###### Abstract  
The acquisition of the resistance determinant _mecA_ by _Staphylococcus aureus_ is of major clinical importance, since it confers a resistant phenotype to virtually the entire large family of structurally diverse \(\beta\)-lactam antibiotics. While the common resistance determinant _mecA_ is essential, the optimal expression of the resistance phenotype also requires additional factors. Previous studies showed that the great majority of clinical isolates of methicillin-resistant _S. aureus_ (MRSA) have a heterogeneous resistant phenotype, and we observed that strains carrying methicillin genetic determinants other than _mecA_ also produce similar heterogeneous phenotypes. All these strains were able to express high and homogeneous levels of oxacillin resistance when sub-inhibitory concentrations of muiprocin, an effector of the stringent stress response, were added to growth media. Our studies show that the gene _gmk_, involved in guanine metabolism, was one of the first genes to exhibit mutations in homoresistant (H*R) derivatives obtained through serial passages (with increasing concentrations of oxacillin) of the prototype _mecC_-carrying MRSA strain LGA251. All these observations led us to propose that a common molecular mechanism for the establishment of high and homogeneous oxacillin resistance must be present among isolates carrying different methicillin resistance determinants. In this work, we tested this hypothesis using whole-genome sequencing (WGS) to compare isogenic populations differing only in their degrees of oxacillin resistance and carrying various methicillin genetic determinants  
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Footnote †: journal: _antibiotics_  
0000-0001-6600-0000]C.M.Milheirico  
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the evolutionary precursor of the resistance determinant _mecA_ carried by all methicillinresistant _S. aureus_ (MRSA) strains. Although the overwhelming majority of _M. sciuri_ isolates that were initially examined were fully susceptible to \(\beta\)-lactam antibiotics [13], _M. sciuri_ isolates from human clinical specimens were later reported with increased MICs for \(\beta\)-lactam antibiotics [14]. Interestingly, although antibiotic resistance is believed to arise primarily from human antibiotic overuse [15], a recent study [16] making use of time-resolved phylogenies suggested that particular lineages of _mec_-MRSA appeared in European hedgehogs 200 years ago, i.e., more than a century before the first description of MRSA in humans. These lineages emerged in hedgehogs colonized with _S. aureus_ and co-infected with a \(\beta\)-lactam-producer dermatophyte, _Trichophyton erinacci_. The _mec_-MRSA lineages developed to resist the penicillin produced by _T. erinacci_ and eventually spread within local hedgehog populations and between hedgehogs and secondary hosts, such as livestock and humans [16]. The _mecA_ gene is widely disseminated among staphylococcal species, and its expression is essential for the methicillin-resistant phenotype. However, the mechanism involved in the strain-to-strain variation of the phenotypic expression of methicillin resistance is still not well understood. Most MRSA strains exhibit a heterogeneous phenotype of \(\beta\)-lactam resistance under routine growth conditions: the majority of cells exhibiting variable but typically low-level resistance coexist with a few cells displaying homogeneous and high-level oxacillin resistance (H*R) [17; 18]. This phenotype may have considerable clinical relevance since, upon being challenged by \(\beta\)-lactam agents, a homogeneous and highly oxacillin-resistant subpopulation may rapidly be selected. Several factors are known to contribute to \(\beta\)-lactam resistant phenotype. Studies with MRSA strain COL have shown that the optimal expression of the resistance phenotype requires not only the presence of _mecA_ but also a large number of key additional chromosomal determinants--\"auxiliary genes\", identified by Tn551 mutagenesis [19; 20]. TCA cycle has also been implicated in \(\beta\)-lactam H*R phenotype [21; 22].