Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic

Abstract

An emerging theme in medical microbiology is that extensive variation exists in gene content among strains of many pathogenic bacterial species. However, this topic has not been investigated on a genome scale with strains recovered from patients with well-defined clinical conditions. Staphylococcus aureus is a major human pathogen and also causes economically important infections in cows and sheep. A DNA microarray representing >90% of the S. aureus genome was used to characterize genomic diversity, evolutionary relationships, and virulence gene distribution among 36 strains of divergent clonal lineages, including methicillin-resistant strains and organisms causing toxic shock syndrome. Genetic variation in S. aureus is very extensive, with approximately 22% of the genome comprised of dispensable genetic material. Eighteen large regions of difference were identified, and 10 of these regions have genes that encode putative virulence factors or proteins mediating antibiotic resistance. We find that lateral gene transfer has played a fundamental role in the evolution of S. aureus. The mec gene has been horizontally transferred into distinct S. aureus chromosomal backgrounds at least five times, demonstrating that methicillin-resistant strains have evolved multiple independent times, rather than from a single ancestral strain. This finding resolves a long-standing controversy in S. aureus research. The epidemic of toxic shock syndrome that occurred in the 1970s was caused by a change in the host environment, rather than rapid geographic dissemination of a new hypervirulent strain. DNA microarray analysis of large samples of clinically characterized strains provides broad insights into evolution, pathogenesis, and disease emergence.

Figure 1

S. aureus genetic diversity. (A) Genome-wide distribution of absent ORFs among the 36 S. aureus strains studied. Red and black denote presence or absence of ORFs, respectively. RDs are marked by black arrows and were numbered arbitrarily, because the genome sequence of strain COL is not complete. (B) Dendrogram showing estimates of genomic relationships of the 36 strains constructed by complete linkage hierarchical cluster analysis. Strains were grouped with the cluster program on the basis of the presence or absence of ORFs with RDs treated as single loci, and the output was displayed with the computer program treeview. Phylogenetic lineage and ET of each strain are presented. Red text signifies MRSA strains. The scale represents the value of the Pearson correlation coefficient at each node. For a pairwise comparison, a coefficient of 1 would denote absolute identity, and zero would indicate complete independence.

Figure 2

Presence or absence of RDs in the 36 S. aureus isolates studied. Filled square denotes presence of an RD and an empty space, its absence. Hatched squares indicate presence of RDs in MRSA strains. Red signifies ET 234 strains.

Figure 3

Variation in gene content of chromosomal RDs. (A) RDs with extensive variation in gene content or size among the 36 strains examined. Genes that encode proteins containing homology to proteins of known function are denoted by squares. Horizontal black lines represent regions containing ORFs that encode hypothetical proteins of unknown function. The hypothetical proteins encoded by the genes designated are as follows: lukD, E, leukotoxins D and E; tra, transposase; bla, β-lactamase; splA-F, serine protease A–F; r/m, restriction and modification proteins; gdp, glycerophosphoryl diester phosphodiesterase; mecA, penicillin-binding protein 2′; mecR1, signal transducer protein; hsdR, type 1 restriction endonuclease; ccrA, B, cassette chromosome recombinase A, B; plsl, Pls-like protein; tagE, poly(glycerol-phosphate)alpha-glucosyltransferase homologue; efp, efflux protein homologue; int, integrase; tss, phage terminase small subunit homologue; fbp, ferrichrome binding protein homologue; tcmp, tetracenomycin C synthesis protein homologue; sddA, B, succinyl-diaminopimelate desuccinylase-related proteins A, B; lukY, Z, leukotoxin F, S homologues; set genes, staphylococcal exotoxin protein homologues. (B) The major variants of RD1 identified among the 36 strains studied. In B, different colors are used to differentiate among genes encoding proteins with distinct functions.