Polymorphism of theStaphylococcus aureusPanton‐Valentine Leukocidin Genes and Its Possible Link with the Fitness of Community‐Associated Methicillin‐ResistantS. aureus

To the Editor—We read with interest the recent article by O'Hara et al. [1] in which nucleotide polymorphism analysis of Staphylococcus aureus lukSF-PV genes showed 12 variable positions scattered along a highly conserved nucleotide sequence. Variation at position 527 resulted in a histidine (H) to arginine (R) substitution at aa 176 with potential functional implications. The authors defined 2 groups on the basis of this amino acid change, the “R variant” group and the “H variant” group. The major finding was that H176R correlated strongly with the geography of the isolates analyzed and also with the presence of mecA. Thus, the R variant was recovered mainly from mecA+ USA300 strains found in the United States, whereas the H haplotypes were carried by mecA− isolates found outside the United States. Furthermore, phylogenetic analysis of these Panton-Valentine leukocidin (PVL) gene sequences suggested that the H variant was the ancestor of the R variant. The authors hypothesize that the H variant emerged first and gave rise to the R variant harbored by strain MW2, which then transferred to the clonal complex 8, leading to the USA300 strains that rapidly spread throughout the United States. This attractive model of the origin of community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 and 400 does not consider the evolutionary history of other major CA-MRSA clones that emerged in Europe concomitantly [2]. We have studied 48 clinical isolates of PVL+S. aureus, both mecA− and mecA+ from the most prevalent CA-MRSA clones collected in Europe, Asia, Australia, North Africa, and the United States. Haplotypes of lukSF-PV nucleotide sequences were assigned to the 4 groups described by O'Hara et al. (figure 1). The R variant was found in 15 strains belonging to CC8 (USA300 strain) (6 mecA+ and 5 mecA− strains), CC1 (USA400 strain) (2 mecA+ and 1 mecA− strain) from the USA, and to CC93 (Queensland clone) (1 mecA+ strain) recovered from Australia. There were 14 strains that harbored the highly conserved H1 variant; these were mainly recovered from Europe (10 strains), but were also recovered from the United States (2 strains) and Asia (2 strains); 4 mecA− strains belonged to clonal complexes CC5, CC25, and CC30, whereas 10 mecA+ strains belonged to clonal complexes CC1, CC5, CC22, CC30, and CC59, accounting for a large proportion of the worldwide diversity of CA-MRSA. The H3 variant was found in 2 mecA− strains that belonged to CC121, as already reported by O'Hara et al. [1] The H2 variant displayed maximal polymorphism (6 haplotypes), and was present in 7 mecA− strains from several clonal complexes (CC1, CC5, CC30, and CC121), as well as in 9 mecA+ strains that belonged to the CA-MRSA ST80 clone currently spreading in North Africa and in Europe and to the ST377 clone, which emerged simultaneously in Europe and Australia [3]. lukSF-PV sequence variants found in 48 clinical Staphylococcus aureus isolates, shown as a summary of nucleotide variation observed among methicillin-resistant and methicillin-susceptible S. aureus (left table) and comparison with the sequence reported from the USA300 lukSF-PV. Black horizontal line, USA300 genomic sequence; short vertical lines above and below indicate the relative positions of variable sites. The nucleotide residue from the USA300 genome is indicated. Long vertical lines, 5′ and 3′ ends of the sequenced fragment. Colored horizontal lines, unique haplotypes observed in our samples (sequences of different haplotypes have been submitted to GenBank under accession numbers EU518760–EU518770). Short vertical lines on the colored lines indicate the positions of sites at which the haplotype differs from that of USA300. Alternate nucleotide residues are indicated, and substitutions resulting in an amino acid change are indicated in bold. Numbers to the right of the horizontal lines indicate the frequency with which the haplotype was observed, and brackets denote the 4 major haplotype groups identified by O'Hara et al. [1] At left, mecA status, sequence type (ST), and clonal complex (CC) (defined on the basis of at least 5 identical loci) as determined by multilocus sequence typing and eBURST analysis of the 48 strains. Our results are consistent with the work of O'Hara et al. [1], showing that in comparison with the R variant, the H variant has a broader geographic distribution, has spread among several clonal complexes, and contains more genetic variation (especially the H2 group). In addition to the finding of O'Hara et al. [1] that community-associated methicillin-susceptible S. aureus strains belong mainly to the H groups (only 5% of strains in the H-group being mecA+), we show that almost all PVL+ CA-MRSA clones that have spread outside the Unites States also carry the H variant. O'Hara et al. [1] suggest that H-R substitution might have functional implications that result in better fitness for the R variant, thus contributing to the successful spread and high virulence of the USA300 strain. However, direct link between virulence and successful spread holds only when the pathogen's overall transmission potential is enhanced by virulence [4]. PVL gene expression has been associated with severe skin infections, but also with high-mortality pathologies such as necrotizing pneumonia [5]. USA300 strains harboring the R variant may, therefore, have evolved by selection of strains that cause durable cutaneous lesions, which would improve transmissibility of the bacteria, but not (or at least infrequently) produce high-mortality pathologies such as necrotizing pneumonia. In this case, being too virulent seems detrimental for the pathogen, because it kills and immobilizes the host before being successfully transmitted to another host [4]. We hypothesize that the R variant has evolved towards a well-balanced host-pathogen association in which pathogens succeed best by causing severe, highly transmissible cutaneous infections but very rarely causing lethal disease [6, 7]. This is consistent with the observation made by Labandeira-Rey et al. that the H2 variant of PVL harbored by phiSLT phage increased the virulence of a laboratory strain (strain RN6390 lysogenized with phiSLT from A980470) in a mouse pneumonia model [8], whereas Bubeck Wardenburg et al. failed to observe a significant difference in virulence in a mouse pneumonia model that examined R-variant PVL in isogenic USA300 strains [9]. The relevance of amino acid substitution at position 176 of S. aureus lukSF-PV with respect to human infection needs further investigation. However, it supplies a logical basis for future understanding of the evolutionary history of CA-MRSA clones emerging and spreading all over the world.