Brian J. Werth

Ceftaroline Increases Membrane Binding and Enhances the Activity of Daptomycin against Daptomycin-Nonsusceptible Vancomycin-Intermediate Staphylococcus aureus in a Pharmacokinetic/Pharmacodynamic Model

ABSTRACT New antimicrobial agents and novel combination therapies are needed to treat serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to daptomycin and vancomycin. The purpose of this study was to evaluate the combination of ceftaroline plus daptomycin or vancomycin in an in vitro pharmacokinetic/pharmacodynamic model. Simulations of ceftaroline-fosamil at 600 mg per kg of body weight every 8 h (q8h) (maximum free-drug concentration in serum [fCmax], 15.2 mg/liter; half-life [t1/2], 2.3 h), daptomycin at 10 mg/kg/day (fCmax, 11.3 mg/liter; t1/2, 8 h), vancomycin at 2 g q12h (fCmax, 30 mg/liter; t1/2, 6 h), ceftaroline plus daptomycin, and ceftaroline plus vancomycin were evaluated against a clinical, isogenic MRSA strain pair: D592 (daptomycin susceptible and heterogeneous vancomycin intermediate) and D712 (daptomycin nonsusceptible and vancomycin intermediate) in a one-compartment in vitro pharmacokinetic/pharmacodynamic model over 96 h. Therapeutic enhancement of combinations was defined as ≥2 log10 CFU/ml reduction over the most active single agent. The effect of ceftaroline on the membrane charge, cell wall thickness, susceptibility to killing by the human cathelicidin LL37, and daptomycin binding were evaluated. Therapeutic enhancement was observed with daptomycin plus ceftaroline in both strains and vancomycin plus ceftaroline against D592. Ceftaroline exposure enhanced daptomycin-induced depolarization (81.7% versus 72.3%; P = 0.03) and killing by cathelicidin LL37 (P < 0.01) and reduced cell wall thickness (P < 0.001). Fluorescence-labeled daptomycin was bound over 7-fold more in ceftaroline-exposed cells. Whole-genome sequencing and mutation analysis of these strains indicated that change in daptomycin susceptibility is related to an fmtC (mprF) mutation. The combination of daptomycin plus ceftaroline appears to be potent, with rapid and sustained bactericidal activity against both daptomycin-susceptible and -nonsusceptible strains of MRSA.

Novel Combinations of Vancomycin plus Ceftaroline or Oxacillin against Methicillin-Resistant Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA

ABSTRACT We demonstrated a significant inverse correlation between vancomycin and beta-lactam susceptibilities in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates. Using time-kill assays, vancomycin plus oxacillin or ceftaroline was synergistic against 3 of 5 VISA and 1 of 5 hVISA isolates or 5 of 5 VISA and 4 of 5 hVISA isolates, respectively. Beta-lactam exposure reduced overall vancomycin-Bodipy (dipyrromethene boron difluoride [4,4-difluoro-4-bora-3a,4a-diaza-s-indacene] fluorescent dye) binding but may have improved vancomycin-cell wall interactions to improve vancomycin activity. Further research is warranted to elucidate the mechanism behind vancomycin and beta-lactam synergy.

Evaluation of Ceftaroline Activity against Heteroresistant Vancomycin-Intermediate Staphylococcus aureus and Vancomycin-Intermediate Methicillin-Resistant S. aureus Strains in an In Vitro Pharmacokinetic/Pharmacodynamic Model: Exploring the “Seesaw Effect”

ABSTRACT A “seesaw effect” in methicillin-resistant Staphylococcus aureus (MRSA) has been demonstrated, whereby susceptibility to β-lactam antimicrobials increases as glyco- and lipopeptide susceptibility decreases. We investigated this effect by evaluating the activity of the anti-MRSA cephalosporin ceftaroline against isogenic pairs of MRSA strains with various susceptibilities to vancomycin in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model. The activities of ceftaroline at 600 mg every 12 h (q12h) (targeted free maximum concentration of drug in serum [fCmax], 15.2 μg/ml; half-life [t1/2], 2.3 h) and vancomycin at 1 g q12h (targeted fCmax, 18 μg/ml; t1/2, 6 h) were evaluated against 3 pairs of isogenic clinical strains of MRSA that developed increased MICs to vancomycin in patients while on therapy using a two-compartment hollow-fiber PK/PD model with a starting inoculum of ∼107 CFU/ml over a 96-h period. Bacterial killing and development of resistance were evaluated. Expression of penicillin-binding proteins (PBPs) 2 and 4 was evaluated by reverse transcription (RT)-PCR. The achieved pharmacokinetic parameters were 98 to 119% of the targeted values. Ceftaroline and vancomycin were bactericidal against 5/6 and 1/6 strains, respectively, at 96 h. Ceftaroline was more active against the mutant strains than the parent strains, with this difference being statistically significant for 2/3 strain pairs at 96 h. The level of PBP2 expression was 4.4× higher in the vancomycin-intermediate S. aureus (VISA) strain in 1/3 pairs. The levels of PBP2 and PBP4 expression were otherwise similar between the parent and mutant strains. These data support the seesaw hypothesis that ceftaroline, like traditional β-lactams, is more active against strains that are less susceptible to vancomycin even when the ceftaroline MICs are identical. Further research to explore these unique findings is warranted.

Evaluation of the novel combination of daptomycin plus ceftriaxone against vancomycin-resistant enterococci in an in vitro pharmacokinetic/pharmacodynamic simulated endocardial vegetation model

OBJECTIVES Daptomycin has demonstrated synergy with β-lactams against Enterococcus faecium and this combination has been used successfully to treat infections refractory to daptomycin. We investigated daptomycin alone and combined with ceftriaxone against vancomycin-resistant enterococci (VRE) in an in vitro pharmacokinetic/pharmacodynamic simulated endocardial vegetation (SEV) model. METHODS Daptomycin (6 and 12 mg/kg/day) with and without 2 g of ceftriaxone every 24 h were evaluated against two clinical E. faecium strains (8019 and 5938) and one Enterococcus faecalis (6981) in a 96 h in vitro pharmacokinetic/pharmacodynamic SEV model. FITC-labelled poly-l-lysine was used to assess β-lactam-induced changes in cell surface charge. RESULTS For 8019 and 6981, daptomycin 6 mg/kg with ceftriaxone and daptomycin 12 mg/kg alone and in combination with ceftriaxone displayed significantly more activity than daptomycin 6 mg/kg alone from 48 to 96 h (P ≤ 0.005). The addition of ceftriaxone significantly enhanced activity of daptomycin 6 mg/kg against both strains at 96 h (8019, reductions -0.55 versus 3.64 log10 cfu/g; 6981, reductions 1.11 versus 5.67 log10 cfu/g; P < 0.001) and improved daptomycin 12 mg/kg against 8019 at 96 h. Daptomycin 12 mg/kg plus ceftriaxone displayed no appreciable activity against 5938 (daptomycin MIC 32 mg/L). Daptomycin non-susceptibility developed in 8019 and 6981 versus daptomycin 6 mg/kg by 96 h. Ampicillin or ceftriaxone exposure reduced daptomycin surface charge in 8019, resulting in significantly increased FITC-poly-l-lysine binding. CONCLUSIONS The combination of daptomycin and ceftriaxone may be promising for eradicating high-inoculum, deep-seated enterococcal infections. Further research is warranted to examine the enhancement of daptomycin and innate immunity killing of VRE by ceftriaxone and other β-lactams.

Evaluation of the Novel Combination of High-Dose Daptomycin plus Trimethoprim-Sulfamethoxazole against Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus Using an In Vitro Pharmacokinetic/Pharmacodynamic Model of Simulated Endocardial Vegetations

ABSTRACT Daptomycin-nonsusceptible (DNS) Staphylococcus aureus is found in difficult-to-treat infections, and the optimal therapy is unknown. We investigated the activity of high-dose (HD) daptomycin plus trimethoprim-sulfamethoxazole de-escalated to HD daptomycin or trimethoprim-sulfamethoxazole against 4 clinical DNS methicillin-resistant S. aureus (MRSA) isolates in an in vitro pharmacokinetic/pharmacodynamic model of simulated endocardial vegetations (109 CFU/g). Simulated regimens included HD daptomycin at 10 mg/kg/day for 14 days, trimethoprim-sulfamethoxazole at 160/800 mg every 12 h for 14 days, HD daptomycin plus trimethoprim-sulfamethoxazole for 14 days, and the combination for 7 days de-escalated to HD daptomycin for 7 days and de-escalated to trimethoprim-sulfamethoxazole for 7 days. Differences in CFU/g (at 168 and 336 h) were evaluated by analysis of variance (ANOVA) with a Tukeys post hoc test. Daptomycin MICs were 4 μg/ml (SA H9749-1, vancomycin-intermediate Staphylococcus aureus; R6212, heteroresistant vancomycin-intermediate Staphylococcus aureus) and 2 μg/ml (R5599 and R5563). Trimethoprim-sulfamethoxazole MICs were ≤0.06/1.19 μg/ml. HD daptomycin plus trimethoprim-sulfamethoxazole displayed rapid bactericidal activity against SA H9749-1 (at 7 h) and R6212 (at 6 h) and bactericidal activity against R5599 (at 72 h) and R5563 (at 36 h). A ≥8 log10 CFU/g decrease was observed with HD daptomycin plus trimethoprim-sulfamethoxazole against all strains (at 48 to 144 h), which was maintained with de-escalation to HD daptomycin or trimethoprim-sulfamethoxazole at 336 h. The combination for 14 days and the combination for 7 days de-escalated to HD daptomycin or trimethoprim-sulfamethoxazole was significantly better than daptomycin monotherapy (P < 0.05) and trimethoprim-sulfamethoxazole monotherapy (P < 0.05) at 168 and 336 h. Combination therapy followed by de-escalation offers a novel bactericidal therapeutic alternative for high-inoculum, serious DNS MRSA infections.

Ceftobiprole and ampicillin increase daptomycin susceptibility of daptomycin-susceptible and -resistant VRE

OBJECTIVES The synergistic combination of daptomycin plus ampicillin has proven to be effective against VRE including daptomycin-non-susceptible strains. Ceftobiprole is a cephalosporin with broad binding affinity for enterococcal PBP subtypes including PBP5. Given the synergy between β-lactams and daptomycin against VRE, it was of interest to determine whether ceftobiprole offered any synergistic advantage with daptomycin compared with ampicillin. METHODS MICs were determined by broth microdilution in the presence and absence of ampicillin or ceftobiprole for 20 ampicillin-resistant VRE. Six strains, including two isogenic pairs of vancomycin-resistant Enterococcus faecium and two vancomycin-resistant Enterococcus faecalis, were evaluated for synergy using time-kill methods. Synergy was defined as a ≥2 log10 cfu/mL reduction of the combination over the most active single agent. Binding of daptomycin-bodipy in the presence and absence of ceftobiprole was quantified. RESULTS Daptomycin MICs ranged from 2 to 256 mg/L. The addition of ceftobiprole and ampicillin reduced daptomycin MICs by a median of 3 and 4 log2 dilutions, respectively. In time-kill studies, daptomycin plus either ceftobiprole or ampicillin was synergistic against four of six strains, but not the same strains. Both combinations were synergistic against the vancomycin-resistant E. faecalis strains. Ceftobiprole exposure increased daptomycin-bodipy binding by 2.8 times (P<0.0001). CONCLUSIONS Ceftobiprole appears to offer a similar degree of synergistic activity to ampicillin when combined with daptomycin against VRE. Further research should explore the genetic and phenotypic qualities of strains that respond preferentially to ceftobiprole as opposed to ampicillin.

Defining Daptomycin Resistance Prevention Exposures in Vancomycin-Resistant Enterococcus faecium and E. faecalis

ABSTRACT Daptomycin is used off-label for enterococcal infections; however, dosing targets for resistance prevention remain undefined. Doses of 4 to 6 mg/kg of body weight/day approved for staphylococci are likely inadequate against enterococci due to reduced susceptibility. We modeled daptomycin regimens in vitro to determine the minimum exposure to prevent daptomycin resistance (Dapr) in enterococci. Daptomycin simulations of 4 to 12 mg/kg/day (maximum concentration of drug in serum [Cmax] of 57.8, 93.9, 123.3, 141.1, and 183.7 mg/liter; half-life [t1/2] of 8 h) were tested against one Enterococcus faecium strain (S447) and one Enterococcus faecalis strain (S613) in a simulated endocardial vegetation pharmacokinetic/pharmacodynamic model over 14 days. Samples were plated on media containing 3× the MIC of daptomycin to detect Dapr. Mutations in genes encoding proteins associated with cell envelope homeostasis (yycFG and liaFSR) and phospholipid metabolism (cardiolipin synthase [cls] and cyclopropane fatty acid synthetase [cfa]) were investigated in Dapr derivatives. Dapr derivatives were assessed for changes in susceptibility, surface charge, membrane depolarization, cell wall thickness (CWT), and growth rate. Strains S447 and S613 developed Dapr after simulations of 4 to 8 mg/kg/day but not 10 to 12 mg/kg/day. MICs for Dapr strains ranged from 8 to 256 mg/liter. Some S613 derivatives developed mutations in liaF or cls. S447 derivatives lacked mutations in these genes. Dapr derivatives from both strains exhibited lowered growth rates, up to a 72% reduction in daptomycin-induced depolarization and up to 6-nm increases in CWT (P < 0.01). Peak/MIC and AUC0–24/MIC ratios (AUC0–24 is the area under the concentration-time curve from 0 to 24 h) associated with Dapr prevention were 72.1 and 780 for S447 and 144 and 1561 for S613, respectively. Daptomycin doses of 10 mg/kg/day may be required to prevent Dapr in serious enterococcal infections.

A Novel Approach Utilizing Biofilm Time-Kill Curves To Assess the Bactericidal Activity of Ceftaroline Combinations against Biofilm-Producing Methicillin-Resistant Staphylococcus aureus

ABSTRACT Medical device infections frequently require combination therapy. Beta-lactams combined with glycopeptides/lipopeptides are bactericidal against methicillin-resistant Staphylococcus aureus (MRSA). Novel macrowell kill-curve methods tested synergy between ceftaroline or cefazolin plus daptomycin, vancomycin, or rifampin against biofilm-producing MRSA. Ceftaroline combinations demonstrated the most pronounced bacterial reductions. Ceftaroline demonstrated greatest kill with daptomycin (4.02 ± 0.59 log10 CFU/cm2), compared to combination with vancomycin (3.36 ± 0.35 log10 CFU/cm2) or rifampin (2.68 ± 0.61 log10 CFU/cm2). These data suggest that beta-lactam combinations are useful against MRSA biofilms.

Evaluation of Ceftaroline, Vancomycin, Daptomycin, or Ceftaroline plus Daptomycin against Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus in an In Vitro Pharmacokinetic/Pharmacodynamic Model of Simulated Endocardial Vegetations

ABSTRACT Infective endocarditis (IE) caused by methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin and daptomycin has few adequate therapeutic options. Ceftaroline (CPT) is bactericidal against daptomycin (DAP)-nonsusceptible (DNS) and vancomycin-intermediate MRSA, but supporting data are limited for IE. This study evaluated the activities of ceftaroline, vancomycin, daptomycin, and the combination of ceftaroline plus daptomycin against DNS MRSA in a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). Simulations of ceftaroline-fosamil (600 mg) every 8 h (q8h) (maximum concentration of drug in serum [Cmax], 21.3 mg/liter; half-life [t1/2], 2.66 h), daptomycin (10 mg/kg of body weight/day) (Cmax, 129.7 mg/liter; t1/2, 8 h), vancomycin (1 g) q8h (minimum concentration of drug in serum [Cmin], 20 mg/liter; t1/2, 5 h), and ceftaroline plus daptomycin were evaluated against 3 clinical DNS, vancomycin-intermediate MRSA in a two-compartment, in vitro, PK/PD SEV model over 96 h with a starting inoculum of ∼8 log10 CFU/g. Bactericidal activity was defined as a ≥3-log10 CFU/g reduction from the starting inoculum. Therapeutic enhancement of combinations was defined as ≥2-log10 CFU/g reduction over the most active agent alone. MIC values for daptomycin, vancomycin, and ceftaroline were 4 mg/liter, 4 to 8 mg/liter, and 0.5 to 1 mg/liter, respectively, for all strains. At simulated exposures, vancomycin was bacteriostatic, but daptomycin and ceftaroline were bactericidal. By 96 h, ceftaroline monotherapy offered significantly improved killing compared to other agents against one strain. The combination of DAP plus CPT demonstrated therapeutic enhancement, resulting in significantly improved killing versus either agent alone against 2/3 (67%) strains. CPT demonstrated bactericidal activity against DNS, vancomycin-intermediate MRSA at high bacterial densities. Ceftaroline plus daptomycin may offer more rapid and sustained activity against some MRSA in the setting of high-inoculum infections like IE and should also be considered.

Rapid Detection of Vancomycin-Intermediate Staphylococcus aureus by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Vancomycin is the standard of care for the treatment of invasive methicillin-resistant Staphylococcus aureus (MRSA) infections. Infections with vancomycin-nonsusceptible MRSA, including vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA), are clinically challenging and are associated with poor patient outcomes. The identification of VISA in the clinical laboratory depends on standard susceptibility testing, which takes at least 24 h to complete after isolate subculture, whereas hVISA is not routinely detected in clinical labs. We therefore sought to determine whether VISA and hVISA can be differentiated from vancomycin-susceptible S. aureus (VSSA) using the spectra produced by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Strains of MRSA were characterized for vancomycin susceptibility phenotype by broth microdilution and modified population analysis. We tested 21 VISA, 21 hVISA, and 38 VSSA isolates by MALDI-TOF MS. Susceptibility phenotypes were separated by using a support vector machine (SVM) machine learning algorithm. The resulting model was validated by leave-one-out cross validation. Models were developed and validated by using spectral profiles generated under various subculture conditions, as well as with and without hVISA strains. Using SVM, we correctly identified 100% of the VISA and 97% of the VSSA isolates with an overall classification accuracy of 98%. Addition of hVISA to the model resulted in 76% hVISA identification, 100% VISA identification, and 89% VSSA identification, for an overall classification accuracy of 89%. We conclude that VISA/hVISA and VSSA isolates are separable by MALDI-TOF MS with SVM analysis.