JAC Advance Access originally published online on June 4, 2007
Journal of Antimicrobial Chemotherapy 2007 60(2):300-311; doi:10.1093/jac/dkm150
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
In vitro activity of ceftaroline (PPI-0903M, T-91825) against bacteria with defined resistance mechanisms and phenotypes
1 Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK 2 Peninsula Pharmaceuticals Inc., 1751 Harbor Bay Parkway, Alameda, CA 94502, USA
* Corresponding author. Tel: +44-20-8327-7223; Fax: +44-20-8327-6264; E-mail: david.livermore{at}hpa.org.uk
Received 23 March 2007; returned 16 April 2007; revised 19 April 2007; accepted 19 April 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Background: Ceftaroline (PPI-0903M, T-91825) is a novel cephalosporin, administered as an N-phosphono prodrug. We investigated its in vitro activity and resistance selection potential.
Methods: MICs were determined by CLSI agar dilution, but with varied inocula. Mutant selection was investigated in single- and multi-step procedures.
Results: MICs for methicillin-resistant Staphylococcus aureus (MRSA) were 0.5–2 mg/L, compared with 0.12–0.25 mg/L for methicillin-susceptible S. aureus; corresponding values for coagulase-negative staphylococci were 0.25–2 and 0.06–0.12 mg/L, respectively. Even with 2% NaCl added, all MRSA were susceptible at 2 mg/L. MICs for Enterococcus faecalis were from 0.25 to 8 mg/L; E. faecium was resistant. MICs for Escherichia coli, Klebsiella spp., Morganella morganii and Proteeae without acquired resistance were 0.06–0.5 mg/L versus 0.12–1 mg/L for Enterobacter, Serratia and Citrobacter spp. and 2–8 mg/L for Acinetobacter spp. MICs rose to 1–2 mg/L for many Enterobacteriaceae with classical TEM ß-lactamases, and were much higher for those with extended-spectrum ß-lactamases (ESBLs), hyperproduced AmpC or K1 enzymes. MICs for strains with classical TEM/SHV ß-lactamases rose if the inoculum was increased to 106 cfu/spot; this effect was even more marked for those with ESBLs. Resistance due to Class A ß-lactamases was reversed by clavulanate. Geometric mean MICs were 0.005, 0.05 and 0.09 mg/L for penicillin-susceptible, -intermediate and -resistant Streptococcus pneumoniae strains, respectively—lower than for any comparator ß-lactam. Haemophilus influenzae and Moraxella catarrhalis were very susceptible, although with marginally raised MICs for ß-lactamase-positive Moraxella strains and for haemophili with chromosomal ampicillin resistance. Ceftaroline selected AmpC-derepressed Enterobacter mutants similarly to cefotaxime in single-step experiments; in multi-step procedures it selected ESBL variants of blaTEM in E. coli. Resistance selection was not seen with S. aureus, H. influenzae or pneumococci.
Conclusions: Ceftaroline has impressive anti-MRSA and anti-pneumococcal activity. Slight lability to classical TEM and SHV ß-lactamases is exceptional for an oxyimino-cephalosporin, but was reversible with clavulanate, as was the greater resistance mediated by ESBLs. Resistance selection occurred with Enterobacteriaceae, not MRSA.
Keywords: inoculum effects , ESBLs , ß-lactamases
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Ceftaroline, formerly PPI-0903M or T-91825, (Cerexa, Alameda, CA, USA) is a developmental cephalosporin, administered clinically as an N-phosphono prodrug (PPI-0903, TAK-599). Like ceftobiprole,1 S-3578,2 and several now-abandoned analogues, such as RWJ-544283,4 and TOC-50,5 ceftaroline is active against methicillin-resistant staphylococci (MRSA),6–8 having high affinity for PBP2' (PBP2a).8 It is also reportedly active against many Enterobacteriaceae and streptococci, though activity is variable against Enterococcus faecalis and poor against anaerobes and non-fermenters, particularly Pseudomonas aeruginosa.6,7
We examined the in vitro activity of ceftaroline against clinical isolates and laboratory strains with known resistance mechanisms, and investigated whether its activity could be extended by combination with clavulanic acid. In addition, we examined the compound's propensity to select resistance in single- and multi-step procedures.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Bacterial strains
Test panels were largely composed of UK clinical isolates with their resistance mechanisms inferred by interpretative reading of phenotypes,9 or identified by molecular analysis. To ensure epidemiological diversity, they were sourced from a wide range of hospitals. ß-Lactamase production among the isolates used in inoculum effect studies was investigated by isoelectric focusing10 and by PCR for blaTEM, blaSHV and blaOXA, using published conditions and primers.11,12 The AmpC expression mutants of Enterobacteriaceae and Escherichia coli transconjugant series with different ß-lactamases were described previously.13
The strains used in mutant selection included representatives of the predominant UK methicillin-resistant Staphylococcus aureus (MRSA) lineage EMRSA-15;14 the original vancomycin-intermediate S. aureus strain Mu50;15 Enterobacter cloacae strains known to be inducible for AmpC in direct assays16 and a reference pair of E. coli strains comprising a parent and its hypermutable (mutS) derivative, each carrying a blaTEM ß-lactamase gene encoded by the laboratory-constructed plasmid pT1.17
Antibiotics and susceptibility tests
Ceftaroline was from Cerexa (formerly Peninsula Pharmaceuticals, Alameda, CA, USA); other antimicrobial agents were from their respective manufacturers, or were purchased from Sigma (Poole, Dorset, UK).
MICs for non-fastidious bacteria were determined by the CLSI dilution methods on Mueller–Hinton agar (Oxoid, Basingstoke, Hampshire) with variations as follows: (i) we determined MICs with inocula of 106 cfu/spot, as well as 104 cfu/spot for batteries of Gram-negative isolates with established ß-lactamase profiles, and (ii) MICs for staphylococci were additionally determined at 35°C on Mueller–Hinton agar containing 2% NaCl to induce PBP-2' and its contingent resistance. MICs for pneumococci were determined on Mueller–Hinton agar with 5% ovine blood, incubated in 5% CO2; for haemophili, we used Haemophilus Test Medium gelled as agar, rather than as broth. Clavulanate was used at 4 mg/L in combination tests.
Single-step selection. Selection was performed with ceftaroline or cefotaxime at four times MIC in Mueller–Hinton agar plates, supplemented as necessary to support growth of haemophili and pneumococci. Inocula comprised approximately 5 x 108 cfu/plate from overnight nutrient broth cultures; selection experiments were performed in duplicate and incubated for 24 h at 37°C; serial dilutions of the same inoculum were spread on drug-free media, giving a denominator for the calculation of mutation frequencies.
Multi-step selection. Bacteria were grown overnight in 10 mL amounts of Mueller–Hinton broth with 0.5x MIC of ceftaroline. One-hundred-microlitre volumes of these cultures were then transferred to fresh broth with double the original antibiotic concentration and incubated overnight. This process was repeated for up to 14 cycles, with the drug concentration doubled after each passage, or until it was impossible to obtain further growth. After the third, sixth, ninth, 12th and final passages, the broths were sub-cultured onto agar containing the same drug concentration. A smear of the resulting growth, retained at –70°C, was used to determine MICs.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Activity versus staphylococci and enterococci
Staphylococci. MICs of ceftaroline for methicillin-susceptible S. aureus (MSSA) mostly were 0.12–0.25 mg/L and were raised by less than one doubling dilution by inclusion of 2% NaCl in the test medium (geometric mean 0.34 mg/L compared with 0.24 mg/L) (Figure 1). MICs for MRSA—most of them variants of the epidemic -15 and -16 lineages that predominate in the UK14—were 1–2 mg/L under standard test conditions and, again, were little raised if 2% NaCl was added to induce PBP-2' (geometric mean MIC 1.56 mg/L with NaCl versus 1.18 mg/L without). No MRSA isolate required >2 mg/L ceftaroline for inhibition, even in the presence of NaCl. MRSA isolates were resistant to other ß-lactams (oxacillin and ceftriaxone) irrespective of addition of NaCl and—as is typical of EMRSA-15 and -16—also to levofloxacin;14 none was resistant to vancomycin (data not shown). MICs of ceftaroline for methicillin-susceptible coagulase-negative staphylococci (CoNS) generally were lower than for MSSA, clustering around 0.06 mg/L and rising only to a geometric mean of 0.11 mg/L in the presence of 2% NaCl (Figure 1). All the methicillin-resistant CoNS isolates were susceptible to ceftaroline at 2 mg/L or less in the absence of 2% NaCl but one isolate required an MIC of 4 mg/L in its presence; geometric mean MICs were well below these values, at 0.47 and 0.64 mg/L, respectively, without and with NaCl.
|
Enterococci. MICs of ceftaroline for E. faecalis and E. faecium isolates are shown in Figure 2. Ceftaroline was more active than other cephalosporins against E. faecalis with values scattered between 0.25 and 32 mg/L and with some suggestion of bimodal behaviour. There was no obvious relationship to the MICs of ampicillin, which clustered from 0.5 to 2 mg/L. MICs of ceftaroline for E. faecium were mostly 16–64 mg/L (geometric mean 28 mg/L) but the two ampicillin-susceptible strains (MIC
8 mg/L) were more susceptible, with ceftaroline MICs at 1 mg/L. Among the 33 E. faecalis tested, six were vancomycin resistant, all with VanA phenotypes, and five were linezolid resistant; among the 56 E. faecium, all except six were vancomycin resistant, mostly with VanA phenotypes, whereas nine were resistant to linezolid and three to quinupristin/dalfopristin. MICs of ceftaroline, predictably, were unrelated to those of vancomycin, linezolid and quinupristin/dalfopristin (data not shown).
|
Activity versus non-fastidious Gram-negative pathogens
E. coli transconjugants. MICs of ceftaroline for transconjugants with classical TEM (i.e. TEM-1 and TEM-2) and SHV-1 penicillinases were 0.5–2 mg/L at standard inocula, compared with 0.015–0.03 mg/L for their isogenic plasmid-free counterparts (Table 1). MICs were considerably higher (mostly 16–128 mg/L) for transconjugants with extended-spectrum TEM, SHV and PER enzymes. OXA-1, -3 and -4 enzymes had little effect on ceftaroline MICs, but values were raised for transconjugants with rarer Class D types, such as OXA-2, -5 and -7; MICs also were raised for transconjugants with NMC-A (Class A) and IMP (Class B) carbapenemases.
|
MICs of ceftaroline for most producers of classical and extended-spectrum class A (TEM, SHV) and D (OXA) ß-lactamases were brought as low as those for plasmid-free recipients by clavulanic acid at 4 mg/L, confirming the role of these enzymes in conferring resistance or reduced susceptibility. IMP-1 conferred resistance irrespective of clavulanic acid. ESBLs, IMP-1 and a few OXA enzymes raised the MICs of comparator oxyimino-cephalosporins but, predictably (and unlike with ceftaroline), MICs of these agents for producers of classical TEM-1/2 and SHV-1 enzymes were within one doubling dilution of those for non-producers.
AmpC expression mutants. MICs for two AmpC inducibility mutant series each of C. freundii, E. cloacae, M. morganii and S. marcescens are shown in Table 2, along with data for P. vulgaris, which has an inducible Class A ß-lactamase. Analogous data were obtained for one-to-three further mutant series of each species (data not shown).
|
As with third-generation cephalosporins (i.e. cefotaxime, ceftriaxone and ceftazidime), MICs of ceftaroline generally were similarly low for AmpC-inducible and -basal (deficient) organisms within a series, but were markedly raised (often to 16–128 mg/L) for the AmpC-derepressed mutants. Such data imply that ceftaroline—like these third-generation cephalosporins18—is labile to AmpC enzymes, but is a weak inducer of their synthesis, meaning that AmpC is protective only when it becomes derepressed. The activity of cefepime was less impaired by derepression of AmpC, reflecting its greater stability to these enzymes. Chromosomal ß-lactamase derepression in P. vulgaris (or even inducible expression in the VA1 series) raised the MICs of ceftaroline and ceftriaxone, but not those of ceftazidime. Derepression-mediated resistance to ceftaroline was reversed by clavulanate in P. vulgaris, but not in the species with AmpC enzymes.
Enterobacteriaceae clinical isolates.
Summary MIC parameters for Enterobacteriaceae isolates are shown in Table 3, by resistance phenotype. Isolates susceptible to other oxyimino-cephalosporins were consistently susceptible to ceftaroline at 2 mg/L, with lower values for E. coli, Klebsiella spp. and P. mirabilis than for Enterobacter and Serratia spp. Nevertheless, ampicillin-resistant E. coli and P. mirabilis and piperacillin-resistant Klebsiella spp.—i.e. those inferred to have acquired penicillinases only, not ESBLs or hyperproduced AmpC enzymes—typically were about 4-fold less susceptible to ceftaroline than were strains susceptible to these penicillins. This differential, best illustrated by the geometric mean MICs, was reversed by clavulanate. Other oxyimino-cephalosporins retained fuller activity against isolates with these penicillinases, with geometric mean MICs no more than 2-fold higher than for ampicillin- and piperacillin-susceptible isolates.
|
MICs of ceftaroline for many AmpC-inducible (i.e. cephalosporin-susceptible) Enterobacter spp., Serratia spp. and Morganella morganii isolates were raised by one or two doubling dilutions by clavulanate, though with geometric mean values remaining
1.2 mg/L. Such antagonism is common with extended-spectrum cephalosporins and reflects induction of AmpC by the clavulanate.19 In contrast, P. vulgaris isolates, all of which remained susceptible to ceftaroline at 1 mg/L, showed up to 16-fold increased susceptibility in the presence of clavulanic acid, implying that even inducible class A cephalosporinase could confer slight protection in some strains. ESBLs were associated with resistance to ceftaroline and other cephalosporins in E. coli and Klebsiella spp., supporting the transconjugant data (Table 1); in general, MICs for isolates with CTX-M ESBLs were higher (>128 mg/L), than for isolates with TEM and SHV ESBLs (mostly 2–16 mg/L). Derepressed AmpC enzymes conferred resistance to ceftaroline and other cephalosporins except cefepime; hyperproduced K1 enzyme in K. oxytoca conferred resistance to ceftaroline and ceftriaxone. Clavulanate potentiated ceftaroline versus ESBL producers and hyperproducers of K1 enzyme, not against those with hyperproduced AmpC.
Acinetobacter spp. MICs of ceftaroline for the Acinetobacter spp. isolates ranged from 4 mg/L to >128 mg/L, and often were reduced in the presence of clavulanate, though this may reflect antibiotic activity by clavulanate itself, not ß-lactamase inhibition. MICs of other cephalosporins were similar to or, in the case of ceftazidime, slightly lower than those of ceftaroline; but none of these compounds had potent activity. Although some isolates counted as cephalosporin susceptible at CLSI breakpoints (8–16 mg/L), almost none did so at the British Society for Antimicrobial Chemotherapy values (1–2 mg/L).
Bacteroides spp. Fourteen of 15 Bacteroides spp. tested were presumptively resistant to ceftaroline, with MICs from 8–128 mg/L (geometric mean 23 mg/L), the exception being one isolate with an MIC of 0.5 mg/L. This isolate was also susceptible to amoxicillin (MIC 0.25 mg/L, compared with a geometric mean of 32 mg/L) and to cefotaxime (MIC, 2 mg/L, compared with a geometric mean of 58 mg/L). All except for one strain, presumptively with a metalloenzyme, became more susceptible to ceftaroline in the presence of clavulanate, with MICs of 0.25–8 mg/L (geometric mean 2.6 mg/L).
As is typical for cephalosporins, the MICs of ceftaroline for pneumococci rose in parallel with those of penicillin (Table 4). Nevertheless, they remained the lowest for any ß-lactam tested, with geometric mean values of 0.005, 0.05 and 0.1 mg/L for penicillin-susceptible, -intermediate and -resistant organisms, respectively. No individual MIC value exceeded 0.25 mg/L, compared with maxima of 2 and 4 mg/L for cefotaxime and ceftriaxone, respectively; penicillin MICs ranged up to 4 mg/L.
|
In common with the other cephalosporins tested, ceftaroline retained excellent activity against H. influenzae isolates, with MICs consistently
0.06 mg/L (Table 4). Isolates with intrinsic-type ampicillin/co-amoxiclav resistance had slightly raised MICs for ceftaroline, with a geometric mean 0.03 mg/L, compared with 0.015 mg/L for fully susceptible isolates, but this seems of academic interest only. MICs for Moraxella catarrhalis isolates were 1 mg/L; lower than or equal to those of the other cephalosporins tested (Table 4). Most isolates of this species were amoxicillin resistant but co-amoxiclav susceptible, suggesting presence of BRO-1 or -2 enzymes. One exceptional isolate was very susceptible to amoxicillin, with an MIC of 0.12 mg/L, compared with a geometric mean value of 6.0 mg/L, and lacked significant synergy with clavulanate, implying the absence of ß-lactamase. This organism also had increased susceptibility to ceftaroline, with an MIC 0.004 mg/L compared with a geometric mean of 0.32 mg/L; differentials for cefotaxime and ceftriaxone were less marked, with MICs of 0.06 and 0.015 mg/L, respectively, for this isolate, compared with geometric mean values of 0.016 and 0.19 mg/L. Inoculum effects in relation to ß-lactamase expression
We observed that the activity of ceftaroline was slightly reduced against E. coli transconjugants with classical TEM and SHV penicillinases (Table 1). Likewise, activity was slightly reduced against many E. coli, P. mirabilis and K. pneumoniae isolates with resistance patterns implying the production only of classical penicillinases (Table 3). These results led us to undertake inoculum effect studies, with inocula of 104 or 106 cfu/spot (Table 5).
|
Among eight ampicillin-susceptible E. coli (MICs 4–16 mg/L), we found smaller inoculum effects for ceftaroline (geometric mean 1.3-fold) between inocula of 104 and 106 per spot than for cefotaxime (2.5-fold). This pattern reversed among 16 ampicillin-resistant (MIC > 128 mg/L), cefotaxime-susceptible (MIC 0.03–0.06 mg/L) E. coli, where we found larger inoculum effects for ceftaroline (geometric mean 7.3-fold) than for cefotaxime (3.8-fold). These 16 isolates were positive for blaTEM and had a corresponding pI 5.4 band on electrofocusing. More strikingly, the high-inoculum ceftaroline MICs rose to 64–256 mg/L in three out of 16 cases, whereas cefotaxime MICs never rose above 2 mg/L.
For tests with Klebsiella spp., we selected 25 isolates, six with piperacillin MICs of 4–16 mg/L and 19 with MICs >32 mg/L. We anticipated, wrongly, that most of the latter group would have blaTEM although, in fact, only two proved TEM-positive by PCR and isoelectric focusing. All the K. pneumoniae isolates, whether piperacillin resistant or not, were PCR-positive for blaSHV, and most gave a corresponding pI 7.6 band on isoelectric focusing. The K. oxytoca isolates were negative for blaSHV and gave a variety of bands on focusing, believed to correspond to KOXY/K1 enzyme, which has variable electro-focusing behaviour. These SHV and K1/KOXY enzymes are universal in their respective species. Among the piperacillin ‘susceptible’ group, the geometric mean inoculum effects were 2.9-fold for ceftaroline and 5.9-fold for cefotaxime, whereas corresponding values among the piperacillin-resistant group were 12.5-fold for ceftaroline and 5.1-fold for cefotaxime. MICs of ceftaroline for the piperacillin-susceptible group never exceeded 2 mg/L at 106 cfu inocula, and remained 0.25 mg/L in five out of six cases. In contrast, six out of 19 piperacillin-resistant isolates, including both TEM-1/2 producers, required MICs > 128 mg/L at high inoculum. High-inoculum cefotaxime MICs never exceeded 4 mg/L and only exceed 1 mg/L for two strains, both piperacillin resistant.
In the case of P. mirabilis, we tested 12 isolates susceptible to ampicillin (MIC <8 mg/L) and 14 resistant isolates (MIC > 128 mg/L). All of the latter group were PCR-positive for blaTEM though isoelectric focusing only found corresponding bands in 12 out of 14: four having TEM-2 and eight having TEM-1. All 26 isolates were susceptible to cefotaxime. Sizeable inoculum effects were seen for both cephalosporins with both the ampicillin-susceptible and resistant groups but the largest effect was for ceftaroline versus the ampicillin-resistant group (Table 5); only here did high-inoculum MICs ever exceed 4 mg/L, ranging up to 256 mg/L.
Six E. coli and 10 Klebsiella spp. isolates with ESBLs were also tested and, here, major inoculum effects were seen for both ceftaroline and cefotaxime. Finally, we re-tested the reference E. coli transconjugants (Table 1) with copiously-produced TEM-1, -2 and SHV-1 ß-lactamases. In all cases, there were major effects for ceftaroline, with MICs rising from 0.5–2 mg/L at low inoculum to >32 mg/L at high inoculum. Effects for cefotaxime were marginal or absent, with high-inoculum MICs 0.25 mg/L.
Using ceftaroline at 4x MIC, we failed to select mutants at detectable frequencies (i) from any of the three S. aureus strains tested (MSSA, EMRSA-15 and VISA Mu50), (ii) from penicillin-susceptible and -resistant pneumococci or (iii) from amoxicillin-susceptible and -resistant haemophili (Table 6). In contrast, we could readily select resistant mutants of both of two AmpC-inducible E. cloacae strains tested, using either ceftaroline or cefotaxime. The Enterobacter mutants had phenotypes typical of AmpC-derepressed organisms, with clavulanate-independent resistance to ceftaroline and third-generation cephalosporins but retained susceptibility to cefepime (Table 7). Mutants of E. coli 1411 pT1 and 1413 pT1 were obtained under selection with ceftaroline but not cefotaxime; their frequency was higher with 1413 pT1, which has a lesion in the mutS mismatch repair system;17 moreover its mutants, unlike those derived from E. coli 1411 retained resistance to ceftaroline after repeated passage on drug-free agar.
|
|
Ceftaroline-selected E. coli 1413 pT1 mutants regained full susceptibility to ceftaroline in the presence of clavulanate; it was notable also that MICs of cefotaxime, ceftriaxone and ceftazidime for these organisms were no higher than for their parent strain. We therefore suggest that they had some change to TEM ß-lactamase quantity or specificity but had not developed true ESBL activity.
The drug concentrations to which we could ‘train’ growth are shown in Table 8, along with MICs for the mutants selected. No variants with elevated MICs of ceftaroline could be obtained from any of the three S. aureus (MSSA, EMRSA-15 and VISA-Mu50) strains, whereas we could drive the MIC for the TEM-ß-lactamase-negative E. coli strain U up 16-fold from 0.06 to 1 mg/L, and that for the TEM-1 producer E. coli V from 0.25 to 128 mg/L. Mutants of the TEM-negative strain U showed small rises in MICs of ceftaroline itself, other cephalosporins and levofloxacin. This profile, with both ß-lactams and non-ß-lactams affected, implied reduced permeability and/or up-regulated efflux.9 In contrast, ceftaroline-selected mutants of the TEM-producer V had a ceftazidimase ESBL phenotype, and showed strong ceftaroline/clavulanate synergy; only mutants obtained at the highest concentrations—maybe with secondary mutations—showed even a one-tube rise in the levofloxacin MIC, suggesting little or no change in permeability or efflux.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Ceftaroline is among several anti-MRSA ß-lactams now in development; others include ceftobiprole and, potentially, carbapenems. They represent important developments, both because ß-lactams have a favourable toxicity profile compared with other anti-MRSA agents and because no other antibiotic family has such a long and good provenance as the ß-lactams against methicillin-susceptible staphylococci.20
In common with other groups, notably Sader et al.,6 we found that the MICs of ceftaroline for MRSA were 2 mg/L under standard conditions, compared with 0.03–0.25 mg/L for methicillin-susceptible strains. However, standard MIC tests are a poor measure of ß-lactam activity against MRSA, since induction of PBP-2' is unreliable.21 To overcome this limitation, we re-tested ceftaroline in the presence of 2% NaCl, finding that MICs remained 2 mg/L for all the MRSA isolates and for all but one methicillin-resistant CoNS strain (MIC, 4 mg/L). This performance is similar, or marginally superior, to those reported previously for ceftobiprole and RWJ-54 428.1,4,21 Attempts to select higher-level resistance in MSSA, MRSA and VISA were unsuccessful, even under a multi-step ‘training’ procedure. This lack of easy mutant selection is in keeping with previous data for RWJ 54 428.4
Like ceftobiprole and RWJ-54 428, but unlike any cephalosporin now available, ceftaroline also had activity at 1 mg/L against about half the E. faecalis isolates tested, though the remainder were more resistant, with MICs 4–8 mg/L, (Figure 2). The reasons for this dichotomy are unclear and the MICs of ceftaroline were unrelated to those of ampicillin. E. faecium was resistant, as found also by Sader et al.,6 the only exceptions being the few isolates also susceptible to amoxicillin.
Activity was impressive against all three major community respiratory pathogens—S. pneumoniae, H. influenzae and M. catarrhalis, also as found by Sader et al.6 Although MICs were raised for penicillin-non-susceptible pneumococci, they remained 2–4-fold below those of cefotaxime and ceftriaxone. Allowing the incremental upward ‘creep’ of ß-lactam MICs for pneumococci, this may be a significant future advantage, though substantive resistance to available cephalosporins is rare at present.22,23 We observed no propensity for ceftaroline to select resistance in S. pneumoniae and H. influenzae but would caution that resistance in these species partly accrues by step-wise transformation, not reflected in the mutant selection tests used here.24
Ceftaroline had low MICs for many Enterobacteriaceae, similar to those of comparator oxyimino-cephalosporins. However, like these comparators, it lost activity against ESBL producers, with MICs being especially high for isolates with CTX-M enzymes. CTX-M enzymes are now the predominant ESBLs in much of Europe, Asia and South America, though not yet in the USA.25 As with third-generation cephalosporins, but not cefepime, ceftaroline also lost activity against AmpC-derepressed Enterobacteriaceae isolates and mutants and consequently selected AmpC-derepressed E. cloacae mutants from inducible populations. It was compromised too against K. oxytoca strains hyperproducing K1 enzyme, as is ceftriaxone but not ceftazidime.26
More surprisingly, the activity of ceftaroline was slightly reduced, even at standard inocula, against E. coli transconjugants with classical TEM and SHV penicillinases and against many E. coli, P. mirabilis and K. pneumoniae isolates with resistance patterns indicating only classical TEM and SHV penicillinases. These results led us to undertake the inoculum effect studies detailed in Table 5, which confirmed that the MICs of ceftaroline rose markedly with the inoculum for many (but not all) E. coli and P. mirabilis with TEM-1 or -2 enzymes and for piperacillin-resistant Klebsiella spp. Inoculum effects were much less marked for ampicillin-susceptible E. coli and P. mirabilis and for piperacillin-susceptible Klebsiella spp.; comparable effects were not seen for cefotaxime. The fact that the inoculum effects for ceftaroline were not universal among ß-lactamase producers may be because they vary with the amount of enzyme; likewise the occurrence of ß-lactamase-independent inoculum effects for both ceftaroline and cefotaxime for P. mirabilis implies some other contributory factor in this species. Nevertheless, the predominant mechanisms appear to be ß-lactamase-related, as confirmed by its reversal by clavulanate. In some cases, notably for the reference TEM-1, TEM-2 and SHV-1 enzyme producers, the MICs of ceftaroline at high inoculum were >32 mg/L, whereas those of cefotaxime never exceeded 4 mg/L, except when ESBL producers were tested. Taken collectively, these data imply that—very unusually for an oxyimino-cephalosporin—ceftaroline has some lability to classical broad-spectrum penicillinases, though the significance of this (and of ß-lactamase-related inoculum effects in general)27 remains a subject of debate.
Clavulanate, at 4 mg/L, not only restored full susceptibility to TEM-1, -2 and SHV-1 producers but also overcame resistance conferred by ESBLs and K1 enzyme. In addition, it also brought most Bacteroides isolates into the compound's spectrum. The limits of such a combination were, predictably, the failure to overcome resistance due to derepressed AmpC enzymes and weak antagonism against some Serratia, Morganella and Enterobacter strains with inducible AmpC enzymes. Such antagonism occurs also with ticarcillin/clavulanate and other oxyimino-cephalosporin/clavulanate combinations,28 and is of unproven significance almost 20 years after it was first described.
In summary, ceftaroline is a promising new cephalosporin with excellent activity against staphylococci, including methicillin-resistant strains, and with better activity against penicillin-resistant pneumococci than any available cephalosporin. While ceftaroline also has good activity against many Enterobacteriaceae that act as opportunist pathogens, it has significant lability to ESBLs and AmpC enzymes, and some vulnerability also to enterobacterial penicillinases. Much of this lability could potentially be overcome by combination with clavulanate.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
S. M. and M. W., none to declare. Y. G. is employed by the developer of ceftaroline; K. K. was formerly employed by them, but is now employed by Johnson & Johnson. D. M. L. is employed within the UK public sector and is influenced by HPA and NHS policies and attitudes on prescribing; he has received grants and accepted lecture and conference invites from numerous pharmaceutical companies and holds shares in several, with these holdings amounting to less than 5% of a diversified portfolio. He does not believe that his comments in this paper have been materially influenced by these factors, nor that these interests will be materially influenced by his comments in this paper.
|
Footnotes |
|---|
Present address. Cerexa Inc., 1751 Harbor Bay Parkway, Alameda, CA 94502, USA. 
Present address. Peninsula Pharmaceuticals Inc., 1900 Charleston Road, Mountain View, CA 94043, USA.
|
Acknowledgements |
|---|
This work was supported by a grant from Cerexa to the HPA.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
1 Jones RN, Deshpande LM, Mutnick AH, et al. In vitro evaluation of BAL9141, a novel parenteral cephalosporin active against oxacillin-resistant staphylococci. J Antimicrob Chemother (2002) 50:915–32.
2
Fujimura T, Yamano Y, Yoshida I, et al. In vitro activity of S-3578, a new broad-spectrum cephalosporin active against methicillin-resistant staphylococci. Antimicrob Agents Chemother (2003) 47:923–31.
3
Chamberland S, Blais J, Hoang M, et al. In vitro activities of RWJ-54428 (MC-02,479) against multiresistant gram-positive bacteria. Antimicrob Agents Chemother (2001) 45:1422–30.
4
Johnson AP, Warner M, Carter M, et al. In vitro activity of cephalosporin RWJ-54428 (MC-02479) against multidrug-resistant gram-positive cocci. Antimicrob Agents Chemother (2002) 46:321–6.
5 Nomura S, Hanaki H, Unemi N. In vitro antibacterial activity of TOC-50, a new parenteral cephalosporin against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Chemotherapy (1996) 42:253–8.[Web of Science][Medline]
6
Sader HS, Fritsche TR, Kaniga K, et al. Antimicrobial activity and spectrum of PPI-0903M (T-91825), a novel cephalosporin, tested against a worldwide collection of clinical strains. Antimicrob Agents Chemother (2005) 49:3501–12.
7 Iizawa Y, Nagai J, Ishikawa T, et al. In vitro antimicrobial activity of T-91825, a novel anti-MRSA cephalosporin, and in vivo anti-MRSA activity of its prodrug, TAK-599. J Infect Chemother (2004) 10:146–56.[Medline]
8 Ishikawa T, Matsunaga N, Tawada H, et al. TAK-599, a novel N-phosphono type prodrug of anti-MRSA cephalosporin T-91825: synthesis, physicochemical and pharmacological properties. Bioorg Med Chem (2003) 11:2427–37.[CrossRef][Medline]
9 Livermore DM, Winstanley TG, Shannon KP. Interpretative reading: recognizing the universal and inferring resistance mechanisms from resistance phenotypes. J Antimicrob Chemother (2001) 48:87–102.[Abstract]
10 Livermore DM, Williams JD. ß-Lactams: mode of action and mechanisms of bacterial resistance. In: Antibiotics in Laboratory Medicine—Lorian V, ed. (1996) Baltimore: Williams and Wilkins. 502–78.
11
Yuan M, Aucken H, Hall LM, et al. Epidemiological typing of klebsiellae with extended-spectrum ß-lactamases from European intensive care units. J Antimicrob Chemother (1998) 41:527–39.
12 Arlet G, Brami G, Decre D, et al. Molecular characterisation by PCR-restriction fragment length polymorphism of TEM ß-lactamases. FEMS Microbiol Lett (1995) 134:203–8.[Web of Science][Medline]
13
Mushtaq S, Ge Y, Livermore DM. Comparative activities of doripenem versus isolates, mutants, and transconjugants of Enterobacteriaceae and Acinetobacter spp. with characterized ß-lactamases. Antimicrob Agents Chemother (2004) 48:1313–9.
14
Johnson AP, Aucken H, Cavendish S, et al. Dominance of EMRSA-15 and -16 among MRSA causing nosocomial bacteraemia in the UK: analysis of isolates from the European Antimicrobial Resistance Surveillance System (EARSS). J Antimicrob Chemother (2001) 48:144.
15
Hiramatsu K, Hanaki H, Ino T, et al. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother (1997) 40:135–6.
16
Yang YJ, Livermore DM, Williams RJ. Chromosomal ß-lactamase expression and antibiotic resistance in Enterobacter cloacae. J Med Microbiol (1988) 25:227–33.
17 Ellington MJ, Livermore DM, Pitt TL, et al. Development of extended-spectrum activity in TEM ß-lactamases in hyper-mutable, mutS Escherichia coli. Clin Microbiol Infect (2006) 12:800–3.[Web of Science][Medline]
18 Livermore DM. ß-Lactamases in laboratory and clinical resistance. Clin Microbiol Rev (1995) 8:557–84.[Abstract]
19
Akova M, Yang Y, Livermore DM. Interactions of tazobactam and clavulanate with inducibly- and constitutively-expressed Class I ß-lactamases. J Antimicrob Chemother (1990) 25:199–208.
20 Livermore DM. Can ß-lactams be re-engineered to beat MRSA? Clin Microbiol Infect (2006) 12(Suppl. 2):11–6.[CrossRef][Web of Science][Medline]
21 Brown DF. Detection of methicillin/oxacillin resistance in staphylococci. J Antimicrob Chemother (2001) 48:65–70.[Abstract]
22 Felmingham D, Feldman C, Hryniewicz W, et al. Surveillance of resistance in bacteria causing community-acquired respiratory tract infections. Clin Microbiol Infect (2002) 8(Suppl. 2):12–42.[CrossRef][Web of Science][Medline]
23 Karlowsky JA, Jones ME, Mayfield DC, et al. Ceftriaxone activity against Gram-positive and Gram-negative pathogens isolated in US clinical microbiology laboratories from 1996 to 2000: results from The Surveillance Network (TSN®) Database-USA. Int J Antimicrob Agents (2002) 19:413–26.[CrossRef][Web of Science][Medline]
24
Spratt BG. Resistance to antibiotics mediated by target alterations. Science (1994) 264:388–93.
25
Livermore DM, Hawkey PM. CTX-M: changing the face of ESBLs in the UK. J Antimicrob Chemother (2005) 56:451–4.
26 Livermore DM. Clinical significance of ß-lactamase induction and stable derepression in gram-negative rods. Eur J Clin Microbiol (1987) 6:439–45.[CrossRef][Web of Science][Medline]
27 Craig WA, Bhavnani SM, Ambrose PG. The inoculum effect: fact or artifact? Diagn Microbiol Infect Dis (2004) 50:229–30.[CrossRef][Web of Science][Medline]
28
Livermore DM, Akova M, Wu PJ, et al. Clavulanate and ß-lactamase induction. J Antimicrob Chemother (1989) 24(Suppl. B):23–33.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Vidaillac, S. N. Leonard, and M. J. Rybak In Vitro Activity of Ceftaroline against Methicillin-Resistant Staphylococcus aureus and Heterogeneous Vancomycin-Intermediate S. aureus in a Hollow Fiber Model Antimicrob. Agents Chemother., November 1, 2009; 53(11): 4712 - 4717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. I. Llarrull, J. F. Fisher, and S. Mobashery Molecular Basis and Phenotype of Methicillin Resistance in Staphylococcus aureus and Insights into New {beta}-Lactams That Meet the Challenge Antimicrob. Agents Chemother., October 1, 2009; 53(10): 4051 - 4063. [Full Text] [PDF]
|
|
|
|
|
|
C. Vidaillac, S. N. Leonard, H. S. Sader, R. N. Jones, and M. J. Rybak In Vitro Activity of Ceftaroline Alone and in Combination against Clinical Isolates of Resistant Gram-Negative Pathogens, Including {beta}-Lactamase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Antimicrob. Agents Chemother., June 1, 2009; 53(6): 2360 - 2366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Brown and M. M. Traczewski In Vitro Antimicrobial Activity of a New Cephalosporin, Ceftaroline, and Determination of Quality Control Ranges for MIC Testing Antimicrob. Agents Chemother., March 1, 2009; 53(3): 1271 - 1274. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Livermore and M. Warner Effects of low temperature and high salt on the activity of the novel carbapenem PZ-601 (SMP-601) against methicillin-resistant Staphylococcus aureus J. Antimicrob. Chemother., February 1, 2009; 63(2): 411 - 413. [Full Text] [PDF]
|
|
|
|
|
|
A. Fenoll, L. Aguilar, O. Robledo, M.-J. Gimenez, J.-J. Granizo, D. Biek, and D. Tarrago In Vitro Activity of Ceftaroline against Streptococcus pneumoniae Isolates Exhibiting Resistance to Penicillin, Amoxicillin, and Cefotaxime Antimicrob. Agents Chemother., November 1, 2008; 52(11): 4209 - 4210. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Y. Peleg, H. Seifert, and D. L. Paterson Acinetobacter baumannii: Emergence of a Successful Pathogen Clin. Microbiol. Rev., July 1, 2008; 21(3): 538 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. S. Sader, T. R. Fritsche, and R. N. Jones Antimicrobial Activities of Ceftaroline and ME1036 Tested against Clinical Strains of Community-Acquired Methicillin-Resistant Staphylococcus aureus Antimicrob. Agents Chemother., March 1, 2008; 52(3): 1153 - 1155. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||