Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain

Abstract

Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the approximately 2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the approximately 2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis.

FIG. 1.

Circular representation of the sequenced S. aureus and S. epidermidis genomes. Each concentric circle represents genomic data for S. aureus (A) and S. epidermidis (B) and is numbered from the outermost circle to the innermost circle. The outermost circles indicate the genome coordinates in base pairs. The second and third circles represent the predicted S. aureus COL and S. epidermidis RP62a ORFs on the plus and minus strands, respectively, colored by role categories: salmon, amino acid biosynthesis; light blue, biosynthesis of cofactors and prosthetic groups and carriers; light green, cell envelope; red, cellular processes; brown, central intermediary metabolism; yellow, DNA metabolism; green, energy metabolism; purple, fatty acid and phospholipid metabolism; pink, protein fate and synthesis; orange, purines, pyrimidines, nucleosides, and nucleotides; blue, regulatory functions; grey, transcription; teal, transport and binding proteins; black, hypothetical and conserved hypothetical proteins. The fourth (strain COL), fifth (strain Mu50), sixth (strain N315), and seventh (strain MW2) circles in S. aureus and the fourth (strain RP62a) and fifth (strain ATCC 12228) circles in S. epidermidis indicate genome islands involved in virulence (red or yellow), regulatory loci (agr) (blue-green), prophage (black), SSCmec (blue), SSCpbp4 (orange), integrated plasmids (pink), STAR elements (brown), transposable elements (dark green), and CRISPR regions (light blue). The 8th (strain Mu50), 9th (strain N315), and 10th (strain MW2) circles in S. aureus and the sixth (strain ATCC 12228) circle in S. epidermidis represent the number of SNPs per 5 kb compared to S. aureus strain COL and S. epidermidis strain RP62a. Gold ticks, 1 to 75 SNPs; red ticks, 76 to 200 SNPs; dark green ticks, 201 to 300 SNPs; blue ticks, more than 301 SNPs. Complete DNA sequence and annotation for S. aureus MW2, N315, and Mu50 and S. epidermidis ATCC 12228 were obtained from GenBank accession numbers BA000033, BA000018, BA000017, and AE015929, respectively.

FIG. 2.

Novel integrated plasmids and genome islands in S. aureus and S. epidermidis. Shown are the schematic diagrams of integrated plasmids of νSe1 in S. epidermidis RP62a (A), νSe2 in S. epidermidis ATCC 12228 (B), νSaγ in S. aureus COL (C), and νSeγ in S. epidermidis RP62a (D). ORFs are marked in the direction of transcription as arrows and are colored according to functional categories as indicated. Positions within the respective genomes are indicated as genome coordinates on the ends of each schematic. Putative functions of selected ORFs accompany the gene locus number. Putative functions of all ORFs and their locus numbers are presented in Supplementary Table 2.

FIG. 3.

Phylogenetic analysis and organization of the Cap operon in S. epidermidis RP62a. Homologs of S. epidermidis RP62a Cap operon (E) were identified by BLASTP of the WU-BLAST formatted database of all complete bacterial genomes. Each gene in the Cap operon was aligned against respective homologs with ClustalW, and phylogenetic trees were generated with Belvu for gamma-glutamyltranspeptidase (A), CapA (B), CapC (C), and CapB (D). Organization of the S. epidermidis RP62a and B. anthracis Cap operons is shown in schematics E and F, respectively. Positions within the respective genomes are indicted as genome coordinates on the ends of each schematic. BLASTP e values determined from homolog search results are shown between each ORF in schematics E and F. GenBank accession numbers are in parentheses and accompany all matches on the phylogenetic tree.

FIG. 4.

Overview of metabolism and transport in S. aureus and S. epidermidis. Pathways for energy production, metabolism of organic compounds, and synthesis of carotenoids are shown. Orange text, processes unique to S. aureus; green text, processes unique to S. epidermidis. Transporters are grouped by substrate specificity as follows: inorganic cations (green); inorganic anions (pink); carbohydrates and carboxylates (yellow); amino acids, peptides, amines, and purines and pyrimidines (red); and drug efflux and other (black). Question marks indicate uncertainty about the substrate transported. Export or import of solutes is designated by the direction of the arrow through the transporter. The energy-coupling mechanisms of the transporters are also shown: double-headed arrow, solutes transported by channel proteins; two arrows, secondary transporters, indicating both the solute and the coupling ion; single arrow, transporters with an unknown energy coupling mechanism. ATP-driven transporters are indicated by the ATP hydrolysis reactions. Components of transporter systems that function as multisubunit complexes that were not identified are outlined with dotted lines. Where multiple homologous transporters with similar substrate predictions exist, the number of that type of transporter is indicated in parentheses.