Identification of non-PBP2a resistance mechanisms in Staphylococcus aureus after serial passage with ceftaroline: involvement of other PBPs

<span class="paragraphSection"><div class="boxTitle">Objectives</div>Ceftaroline (the active metabolite of ceftaroline fosamil) is a cephalosporin that possesses activity against MRSA due to its differentiating high affinity for PBP2a. It is known that PBP2a sequence variations, including some outside of the transpeptidase-binding pocket, impact ceftaroline susceptibility and recent evidence suggests involvement of non-PBP2a mechanisms in ceftaroline resistance. This study evaluated the potential of ceftaroline to select for resistant <span style="font-style:italic;">Staphylococcus aureus</span> clones during serial passage.<div class="boxTitle">Methods</div>Selection experiments were performed by up to 20 daily passages of three <span style="font-style:italic;">S. aureus</span> isolates (two MRSA and one MSSA) in broth with increasing selective pressure. Mutants that emerged were tested for changes in ceftaroline susceptibility and genetically characterized.<div class="boxTitle">Results</div>The MSSA isolate developed mutations in PBP2 and PBP3 that increased the ceftaroline MIC by 16-fold and increased the MICs of other β-lactams. A Glu₄₄₇Lys substitution in the PBP2a transpeptidase pocket in one MRSA isolate elevated the ceftaroline MIC to 8 mg/L. Selective pressure in a ceftaroline-resistant MRSA isolate generated mutations in LytD, as well as changes in the <span style="font-style:italic;">pbp4</span> promoter previously shown to result in PBP4 overexpression, the one PBP not inhibited by ceftaroline. Elevated ceftaroline MIC was reversed when tested in combination with extremely low levels of methicillin or meropenem that could inhibit the function of PBP4.<div class="boxTitle">Conclusions</div>These studies demonstrate that resistance to ceftaroline can be manifested through numerous mechanisms. Further, they support a hypothesis where PBP4 can functionally provide the essential transpeptidase activity required for MRSA cell wall biogenesis when PBP2a is inhibited.</span>

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