Penicillin-binding protein PBP2a provides variable levels of protection toward different β-lactams in Staphylococcus aureus RN4220.

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to most beta-lactams due to the expression of an extra penicillin-binding protein, PBP2a, with low beta-lactam affinity. It has long been known that heterologous expression of the PBP2a-encoding mecA gene in methicillin-sensitive S. aureus (MSSA) provides protection towards beta-lactams, however, some reports suggest that the degree of protection can vary between different beta-lactams. To test this more systematically, we introduced an IPTG-inducible mecA into the MSSA laboratory strain RN4220. We confirm, by growth assays as well as single-cell microfluidics time-lapse microscopy experiments, that PBP2a expression protects against beta-lactams in S. aureus RN4220. By testing a panel of ten different beta-lactams, we conclude that there is also a great variation in the level of protection conferred by PBP2a. Expression of PBP2a resulted in an only fourfold increase in minimum inhibitory concentration (MIC) for imipenem, while a 32-fold increase in MIC was observed for cefaclor and cephalexin. Interestingly, in our experimental setup, PBP2a confers the highest protection against cefaclor and cephalexin-two beta-lactams that are known to have a high specific affinity toward the transpeptidase PBP3 of S. aureus. Notably, using a single-cell microfluidics setup we demonstrate a considerable phenotypic variation between cells upon beta-lactam exposure and show that mecA-expressing S. aureus can survive beta-lactam concentrations much higher than the minimal inhibitory concentrations. We discuss possible explanations and implications of these results including important aspects regarding treatment of infection.