Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification

Abstract

Emerging and re-emerging infectious diseases, especially those caused by drug-resistant bacteria, are a major problem worldwide. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) appeared rapidly and unexpectedly in the United States, resulting in an epidemic caused primarily by isolates classified as USA300. The evolutionary and molecular underpinnings of this epidemic are poorly understood. Specifically, it is unclear whether there has been clonal emergence of USA300 isolates or evolutionary convergence toward a hypervirulent phenotype resulting in the independent appearance of similar organisms. To definitively resolve this issue and understand the phylogeny of USA300 isolates, we used comparative whole-genome sequencing to analyze 10 USA300 patient isolates from eight states in diverse geographic regions of the United States and multiple types of human infection. Eight of 10 isolates analyzed had very few single nucleotide polymorphisms (SNPs) and thus were closely related, indicating recent diversification rather than convergence. Unexpectedly, 2 of the clonal isolates had significantly reduced mortality in a mouse sepsis model compared with the reference isolate (P = 0.0002), providing strong support to the idea that minimal genetic change in the bacterial genome can have profound effects on virulence. Taken together, our results demonstrate that there has been recent clonal expansion and diversification of a subset of isolates classified as USA300. The findings add an evolutionary dimension to the epidemiology and emergence of USA300 and suggest a similar mechanism for the pandemic occurrence and spread of penicillin-resistant S. aureus (known as phage-type 80/81 S. aureus) in the 1950s.

Fig. 1.

Analysis of USA300 isolates by pulsed-field gel electrophoresis (PFGE). PFGE was performed as described in Materials and Methods. Although there is variation among the strains, the PFGE patterns are typical of USA300. FPR3757 is the reference strain. The white line indicates a merge of two separate images.

Fig. 2.

SNPs in the core genome of USA300 isolates. SNPs were identified by using comparative genome sequencing. Each bar represents a SNP. Green and dark blue vertical lines indicate synonymous and nonsynonymous nucleotide changes, respectively. Light blue indicates a SNP in an intergenic region. The gray shaded regions represent notable genetic elements. Forward (above the line) and reverse (below the line) strand ORFs of the FPR3757 reference strain (Ref.) are illustrated in red at the top of the aligned sequences.

Fig. 3.

SNP matrix and SNP-based phylogenetic analysis. (A) SNP matrix. Red font indicates number of SNPs compared with reference strain FPR3757. (B) Phylogenetic analysis of the 11 USA300 isolates is based upon 578 concatenated core genomic SNPs and includes SNPs present in intergenic regions. (C) Phylogenetic analysis using 472 concatenated amino acid residues containing SNPs, synonymous, and nonsynonymous. (D) Phylogenetic analysis of USA300 isolates and strain COL is based upon 1,063 concatenated core genomic SNPs.

Fig. 4.

Virulence of S. aureus isolates in a mouse sepsis model. Strain-to-strain variation in virulence. Survival after infection (5 × 10⁷ CFU, tail vein) was determined as described in Materials and Methods using 14–15 mice per group. Each isolate is compared with reference strain FPR3757 (gray circle).

Fig. 5.

Variation in S. aureus exoprotein profile. (A) Exoproteins present in the supernatant obtained from overnight cultures were analyzed by 10–20% gradient SDS/PAGE. (B and C) Production of protein A (Spa) (B) and of α-hemolysin (Hla) and LukF-PV (C) in the culture media. Aliquots of media from overnight cultures were analyzed by SDS/PAGE, and the indicated proteins were detected by Western immunoblot analysis. (D) Production of delta-toxin. Aliquots of the indicated media were subjected to LC/MS to quantitate delta-toxin. Data are the mean ± SEM of three experiments.