Warren E. Rose

Impact of biofilm on the in vitro activity of vancomycin alone and in combination with tigecycline and rifampicin against Staphylococcus aureus

OBJECTIVES This study evaluated vancomycin susceptibility and activity alone and in combination with rifampicin and tigecycline against low-biofilm- and high-biofilm-producing methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. METHODS Forty MRSA isolates recovered from bloodstream infections were analysed. Susceptibilities were performed in planktonic and biofilm cultures by microbroth dilution. Biofilm production was determined using an adherent plate assay. Time-kill analysis was performed on six low- and six high-biofilm-producing isolates with 15 mg/L vancomycin alone and in combination with rifampicin or tigecycline at 4x MIC. RESULTS Vancomycin susceptibility displayed a 4-fold and an 8-fold increase in the MIC(50) and MIC(90), respectively, in the presence of biofilm. Rifampicin and tigecycline susceptibilities also increased in biofilms, but still remained within the susceptibility breakpoints except for a tigecycline MIC(90) of 1 mg/L. High biofilm production was detected in 60% of the isolates. In time-kill analysis, 15 mg/L vancomycin achieved bactericidal activity against only low-biofilm-producing strains with a 1.8 log(10) cfu/mL difference in bacterial kill compared with high-biofilm-producing strains (P < 0.001). Rifampicin alone had minimal activity, resulting in resistance. Tigecycline was minimally effective and was not bactericidal, but no difference was observed in the comparison of biofilm-producing strains. Vancomycin in combination with rifampicin or tigecycline was bactericidal against all strains (mean kill 4.5 +/- 0.5 log(10) cfu/mL), regardless of biofilm production. CONCLUSIONS Vancomycin exposures at 15 mg/L may not be adequate in eradicating biofilm-producing S. aureus. Alternative treatments or combination therapy should be explored to optimize outcomes in biofilm-associated infections.

Activities of High-Dose Daptomycin, Vancomycin, and Moxifloxacin Alone or in Combination with Clarithromycin or Rifampin in a Novel In Vitro Model of Staphylococcus aureus Biofilm

ABSTRACT Biofilm formation is an important virulence factor that allows bacteria to resist host responses and antibacterial agents. The aim of the study was to assess the in vitro activities of several antimicrobials alone or in combination against two Staphylococcus aureus isolates in a novel pharmacokinetic/pharmacodynamic (PK/PD) model of biofilm for 3 days. One methicillin-susceptible S. aureus strain (SH1000) and one methicillin-resistant S. aureus strain (N315) were evaluated in a modified biofilm reactor with polystyrene coupons. Simulated regimens included vancomycin (VAN) plus rifampin (RIF), moxifloxacin (MOX), and high doses (10 mg/kg of body weight/day) of daptomycin (DAP) alone or combined with RIF or clarithromycin (CLA). Against viable planktonic bacteria (PB) and biofilm-embedded bacteria (BB) of SH1000, neither DAP nor MOX alone was bactericidal. In contrast, the combination of DAP or MOX with CLA significantly increased the activity of the two agents against both PB and BB (P < 0.01), and DAP plus CLA reached the limit of detection at 72 h. Against PB of N315, DAP alone briefly achieved bactericidal activity at 24 h, whereas sustained bactericidal activity was observed at 32 h with VAN plus RIF. Overall, only a minimal reduction was observed with both regimens against BB (<2.8 log10 CFU/ml). Finally, the combination of DAP and RIF was bactericidal against both PB and BB, achieving the limit of detection at 72 h. In conclusion, we developed a novel in vitro PK/PD model to assess the activities of antimicrobials against mature bacterial biofilm. Combinations of DAP or MOX with CLA were the most effective regimens and may represent promising options to treat persistent infections caused by S. aureus biofilms.

Evaluation of Daptomycin Pharmacodynamics and Resistance at Various Dosage Regimens against Staphylococcus aureus Isolates with Reduced Susceptibilities to Daptomycin in an In Vitro Pharmacodynamic Model with Simulated Endocardial Vegetations

ABSTRACT The need to investigate novel dosing regimens and combinations is essential in combating poor treatment outcomes for Staphylococcus aureus bacteremia and endocarditis. We evaluated the impact of simulated standard- and high-dose daptomycin in combination with gentamicin or rifampin against daptomycin-susceptible and nonsusceptible matched strains of S. aureus. These strains were collected from the daptomycin bacteremia and endocarditis clinical trial and consisted of three susceptible strains (MIC, 0.25 mg/liter) and four nonsusceptible isolates (MICs, 2 to 4 mg/liter). Daptomycin regimens of 6 and 10 mg/kg of body weight daily alone and in combination with gentamicin at 5 mg/kg daily or rifampin at 300 mg every 8 h were evaluated using an in vitro model with simulated endocardial vegetations over 96 h. Rapid bactericidal activity, identified by time to 99.9% kill, was displayed in all regimens with the daptomycin-susceptible strains. Concentration-dependent activity was noted by more-rapid killing with the 10-mg/kg/day dose. The addition of gentamicin improved activity in the majority of susceptible isolates. Daptomycin 6-mg/kg/day monotherapy displayed bactericidal activity for only one of the nonsusceptible isolates and for only two isolates with increased doses of 10 mg/kg/day. Combination regimens demonstrated improvement with some but not all nonsusceptible isolates. Three isolates developed a reduction in daptomycin susceptibility with 6-mg/kg/day monotherapy, but this was suppressed with both combination therapy and high-dose daptomycin. These results suggest that high-dose daptomycin therapy and combination therapy may be reasonable treatment options for susceptible isolates; however, more investigations are needed to confirm the variability of these regimens with nonsusceptible isolates.

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.

Addition of ceftaroline to daptomycin after emergence of daptomycin-nonsusceptible Staphylococcus aureus during therapy improves antibacterial activity.

ABSTRACT Antistaphylococcal beta-lactams enhance daptomycin activity and have been used successfully in combination for refractory methicillin-resistant Staphylococcus aureus (MRSA) infections. Ceftaroline possesses MRSA activity, but it is unknown if it improves the daptomycin potency comparably to other beta-lactams. We report a complex patient case of endocarditis who was treated with daptomycin in combination with ceftaroline, which resulted in clearance of a daptomycin-nonsusceptible strain. An in vitro pharmacokinetic/pharmacodynamic model of renal failure was used to simulate the development of daptomycin resistance and evaluate the microbiologic effects of daptomycin plus ceftaroline treatment. Combination therapy with daptomycin and ceftaroline restored daptomycin sensitivity in vivo and resulted in clearance of persistent blood cultures. Daptomycin susceptibility in vitro was increased in the presence of either ceftaroline or oxacillin. Daptomycin at 6 mg/kg of body weight every 48 h was bactericidal in the model but resulted in regrowth and daptomycin resistance (MIC, 2 to 4 μg/ml) with continued monotherapy. The addition of ceftaroline at 200 mg every 12 h after the emergence of daptomycin resistance enhanced bacterial killing. Importantly, daptomycin plus ceftaroline as the initial combination therapy produced rapid and sustained bactericidal activity and prevented daptomycin resistance. Both in vivo- and in vitro-derived daptomycin resistance resulted in bacteria with more fluid cell membranes. After ceftaroline was added in the model, fluidity was restored to the level of the initial in vivo isolate. Daptomycin-resistant isolates required high daptomycin exposures (at least 10 mg/kg) to optimize cell membrane damage with daptomycin alone. Ceftaroline combined with daptomycin was effective in eliminating daptomycin-resistant MRSA, and these results further justify the potential use of daptomycin plus beta-lactam therapy for these refractory infections.

Antimicrobial Salvage Therapy for Persistent Staphylococcal Bacteremia Using Daptomycin Plus Ceftaroline

PURPOSE Guidelines recommend daptomycin combination therapy as an option for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia after vancomycin failure. Recent data suggest that combining daptomycin with a β-lactam may have unique benefits; however, there are very limited clinical data regarding the use of ceftaroline with daptomycin. METHODS All 26 cases from the 10 medical centers in which ceftaroline plus daptomycin was used for treatment of documented refractory staphylococcal bacteremia from March 2011 to November 2012 were included. In vitro (synergy studies, binding assays, cathelicidin LL-37 killing assays), and in vivo (virulence assays using a murine subcutaneous infection model) studies examining the effects of ceftaroline with daptomycin were also performed. FINDINGS Daptomycin plus ceftaroline was used in 26 cases of staphylococcal bacteremia (20 MRSA, 2 vancomycin-intermediate S aureus, 2 methicillin-susceptible S aureus [MSSA], 2 methicillin-resistant S epidermidis). Bacteremia persisted for a median of 10 days (range, 3-23 days) on previous antimicrobial therapy. After daptomycin plus ceftaroline was started, the median time to bacteremia clearance was 2 days (range, 1-6 days). In vitro studies showed ceftaroline synergy against MRSA and enhanced MRSA killing by cathelicidin LL-37 and neutrophils. Ceftaroline also induced daptomycin binding in MSSA and MRSA to a comparable degree as nafcillin. MRSA grown in subinhibitory concentrations of ceftaroline showed attenuated virulence in a murine subcutaneous infection model. IMPLICATIONS Ceftaroline plus daptomycin may be an option to hasten clearance of refractory staphylococcal bacteremia. Ceftaroline offers dual benefit via synergy with both daptomycin and sensitization to innate host defense peptide cathelicidin LL37, which could attenuate virulence of the pathogen.

β-lactam Antibiotics Targeting PBP1 Selectively Enhance Daptomycin Activity against Methicillin-Resistant Staphylococcus aureus

ABSTRACT The activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA) is enhanced in the presence of subinhibitory concentrations of antistaphylococcal β-lactam antibiotics by an undefined mechanism. Given the variability in the penicillin-binding protein (PBP)-binding profiles of different β-lactam antibiotics, the purpose of this study was to examine the relative enhancement of DAP activity against MRSA by different β-lactam antibiotics to determine if a specific PBP-binding profile is associated with the ability to enhance the anti-MRSA activity of DAP. We determined that both broad- and narrow-spectrum β-lactam antibiotics known to exhibit PBP1 binding demonstrated potent enhancement of DAP anti-MRSA activity, whereas β-lactam antibiotics with minimal PBP1 binding (cefoxitin, ceftriaxone, cefaclor, and cefotaxime) were less effective. We suspect that PBP1 disruption by β-lactam antibiotics affects pathways of cell division in S. aureus that may be a compensatory response to DAP membrane insertion, resulting in DAP hypersusceptibility.

Structure and biosynthesis of the antibiotic bottromycin D.

Drug resistant infectious diseases are quickly becoming a global health crisis. While Streptomyces spp. have been a major source of antibiotics over the past 50 years, efficient methods are needed to identify new antibiotics and greatly improve the rate of discovery. LCMS-based metabolomics were applied to analyze extracts of 50 Streptomyes spp. Using this methodology, we discovered bottromycin D and used whole genome sequencing to determine its biosynthesis by a ribosomal pathway.

Poly(amidoamine) dendrimer-mediated synthesis and stabilization of silver sulfonamide nanoparticles with increased antibacterial activity

UNLABELLED Silver sulfadiazine (AgSD) is a topical antibiotic with limited aqueous solubility. In this study, it was shown that poly(amido amine) (PAMAM) dendrimer complexes with SD (SDZ) and silver (Ag) could be used for a bottom-up approach to synthesize highly-soluble AgSD nanoparticles (NPs). These NPs were stabilized against crystal growth by electrostatic layer-by-layer (LBL) coating with various PAMAM dendrimers. Additionally, AgNPs can be incorporated in the dendrimer shells that augmented AgSD release. NP formulation in a cream base provided a topical drug-delivery platform with enhanced antibacterial properties against burn-wound infections, comprising three nanostructures i.e., nano-AgSD, AgNPs as well as PAMAM dendrimers, in one efficient, elegant nanosystem. FROM THE CLINICAL EDITOR In this paper an elegant silver sulfadiazine-based nanoparticle complex is demonstrated with enhanced antibacterial properties and improved solubility for the treatment of burn-wound infections in a topical crème formulation.

Genotypic and Phenotypic Evaluation of the Evolution of High-Level Daptomycin Nonsusceptibility in Vancomycin-Resistant Enterococcus faecium

ABSTRACT Whole-genome sequencing and cell membrane studies of three clonal Enterococcus faecium strains with daptomycin MICs of 4, 32, and 192 μg/ml were performed, revealing nonsynonymous single nucleotide variants in eight open reading frames, including those predicted to encode a phosphoenolpyruvate-dependent, mannose-specific phosphotransferase system, cardiolipin synthetase, and EzrA. Membrane studies revealed a higher net surface charge among the daptomycin-nonsusceptible isolates and increased septum formation in the isolate with a daptomycin MIC of 192 μg/ml.