MICs (μg/ml) for ceftaroline and comparators for strains used in this study are shown in Table . Affinities of ceftaroline and comparators for the individual PBPs in the studied isolates are indicated as IC50
s and are reported in Table for the isolates of S. aureus
and in Table for S. pneumoniae
. Ceftaroline demonstrated the lowest MICs for all 7 pneumococcal strains tested, with a range of 0.015 to 2 μg/ml. Three S. pneumoniae
isolates (1564, 2688, and 1394), with penicillin G MICs of 16, 8, and 4 μg/ml, respectively, did not show demonstrable PBP profiles due to affected growth (low cells count and early cell lysis occurred) resulting in poor Bocillin FL binding, which may be explained by lytic cell death, as reported by Regev-Yochay and coworkers (53
). PBP binding studies of penicillin G-resistant S. pneumoniae
isolates defined according to current CLSI nonmeningeal penicillin G breakpoints (susceptible, ≤2 μg/ml; intermediate, 4 μg/ml, and resistant, ≥8 μg/ml) (10
) were difficult because of low bacterial cell counts (OD600
< 0.1) and the fact that cell lysis complicated preparation of sufficient amounts of cell material with all active penicillin-binding proteins (only minimal PBP3 affinity was present) for binding studies with Bocillin FL (K. Kosowska-Shick, unpublished information).
Binding affinities of β-lactams for S. aureus PBPs
Ceftaroline had the lowest MICs against the 6 S. aureus
isolates tested among those for cefotaxime, ceftriaxone, and penicillin G. The ceftaroline MIC was 0.5 μg/ml for all isolates except for 1 vancomycin-resistant MRSA isolate (VRSA 510), for which the MIC was 1 μg/ml. In all strains tested, ceftaroline bound to all PBPs with the same or a higher affinity (IC50
of ≤0.5 μg/ml for PBP1, -2, -2a, and -3 in MSSA strain and ≤1 μg/ml for PBP1, -2, -2a, and -3 in MRSA strains, with 4 exceptions: PBP1 in strains ATCC 29213, 873, and 2149A and PBP2 in strain 1287) than those of all β-lactam comparators (Table ). All antibiotics tested had a weak affinity for PBP4 (IC50
> 8 μg/ml). Moisan et al. observed similar weak affinities of ceftaroline and ceftriaxone for PBP4 in strain ATCC 29213 (42
). A fluorogram of PBP resolutions in methicillin-susceptible S. aureus
ATCC 29213 is presented in Fig. , and Fig. shows PBP2a profiles for all MRSA strains tested.
Competition assays for penicillin G binding to PBPs in MSSA (ATCC 29213).
Competition assays for ceftaroline binding to PBP2a in MRSA strains.
Methicillin resistance in S. aureus
is caused by production of the mecA
-encoded protein PBP2a. The affinity of ceftaroline for PBP2a in all 5 MRSA strains was up to >128 times higher than those of other agents. We also observed that the PBP2a affinity levels in MRSA strains differed, depending on the growth phase during which cells were harvested for membrane isolation. PBP2a affinities for cephalosporins were higher for four strains from which membranes were isolated in the exponential growth phase (OD600
= 0.3) than for one strain (strain 25) isolated in the stationary growth phase (OD600
> 2.00). In strain 25, despite five attempts, there was no measurable PBP affinity observed during the exponential growth phase. The ceftaroline IC50
of 0.01 μg/ml for this strain was 50, 25, and 400 times lower than those of cefotaxime, ceftriaxone, and penicillin G, respectively, but the ceftaroline MIC was ≤128 times lower than those of the latter antibiotics. Hence, the higher PBP2a affinity resulted in lower MICs. We did not see a strictly proportional correlation between IC50
and the MIC for the β-lactams used in the study, which is a known phenomenon and reflects many other factors involved in the resistance level (7
). We observed such differences between PBP2a affinity and the MIC for strain 25 for all antibiotics tested. This lack of correlation was also seen for strain 510 (the vanA
-containing Hershey VRSA strain [4
]) and ceftriaxone (MIC > 64 μg/ml; IC50
= 1 μg/ml), for penicillin G and linezolid-resistant strain 2419A (28
) (MIC = 64 μg/ml; IC50
= 2 μg/ml) or for ceftriaxone and this strain (MIC = 8 μg/ml; IC50
= 2 μg/ml), and for penicillin G (MIC 32 μg/ml; IC50
= 4 μg/ml) and strain 1287 (a VISA strain isolated at Hershey Medical Center [33
]). Many factors may help to explain these differences. One factor could be that β-lactams, especially penicillin G, may have a relatively good affinity for PBP2a but that their susceptibility to hydrolysis by penicillinase results in high MICs (19
). This may explain lower affinities of penicillin G for PBP2a in strains 2149A and 1287, which are β-lactamase positive (Table ).
There is no reported direct relationship between the amount of expressed PBP2a protein and the β-lactam MIC (49
). Additionally, not only PBP2a production but also a sufficient supply of peptidoglycan precursors would be required to mediate β-lactam resistance in MRSA. Also, altered peptidoglycan composition indicates the presence of factors other than PBP2a (12
) which are involved in methicillin resistance, such as mutations in femA
or genes involved in staphylococcal cell wall synthesis (12
). In our previous study, we analyzed the cell wall composition of strain 25 and demonstrated reduced muropeptide cross-linking and a reduction in muramic acid O-acetylation (29
). This strain was isolated in our hospital from a patient whose blood MRSA isolate developed vancomycin and then daptomycin resistance while the patient was on therapy with both agents (29
). These facts may contribute to the observed high MICs for cefotaxime, ceftriaxone, and penicillin G, with relatively low PBP2a affinities. Many changes in the peptidoglycan composition and thickness have been noted in other VISA and VRSA strains (1
), and in our collection, four strains (873, 25, 1287, and 510) had an hVISA, VISA, or VRSA phenotype, which may indirectly suggest the presence of β-lactam resistance mechanisms other than PBP2a. In strain 510, the PBP2a affinities for ceftriaxone (1 μg/ml) and cefotaxime (>128 μg/ml) varied compared to the MICs (>64 μg/ml), and this observation cannot be explained without additional experiments. The above-mentioned facts (a sufficient supply of peptidoglycan precursors and differences in peptidoglycan composition caused by altered gene expression involved in cell wall synthesis) may partially explain the differences as well as the possible mutations in the mecA
gene encoding the PBP2a protein (30
). It is important that affinity studies of this nature have not, to our knowledge, been published for hVISA and VISA strains, so there is no current basis for comparison of the results. These strains may well represent heterogeneous groups, each with its own specific resistance mechanisms. Villegas-Estrada et al. (61
), using different determination and calculation methodology, tested the same (Hershey) VRSA strain examined in the current study as well as one different linezolid-resistant MRSA strain. Ceftaroline was found to be very active, with MICs of 0.25 to 2 μg/ml. Comparison between the IC50
s found in the latter study and those found in the current study are not valid because of differing methodologies.
The range of IC50
s for ceftaroline in our study with PBP2a was 0.5 to 1 μg/ml for membranes isolated in exponential growth phase and 0.01 μg/ml for strain 25, whose PBPs could be harvested only in stationary growth phase. Previous papers using comparable techniques reported average IC50
s of 0.16 to 0.9 μg/ml for ceftaroline in MRSA strains (27
). Differences between our results and those reported previously (27
) may be explained at least partially by differences in strains and culture conditions. The higher affinity of ceftaroline for PBP2a than those of other β-lactams may be explained by the presence of longer side chains in the chemical structure, which increase interactions with the active-site groove of PBP2a (6
) and/or facilitate allosteric interactions that promote access to the active site (61
In MRSA strains, it is known that PBP2a may replace the transpeptidase function of PBP2, although the transglycosylase function of the latter becomes critical for MRSA growth in the presence of β-lactams (51
). In our study, ceftaroline showed very good affinity for PBP2, and in only 1 VISA strain (1287) was its affinity 2-fold lower than those of other cephalosporins (Table ). The mechanisms of low affinity for PBP2 of penicillin G in strains 510 (VRSA) and 2149A (linezolid-resistant MRSA) compared to those of cephalosporins are unknown and may reflect the sequence mutations present in the gene encoding PBP2, which may affect binding (20
PBP4 has been reported to be responsible for production of more highly cross-linked oligomers (36
). The impaired function of PBP4 may be compensated by PBP2 activity (36
). Also, PBP4 is linked to low-level methicillin resistance in strains lacking PBP2a (36
). PBP4 significance in β-lactam resistance has been reported to be more important in community-acquired MRSA (CA-MRSA) strains (41
), which were not tested in the current study. In our study, PBP4 binding was weak (IC50
s > 8 to 128 μg/ml) for all tested β-lactams, with the exception of VISA isolate 1287 with penicillin G (IC50
of 4 μg/ml) (Table ). For VISA strain 25 (resistant to daptomycin and isolated from the blood of a patient who failed vancomycin and daptomycin therapy [29
]), we could not obtain visible binding of Bocillin FL to PBP4; this phenomenon cannot be explained without additional investigations.
PBP1 may play a role in cell division of S. aureus
) and is essential for growth in both MSSA and MRSA, and its function cannot be replaced by PBP2a (50
). Ceftaroline showed good affinity for PBP1, with IC50
s of ≤1 μg/ml for all isolates whose PBPs were harvested in exponential growth phase, with the exception of hVISA 873, for which the IC50
was 8 μg/ml. The IC50
of PBP1 in strain 25, whose PBPs were harvested in the stationary growth phase, was 0.5 μg/ml.
PBP3 is an essential PBP, and β-lactam binding to PBP3 results in cell enlargement and the cessation of septation (17
). Ceftaroline had high PBP3 binding affinity relative to those of comparator β-lactams for 4 of 5 MRSA isolates tested, and in the remaining MRSA strain, hVISA strain 873, the affinity was only 2-fold lower than those of other cephalosporins.
Cephalosporin resistance in pneumococci is due to specific alterations and undefined changes in PBPs and may be influenced by mutations in other genes (14
). As expected, the penicillin G-susceptible strain 1076 had no changes in the penicillin-binding domains of PBP1A (99% homology to strain R6), -2X (99% homology to strain R6), or -2B (96% homology to strain R6), which are known to be associated with β-lactam resistance or effects on PBP binding (2
SDS-PAGE gels of competitive PBP binding experiments for ceftaroline are shown in Fig. for all S. pneumoniae
strains. In our studies on S. pneumoniae
, the order of ceftaroline binding affinities for PBPs in penicillin-susceptible strain 1076 was PBP2X > PBP1A, -1B, and -2A > PBP3 > PBP2B, and ceftaroline had ≥2-fold higher IC50
s for PBP1A and -1B than those of ceftriaxone and cefotaxime and lower IC50
s for the other PBPs in this isolate (Table ), against which all 3 cephalosporins had low MICs (≤0.03 μg/ml). PBP1A and -2X are known to be primary targets for cephalosporins in pneumococci, and all 3 cephalosporins tested showed high affinities for these PBPs, with IC50
s of <0.25 μg/ml (18
). Ceftaroline showed a higher affinity for PBP2B than those of ceftriaxone and cefotaxime in the penicillin G-susceptible pneumococcal strain 1076. Low affinities of cefotaxime and ceftriaxone for PBP2B have been described previously (18
Competition assays for ceftaroline binding to PBPs from Streptococcus pneumoniae strains.
Among 3 penicillin G-resistant strains, ceftaroline had the lowest MICs (≤0.25 μg/ml) and higher or equal affinities for PBP1A, -2B, -2A, and -1B relative to those of the comparators (Table ). The affinity of ceftaroline for PBP2X was higher than or equal to that for the most active comparator, cephalosporin, for any given isolate. The IC50
s of ceftaroline for PBP3 ranged from 0.1 to 0.25 μg/ml, similar to those of comparators, although S. pneumoniae
PBP3 has not been implicated in the killing action of β-lactam antibiotics (59
). Ceftaroline showed the same or improved affinity for PBP2B relative to those of cefotaxime and ceftriaxone (Table ), despite the presence of a T446A substitution in all penicillin-resistant strains (34
). In penicillin-resistant strain 24, the ceftaroline MIC was 0.25 μg/ml, 2- to 4-fold lower than those of cefotaxime and ceftriaxone, with the same affinity for PBP2X (IC50
= 1 μg/ml). The lower MICs in this strain may result from improved PBP2B ceftaroline affinity, which was 16-fold higher than those of ceftriaxone and cefotaxime. In strain 3413, the ceftaroline MIC was 8-fold higher than those of cefotaxime and ceftriaxone, which may reflect improved ceftaroline affinity (4- to 8-fold lower IC50
) for PBP2X and PBP2B. In penicillin-resistant strain 2527, for which all cephalosporins had MICs of ≤0.03 μg/ml, cephalosporin affinities for PBP2X and PBP2B were identical (IC50
, 0.1 and 4 μg/ml, respectively). It should be noted that the role of individual PBP IC50
s in contributing to the pneumococcal MIC is potentially complex and may be influenced by the constellation of mutations present in PBP genes and other genes, as well as the accessibility of the PBPs in whole bacteria (22
In summary, ceftaroline demonstrated potent binding to multiple PBPs in S. aureus, including PBP2a, responsible for methicillin resistance of MRSA, and to PBPs in S. pneumoniae, including PBP2B, PBP2X, and PBP1A, which are important in penicillin resistance. Our results help to explain the improved in vitro activity of this cephalosporin against S. aureus, including MRSA with impaired vancomycin susceptibility, and multidrug-resistant S. pneumoniae. These findings have important potential implications and support the use of ceftaroline for treatment of infections caused by resistant staphylococci and pneumococci.