Free Peak Concentrations Research Articles

1573. Daptomycin Resistant Enterococcus faecium: Combination Therapy Screening

Background Enterococcus faecium infections are difficult to treat and there is a growing concern regarding the rising occurrence of daptomycin resistance. We have previously demonstrated that only daptomycin plus ampicillin combination was effective against DAP-R E. faecium R497. The efficacy and systematic screening DAP plus β-lactams and DAP plus other combinations against daptomycin-resistant strains of E. faecium has not been investigated. Here, we evaluated 40 selected single, dual and triple combinations of antibacterial regimens against two clinical isolates of DAP-R E. faecium (R497 and R496 (with daptomycin MIC of 16 and 32 µg/ml, respectively).Methods E.faecium R497 and R496 were tested against an array of antibacterial agents including daptomycin, tigecycline, linezolid, ertapenem, ceftaroline and ceftriaxone using MIC susceptibility tests and 24h time-kill curves (TKC). All susceptibility tests and TKCs were performed in MHB broth containing 50 mg/L calcium. TKCs were performed at half MIC or free peak concentration of each antibacterial (whichever was lower). Synergy was defined as >2 log10 CFU/ml decrease compared to the most potent antibacterial agent.ResultsSusceptibility tests demonstrated resistance to all listed β-lactams for both organisms. TKCs demonstrated that combination of daptomycin-ertapenem, daptomycin-ceftriaxone and daptomycin-ceftaroline was not effective against R497. However, addition of ceftriaxone or linezolid to either daptomycin-ertapenem or daptomycin-ceftaroline combinations resulted in synergy against this organism. Combinations of daptomycin-ertapenem and daptomycin-ceftaroline were synergistic against R496. Addition of linezolid, ceftriaxone or tigecycline to either daptomycin-ceftaroline or daptomycin-ertapenem combination did not increase killing activity against R496.ConclusionDifferential affinity of β-lactams to specific PBP isotypes seems to be a key parameter for the success of daptomycin- β-lactam combinations against multi-drug resistant E. faecium . The optimized use of double β-lactam therapy in addition to daptomycin can potentially lead to improved patient outcomes and preserving antibiotic therapy for serious enterococcus infections.Disclosures Cesar A. Arias, MD, MSc, PhD, FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support) Michael J. Rybak, PharmD, MPH, PhD, Paratek (Grant/Research Support)

2391. Liposomal Vancomycin and Cefazolin Combinations for S. aureus Biofilms

BackgroundBiofilms are sophisticated communities of matrix-encased and surface-attached bacteria that exhibit a distinct and specific resistant/tolerant phenotype to almost all antibacterial agents, with activity reduced 10- to 1,000-fold. Interestingly, this augmented resistance rapidly reverts when bacteria detach from the biofilm and return to a planktonic state. However, in this in vitro pharmacokinetic and pharmacodynamic (PK/PD) model we are able to expose biofilms to shear rates that are consistent with human interface and mimic antibiotic penetration and diffusion pathways from serum antibiotic concentration in humans.MethodsMethicillin-susceptible ATCC 29213 and MRSA 494 strains were evaluated. Initial susceptibility tests were performed by broth microdilution method. Time kill studies were performed to identify synergy patterns for liposomal and commercial antibiotics. Biofilm eradication was investigated using antibiotics vancomycin (VAN) (commercial) vs. liposomal VAN (VAN-L) (Patent#17-1460) and also combination of VAN- cefazolin (commercial) vs. liposomal vancomycin and liposomal cefazolin (CFZ-L) (Patent# 17-1460) in biofilms for strain MRSA 494. Biofilms were generated overnight using the BioFlux Microfluidic system (Fluxion BioSciences) at constant and continuous shear rates to optimize biofilm attachment and creation. Perfusion of antibiotic solutions (free peak concentration) was applied over a 24 h time period. Time lapse pictures were recorded to determine antibiotic biofilm eradication rates over 18h of incubation and pictures were analyzed using Bioflux Montage software.ResultsMIC values demonstrated a 2-fold reduction for liposomal vancomycin vs. commercial vancomycin. Also, combination of liposomal VAN MIC in presence of CFZ-L showed a 15.87-fold reduction in comparison to commercial VAN for 494. Overall, our biofilm results demonstrated a 43.6% improved eradication using VAN-L and CFZ-L combination in comparison to commercial VAN-CFZ combination. We also observed 5.7% improved eradication using VAN-L vs. commercial VAN.ConclusionLiposomal form of VAN and CFZ combinations are a promising approach to improved efficacy and reduced VAN resistance in S. aureus biofilms.Disclosures M. J. Rybak, Allergan: Consultant, Grant Investigator and Speaker’s Bureau, Research grant and Research support. Achaogen: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support. Bayer: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support. Melinta: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support. Merck: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support. Theravance: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support. Sunovian: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support. Zavante: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support. NIAID: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support.

2403. Comparison of Daptomycin Combination Therapy With Ceftaroline or Oxacillin Against Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates Causing Persistent Bacteremia

BackgroundIncreasing evidence suggests that daptomycin (DAP) demonstrates in vitro synergy in combination with other anti-staphylococcal agents, including ceftaroline (CPT) and oxacillin (OXA), against MRSA. Nevertheless, optimal combinations remain undefined. Here, our objective was to compare DAP in combination with CPT or OXA against MRSA bloodstream isolates collected from patients with persistent bacteremia despite >7 days of vancomycin treatment.MethodsMinimum inhibitory concentrations (MICs) for DAP, CPT, and OXA were determined in duplicate by reference broth microdilution methods. We used time-kill analyses (TKA) to test free peak concentrations (fCmax) of DAP (8 µg/mL), CPT (16 µg/mL), and OXA (4 µg/mL) alone and in combination against 1 × 108 CFU/mL to simulate high-inocula infections. Bactericidal and synergistic activity were defined as a ≥3-log10 decrease in CFU/mL and >2-log10 decrease in CFU/mL in combination compared with the most active single agent, respectively, at 24 hours.ResultsA representative isolate was selected from 12 patients with persistent MRSA bacteremia. Median (range) MICs were 0.5 (0.5–1), 0.5 (0.5–1), and 64 (64–≥128) µg/mL for DAP, CPT, and OXA, respectively. By TKA (n = 5 isolates), median log-kills were −3.81, −1.90, and +1.99 log10CFU/mL for DAP, CPT, OXA, respectively. Corresponding rates of bactericidal activity were 80%, 20%, and 0%, respectively. In combination, median log-kills were −7.83 and −4.82 log10CFU/mL for DAP+CPT and DAP+OXA, respectively (P = 0.111; Figure 1). DAP was synergistic in combination with CPT or OXA against 80% and 60% of isolates, respectively. Median log-kills in combination with CPT or OXA were higher than DAP alone (P = 0.003 and P = 0.0497, respectively). At 24 hours, colony counts were below the lower limit of detection (50 CFU/mL) against 60% and 20% of isolates exposed to DAP+CPT or DAP+OXA, respectively.ConclusionAmong persistent MRSA bloodstream isolates, combinations of DAP + CPT or OXA demonstrates synergy and statistically greater killing effects in vitro at fCmax concentrations than DAP alone. Log-kills were greatest with DAP+CPT, which merits further validation in pre-clinical models. Disclosures All authors: No reported disclosures.

Antibacterial activity of ceftolozane/tazobactam alone and in combination with other antimicrobial agents against MDR Pseudomonas aeruginosa.

Broad-spectrum antimicrobial resistance in Pseudomonas aeruginosa (PSA) isolates is a growing concern as our therapeutic options have become significantly limited. Although ceftolozane/tazobactam (C/T) has been shown to be highly active against MDR PSA pathogens, combination regimens are often employed in real-world settings. To assist the clinical decision-making process regarding the selection of combination antibiotics and dosages for this pathogen, we performed time-kill studies assessing clinical free peak and trough C/T concentrations alone and in combination with eight anti-pseudomonal agents against four clinical MDR PSA isolates. Time-kill analyses were performed over 24 h in duplicate using C/T concentrations reflective of the free peak concentrations of a 3 g dose every 8 h (q8h; 120/25.2 mg/L) and the peak and trough of a 1.5 g q8h dose (60/12.6 and 7.5/1.6 mg/L) in humans. The activity of C/T 120, 60 and 7.5 mg/L alone and C/T 7.5 mg/L in combination with free peak and trough concentrations of clinical doses for cefepime, ciprofloxacin, colistin, aztreonam, meropenem, piperacillin/tazobactam, fosfomycin and amikacin was tested for all isolates. C/T 3 and 1.5 g q8h peak concentrations demonstrated killing against the MDR PSA. Colistin and fosfomycin were synergistic with C/T as dual therapy and triple therapy regimens. As a result of escalating resistance, PSA is an increasingly challenging pathogen in the clinical setting. Our findings aid in the identification of novel treatment options using achievable drug exposures for the treatment of MDR PSA.

Time-Kill Analysis of Ceftolozane/Tazobactam Efficacy Against Mucoid Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients.

IntroductionMucoid Pseudomonas aeruginosa (MP) strains in cystic fibrosis (CF) patients are thought to initiate the chronic infection stage of CF and are associated with pulmonary function decline.ObjectivesThe purpose of this study was to assess the susceptibility of MP strains to ceftolozane/tazobactam and the efficacy of ceftolozane/tazobactam against MP strains compared with those for standard-of-care antipseudomonal antibiotics.MethodsTen clinical isolates of MP from CF patients were tested for susceptibility with Etest and time–kill analysis with ceftolozane/tazobactam compared with ceftazidime, cefepime, ciprofloxacin, meropenem, tobramycin, and polymyxin B. The physiologic free peak concentrations were used in the time–kill experiments.ResultsCeftolozane/tazobactam minimum inhibitory concentrations ranged from 0.032 to 1.5 mg/L. In the time–kill analysis, the mean starting inoculum for the isolates was 6.29 ± 0.22 log10 colony forming units (CFU) per milliliter. On average, ceftolozane/tazobactam, cefepime, ciprofloxacin, meropenem, tobramycin, and polymyxin B all demonstrated bactericidal activity. With all isolates taken into account, polymyxin B, tobramycin, meropenem, and ceftolozane/tazobactam 3 g were the most potent, with reductions in inoculum of 5.07 ± 0.45, 4.58 ± 2.2, 4.76 ± 0.71, and 4.17 ± 0.94 log10 CFU/mL, respectively. Ceftolozane/tazobactam 1.5 g, cefepime, and ciprofloxacin reduced the starting inoculum by 3.74 ± 0.99, 3.42 ± 1.4, and 3.23 ± 2.0 log10 CFU/mL, respectively. Despite 90% susceptibility, ceftazidime was bactericidal against seven of ten strains, with an average reduction in starting inoculum of 2.91 ± 2.2 log10 CFU/mL.ConclusionCeftolozane/tazobactam activity against MP strains derived from CF patients was comparable to that of standard-of-care agents at both the 1.5-g dose and the 3-g dose. Further in vitro modeling and clinical trials are warranted.

In Vitro Antibacterial Activity of Ceftolozane/Tazobactam (C/T) Alone and in Combination with other Antimicrobial Agents against Multidrug-Resistant (MDR) Pseudomonas aeruginosa (PSA)

Abstract Background Broad-spectrum antimicrobial resistance in MDR PSA isolates significantly limits our therapeutic options. C/T has been shown to be highly active against MDR PSA isolates. To assist the clinical decision-making process regarding the selection of agents and dosages for this pathogen, we performed time-kill studies assessing various C/T concentrations alone and in combination with other anti-pseudomonal agents. Methods Four clinical MDR P. aeruginosa isolates were selected. MICs were determined via broth microdilution methods according to CLSI. Time-kill analyses were performed in duplicate using C/T free concentrations reflective of the peak and trough of a 3g q8h dose (120/25.2 µg/mL,7.5/1.6 µg/mL) and the peak of a 1.5g q8h dose (60/12.6 µg/mL) in humans. The activity of C/T 120, 60, and 7.5 alone and C/T 7.5 in combination with free peak concentrations of FEP, CIP, CST, ATM, MEM, TZP, FOF, or AMK was tested for all isolates. Colony counts were determined at 0, 3, 6, and 24h by serial dilution plating. Synergy was defined at ≥ 2 log10 CFU reduction from the most active agent. Results MICs of the 4 MDR P. Aeruginosa isolates are in Table 1. As the C/T concentrations increased, bacterial reduction improved, achieving a mean (±SD) log10CFU change from 0 h of 0.03 (±0.67), −1.19 (±1.03), −2.59 (±0.86) with C/T 7.5, 60, 120, respectively. C/T 7.5 was synergistic with CST (PSA C8-21, PSA C45-10) and FOF (PSA C28-5, PSA C14-22) in two of four isolates. No synergy was observed with double β-lactam therapy or CIP. AMK alone achieved maximal bacterial kill; therefore, synergy could not be assessed. Conclusion C/T 3g and 1.5g q8h peak concentrations demonstrate killing against the MDR PSA. CST and FOF were synergistic with C/T in vitro. Our findings aide in identification of novel treatment options and dosing regimens for the treatment of MDR P. Aeruginosa. Disclosures D. P. Nicolau, Merck: Investigator and Speaker’s Bureau, Research support.

Oritavancin Combinations with β-Lactams against Multidrug-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococci.

Oritavancin possesses activity against vancomycin-resistant enterococci (VRE) and methicillin-resistantStaphylococcus aureus(MRSA).In vitrodata suggest synergy between beta-lactams (BLs) and vancomycin or daptomycin, agents similar to oritavancin. We evaluated the activities of BLs combined with oritavancin against MRSA and VRE. Oritavancin MICs were determined for 30 strains, 5 each of MRSA, daptomycin-nonsusceptible (DNS) MRSA, vancomycin-intermediate MRSA (VISA), heteroresistant VISA (hVISA), vancomycin-resistantEnterococcus faecalis, and vancomycin-resistantEnterococcus faecium Oritavancin MICs were determined in the presence of subinhibitory concentrations of BLs. Oritavancin combined with ceftaroline, cefazolin, or nafcillin was evaluated for lethal synergy against MRSA, and oritavancin combined with ceftaroline, ampicillin, or ertapenem was evaluated for lethal synergy against VRE in 24-h time-kill assays. Oritavancin at 0.5× the MIC was combined with BLs at 0.5× the MIC or the biological free peak concentration, whichever one was lower. Synergy was defined as a ≥2-log10-CFU/ml difference between the killing achieved with the combination and that achieved with the most active single agent at 24 h. Oritavancin MICs were ≤0.125 μg/ml for all MRSA isolates except three VISA isolates with MICs of 0.25 μg/ml. Oritavancin MICs for VRE ranged from 0.03 to 0.125 μg/ml. Oritavancin in combination with ceftaroline was synergistic against all MRSA phenotypes and statistically superior to all other combinations against DNS MRSA, hVISA, and MRSA isolates (P< 0.02). Oritavancin in combination with cefazolin and oritavancin in combination with nafcillin were also synergistic against all MRSA strains. Synergy between oritavancin and all BLs was revealed against VRE strain 8019, while synergy between oritavancin and ampicillin or ertapenem but not ceftaroline was demonstrated against VRE strain R7164. The data support the potential use of oritavancin in combination with BLs, especially oritavancin in combination with ceftaroline, for the treatment of infections caused by MRSA. The data from the present study are not as strong for oritavancin in combination with BLs for VRE. Further study of both MRSA and VRE in more complex models is warranted.

In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene

Meticillin-resistant Staphylococcus aureus (MRSA) is routinely detected by amplification of the mecA gene. Recently, MRSA isolates harbouring a novel mec gene (mecC) that is not detected by mecA amplification have been reported. In this study, the activities of the lipoglycopeptide oritavancin as well as the comparators vancomycin, daptomycin and linezolid against 14 mecC MRSA isolates were studied by broth microdilution minimum inhibitory concentration (MIC) and time–kill assays at clinically relevant concentrations of each antibacterial agent. Oritavancin, vancomycin, daptomycin and linezolid MIC90 values (MIC required to inhibit 90% of the isolates) against the mecC isolates were 0.06, 1, 1 and 2mg/L, respectively. In time–kill assays, oritavancin at concentrations reflective of its free peak in plasma of patients receiving a single 1200mg intravenous dose and the level 24h thereafter was bactericidal against all isolates tested, attaining 3 log kill relative to the starting inoculum between 5min and 15min. Vancomycin both at its free peak and free trough concentrations was also bactericidal against all isolates, attaining bactericidal activity between 6h and 24h. Daptomycin was bactericidal only at its free peak concentration, attaining bactericidal activity between 30min and 4h against the tested isolates. Linezolid was bacteriostatic (<3 log kill relative to the starting inoculum) against the tested isolates. Oritavancin's in vitro activity against mecC MRSA isolates was indistinguishable from that against mecA MRSA isolates both in MIC and time–kill assays.

Susceptibility Breakpoints for Amphotericin B and Aspergillus Species in an In Vitro Pharmacokinetic-Pharmacodynamic Model Simulating Free-Drug Concentrations in Human Serum

Although conventional amphotericin B was for many years the drug of choice and remains an important agent against invasive aspergillosis, reliable susceptibility breakpoints are lacking. Three clinical Aspergillus isolates (Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus) were tested in an in vitro pharmacokinetic-pharmacodynamic model simulating the biphasic 24-h time-concentration profile of free amphotericin B concentrations in human serum with free peak concentrations (fCmax) of 0.1, 0.3, 0.6, 1.2, and 2.4 mg/liter administered once daily. Drug concentrations were measured with a bioassay, and fungal growth was monitored for 72 h with galactomannan production. The fCmax/MIC corresponding to half-maximal activity (P50) was determined for each species, and the percentage of target attainment was calculated for different MICs for the standard (1 mg/kg of body weight) and a lower (0.6-mg/kg) dose of amphotericin B with Monte Carlo simulation analysis. The fCmax/MICs (95% confidence intervals) corresponding to P50 were 0.145 (0.133 to 0.158), 0.371 (0.283 to 0.486), and 0.41 (0.292 to 0.522) for A. fumigatus, A. flavus, and A. terreus, respectively. The median percentages of P50 attainment were ≥88%, 47%, and 0% for A. fumigatus isolates with MICs of ≤0.5, 1, and ≥2 mg/liter, respectively, and ≥81%, 24%, and 0% and ≥75%, 15%, and 0% for A. flavus and A. terreus isolates with MICs of ≤0.25, 0.5, and ≥1 mg/liter, respectively. The lower dose of 0.6 mg/kg would retain efficacy for A. fumigatus, A. flavus, and A. terreus isolates with MICs of ≤0.25, ≤0.125, and ≤0.125 mg/liter, respectively. The susceptibility, intermediate susceptibility, and resistance breakpoints of ≤0.5, 1, and ≥2 mg/liter for A. fumigatus and ≤0.25, 0.5, and ≥1 mg/liter for A. flavus and A. terreus were determined for conventional amphotericin B with a pharmacokinetic-pharmacodynamic model simulating free-drug serum concentrations.

Plectasin Shows Intracellular Activity against Staphylococcus aureus in Human THP-1 Monocytes and in a Mouse Peritonitis Model

Antimicrobial therapy of infections with Staphylococcus aureus can pose a challenge due to slow response to therapy and recurrence of infection. These treatment difficulties can partly be explained by intracellular survival of staphylococci, which is why the intracellular activity of antistaphylococcal compounds has received increased attention within recent years. The intracellular activity of plectasin, an antimicrobial peptide, against S. aureus was determined both in vitro and in vivo. In vitro studies using THP-1 monocytes showed that some intracellular antibacterial activity of plectasin was maintained (maximal relative efficacy [E(max)], 1.0- to 1.3-log reduction in CFU) even though efficacy was inferior to that of extracellular killing (E(max), >4.5-log CFU reduction). Animal studies included a novel use of the mouse peritonitis model, exploiting extra- and intracellular differentiation assays, and assessment of the correlations between activity and pharmacokinetic (PK) parameters. The intracellular activity of plectasin was in accordance with the in vitro studies, with an E(max) of a 1.1-log CFU reduction. The parameter most important for activity was fC(peak)/MIC, where fC(peak) is the free peak concentration. These findings stress the importance of performing studies of extra- and intracellular activity since these features cannot be predicted from traditional MIC and killing kinetic studies. Application of both the THP-1 and the mouse peritonitis models showed that the in vitro results were similar to findings in the in vivo model with respect to demonstration of intracellular activity. Therefore the in vitro model was a good screening model for intracellular activity. However, animal models should be applied if further information on activity, PK/pharmacodynamic parameters, and optimal dosing regimens is required.

Multiple-dose pharmacokinetics of telithromycin in peripheral soft tissues

Based on clinicians’ expectations of high concentrations of telithromycin (TEL) in tissues, combined with its excellent in vitro antimicrobial characteristics, TEL is casually considered as a potential therapeutic option for the therapy of minor cases of soft tissue or bite-wound infections. To clarify this clinically important issue, the present investigation was carried out to measure the pharmacokinetic profile of TEL in the interstitial space fluid (ISF) of skeletal muscle and subcutaneous adipose tissue by means of the microdialysis technique in 10 healthy subjects following repetitive daily doses of 800 mg TEL. These data were compared with free concentrations of TEL determined in plasma. External controls for the present examination were the use of historic, single-dose data collected by our study group utilising identical methods and the same trial subjects. Despite an increase in the median half-life from ca. 3 h after a single dose to ca. 10 h at steady-state conditions in all compartments, accumulation of TEL in ISF of soft tissues and plasma was clinically non-relevant. Median free peak concentrations in plasma, skeletal muscle and subcutis were 0.52, 0.13 and 0.19 mg/L, respectively. The median ratios of the tissue to plasma free areas under the concentration–time curves from 0 to 24 h ( fAUC 0–24 tissue/ fAUC 0–24 plasma) were 0.27 and 0.58 for muscle and subcutis, respectively ( P > 0.05). The present multiple-dose investigation of TEL is in line with a previous single-dose study confirming that TEL 800 mg/day may not be optimally effective in the therapy of soft tissue infections.

Concentration-Dependent Mycobacterium tuberculosis Killing and Prevention of Resistance by Rifampin

Rifampin is a cornerstone of modern antituberculosis therapy. However, rifampin's half-life of 3 h is believed to limit its utility for intermittent therapy, so new congeners with long half-lives are being developed. Using an in vitro pharmacokinetic-pharmacodynamic model of tuberculosis, we examined the relationships between rifampin exposure, microbial killing of log-phase-growth Mycobacterium tuberculosis, and suppression of resistance. Rifampin's microbial killing was linked to the area under the concentration-time curve-to-MIC ratio. The suppression of resistance was associated with the free peak concentration (C(max))-to-MIC ratio and not the duration that the rifampin concentration was above MIC. Rifampin prevented resistance to itself at a free C(max)/MIC ratio of > or =175. The postantibiotic effect duration was > or =5.2 days and was most closely related to the C(max)/MIC ratio (r(2) = 0.96). To explain rifampin's concentration-dependent effect, we examined the kinetics of rifampin entry into M. tuberculosis. Rifampin achieved concentration-dependent intracellular steady-state concentrations within 15 min. Our results suggest that doses of rifampin higher than those currently employed would optimize the effect of rifampin, if patients could tolerate them. Another major implication is that in the design of new rifampin congeners for intermittent therapy, the important properties may include (i) the efficient entry of the rifamycin into M. tuberculosis, (ii) the achievement of a free C(max)/MIC of >175 that can be tolerated by patients, and (iii) a long postantibiotic effect duration.