The opportunistic human pathogen Enterococcus faecium overproduces the low-affinity PBP5. In clinical strains, mutations in PBP5 further reduce its acylation rate by ␤-lactams. Previous studies have reported that ceftaroline had poor inhibitory activity against ␤-lactam-resistant E. faecium strains. In this study, we show that ceftaroline exhibits killing activity against our laboratory-derived ampicillin-resistant E. faecium mutant that overproduces a wild-type PBP5 and that ceftaroline inactivates PBP5 much faster than benzylpenicillin and faster than ceftobiprole. ␤ -Lactam antibiotics (penicillins, cephalosporins, carbapenems, and monobactams) represent the most important group of drugs prescribed to treat bacterial infections. They form stable acyl-enzymes with their targets, the membrane-bound D, D-transpeptidases, which are essential enzymes in peptidoglycan biosynthesis. These proteins are usually referred to as penicillin-binding proteins (PBPs) (1-3). The presence or overproduction of loweraffinity PBPs is responsible for the resistance of Gram-positive cocci against ␤-lactam antibiotics. It is known that the opportunistic human pathogen Enterococcus faecium overproduces the low-affinity PBP5 and that mutations further reduce the acylation rate of PBP5 by altering its amino acid sequence (4, 5). Ceftaroline is a cephalosporin with broad-spectrum activity against Grampositive organisms, including methicillin-resistant Staphylococcus aureus (MRSA), and many common Gram-negative bacteria except those producing extended-spectrum ␤-lactamase (ESBL) or AmpC ␤-lactamase (6-8). The Staphylococcus aureus low-affinity PBP2a is closely related to PBP5 (9). It is inhibited by ceftaroline, the active metabolite of the prodrug ceftaroline fosamil, which was recently approved by the U.S. Food and Drug Administration (FDA) for use in adult patients with acute bacterial skin and skinstructure infections and community-acquired bacterial pneumonia and more recently approved by the European Medicines Agency (EMA) for similar indications (10, 11). Previous studies have reported that ceftaroline had poor inhibitory activity against ␤-lactam-resistant E. faecium strains, all of which were expected to possess a PBP5 protein (6,12). To better understand these differences, we have characterized the interaction between a wild-type form of PBP5 and ceftaroline.Ceftaroline MIC values determined by the microdilution method (13) for E. faecium D63r and D63 strains (5) were 2 and 0.25 mg · liter Ϫ1 , respectively. These values contrasted with those reported by others (10-12), who have found that most ampicillinresistant E. faecium clinical isolates were resistant to ceftaroline and concluded that ceftaroline had little activity against most isolates of E. faecium. The killing effects (14) of ceftaroline on E. faecium D63 and D63r strains were studied by exposing exponentially growing cultures of both strains to increasing concentrations of antibiotics corresponding to 1 and 4 times their respective MICs (Fig. 1). Ceftaroline showed k...