Cutaneous lymphomas and COVID ‐19: What is known so far?

J Antimicrob Chemother 2013; 68: 648 – 658 doi:10.1093/jac/dks442 Advance Access publication 27 November 2012 Activity of ceftaroline against extracellular (broth) and intracellular (THP-1 monocytes) forms of methicillin-resistant Staphylococcus aureus: comparison with vancomycin, linezolid and daptomycin Aurélie Mélard1, Laetitia G. Garcia1, Debaditya Das1, Raoul Rozenberg2, Paul M. Tulkens1*, Françoise Van Bambeke1 and Sandrine Lemaire1 1 Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium; 2Analyse structurale moléculaire, Institute of Condensed Matter and Nanosciences, Université catholique de Louvain, Louvain-la-Neuve, Belgium *Corresponding author. Pharmacologie cellulaire et moléculaire, Université catholique de Louvain, Avenue E. Mounier 73 B1.73.05, B-1200 Brussels, Belgium. Tel: +32-2-7647371; Fax: +32-2-7647373; E-mail: [email protected] Received 22 August 2012; returned 25 September 2012; revised 26 September 2012; accepted 11 October 2012 Background: Ceftaroline fosamil is approved for treatment of acute bacterial skin and skin structure infections caused by methicillin-resistant Staphylococcus aureus (MRSA). We examined the activity of its active metabolite (ceftaroline) against intracellular forms of S. aureus in comparison with vancomycin, daptomycin and linezolid. Methods: Two methicillin-susceptible S. aureus (MSSA) and 11 MRSA strains with ceftaroline MICs from 0.125 to 2 mg/L [two strains vancomycin- and one strain linezolid-resistant (EUCAST interpretative criteria); VISA and cfr+] were investigated. The activity was measured in broth and after phagocytosis by THP-1 monocytes in concentration-dependent experiments (24 h of incubation) to determine: (i) relative potencies (EC50) and static concentrations (Cs) (mg/L and ×MIC); and (ii) relative activities at human Cmax (ECmax) and maximal relative efficacies (Emax) (change in log10 cfu compared with initial inoculum). Ceftaroline stability and cellular accumulation (at 24 h) were measured by mass spectrometry. Results: Ceftaroline showed similar activities in broth and in monocytes compared with vancomycin, daptomycin and linezolid, with no impact of resistance mechanisms to vancomycin or linezolid. For all four antibiotics, intracellular ECmax and Emax were considerably lower than in broth (≏0.5 log10 versus 4– 5 log10 cfu decrease), but the EC50 and Cs showed comparatively little change (all values between ≏0.3 and ≏6× MIC). The mean cellular to extracellular ceftaroline concentration ratios (20 mg/L; 24 h) were 0.66+0.05 and 0.90+0.36 in uninfected and infected cells, respectively. Conclusion: In vitro, ceftaroline controls the growth of intracellular MRSA to an extent similar to that of vancomycin, linezolid and daptomycin for strains with a ceftaroline MIC ≤2 mg/L. Keywords: Hill equation, maximal relative efficacy, static concentration, recursive partitioning analysis, mass spectrometry, VISA, linezolid resistant, cfr Introduction Ceftaroline,1 originally known as T-91825, is a novel cephalosporin with in vitro activities against methicillin-resistant Staphylococcus aureus (MRSA) comparable to those of vancomycin, linezolid and daptomycin towards susceptible strains,2,3 and with unimpaired activity against strains non-susceptible (VISA) or resistant (VRSA) to vancomycin.4 In in vitro time–kill studies, ceftaroline is also more rapidly cidal against MRSA than vancomycin and linezolid.2 Developed for clinical use as a water-soluble N-phosphono prodrug [ceftaroline fosamil (TAK-599)],5 it has proven efficacious, thus receiving approval in the USA and the EU for the treatment of acute bacterial skin and skin structure infections caused by susceptible organisms including MRSA.6 – 9a In these studies a numerically higher clinical response was achieved for ceftaroline over a vancomycin/aztreonam combination at an early stage of treatment.10 Ceftaroline fosamil has also been approved for the treatment of community-acquired bacterial pneumonia caused by susceptible organisms including methicillinsusceptible S. aureus (MSSA) and Streptococcus pneumoniae. These studies compared ceftaroline with ceftriaxone and again # The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected] 648 Ceftaroline and intracellular MRSA ceftaroline clinical response rates were numerically superior to those with the comparator agent. While historically considered as an extracellular organism only, there is now increasing evidence that S. aureus invades, sojourns and thrives intracellularly.11,12 This creates a potential therapeutic challenge for clinicians, since it has been clearly documented using in vitro and in vivo models that most antistaphylococcal agents are considerably less active against intracellular S. aureus than expected given their respective intrinsic activity and/or level of cellular accumulation.13,14 Evaluation of novel antistaphylococcal agents must therefore include an assessment of their ability to control intracellular infections. In this context, we observed that, contrary to most assumptions, b-lactams actually display significant intracellular activity against S. aureus, despite a reported poor cellular accumulation.15,16 In a previous study we also showed that ceftobiprole, another anti-MRSA cephalosporin,17 was equally active against intracellular forms of MSSA and MRSA, regardless of origin (community or hospital acquired) and resistance phenotype towards vancomycin.18 The present study extends these observations to ceftaroline and compares it with vancomycin, daptomycin and linezolid using a panel of strains with increasing MICs towards these antibiotics [including VISA and linezolid resistant (LZDR)]. Our study used a previously established pharmacodynamic model of infected human THP-1 monocytes that allows a quantitative assessment of key properties such as intracellular maximal efficacy and potency of antibiotics.15 We show that all four antibiotics display similar intracellular efficacy, with the activity of ceftaroline remaining essentially unimpaired across all strains investigated up to the highest MIC of ceftaroline observed (2 mg/L). Materials and methods Materials Ceftaroline (potency 85.3%; lot no. FMD-CEF-035) was provided by Forest Laboratories, Inc. (New York, NY, USA). Oxacillin was purchased from Sigma– Aldrich (St Louis, MO, USA). Other antibiotics were obtained as the corresponding branded products for human parenteral use distributed for clinical use in Belgium (gentamicin as Geomycinew, GlaxoSmithKline, Wavre, Belgium; vancomycin as Vancomycin Sandozw, Sandoz n.v., Vilvoorde, Belgium; and linezolid as Zyvoxidw, Pfizer s.a., Brussels, Belgium) or in France (daptomycin as Cubicinw, Novartis Europharm Ltd, Horsham, UK). Culture media and sera were from Invitrogen Corporation (Carlsbad, CA, USA) and Becton Dickinson (Franklin Lakes, NJ, USA), and other reagents were from Sigma–Aldrich or Merck KGaA (Darmstadt, Germany). Bacterial strains and susceptibility testing The clinical isolates used in the present study are listed in Table 1, with information on their origin and their resistance phenotypes. MICs were determined following the general recommendations of the CLSI19 for vancomycin, linezolid and daptomycin (with addition of Ca2+ for daptomycin). For ceftaroline, we used both plain and cation-adjusted Mueller– Hinton (MH) broth, but no difference was observed between these two media. There was also no effect of the addition of 2% NaCl to MH broth. MICs were also measured in broth adjusted to pH 5.5 to mimic the phagolysosomal environment where S. aureus sojourns after phagocytosis.20,21 Strains for which ceftaroline showed an MIC ≥1 mg/L were retested using arithmetic dilutions (0.25 mg/L intervals) between JAC 1 and 4 mg/L to determine their MICs in a more accurate fashion in that range than with the conventional geometric (log2) dilution method. Cell lines, cell infection and determination of cell viability Experiments were performed with THP-1 cells (ATCC TIB-202; supplied through LGC Promochem Ltd, Teddington, UK), a human myelomonocytic cell line displaying macrophage-like activity,22 maintained in our laboratory as previously described.23 Cell infection was performed as previously described.15,16,18 Briefly, S. aureus in the stationary phase (overnight culture) were opsonized in the presence of 10% human serum (Lonza Ltd, Basel, Switzerland) in RPMI-1640 medium and mixed with THP-1 cells (0.5×106 cells/mL) for 1 h at a ratio of four bacteria per macrophage after which extracellular and non-internalized bacteria were eliminated by washing and exposure to gentamicin (100× MIC; 45 min). This yielded a typical post-phagocytosis bacterial load of 1 –3×106 cfu/mg of cell protein. Cells were thereafter incubated for 24 h at 378C. Cell viability was checked by measuring the release of lactate dehydrogenase and trypan blue exclusion assay. Determination of the extracellular and intracellular activities of antibiotics (concentration– response curves) and pharmacodynamic descriptors Extracellular and intracellular activities were measured at a fixed timepoint (24 h) using a large array of antibiotic concentrations (typically from 0.01 to 100× MIC) to obtain a complete description of the concentration-dependent response, as described in detail in our previous publications.15,16,18 For extracellular activity, experiments were performed in MH broth (supplemented with Ca2+ for daptomycin) with an initial inoculum of 106 cfu/mL and results expressed as the change in cfu/mL from the initial inoculum as measured by colony counting. Bactericidal activity was defined as a reduction of 99.9% (≥3 log10 cfu/mL decrease) of the total counts. For intracellular activity, infected THP-1 cells were collected by centrifugation, washed once in PBS and lysed in distilled water. The resulting solution was analysed for protein content (using the Folin-Ciocalteu/biuret method)24 and were plated on TrypticaseTM soy agar (Becton Dickinson) to enumerate bacteria as described in detail in a previous publication (including validation and determination of the lowest limit of detection).15 Results were expressed as the change in cfu/mg of cell protein. Data were used to fit a sigmoidal function (Hill equation; slope factor¼1) by non-linear regression (GraphPad Prismw version 4.03; GraphPad Software, La Jolla, CA, USA) to obtain for each condition numeric values of five key pharmacodynamic descriptors, namely: (i) the increase in the number of cfu for an infinitely low antibiotic concentration [relative minimal efficacy (Emin; in log10 cfu units)] compared with the original inoculum; (ii) the decrease in the number of cfu for an infinitely large concentration of antibiotic [relative maximal efficacy (Emax; in log10 cfu units); limit of detection ≏5.5 log10 cfu decrease from the original inoculum]; (iii) the decrease in the number of cfu at a concentration corresponding to the maximal serum concentration (Cmax) of the drug as observed in humans receiving standard therapies (ECmax; in log10 cfu); (iv) the concentration of antibiotic yielding a response halfway between Emin and Emax [relative potency (EC50; in mg/L or in multiples of MIC)]; and (v) the concentration of antibiotic resulting in no apparent bacterial growth compared with the original inoculum [static concentration (Cs; in mg/L or in multiples of MIC)]. Stability of ceftaroline and measurement of its cellular to extracellular concentration ratio Ceftaroline stability at 378C over 24 h in water, broth (adjusted to pH 7.4 and 5.5) and in the cell culture medium (pH ≏7.4) was checked by 649 Mélard et al. measuring its MIC for S. aureus ATCC 25923 using an arithmetic dilution progression (0.1 mg/L intervals) starting from media containing 10 or 100 mg/L. In parallel, broth and culture medium samples incubated with 20 mg/L ceftaroline and cell samples from uninfected and infected THP-1 cells incubated with 20 mg/L ceftaroline for 24 h (collected by centrifugation, followed by washing in PBS and final resuspension in distilled water) were used for measurement of ceftaroline concentrations using liquid chromatography (LC) and tandem mass spectrometry (MS/MS). In brief, samples (100 mL) were mixed with 1.125 mL of methanol/acetonitrile (4: 21, v/v), stored at 2208C for 30 min (to facilitate protein denaturation), thawed and centrifuged. The supernatant was collected, evaporated to dryness under a gentle stream of nitrogen and resuspended in 100 mL of methanol/water (1: 1) with care to obtain full dissolution of any visible material. Samples were then subjected to LC separation using a ThermoFischer LC system equipped with a C18 XBridge column (150×2.1 mm, i.d. 3.5 mm) (Waters Corp., Milford, MA, USA) and using 100 mM ammonium formate/water/methanol/isopropanol (100: 780: 80: 40, v/v/v/v) as eluent and a flow rate of 0.2 mL/min. Chromatography was performed at 308C, but samples were maintained at 78C prior to injection (10 mL). High collision dissociation spectra were recorded with a Q-Exactive in LC-MS/MS [quadrupole precursor selection with accurate mass (HR/AM) Orbitrap detection; ThermoFisher Scientific, Waltham, MA, USA] at relative collision energy of 40%. Multiple reaction monitoring mode was used for the quantification of the analytes by monitoring the transition m/z 605 208 by high-resolution mass spectrometry. Calculation of the actual concentration was made using a calibration curve [external standard; linearity 1– 5000 ng/mL (R 2 ¼0.9976); limit of detection and limit of quantification: 0.1 and 0.5 ng/mL, respectively] and corrected for actual extraction efficiency from cells by running samples of control cells to which a known amount of ceftaroline had been added and which were then treated exactly as the samples from incubated cells. The cell content in ceftaroline was expressed as ng/mg of cell protein and the ratio of the apparent cellular to extracellular concentrations was calculated using a conversion factor of 5 mL of cell volume per mg of cell protein, as in our previous publications.15 Statistical analyses Statistical analyses of the differences between values of the pharmacological descriptors were made with GraphPad Instat version 3.06 (GraphPad Software). Recursive partitioning analysis was made with JMP version 9.0.3 (SAS Institute, Cary, NC, USA) using a single-pass decision tree method with node splitting based on the LogWorth statistic (see details and justification in the white paper ‘Monte Carlo Calibration of Distributions of Partition Statistics’, available at http://www.jmp.com). Results Strains and susceptibility to ceftaroline and comparator antibiotics at neutral and acid pH Table 1 shows that the MICs of ceftaroline at pH 7.4 for the strains used in this study ranged from 0.125 to 0.25 mg/L for MSSA and from 0.25 to 2 mg/L for MRSA irrespective of their resistance phenotype to vancomycin (range 0.5–4 mg/L), Table 1. Strains used in this study (origin, resistance phenotype and MICs in broth at neutral and acidic pH) MIC (mg/L)a ceftaroline Strain ATCC 25923b 34843/33134c ATCC 33591b 19210/18057c SA 555d SA 1984d 36065/34090c NRS18e 35165/33258c 48046/44800c CM05g 062-13091 Ac 062-13101 Ac vancomycin daptomycin linezolid Resistance phenotype pH 7.4 pH 5.5 pH 7.4 pH 5.5 pH 7.4 pH 5.5 pH 7.4 pH 5.5 MSSA MSSA MRSA MRSA MRSA/VISA MRSA MRSA MRSA/VISA MRSA MRSA MRSA/LZDR MRSA MRSA 0.125 0.25 0.5 0.5 0.5 0.5 0.25 0.5–1 1 (0.75)f 2 (2.25)f 2 (1.75)f 2 (1.75)f 2 (2)e 0.125 0.125–0.25 0.25 0.25 0.125–0.25 0.25 0.125–0.25 0.25 0.25 0.5 0.5–1 1 0.5 0.5 1 0.5 0.5 4 0.5 2 2 –4 0.5 1 1 –2 1 1 0.5– 1 1 1 1 2 0.5 2 2 1 1 0.5– 1 2 2 0.125– 0.25 1 0.25– 0.5 0.5 4 0.25– 0.5 1 1 –2 0.5 1 0.5–1 0.25 0.25 0.5 1 0.5 1 ND 2 2 2 1–2 2 0.5– 1 1 1 1 4 1– 2 2 0.5 1 4 0.5–2 4 2 4– 8 2 1– 2 1 –2 4 2 2 0.5 0.5 2 0.5 2 1 4 2 2 LZDR, linezolid resistant; ND, not determined. Triplicate determinations using conventional 1 log2 dilution progression unless specified otherwise (lower and higher values shown in case of divergence). b From the ATCC collection (Manassas, VA, USA). c From JMI Laboratories (North Liberty, IA, USA); the first number is the strain number and the second is the bank number. d From K. Kosowska-Shick and P. C. Appelbaum (Hershey Medical Center, Hershey, PA, USA); VISA phenotype based on vancomycin MIC determination. e From the Network on Antimicrobial Resistance in S. aureus (managed by the Eurofins Global Central Laboratory, Chantilly, VA, USA; supported under NIAID/NIH contract no. HHSN2722007 00055C); SCCmec group II. f Values in parentheses refer to MICs measured using arithmetic dilutions (0.25 mg/L intervals over the 1 –4 mg/L range). g From J. Quinn (Pfizer, Groton, CT, USA); cfr+ mechanism of resistance. a 650 JAC Ceftaroline and intracellular MRSA Pharmacological descriptors of the activity of ceftaroline and comparator antibiotics against extracellular and intracellular forms of MRSA strain ATCC 33591 daptomycin (range 0.125–4 mg/L) or linezolid (range 0.5–8 mg/L). Strains 35165, 48046, CM05, 062-13091 A and 062-13101 A were retested using arithmetic dilutions (0.25 mg/L intervals between 1 and 4 mg/L), but the values (reported in Table 1) were always within the corresponding +1 log2 range of the progression scale of the conventional assay method. The MICs of ceftaroline for MRSA were lower (1 –2 log2 dilutions) when assayed at pH 5.5, especially for strains with a higher MIC. Conversely, acid pH caused an increase in the MICs of daptomycin, no or discordant changes for vancomycin and nonsystematic decreases for linezolid. Of note, strains 062-13091 A and 062-13101 A, which have previously been reported to display ceftaroline MICs of 4 mg/L, consistently showed an MIC of 2 mg/L in our assays. In this first series of experiments, 24 h concentration –responses were examined for ceftaroline in comparison with vancomycin, daptomycin and linezolid against both extracellular and intracellular forms of the reference MRSA strain ATCC 33591. Data are presented graphically in Figure 1 with the corresponding pharmacological descriptors and regression parameters shown in Table 2. In all cases, a single sigmoidal function could be fitted to the data, in accordance with the pharmacological model previously described for the fully susceptible MSSA strain ATCC 25923 and various antibiotics.15 In broth (Figure 1, left panel), maximal or close to maximal effects (Emax) were observed for all four antibiotics when their concentration reached a value corresponding to their serum peak concentrations in patients (Cmax; total drug). Ceftaroline, vancomycin and daptomycin were highly bactericidal, yielding calculated Emax values corresponding to the actual limit of detection. In contrast, linezolid did not achieve a mean 3 log10 cfu/mL decrease. As expected for this bacteriostatic agent, linezolid was statistically significantly inferior to the other antibiotics when evaluating the rate of bacterial kill. Moving to the intracellular forms (Figure 1, right panel), maximal relative activities (Emax) were considerably lower (less negative) for all four antibiotics, as As b-lactams in general, and ceftaroline in particular, are known to be potentially unstable when exposed to 378C, we checked for recovery of the antibiotic from broth and culture media after 24 h incubation using both microbiological and analytical (LC-MS/MS) assays. Recovery was ≏66% using LC-MS/MS determination, and .75% and ≏50% by the microbiological method for water or broth and the cell culture medium, respectively. Ceftaroline Vancomycin D Log10 cfu from time 0 (24 h) (a) 4 Daptomycin Linezolid (b) 3 2 2 0 1 –2 0 –4 –6 Broth –3 –1 THP–1 –2 –1 0 1 2 3 –3 –2 Log10 concentration (mg/L) D Log10 cfu from time 0 (24 h) Stability of ceftaroline –1 0 1 2 3 Figure 1. Concentration-dependent activities of four antistaphylococcal antibiotics against extracellular [MHB broth pH 7.4 (a)] and intracellular [THP-1 monocytes (b)] forms of S. aureus strain ATCC 33591 (MRSA). For these experiments, broths or infected cells were incubated for 24 h in the presence of increasing concentrations of antibiotic (total drug; abscissa). The ordinates show the change in the number of cfu (log10) per mL of medium (broth) or per mg or cell protein (THP-1). Note that because of the marked difference in the amplitude of the change between bacteria in broth versus bacteria in THP-1 cells, the scale extends from 26 to 4 in panel (a) and from 21 to 3 in panel (b), with the broken horizontal line showing the zero value (no apparent change from the initial, post-phagocytosis inoculum). All values are means+SD (n¼2 or 3; when not visible, the SD bars are smaller than the size of the symbols). The lowest limit of detection corresponds to a cfu decrease of 5 log10 units compared with the original inoculum. The grey zone shows the range of maximal serum concentrations observed in humans for the antibiotics (20– 57 mg/L based on the following reported Cmax values: ceftaroline, 21 mg/L; vancomycin, 20– 50 mg/L; daptomycin, 57 mg/L; and linezolid, 15– 20 mg/L; see footnote c in Table 2). 651 Mélard et al. 652 Table 2. Pharmacological descriptors, goodness of fit and statistical analysis of the concentration–response studies of the antibiotics against strain ATCC 33591 (MRSA) in broth and in THP-1 monocytes (24 h incubation) Pharmacological descriptor EC50d Condition/antibiotic MH broth (extracellular ceftaroline vancomycin daptomycin linezolid Emina Emaxb ECmaxc bacteria) 3.28 A;a (1.91– 4.64) 2.92 A;a (2.08– 3.76) 3.17 A;a (2.27– 4.06) 3.10 A;a (2.40– 3.80) 25.37 A;a (26.50 to 24.23) 25.10 A;a (26.20 to 23.99) 25.09 A;a (25.94 to 24.25) 22.89 B;a (23.48 to 22.28) 25.04 24.55 24.72 22.34 TH P-1 monocytes (intracellular bacteria) ceftaroline 2.60 A;a (1.92– 3.27) vancomycin 2.43 A;a (1.70– 3.16) daptomycin 2.28 A;b (1.76– 2.79) linezolid 2.33 A;b (2.10– 2.55) 20.56 A;b (20.82 to 20.29) 20.65 A;b (21.08 to 20.21) 20.99 B;b (21.27 to 20.71) 20.32 A,C;b (20.47 to 20.17) 20.53 20.59 20.96 20.26 mg/L 0.82 A;a 2.62 B;a 2.70 B;a 1.74 A,B;a (0.33 –2.06) (1.43 –4.81) (1.34 –5.42) (0.92 –3.28) 0.16 A;b (0.08 –0.35) 0.67 B;b (0.23 –1.9) 0.61 B;b (0.32 –1.18) 0.42 B;b (0.27 –0.63) Cse ×MICf mg/L ×MICf Goodness of fit (R 2) 1.64 A;a (0.65 –4.12) 5.24 B;a (2.85 –9.61) 5.39 B;a (2.69 –10.84) 1.74 A;a (0.92 –3.28) ≏0.48 ≏1.54 ≏1.67 ≏1.98 ≏1.01 ≏3.04 ≏3.28 ≏1.89 0.934 0.970 0.978 0.970 0.32 A;b (0.14 –0.71) 1.35 B;b (0.47 –3.88) 2.46 B;b (1.28 –4.70) 0.38 A;b (0.23 –0.61) ≏0.76 ≏2.61 ≏1.40 ≏3.55 ≏1.45 ≏5.22 ≏6.02 ≏3.41 0.969 0.943 0.963 0.990 Data are from Figure 1. Statistical analysis: comparison of regression parameters (Emax and EC50). Figures with different letters are significantly different (P≤0.05) from all others in the same group. Uppercase letters: comparison between antibiotics (i) in broth (upper four rows) or (ii) in THP-1 monocytes (lower four rows) by analysis of variance (with Tukey –Kramer multiple comparisons test if P,0.05) comparing the four values; lowercase letters: comparison between broth and THP-1 monocytes for the same antibiotic (unpaired two-tailed t-test comparing the two values). a cfu increase (in log10 units) at 24 h from the corresponding initial inoculum as extrapolated for an infinitely low antibiotic concentration. b cfu decrease (in log10 units) at 24 h from the corresponding initial inoculum as extrapolated for an infinitely large antibiotic concentration. c cfu decrease (in log10 units) at 24 h from the corresponding initial inoculum as intrapolated (using the Hill equation) for a concentration of antibiotic corresponding to the maximal serum concentration observed in humans receiving conventional therapy (Cmax). Values chosen for this table are: ceftaroline, 21 mg/L; vancomycin, 35 mg/L; daptomycin, 57 mg/L; and linezolid, 17.5 mg/L (based on mean values in the corresponding US labelling for ceftaroline at its registered dosage of 600 mg every 12 h in the USA and the corresponding rationale document from EUCAST for the most common registered dosages of the other antibiotics in Europe). d Concentration [in mg/L or ×MIC (total drug)] causing a reduction halfway between Emin and Emax, as obtained from the Hill equation (slope factor of 1). e Concentration (in mg/L or ×MIC [total drug]) resulting in no apparent bacterial growth, as determined by graphical interpolation. f Measured at pH 7.4 in broth (see Table 1; for linezolid, an MIC of 1 mg/L was used for calculations). JAC Ceftaroline and intracellular MRSA D Log cfu from time 0 (24 h) (a) MIC = 2 mg/L (n = 4) Broth (b) 3 THP-1 (c) 3 3 2 2 1 1 2 0 0 –1 –1 1 –2 –2 –3 –3 0 –4 –5 Broth –6 –4 –3 –2 –4 –5 THP-1 –1 0 1 2 3 –1 –4 D Log cfu from time 0 (24 h) MIC = 0.5 mg/L (n = 3–4) MIC = 1 mg/L (n = 2) MIC = 0.125 (n = 1) MIC = 0.25 mg/L (n = 1) –6 –3 –2 –1 Log10 concentration (mg/L) 0 1 2 3 –4 –3 –2 –1 0 1 2 3 4 Log10 concentration (×MIC) Figure 2. 24 h concentration-dependent activity of ceftaroline against S. aureus isolates with differing susceptibilities. Left (a) and middle (b) panels: activity in broth and in THP-1 monocytes, respectively, as a function of drug weight concentration [mg/L (total drug)]: diamonds, MIC¼0.125 mg/L (strain ATCC 25923; MSSA); squares, MIC¼0.25 mg/L (strain 34843; MSSA); triangles, MIC¼0.5 mg/L [strains ATCC 33591 (MRSA), 19210 (MRSA; not tested in broth), SA 555 (MRSA/VISA) and SA 19834 (MRSA); all MRSA]; inverted triangles, MIC¼1 mg/L [strains NRS18 (MRSA/VISA) and 35165 (MRSA)]; circles, MIC¼2 mg/L [strains 48046 (MRSA), CM05 (MRSA/LZDR), 062-13101 A (MRSA) and 062-13091 A (MRSA)]; see Table 1 for more details. Right panel (c): activity in broth (circles) and in THP-1 monocytes (squares) as a function of multiples of MIC (total drug). The ordinates of all graphs show the change in cfu (log10) per mL of medium (broth) or per mg of cell protein (THP-1) at 24 h compared with the original post-phagocytosis inoculum (horizontal broken line). Note that because of the marked difference in the amplitude of the change between bacteria in broth versus bacteria in THP-1 cells, the scale extends from 26 to 3 in panel (a) and from 21 to 3 for THP-1 cells in panel (b). All values are means+SD (with each strain tested in duplicate); when not visible, the SD bars are smaller than the size of the symbols). The lowest limit of detection corresponds to a cfu decrease of 5 log10 units compared with the original inoculum. The vertical continuous line in the left and middle panels indicates the maximal serum concentration of ceftaroline commonly observed in humans (Cmax). these achieved only a ≏0.5 to ≏1 log cfu decrease compared with the original inoculum. Statistically significant but quite small differences of intracellular Emax were observed, with daptomycin being more active than ceftaroline and vancomycin, and linezolid being the least active. Considering the relative potencies (EC50) and the static concentrations (Cs), ceftaroline appeared to be the most potent, whether in broth or in cells, with values systematically about 2- to 4-fold less than for the other antibiotics, whether expressed as weight concentration (mg/L) or, except for linezolid, as multiples of the MIC in broth (pH 7.4). Interestingly enough, all values of EC50 and Cs were quite similar or lower for intracellular bacteria compared with bacteria in broth, denoting an unimpaired potency in the intracellular milieu. The numerical values of these parameters were also close to the MICs of the corresponding antibiotics. Extracellular and intracellular activity of ceftaroline against S. aureus isolates with differing susceptibilities In these experiments we compared the 24 h concentration– responses of a series of strains of S. aureus with ceftaroline MICs ranging from 0.125 to 2 mg/L (strains with higher MICs could not be identified). The results are presented graphically in Figure 2 with changes in cfu/mL shown as a function of the drug weight concentration for bacteria in broth (left panel) and in THP-1 monocytes (middle panel), and as a function of multiples of MIC for both (right panel). Considering first cfu/mL changes as a function of weight concentrations (mg/L), there was a gradual shift of the curves to higher concentrations as a function of the MIC for the strains, resulting in increases in the EC50 and Cs parameters. This was accompanied by a small (but variable among strains) decrease in the activity (less-negative values) observed at a concentration corresponding to the drug Cmax (ECmax) or of the maximal relative activities (Emax). These changes are illustrated in Figure S1 (available as Supplementary data at JAC Online). The data were then used for recursive partitioning analysis of each descriptor on the basis of the MIC (broth; pH 7.4; log2 dilution) of the strains, with the results shown in Table 3 (and individual graphs shown in Figure S2, available as Supplementary data at JAC Online). The method used (single-pass decision tree) yielded a dichotomous split at an MIC of 1 mg/L (split between ,1 and ≥1) for all four descriptors, but yielded statistically significant differences for the EC50 and Cs parameters with bacteria in broth only (differences were at the limit of statistical significance for ECmax with bacteria in THP-1 cells). Using MICs determined by arithmetic dilutions (0.25 mg/L intervals) for strains with MICs ≥1 mg/L (see Table 1) did not significantly change the results of the analysis and its conclusions. Lastly, when changes in cfu/mL data were plotted as a function of multiples of the MIC of the corresponding strains, data for all strains could be analysed as single functions for bacteria in broth and bacteria in THP-1 cells, respectively, as presented graphically in the right panel of Figure 2 with the corresponding 653 Mélard et al. Table 3. Recursive partitioning analysis of the values of the pharmacological descriptors of the concentration-dependent responses of bacterial strains with increasing MICs of ceftaroline in broth or in THP-1 cells as a function of the corresponding MIC (pH 7.4) Optimal candidate split value [MIC (mg/L)]a Parameter values (below/above MIC split value) LogWorthb/P valuec 1. Broth (n ¼11) Emax (Dlog10 cfu)d ECmax (Dlog10 cfu)e EC50 (mg/L)f Cs (mg/L)g ,1/≥1 ,1/≥1 ,1/≥1 ,1/≥1 25.24+0.10/24.89+0.88 25.04+0.12/24.14+0.79 0.54+0.16/2.41+0.50 0.28+0.12/1.24+0.40 0.09/0.81 0.97/0.11 8.05/,0.01* 3.56/,0.01* 2. THP-1 (n¼12) Emax (Dlog10 cfu) ECmax (Dlog10 cfu) EC50 (mg/L) Cs (mg/L) ,1/≥1 ,1/≥1 ,1/≥1 ,1/≥1 20.68+0.14/20.55+0.22 20.62+0.16/20.36+0.15 0.38+0.21/1.44+1.11 1.83+1.55/6.51+4.35 0.19/0.64 1.35/0.04* 0.85/0.14 1.00/0.10 Descriptor Analysis was made on the basis of the data shown in Figure 2 (left and middle panels), but considering the individual values of each strain. Asterisks indicate results considered statistically significant on the basis of the P value. a Values of MIC separating datasets in two categories based on minimization of the sum of squared errors across the whole data as a function of the MIC (further splitting was unsuccessful because of the limited number of independent values). b Node splitting is based on the LogWorth statistic (values .2 indicate that the variable used in the branch is significant and should be included in the decision tree. c Calculated based from LogWorth value [P = 10(−LogWorth) ]. d cfu decrease (in log10 units) at 24 h from the corresponding initial inoculum, as extrapolated from infinitely large concentrations of antibiotics. e cfu decrease (in log10 units) at 24 h from the corresponding initial inoculum, as intrapolated (using the Hill equation) for a concentration of antibiotic corresponding to the maximal serum concentration observed in humans receiving conventional therapy (Cmax). f Concentration (total drug) causing a reduction halfway between Emin and Emax, as obtained from the Hill equation (slope factor of 1). g Concentration (total drug) resulting in no apparent bacterial growth, as determined by graphical interpolation. Table 4. Pharmacological descriptors, goodness of fit and statistical analysis of the concentration– response studies of ceftaroline against strains with increasing MICs (from 0.125 to 2 mg/L; see list and MICs in the caption of Figure 2) in broth and in THP-1 monocytes (24 h incubation) Pharmacological descriptor Condition Emaxa MH broth (extracellular bacteria), n¼11 25.09 a (25.34 to 24.84) THP-1 monocytes (intracellular bacteria), n¼12 20.58 b (20.74 to 20.43) ECmaxb EC50 (×MIC)c,d Cs (×MIC)d,e Goodness of fit (R 2) 24.60 1.44 a (1.14– 1.83) 0.71 0.938 20.46 0.81 a (0.57– 1.16) 3.62 0.855 Data shown are from Figure 2 (right panel). Statistical analysis: comparison of parameters (Emax and EC50) between broth and THP-1 monocytes. Figures with different letters are significantly different (P≤0.05) from each other (unpaired t-test two-tailed). a cfu decrease (in log10 units) at 24 h from the corresponding initial inoculum as extrapolated from infinitely large concentrations of antibiotics. b cfu decrease (in log10 units) at 24 h from the corresponding initial inoculum as interpolated for an antibiotic concentration (total drug) corresponding to the reported human Cmax of ceftaroline (21 mg/L). c Concentration [multiples of MIC (total drug)] causing a reduction halfway between Emin and Emax, as obtained from the Hill equation (slope factor of 1). d Concentration (multiple of MIC [total drug]) resulting in no apparent bacterial growth, as determined by graphical interpolation. e MIC determined in broth at pH 7.4. pharmacological descriptors shown in Table 4. While this further confirmed the major differences in efficacy (Emax and ECmax) of ceftaroline against bacteria in broth versus bacteria in THP-1 monocytes, the corresponding relative potencies (EC50) were 654 not markedly different from each other. The static concentration (Cs) value was close to 1× the MIC for bacteria in broth, as anticipated, and was only about 4-fold higher for bacteria in THP-1 monocytes. Ceftaroline and intracellular MRSA Cellular accumulation of ceftaroline The accumulation of ceftaroline was first measured after 24 h incubation at 20 mg/L in both uninfected and infected (strain ATCC 33591) cells using an LC-MS/MS assay. The apparent cellular to extracellular concentration ratio was 0.66+0.05 (n¼ 6) and 0.90+0.36 (n¼ 3) in uninfected and infected cells, respectively. Discussion There is a clear need to discover and develop novel antibiotics to meet the increased resistance of bacteria to currently registered drugs.25,26 Although the most blatant lack of progress is for anti-Gram-negative agents, the situation with Gram-positive organisms remains of concern since several new, potentially promising compounds were not approved by the regulatory authorities,27 – 29 are facing toxicity and emergence of resistance issues30 – 33 or may require higher doses than originally foreseen.34 In this context, ceftaroline fosamil may represent a useful alternative, especially in light of the decreased susceptibility of contemporary isolates to vancomycin (including heteroresistance),35,36 and the emergence of both chromosomal and transferable resistance to linezolid.37,38 Ceftaroline in vitro activity against MRSA is related to its high affinity for PBP2a,39,40 an affinity that is enhanced in the presence of a cell wall structural surrogate.41 In the present study we confirm the unimpaired in vitro activity of ceftaroline against MRSA with non-susceptibility or resistance mechanisms to vancomycin and linezolid, as previously reported by others.4,42 In this context, the present study adds important information about the intracellular activity of ceftaroline in comparison with vancomycin, linezolid and daptomycin. We first show that ceftaroline compares in almost every respect to these antibiotics for a susceptible reference strain (ATCC 33591), but that their relative efficacy is considerably reduced compared with what is observed in broth. This is based on a comprehensive pharmacodynamic analysis comparing static concentrations, relative potencies, effects at concentrations corresponding to the Cmax in humans and maximal effects. Of note, daptomycin, reported to be highly bactericidal in vitro,43 did not prove superior to ceftaroline in THP-1 monocytes or in broth. Thus, for all four agents tested here, it clearly appears that intracellular bacteria are protected to some degree against the antibacterial activities of these different classes of antibiotics (a similar observation was made for ceftobiprole, another cephalosporin with activity against MRSA).18 This cannot be due to a lack of bacterial growth in cells (as Emin values show that there is an ≏100-fold growth of bacteria in cells in the absence of antibiotic) and is not related to a loss of potency (the EC50 and Cs parameters being essentially similar for bacteria in broth and in THP-1 monocytes). Thus, ceftaroline as well as vancomycin and daptomycin seem to become essentially bacteriostatic against intracellular bacteria and compare, in this respect, to linezolid. This is in contrast to lipoglycopeptides (telavancin and oritavancin), fluoroquinolones or quinupristin/dalfopristin, which show maximal relative efficacy (Emax) values between 22 and 23 log10 cfu/mL in the same model44 – 48 and may therefore be considered as exerting a near-to-bactericidal intracellular activity based on commonly accepted criteria of cidality.19 JAC In our study we also examined whether a decrease in the susceptibility of S. aureus to ceftaroline (as measured by its MIC in broth) would be accompanied by a decrease or loss of intracellular activity. This approach was triggered by a previous successful attempt to define an intracellular breakpoint for moxifloxacin in the same model.47 With ceftaroline, we see that the intracellular activity of antibiotics is primarily driven by the MIC for the phagocytosed organism, confirming previous observations made with other antibiotics.18,48 – 50 However, the changes in the values of the pharmacodynamic descriptors of ceftaroline over the range of MICs investigated (0.125– 2 mg/L) were small. Thus, although the recursive partitioning analysis suggested a breakpoint at an MIC value of 1 mg/L, this was only significant for bacteria grown in broth and for parameters related to potency (EC50 and Cs), while efficacy parameters (ECmax and Emax) remained unaffected. This may be due to the too narrow MIC range investigated (4 log2 dilutions only) or may simply indicate that changes in efficacy parameters will only become visible at higher MICs. However, strains displaying increased ceftaroline MICs seem very difficult to raise51 and, when observed, may show only limited changes52 as those we used here (mecAindependent high-level ceftaroline-resistant mutants have been recently generated by serial passage in vitro,53 but these were not available to us during our study). The fact that the parameters of intracellular activity are even less affected by an increase in MIC may also be related to the fact that the MIC of ceftaroline is lowered by about 1 –2 log2 dilutions when tested at pH 5.5 (to mimic the phagolysosomal environment),20,21,44 especially for strains against which ceftaroline is less active [thus further narrowing the MIC range (3 log2 dilutions only)]. Interestingly enough, this increased activity at acid pH may partly compensate for the weak cellular accumulation of ceftaroline, and account for its effect on intracellular bacteria as previously observed for other b-lactams.54 This is also consistent with our observation that acidic pH causes conformational changes of PBP2a that improve its acylation by b-lactams within a time frame relevant to the growth rate of MRSA.55 Thus, the present data suggest that ceftaroline activity against intracellular S. aureus may remain unimpaired for organisms with an MIC of 2 mg/L, even though it becomes adversely affected for extracellular bacteria when the MIC exceeds 1 mg/L. The present study also has several limitations that need to be underlined. First, assessment of intracellular activity was made with cells (THP-1 monocytes) that, in contrast to fully differentiated macrophages, do not quickly kill phagocytosed S. aureus. This was by design, as we aimed to analyse the pharmacodynamic properties of ceftaroline without undue interference from host defence mechanisms. Future studies may need to examine how and to what extent ceftaroline cooperates with these mechanisms. Other cell types also capable of harbouring S. aureus, such as keratinocytes or endothelial cells, could also be used. Second, we did not examine the effect of time on the response to ceftaroline (all experiments used a fixed 24 h timepoint), which, again, could be the subject of future studies similar to those made recently with extracellular bacteria.56 However, we know from previous studies15 that killing of intracellular S. aureus by b-lactams is a slow process. Thus, shorter exposure time would yield only minimal changes in cfu that prove non-significant. Third, we did not monitor the expression of Panton-Valentine leucocidin toxin (PVL), which could affect 655 Mélard et al. the host cell viability. However: (i) our previous studies failed to find evidence of an impact of the presence of the PVL-encoding genes on the intracellular behaviour and antibiotic susceptibility of S. aureus in the model used;47 (ii) the production of PVL and other toxins is maximal at the stationary stage,57 which is not reached for intracellular bacteria under the conditions of our experiment; and (iii) PVL presence was not a primary determinant of outcome in patients with complicated skin and skin structure infections due to either MRSA or MSSA in the clinical studies assessing the efficacy of ceftaroline.58 Lastly, all comparisons were made using total drug concentrations and using nominal ones. We know that only free concentrations are usually considered for in vivo activity assessment and for clinical breakpoint setting.59 However, our culture medium contains only 10% serum, which means that most drugs will be free, as previously documented for b-lactams with high protein-binding.16 But this will not influence much the behaviour of ceftaroline since it has a low protein binding (≏20%) in 100% serum.1 Conversely, our model may lead to an overestimation of daptomycin activity, as it is impaired in human serum in comparison with broth.60 Loss of activity during incubation was also not taken into account because it is likely to be progressive and did not, over the 24 h duration of our experiments, exceed 50%, which is less than the 2-fold MIC change that is considered significant in conventional susceptibility testing. In conclusion, we present in vitro evidence that ceftaroline is capable of controlling the growth of intracellular S. aureus to an extent similar to that of vancomycin, daptomycin and linezolid, irrespective of the presence of resistance mechanisms to conventional b-lactams (methicillin resistance), vancomycin (VISA) or linezolid (cfr), and for strains for which ceftaroline shows an MIC ≤2 mg/L. These results may now trigger the performance of in vivo animal and human studies aimed at better delineating the potential use of ceftaroline in difficult-to-treat infections where the persistence of an intracellular inoculum may be a critical determinant.12 Acknowledgements We thank K. Kosowska-Shick, P. C. Appelbaum, J. Quinn, JMI Laboratories and the Network on Antimicrobial Resistance in S. aureus (NARSA) for the gift of bacterial strains. We are grateful to M.-C. Cambier for dedicated technical assistance. Funding This work was supported by Belgian Fonds de la Recherche Scientifique Médicale (FRSM; grant nos. 3.4.597.06 and 3.4530.12), the Belgian Fonds de la Recherche Scientifique (FRS-FNRS; grant no. 1.5118.11) and a grant-in-aid from Cerexa, Inc., a wholly owned subsidiary of Forest Laboratories, Inc. Editorial assistance was funded by Forest Research Institute, Inc. L. G. G. is Boursière of the Belgian Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture (FRIA). D. D. is the recipient of a post-doctoral fellowship of the Belgian Fonds de la Recherche Scientifique (FRS-FNRS; grant no. 1.5118.11). S. L. and F. V. B. are Chargé de Recherche and Maı̂tre de Recherche of the FRS-FNRS, respectively. 656 Transparency declarations No conflicts of interest to declare. Cerexa, Inc. was involved in the design and decision to present these results, but had no involvement in the collection, analysis or interpretation of data. Editorial assistance was provided by Scientific Therapeutics Information, Inc. Supplementary data Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/). References 1 Biek D, Critchley IA, Riccobene TA et al. Ceftaroline fosamil: a novel broad-spectrum cephalosporin with expanded anti-Gram-positive activity. J Antimicrob Chemother 2010; 65 Suppl 4: iv9– 16. 2 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. 3 Flamm RK, Sader HS, Farrell DJ et al. Summary of ceftaroline activity against pathogens in the United States, 2010: Report from the Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) Surveillance Program. Antimicrob Agents Chemother 2012; 56: 2933 –40. 4 Zhanel GG, Rossnagel E, Nichol K et al. Ceftaroline pharmacodynamic activity versus community-associated and healthcare-associated methicillin-resistant Staphylococcus aureus, heteroresistant vancomycinintermediate S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant S. aureus using an in vitro model. J Antimicrob Chemother 2011; 66: 1301–5. 5 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. 6 Corrado ML. Integrated safety summary of CANVAS 1 and 2 trials: Phase III, randomized, double-blind studies evaluating ceftaroline fosamil for the treatment of patients with complicated skin and skin structure infections. J Antimicrob Chemother 2010; 65 Suppl 4: iv67–71. 7 Wilcox MH, Corey GR, Talbot GH et al. CANVAS 2: the second Phase III, randomized, double-blind study evaluating ceftaroline fosamil for the treatment of patients with complicated skin and skin structure infections. J Antimicrob Chemother 2010; 65 Suppl 4: iv53–65. 8 Corey GR, Wilcox MH, Talbot GH et al. CANVAS 1: the first Phase III, randomized, double-blind study evaluating ceftaroline fosamil for the treatment of patients with complicated skin and skin structure infections. J Antimicrob Chemother 2010; 65 Suppl 4: iv41–51. 9 Anonymous. TEFLARO Prescribing Information. Forest Pharmaceuticals, Inc., subsidiary of Forest Laboratories, Inc., St Louis, MO, USA. http://www. frx.com/pi/Teflaro_pi.pdf (11 October 2012, date last accessed). 9a Anonymous. ZINFORO Summary of Product Characteristics. European Medicines Agency, London, UK. http://www.ema.europa.eu/ (26 October 2012, date last accessed). 10 Friedland HD, O’Neal T, Biek D et al. CANVAS 1 and 2: analysis of clinical response at day 3 in two Phase 3 trials of ceftaroline fosamil versus vancomycin plus aztreonam in treatment of acute bacterial skin and skin structure infections. Antimicrob Agents Chemother 2012; 56: 2231– 6. Ceftaroline and intracellular MRSA 11 Lowy FD. Is Staphylococcus aureus an intracellular pathogen? Trends Microbiol 2000; 8: 341– 3. 12 Garzoni C, Kelley WL. Staphylococcus aureus: new evidence for intracellular persistence. Trends Microbiol 2009; 17: 59–65. 13 Van Bambeke F, Barcia-Macay M, Lemaire S et al. Cellular pharmacodynamics and pharmacokinetics of antibiotics: current views and perspectives. Curr Opin Drug Discov Devel 2006; 9: 218–30. 14 Sandberg A, Lemaire S, Van Bambeke F et al. Intra- and extracellular activities of dicloxacillin and linezolid against a clinical Staphylococcus aureus strain with a small-colony-variant phenotype in an in vitro model of THP-1 macrophages and an in vivo mouse peritonitis model. Antimicrob Agents Chemother 2011; 55: 1443–52. 15 Barcia-Macay M, Seral C, Mingeot-Leclercq MP et al. Pharmacodynamic evaluation of the intracellular activities of antibiotics against Staphylococcus aureus in a model of THP-1 macrophages. Antimicrob Agents Chemother 2006; 50: 841– 51. 16 Lemaire S, Van Bambeke F, Mingeot-Leclercq MP et al. Activity of three b-lactams (ertapenem, meropenem and ampicillin) against intraphagocytic Listeria monocytogenes and Staphylococcus aureus. J Antimicrob Chemother 2005; 55: 897–904. 17 Page MG. Emerging cephalosporins. Expert Opin Emerg Drugs 2007; 12: 511–24. 18 Lemaire S, Glupczynski Y, Duval V et al. Activities of ceftobiprole and other cephalosporins against extracellular and intracellular (THP-1 macrophages and keratinocytes) forms of methicillin-susceptible and methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2009; 53: 2289 –97. 19 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twentieth Informational Supplement M100-S20. CLSI, Wayne, PA, USA, 2010. 20 Ohkuma S, Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci USA 1978; 75: 3327– 31. 21 Lemaire S, Van Bambeke F, Mingeot-Leclercq MP et al. Role of acidic pH in the susceptibility of intraphagocytic methicillin-resistant Staphylococcus aureus strains to meropenem and cloxacillin. Antimicrob Agents Chemother 2007; 51: 1627 –32. 22 Tsuchiya S, Yamabe M, Yamaguchi Y et al. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 1980; 26: 171– 6. 23 Carryn S, Van d V, Van Bambeke F et al. Impairment of growth of Listeria monocytogenes in THP-1 macrophages by granulocyte macrophage colony-stimulating factor: release of tumor necrosis factor-a and nitric oxide. J Infect Dis 2004; 189: 2101 –9. 24 Lowry OH, Rosebrough NJ, Farr AL et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–75. 25 Livermore DM. Has the era of untreatable infections arrived? J Antimicrob Chemother 2009; 64 Suppl 1: i29–36. 26 Bush K, Courvalin P, Dantas G et al. Tackling antibiotic resistance. Nat Rev Microbiol 2011; 9: 894– 6. 27 Arias CA, Mendes RE, Stilwell MG et al. Unmet needs and prospects for oritavancin in the management of vancomycin-resistant enterococcal infections. Clin Infect Dis 2012; 54 Suppl 3: S233–8. 28 Sincak CA, Schmidt JM. Iclaprim, a novel diaminopyrimidine for the treatment of resistant gram-positive infections. Ann Pharmacother 2009; 43: 1107– 14. 29 Bush K, Heep M, Macielag MJ et al. Anti-MRSA b-lactams in development, with a focus on ceftobiprole: the first anti-MRSA b-lactam to demonstrate clinical efficacy. Expert Opin Investig Drugs 2007; 16: 419–29. JAC 30 Rubinstein E, Prokocimer P, Talbot GH. Safety and tolerability of quinupristin/dalfopristin: administration guidelines. J Antimicrob Chemother 1999; 44 Suppl A: 37–46. 31 Falagas ME, Karageorgopoulos DE, Dimopoulos G. Clinical significance of the pharmacokinetic and pharmacodynamic characteristics of tigecycline. Curr Drug Metab 2009; 10: 13 –21. 32 Di Paolo A, Malacarne P, Guidotti E et al. Pharmacological issues of linezolid: an updated critical review. Clin Pharmacokinet 2010; 49: 439–47. 33 Smith WJ, Drew RH. Telavancin: a new lipoglycopeptide for grampositive infections. Drugs Today (Barc) 2009; 45: 159– 73. 34 Seaton RA. Daptomycin: rationale and role in the management of skin and soft tissue infections. J Antimicrob Chemother 2008; 62 Suppl 3: iii15 –23. 35 Sakoulas G, Moise-Broder PA, Schentag J et al. Relationship of MIC and bactericidal activity to efficacy of vancomycin for treatment of methicillin-resistant Staphylococcus aureus bacteremia. J Clin Microbiol 2004; 42: 2398– 402. 36 Haque NZ, Zuniga LC, Peyrani P et al. Relationship of vancomycin minimum inhibitory concentration to mortality in patients with methicillinresistant Staphylococcus aureus hospital-acquired, ventilator-associated, or health-care-associated pneumonia. Chest 2010; 138: 1356–62. 37 Ikeda-Dantsuji Y, Hanaki H, Sakai F et al. Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan. J Infect Chemother 2011; 17: 45–51. 38 Witte W, Cuny C. Emergence and spread of cfr-mediated multiresistance in staphylococci: an interdisciplinary challenge. Future Microbiol 2011; 6: 925– 31. 39 Moisan H, Pruneau M, Malouin F. Binding of ceftaroline to penicillinbinding proteins of Staphylococcus aureus and Streptococcus pneumoniae. J Antimicrob Chemother 2010; 65: 713–6. 40 Kosowska-Shick K, McGhee PL, Appelbaum PC. Affinity of ceftaroline and other b-lactams for penicillin-binding proteins from Staphylococcus aureus and Streptococcus pneumoniae. Antimicrob Agents Chemother 2010; 54: 1670– 7. 41 Villegas-Estrada A, Lee M, Hesek D et al. Co-opting the cell wall in fighting methicillin-resistant Staphylococcus aureus: potent inhibition of PBP 2a by two anti-MRSA b-lactam antibiotics. J Am Chem Soc 2008; 130: 9212– 3. 42 Vidaillac C, Leonard SN, Rybak MJ. In vitro activity of ceftaroline against methicillin-resistant Staphylococcus aureus and heterogeneous vancomycin-intermediate S. aureus in a hollow fiber model. Antimicrob Agents Chemother 2009; 53: 4712 –7. 43 Stratton CW, Liu C, Weeks LS. Activity of LY146032 compared with that of methicillin, cefazolin, cefamandole, cefuroxime, ciprofloxacin, and vancomycin against staphylococci as determined by kill-kinetic studies. Antimicrob Agents Chemother 1987; 31: 1210– 5. 44 Seral C, Van Bambeke F, Tulkens PM. Quantitative analysis of gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin (LY333328) activities against intracellular Staphylococcus aureus in mouse J774 macrophages. Antimicrob Agents Chemother 2003; 47: 2283– 92. 45 Barcia-Macay M, Lemaire S, Mingeot-Leclercq MP et al. Evaluation of the extracellular and intracellular activities (human THP-1 macrophages) of telavancin versus vancomycin against methicillinsusceptible, methicillin-resistant, vancomycin-intermediate and vancomycin-resistant Staphylococcus aureus. J Antimicrob Chemother 2006; 58: 1177– 84. 46 Baudoux P, Lemaire S, Denis O et al. Activity of quinupristin/ dalfopristin against extracellular and intracellular Staphylococcus aureus 657 Mélard et al. with various resistance phenotypes. J Antimicrob Chemother 2010; 65: 1228– 36. 47 Lemaire S, Kosowska-Shick K, Appelbaum PC et al. Activity of moxifloxacin against intracellular community-acquired methicillinresistant Staphylococcus aureus: comparison with clindamycin, linezolid and co-trimoxazole and attempt at defining an intracellular susceptibility breakpoint. J Antimicrob Chemother 2011; 66: 596–607. 48 Vallet CM, Marquez B, Ngabirano E et al. Cellular accumulation of fluoroquinolones is not predictive of their intracellular activity: studies with gemifloxacin, moxifloxacin and ciprofloxacin in a pharmacokinetic/ pharmacodynamic model of uninfected and infected macrophages. Int J Antimicrob Agents 2011; 38: 249– 56. 49 Lemaire S, Van Bambeke F, Tulkens PM. Cellular accumulation and pharmacodynamic evaluation of the intracellular activity of CEM-101, a novel fluoroketolide, against Staphylococcus aureus, Listeria monocytogenes, and Legionella pneumophila in human THP-1 macrophages. Antimicrob Agents Chemother 2009; 53: 3734–43. 50 Lemaire S, Van Bambeke F, Appelbaum PC et al. Cellular pharmacokinetics and intracellular activity of torezolid (TR-700): studies with human macrophage (THP-1) and endothelial (HUVEC) cell lines. J Antimicrob Chemother 2009; 64: 1035– 43. 51 Mushtaq S, Warner M, Ge Y et al. In vitro activity of ceftaroline (PPI0903M, T-91825) against bacteria with defined resistance mechanisms and phenotypes. J Antimicrob Chemother 2007; 60: 300– 11. 52 Clark C, McGhee P, Appelbaum PC et al. Multistep resistance development studies of ceftaroline in gram-positive and -negative bacteria. Antimicrob Agents Chemother 2011; 55: 2344– 51. 53 Chan L, Chambers H. MecA-independent high-level b-lactam resistance in Staphylococcus aureus strains passaged in ceftobiprole and ceftaroline. In: Abstracts of the Fifty-second Interscience Conference 658 on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, 2012. Abstract C1-1351a, p. 300. American Society for Microbiology, Washington, DC, USA. 54 Baudoux P, Bles N, Lemaire S et al. Combined effect of pH and concentration on the activities of gentamicin and oxacillin against Staphylococcus aureus in pharmacodynamic models of extracellular and intracellular infections. J Antimicrob Chemother 2007; 59: 246–53. 55 Lemaire S, Fuda C, Van Bambeke F et al. Restoration of susceptibility of methicillin-resistant Staphylococcus aureus to b-lactam antibiotics by acidic pH: role of penicillin-binding protein PBP 2a. J Biol Chem 2008; 283: 12769–76. 56 Marconescu P, Graviss EA, Musher DM. Rates of killing of methicillinresistant Staphylococcus aureus by ceftaroline, daptomycin, and telavancin compared to that of vancomycin. Scand J Infect Dis 2012; 44: 620–2. 57 Graves SF, Kobayashi SD, Braughton KR et al. Relative contribution of Panton-Valentine leukocidin to PMN plasma membrane permeability and lysis caused by USA300 and USA400 culture supernatants. Microbes Infect 2010; 12: 446–56. 58 Tong A, Tong SY, Zhang Y et al. Panton-Valentine leukocidin is not the primary determinant of outcome for Staphylococcus aureus skin infections: evaluation from the CANVAS studies. PLoS One 2012; 7: e37212. 59 Mouton JW, Brown DF, Apfalter P et al. The role of pharmacokinetics/ pharmacodynamics in setting clinical MIC breakpoints: the EUCAST approach. Clin Microbiol Infect 2012; 18: E37–45. 60 Stratton CW, Weeks LS. Effect of human serum on the bactericidal activity of daptomycin and vancomycin against staphylococcal and enterococcal isolates as determined by time-kill kinetic studies. Diagn Microbiol Infect Dis 1990; 13: 245–52. Activity of ceftaroline against extracellular (broth) and intracellular (THP-1 monocytes) forms of methicillinresistant Staphylococcus aureus: comparison with vancomycin, linezolid and daptomycin Mélard et al - J Antimicrob Chemother 2013; 68: 648–658 Supplementary data Figure S1 broth THP-1 efficacy potency 4 -1 3 EC 50 (mg/L) Emax ( log10 cfu) 0 -2 -3 -4 2 1 -5 -6 0 0.125 0.25 0.5 1 2 0.125 0.25 0.5 1 2 1 2 MIC (mgL) 0 15.0 -1 12.5 -2 10.0 Cs (mg/L) ECmax ( log10 cfu) MIC (mgL) -3 -4 -5 7.5 5.0 2.5 -6 0.0 0.125 0.25 0.5 MIC (mgL) 1 2 0.125 0.25 0.5 MIC (mgL) Figure S1: Pharmacological descriptors of the concentration-dependent responses of Staphylococcus aureus strains with increasing MICs to ceftaroline. Circles: bacteria in broth (MHB pH 7.4: 11 strains); squares: bacteria in THP-1 monocytes (12 strains). Data are from the experiments illustrated in Figure 2 (24 h incubation). Emax (upper left): change in cfu (in log10 units) from the original inoculum for an infinitely large antibiotic concentration; ECmax (lower left): change in cfu (in log10 units) for a ceftaroline concentration corresponding to its maximal serum concentration in patients receiving standard therapy (21 mg/L); EC50 (upper right): concentration (in mg/L) yielding a change in cfu half way between Emin (change in cfu from the original inoculum for an infinitely low antibiotic concentration) and Emax; Cs (lower right): concentration (in mg/L) resulting in an apparent static effect (no change from the original inoculum). Emax and EC50 are the parameters of the sigmoidal function (Hill equation; slope factor = 1) fitted to the data (non-linear regression); ECmax and Cs are determined by graphical intrapolation using the corresponding Hill equations (each individual point corresponds to the value observed for one strain). Curves are "best fitting" with no specific underlying model. Activity of ceftaroline against extracellular (broth) and intracellular (THP-1 monocytes) forms of methicillinresistant Staphylococcus aureus: comparison with vancomycin, linezolid and daptomycin Mélard et al - J Antimicrob Chemother 2013; 68: 648–658 Figure S2 Figure S2: Graphical representation of the results of the recursive partitioning analysis of the pharmacological descriptors derived from the concentration-dependent responses experiments using bacterial strains with increasing MIC to ceftaroline in broth (left) or in THP-1 cells (right). The ordinate of each graph shows the values of the corresponding pharmacological descriptor (Emax [upper row]: cfu decrease [in log10 units] at 24 h from the corresponding initial inoculum, as extrapolated from infinitely large concentrations of antibiotic; ECmax [2d row]: cfu decrease [in log10 units] at 24 h from the corresponding initial inoculum, as intrapolated (using the Hill equation) for a concentration of antibiotic corresponding to the maximal serum concentration observed in humans receiving conventional therapy (Cmax [21 mg/L total drug]); EC50 [3d row]: concentration [in mg/L; total drug]) causing a reduction halfway between Emin (cfu change at 24 h [in log10 units] from the corresponding initial inoculum as exatrapolated for an infinitely low antibiotic concentration) and Emax; Cs [lower row]: concentration [in mg/L; total drug] resulting in no apparent bacterial growth, as determined by graphical interpolation (using the Hill equation). The abscissa separates the data in two groups according to the MIC values (broth; pH 7.4) of the corresponding strains as falling below or above the best split value (1 mg/L). The horizontal bars show the mean of the values for each group. Data points are coloured (from green to black to red) according to the scale on the right of each graph to highlight the differences between them (points with similar colour are numerically close to each other).