genes are indicated. (3) Alignment of inferred primary translation products of genes encoding the specificity (HsdS) subunits in the νSaβ island. No polymorphisms occur in the νSaβ HsdM-encoding gene (not shown). (4) Alignment of inferred primary translation products of genes encoding the type III-like restriction system compared to those of N315 and the CC8 S. aureus NCTC8325 prototype sequence. (B) Alignment of inferred primary translation products of dprA genes of VRSA. VRS3a is the only isolate that encodes a dprA product identical to that encoded by the Japanese CC5 isolate N315. All other VRSA strains possess a truncating frameshift mutation predicted to generate a premature termination, and possibly overlapping reinitiation product, as generically designated in the figure as VRSAa and VRSAb.
- FIG 5
Variation in the νSAβ island of VRSA strains compared to other lineages. The gray shading for the schematic representations of the νSAβ islands shown to the right of the figure corresponds to the position within the phylogeny showing the relationship of VRSA genomes to other completely sequenced S. aureus genomes shown to the left of the figure. The asterisk in the top right-hand corner of the schematic representation in panel b indicates that S. aureus COL contains an IS1811 transposase upstream of bsaA2. The pair of asterisks in the top right-hand corner of the schematic representation in panel d indicate that the far right endpoint for RF122 is not shown because of phage insertion.
- FIG 6
Consensus tree for CC5 under relaxed clock conditions. North American CC5 strains are shown in black, while other strains are shown in gray. Blue bars indicate range of 95% highest posterior density interval (95% confidence interval, 1.76 × 10−6 to 5.22 × 10−6). The time of penicillin and methicillin introduction, as well as the emergence of resistances are shown for reference (PRSA, penicillin-resistant S. aureus). The arrow indicates the estimated time of divergence of the most distantly related VRSA genome, that of VRS3a (USA800 branch) from the remaining VRSA (mainly USA100) branch of the CC5 clade, showing that strain divergence occurred far earlier than vancomycin resistance acquisition, supporting the model of independent Tn1546 acquisition.
Supplemental Material
Figure S1
SCCmec cassettes of VRSA and polymorphisms in agr loci. (a) Occurrence of a sorbitol utilization operon adjacent to the type IV SCCmec cassette in strain VRS3a compared to a prototype SCCmec IV from strain USA300_FPR3757 and the ancestral sorbitol operon as it occurs in Staphylococcus carnosus TM300. Double solid lines indicate the end of VRS3a contig on which the SCCmec IV cassette is found. USA300_FPR3757 nomenclature is used in the labeling of the genes for reference. The bar graph shows verification that strain VRS3a can utilize sorbitol compared to strain VRS1 as a representative lacking the sorbitol operon. (b) PCR-confirmed deletion in VRS6 SCCmec II. Deletion results in juxtaposition of reading frames SA0052 (marked with an asterisk) and SA0080 (sequence designations from N315). (c) Alignment of agr-encoded primary translation products. AgrA sequence alignment illustrating a truncation in strain VRS8. Periods indicate stretches of identical sequence not illustrated for brevity. AgrB alignment illustrating an 18-amino-acid amino-terminal extension and high-level sequence conservation among all strains, with strain VRS5 also possessing a 6-amino-acid C-terminal extension. AgrC sequence comparison showing truncation of VRS1 after amino acid 310, followed by a possible restart. AgrD sequence comparisons highlighting a truncation of VRS2 as a result of insertion of an Rve_3 superfamily integrase-bearing insertion element after codon 13. (C1) Hemolytic phenotype correlates with mutations in Agr components. Strains Newman (positive control), RN4220 (β-hemolysin control), and SA564 Δagr mutant are shown. (C2) Cross-streak assay of strains VRS1, VRS2, and VRS8 to further characterize hemolysin production. The arrow denotes δ hemolysin contribution to lysis in the presence of the β lysin produced by the RN4220 cross-streak. Download Figure S1, EPS file, 5.7 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
Characteristics of strains used in this study. (a) Typing of VRSA by protein A (spa), clumping factor B (clfB), and coagulase (coa) sequences. (b) VRSA genomes. (c) Completely sequenced genomes used for comparison Table S1, DOC file, 0.1 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S5
Presumptive plasmid contigs. Contigs that either did not map to a reference genome or mapped to known enterococcal or staphylococcal plasmids. Values indicate fractional content of plasmid occurring in each strain. Table S5, DOC file, 0.1 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
VRS11a/b plasmid. Green reading frames indicate enterococcal pCF10, while yellow reading frames indicate staphylococcal plasmid pLUH02 and gray reading frames indicate aminoglycoside resistance common to both. Blue reading frames indicate identity with insertion sequences. Download Figure S2, EPS file, 0.4 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
Nonsynonymous SNPs found in all North American CC5 strains except for strain VRS3a. Table S2, DOCX file, 0.1 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Nonsyntenic regions of the chromosome between CC5 strains and other S. aureus lineages. Gene organization of CC5 strains is shown against a gray background, and that of comparator strains is shown against a light orange background. Reading frame categories are shown as follows. (i) Open reading frames (ORFs) unique to CC5 and common to all members of that lineage are shown in dark blue. (ii) ORFs occurring in some CC5 strains with orthlogs located elsewhere in the comparator strains are shown in aqua. (iii) ORFs present in the comparator strains but lacking in CC5 strains are shown in orange. (iv) ORFs occurring in non-CC5 strains with nonsyntenic orthologs in all CC5 strains are shown in yellow. Regions high in nonsyntenic genes are shown as follows. (A) Clusters of distinct hypothetical ORFs differing between CC5 (dark blue and aqua) and other lineages (orange) in a region that also contains an IS200 transposase family protein in ST398 (yellow). (b) Area generally containing genes for drug efflux and Mar family transcription regulator (orange) in comparator strains but lacking in CC5. (c) Second cluster of hypothetical ORFs mainly occurring in non-CC5 strains (orange). (d) Location of a hypothetical ORF/major facilitator transporter common to CC5 strains (aqua) but located elsewhere in comparator strains. Strain RF122 possesses other transporters and genes derived from the streptolysin S operon at this site (orange). (e) Region of hypothetical ORFs absent in non-CC5 strains (orange). (f) Differences in νSaβ between CC5 and comparator strains. In place of the bsa lantibiotic operon and associated genes (orange) in non-CC5 strains are two ORFs encoding hypothetical proteins in CC5 (dark blue). Comparator hospital isolates United Kingdom EMRSA15 and MRSA252 lack the lantibiotic gene cluster, as does ST398. (g) Variation in lipoprotein content in νSaα between CC5 and other lineages. (h) Hypothetical ORFs unique (orange) to non-CC5 or nonsyntenic (yellow). (i) Hypothetical ORFs (orange) in comparator strains most closely related by BLAST to genes of a retrotransposon. (j) Glycosyltransferase (orange) not present in the CC5 or the United Kingdom strain MRSA252. Download Figure S3, PDF file, 6.8 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S3
Distinguishing features of CC5 strains based on ortholog cluster comparative analysis. Table S3, DOC file, 0.1 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
Lipoproteins enriched in CC5 strains. (a) Distribution of lipoproteins across S. aureus genomes (yellow, present in a single copy; black, absent; orange, present in two copies; red, present in more than two copies). CC5 enrichment indicated by a red asterisk. A black asterisk indicates that sequences in CC5 were not predicted to be lipoproteins by PRED-LIPO, but clustering in orthogroups was identified as including putative lipoproteins and occurring within one of the tandem lipoprotein cluster )e.g., JH1_2565 and JH1_2564 in JH1 genome). (b) Identities of lipoproteins enriched in CC5 strains (branch indicated by a red asterisk in panel A, rotated 90° counterclockwise) using JH1 naming convention. For lipoproteins not occurring in CC5, the naming convention for Newman (CC8) is provided. One putative lipoprotein occurring in CC5 (SAV_0382) is not annotated in the genome of JH1, and the nomenclature for ED98 is used to represent it. ORFs with two asterisks were not confirmed by PRED-LIPO prediction to be lipoproteins. Also present is a diagram showing the genetic organization of lipoprotein clusters enriched in CC5. (c) Pheromone sequences produced from the processing of putative lipoprotein signal peptides identified in the North American CC5 S. aureus genomes. Download Figure S4, PDF file, 1.5 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S4
PCR primers. Primers used for closure, sequence verification, and typing based on proteins of repetitive structure. Table S4, DOC file, 0.1 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Text S1
Details of bioinformatic methods and surface protein and SCCmec cassette analysis. Additional bioinformatic analysis details are also included. Download Text S1, DOC file, 0.2 MB.
Copyright © 2012 Kos et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
Files in this Data Supplement:
- Figure sf03, PDF - Figure sf03, PDF
- Figure sf04, PDF - Figure sf04, PDF
- Text s1, DOC - Text s1, DOC
- Figure sf01, EPS - Figure sf01, EPS
- Figure sf02, EPS - Figure sf02, EPS
- Table st1, DOC - Table st1, DOC
- Table st2, DOCX - Table st2, DOCX
- Table st3, DOC - Table st3, DOC
- Table st4, DOC - Table st4, DOC
- Table st5, DOC - Table st5, DOC
