Evolution of virulence in epidemic community-associated methicillin-resistant Staphylococcus aureus

Abstract

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged worldwide. The United States, in particular, is experiencing a serious epidemic of CA-MRSA that is almost entirely caused by an extraordinarily infectious strain named USA300. However, the molecular determinants underlying the pathogenic success of CA-MRSA are mostly unknown. To gain insight into the evolution of the exceptional potential of USA300 to cause disease, we compared the phylogeny and virulence of USA300 with that of closely related MRSA clones. We discovered that the sublineage from which USA300 evolved is characterized by a phenotype of high virulence that is clearly distinct from other MRSA strains. Namely, USA300 and its progenitor, USA500, had high virulence in animal infection models and the capacity to evade innate host defense mechanisms. Furthermore, our results indicate that increased virulence in the USA300/USA500 sublineage is attributable to differential expression of core genome-encoded virulence determinants, such as phenol-soluble modulins and alpha-toxin. Notably, the fact that the virulence phenotype of USA300 was already established in its progenitor indicates that acquisition of mobile genetic elements has played a limited role in the evolution of USA300 virulence and points to a possibly different role of those elements. Thus, our results highlight the importance of differential gene expression in the evolution of USA300 virulence. This finding calls for a profound revision of our notion about CA-MRSA pathogenesis at the molecular level and has important implications for design of therapeutics directed against CA-MRSA.

Fig. 1.

Evolutionary relation of CC8 subclones. The tree architecture was inferred by analysis of 7 housekeeping and 7 surface protein genes (Table S1). Presence of virulence genes was determined by analytical PCR (Table S2). Strains analyzed in this study are shaded in gray, blue, and red, representing the 3 different CC8 sublineages, and are labeled with the specific strain designations. *Single-nucleotide polymorphisms in housekeeping or surface protein-encoding genes used to infer evolutionary relation (Table S1).

Fig. 2.

Virulence assessment of CC8 subclones by animal infection models. (A) Bacteremia model. CD1 Swiss female mice were infected with 10⁸ cfus of the indicated MRSA strains. Survival curves were compared using log-rank (Mantel-Cox) tests. Concentration of TNF-α (B) and IFN-γ (C) in mouse blood at death. Strains for which statistically significant differences were achieved are named above bars. Compared with PBS, values were significantly different for BD02-25, SF8300, HS-522, and BAA43 (TNF-α) and for BD02-25 and SF8300 (IFN-γ). n.s., not significant. (D) Abscess model. As a control, SKH1-hrBR hairless mice were infected with 10⁷ cfus of the indicated MRSA strains, and abscess or dermonecrosis areas were measured each day.

Fig. 3.

Neutrophil lysis by CC8 subclones. Lysis of human neutrophils by culture filtrates from CC8 strains was determined by release of lactate dehydrogenase (LDH). Values were significantly different for all comparisons except BD02-25 vs. SF8300 and all among HS-522, BAA-43, BAA-43, and COL.

Fig. 4.

Expression of virulence determinants in CC8 subclones. (A) Presence of cytolysin genes by analytical PCR. (B) Western blot of α-toxin production. (C) PSM production. PSM production in culture filtrates was determined by RP-HPLC/electrospray ionization-MS. Relative production of δ-toxin (pink), other α-type (red), and β-type (blue) PSMs is shown in pie diagrams. (D) Hemolysis. Lysis of red blood cells was determined by dropping equal amounts of cells on sheep blood agar plates, incubating at 37 °C, and measuring clearing zones after 24 h. Values were significantly different for all comparisons except BD02-25 vs. SF8300 and BAA-44 vs. COL. (E) Proteolysis. Proteolysis of supernatants concentrated by lyophilization was measured by an agar diffusion assay. (F) RNAIII expression by qRT-PCR. (E and F) Values were significantly different for all comparisons except BD02-25 vs. SF8300 and all among HS-522, BAA-43, BAA-43, and COL.

Fig. 5.

PSM production in HA-MRSA, CA-MRSA, and ST8 MSSA strain collections. Strains collected at hospitals in the San Francisco area were analyzed by RP-HPLC/electrospray ionization-MS for PSM production. Carriage isolates are from nonhospitalized homeless youth and urban poor in the same area. PSMα3 and δ-toxin levels are shown. Horizontal bars depict the mean. Statistical analysis is by unpaired t tests or one-way ANOVA for comparison of USA300, USA500, and MSSA carriage or infection isolates, respectively, which showed no statistically significant differences. N.S., not significant. ∗∗P < 0.01, ∗∗∗P < 0.001.