Borderline oxacillin-resistant S. aureus (BORSA) have reduced oxacillin susceptibility but lack the mecA gene. The BORSA phenotype has been linked to beta-lactamase hyperproduction or alterations of penicillin-binding proteins. By using whole genome sequencing, we have identified non-synonymous penicillin-binding protein (pbp) 2 and 3 mutations as well as rpoB mutations that emerged during flucloxacillin and rifampicin treatment in two cases of persistent, device-associated, methicillin-susceptible S. aureus (MSSA) infection (oxacillin MIC increase from 0.38 and 0.75 to 4 and 6 ug/ml, rifampicin MIC increase to > 32 ug/ml). Here, we present an investigation of the genetic basis of this secondary emergence of the BORSA phenotype by site-directed mutagenesis to construct mutants carrying the pbp2, pbp3 and rpoB mutations that have been identified during in vivo emergence of the BORSA phenotype. Since beta-lactamase production may in part explain higher oxacillin MIC, mutagenesis will be carried out both in betalactamase positive and negative background. These experiments will clarify the role of pbp and possibly rpoB mutations as opposed to beta-lactamase hyperproduction in determining the BORSA phenotype. Low-level oxacillin resistance may be associated with flucloxacillin failure in persistent MSSA infections. Whole genome sequencing has identified mutations associated with secondary emergence of oxacillin resistance that can now be tested by mutagenesis.