The rationale for this study comes from several observations strongly suggesting a role of bla genes in the acquisition, stabilization and regulation of mecA gene, the central element of "broad-spectrum" β-lactam resistance characteristic of MRSA strains. The purpose of this study was to evaluate the allelic variability of the bla locus in a representative collection of international epidemic MRSA clones and also, for comparative purposes, in a diverse collection of MSSA strains, in an attempt to establish evolutionary correlations between bla allotypes and β-lactam resistance phenotypes (i.e. between MRSA and MSSA), SCCmec types (i.e. polymorphisms in the mecA regulatory locus) and/or genetic lineages.
MRSA lineages are much less diverse than MSSA lineages in terms of their genome content, a consequence of their more recent evolutionary history [19, 20] and, apparently, also due to some "host barrier" to the SCCmec acquisition . These differences in genetic background variability were well illustrated in our collections since the international MRSA collection comprised eight lineages as defined by MLST clonal complexes, whereas in the smaller and local MSSA collection 15 lineages were represented.
In contrast to the genetic background diversity, we could not detect significant differences between MSSA and MRSA in terms of the bla locus allelic variability. Actually, there were disparate subtle differences in terms of number of allotypes and number of point mutations per allotype: e.g. 11 vs 9 blaZ allotypes and 11.4 vs 14.7 SNP/allele in MRSA and MSSA, respectively. These subtle differences may reflect the more ancient evolutionary history of MSSA or a selective pressure to improve the bla locus activity in these strains. That is to say, although fewer bla types have been retained by the natural selection in MSSA, on average, these allotypes seem to have accumulated more adaptive mutations, in comparison to MRSA strains. In particular for blaZ, for which differences in terms of number of alleles and SNP/allele were more significant, the presence of the alternative β-lactam resistance mechanism mediated by the mecA gene in MRSA strains might have allowed a release in the selective pressure to keep blaZ with optimal activity, in contrast to MSSA, which rely only on blaZ-mediated resistance to β-lactams.
No correlation could be established between bla allotypes and strain backgrounds, β-lactam resistance phenotypes, strain origin and/or isolation dates, indicating that bla genes have evolved independently from S. aureus clonal lineages. This is particularly striking for MRSA strains, which have a very strong clonal structure. These observations may be explained either by differences in evolutionary clock speeds between the genetic background and the bla locus or may result from the horizontal transfer of bla genes between different lineages, which are usually integrated in mobile elements (plasmids and composite transposons). Interestingly, based on the characterization of a collection of several staphylococcal species, Olsen et al, suggested that there is little exchange of bla genes between strains or species , which somehow contradicts our findings. In our study, the most parsimony explanation for the presence of the same bla type in different genetic lineages either MRSA or MSSA or the presence of several bla types in the same lineage, is indeed a high frequency for the horizontal transfer of bla genes across S. aureus clonal clusters.
In spite of the lack of evolutionary links between bla allotypes and genetic lineages, our data strongly suggests a selective pressure to keep the bla locus fully functional, as illustrated by the calculated average dN/dS values well below 1. This observation is valid even on MRSA for which one could expect the accumulation of nonsense or frameshift mutations that would render the bla locus non-functional, due to presence of the mecA gene. Actually, the majority of the mutational events detected in this study were either silent or neutral mutations, being the blaR1 the gene with the highest mutational rate and the blaI the one with the lowest. The increased allelic variability detected for blaR1 (in terms of number of alleles, Simpson's index of diversity, average SNP/allele, and dN/dS values) may suggest that this sensor-inducer gene is the primary target for the evolutionary adaptive mechanisms in the bla locus, presumably to improve the induction efficiency of blaZ expression or even mecA expression, in the case of MRSA strains with no functional mecI-mecR1 regulatory system. In contrast, the relatively lower variability of the much smaller blaI gene, may suggest a fine-tuned repressor activity and a selective pressure to maintain the repressor activity; i.e to maintain the blaZ expression inducible.
Despite the cross-resistance to virtually all β-lactam antibiotics provided by mecA, most contemporary MRSA strains still carry, besides the SCCmec element, the β-lactamase locus. This might be due to the fact that not enough time has elapsed since the mecA acquisition for MRSA strains start loosing the bla genes, because there is a little or no fitness cost associated to the bla genes, or because these genes may be linked to other positively selected genes (e.g. the cadmium resistance genes present in some β-lactamase plasmids). Alternatively, the bla locus may be involved in the "domestication" of the mecA gene, as bla genes have been shown to stabilize the in vitro mecA acquisition [12, 13] and efficiently control mecA transcription [9, 10], explaining the "retention" of a functional bla regulatory system by most contemporary MRSA strains . Interestingly, as no correlation could be established between bla allotypes and SCCmec types, which have polymorphisms in the mecA regulatory locus, this maintenance of functional blaI-blaR1 genes seems to be independent of the functional status of the mecA "natural" regulators mecI-mecR1.
Concerning the maintenance of a functional blaZ gene in MRSA strains one can speculate that, even in the presence of mecA, it might be useful for the bacteria to keep blaZ as a "first-line defense" against β-lactams. In fact, first generation β-lactams (i.e. penicillins) are still widely prescribed either empirically or for the treatment of specific infections (e.g. streptococcal infections). Moreover, penicillins have also been widely used prophylactically in the livestock industry. This means that, both in the nosocomial and community settings, MRSA are still exposed to penicillins and, under these circumstances, expression of β-lactamase is enough for survival under antibiotic pressure. From a physiological perspective, this ability to choose between the expression of two resistance genes may be advantageous for the bacteria since the expression of β-lactamase is likely to impose a smaller fitness cost than the expression of PBP2a. In fact, besides being much smaller than PBP2a (257 vs 668 amino acids), BlaZ is a secreted enzyme whereas PBP2a is a transpeptidase protein, which must be incorporated into the complex cell-wall metabolism.