Adam Belley

Oritavancin Kills Stationary-Phase and Biofilm Staphylococcus aureus Cells In Vitro

ABSTRACT Slow-growing bacteria and biofilms are notoriously tolerant to antibiotics. Oritavancin is a lipoglycopeptide with multiple mechanisms of action that contribute to its bactericidal action against exponentially growing gram-positive pathogens, including the inhibition of cell wall synthesis and perturbation of membrane barrier function. We sought to determine whether oritavancin could eradicate cells known to be tolerant to many antimicrobial agents, that is, stationary-phase and biofilm cultures of Staphylococcus aureus in vitro. Oritavancin exhibited concentration-dependent bactericidal activity against stationary-phase inocula of methicillin-susceptible S. aureus (MSSA) ATCC 29213, methicillin-resistant S. aureus (MRSA) ATCC 33591, and vancomycin-resistant S. aureus (VRSA) VRS5 inoculated into nutrient-depleted cation-adjusted Mueller-Hinton broth. As has been described for exponential-phase cells, oritavancin induced membrane depolarization, increased membrane permeability, and caused ultrastructural defects including a loss of nascent septal cross walls in stationary-phase MSSA. Furthermore, oritavancin sterilized biofilms of MSSA, MRSA, and VRSA at minimal biofilm eradication concentrations (MBECs) of between 0.5 and 8 μg/ml. Importantly, MBECs for oritavancin were within 1 doubling dilution of their respective planktonic broth MICs, highlighting the potency of oritavancin against biofilms. These results demonstrate a significant activity of oritavancin against S. aureus in phases of growth that exhibit tolerance to other antimicrobial agents.

Time–kill kinetics of oritavancin and comparator agents against Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium

OBJECTIVES Oritavancin, a lipoglycopeptide, possesses bactericidal activity against Gram-positive bacteria including vancomycin-resistant Staphylococcus aureus and enterococci. To understand the time dependence of oritavancin activity, we have undertaken time-kill experiments against isolates of S. aureus, Enterococcus faecalis and Enterococcus faecium, including recent antibiotic-resistant strains. METHODS Six strains of S. aureus [methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA)] and five strains of enterococci [vancomycin-susceptible enterococci (VSE) and vancomycin-resistant enterococci (VRE; both VanA and VanB)] were tested in time-kill assays; oritavancin assays included 0.002% polysorbate-80 to ensure quantitative drug recovery. Oritavancin and comparators vancomycin, teicoplanin, linezolid and daptomycin were tested at static concentrations approximating their free peak (fC(max)) and free trough (fC(min)) in plasma when administered at standard doses for complicated skin and skin structure infections. RESULTS Oritavancin showed concentration-dependent killing of all strains tested: at its fC(max) predicted from a 200 mg dose in humans, oritavancin exerted bactericidal activity (> or =3 log kill relative to starting inoculum) against MSSA, MRSA and VRSA within 1 h and against VSE between 11 and 24 h. At predicted fC(max) from an 800 mg dose, oritavancin was bactericidal against VISA strains at 24 h and against VRE at 10 h. CONCLUSIONS Oritavancin displayed concentration-dependent killing of MSSA, MRSA, VRSA, VISA, VSE and VRE. Oritavancin was more rapidly bactericidal against all strains tested than were vancomycin, teicoplanin, linezolid or daptomycin at physiologically relevant concentrations. These data support the conclusion that oritavancin exerts concentration-dependent bactericidal activity on recent, drug-resistant isolates of S. aureus and enterococci.

Effect of Polysorbate 80 on Oritavancin Binding to Plastic Surfaces: Implications for Susceptibility Testing

ABSTRACT Oritavancin, a semisynthetic lipoglycopeptide with activity against gram-positive bacteria, has multiple mechanisms of action, including the inhibition of cell wall synthesis and the perturbation of the membrane potential. Approved guidelines for broth microdilution MIC assays with dalbavancin, another lipoglycopeptide, require inclusion of 0.002% polysorbate 80. To investigate the potential impact of polysorbate 80 on oritavancin susceptibility assays, we quantified the recovery of [14C]oritavancin from susceptibility assay plates with and without polysorbate 80 and examined the effect of the presence of polysorbate 80 on the oritavancin MICs for 301 clinical isolates from the genera Staphylococcus, Enterococcus, and Streptococcus. In the absence of polysorbate 80, [14C]oritavancin was rapidly lost from solution in susceptibility assay test plates: 9% of the input drug was recovered in broth at 1 h when [14C]oritavancin was tested at 1 μg/ml. Furthermore, proportionately greater losses were observed at lower oritavancin concentrations, suggesting saturable binding of oritavancin to surfaces. The inclusion of 0.002% polysorbate 80 or 2% lysed horse blood permitted the recovery of 80 to 100% [14C]oritavancin at 24 h for all drug concentrations tested. Concordantly, oritavancin MIC90s for streptococcal isolates, as determined in medium containing 2% lysed horse blood, were identical with and without polysorbate 80. In stark contrast, polysorbate 80 reduced the oritavancin MIC90s by 16- to 32-fold for clinical isolates of enterococci and staphylococci, which are typically cultured without blood. The results presented here provide evidence that the MIC data for oritavancin in the current literature significantly underestimate the potency of oritavancin in vitro. Moreover, the combination of data from MIC and [14C]oritavancin recovery studies supports the revision of the oritavancin broth microdilution method to include polysorbate 80 throughout the assay.

Oritavancin Disrupts Membrane Integrity of Staphylococcus aureus and Vancomycin-Resistant Enterococci To Effect Rapid Bacterial Killing

ABSTRACT Oritavancin is an investigational lipoglycopeptide in clinical development for the treatment of acute bacterial skin and skin structure infections. In this study, we demonstrate that oritavancin causes bacterial membrane depolarization and permeabilization leading to cell death of Gram-positive pathogens and that these effects are attributable to the 4′-chlorobiphenylmethyl group of the molecule.

Pharmacodynamics of a Simulated Single 1,200-Milligram Dose of Oritavancin in an In Vitro Pharmacokinetic/Pharmacodynamic Model of Methicillin-Resistant Staphylococcus aureus Infection

ABSTRACT The safety and efficacy of a single 1,200-mg dose of the lipoglycopeptide oritavancin are currently being investigated in two global phase 3 studies of acute bacterial skin and skin structure infections. In this study, an in vitro pharmacokinetic/pharmacodynamic model was established to compare the free-drug pharmacodynamics associated with a single 1,200-mg dose of oritavancin to once-daily dosing with daptomycin at 6 mg/kg of body weight and twice-daily dosing with vancomycin at 1,000 mg against three methicillin-resistant Staphylococcus aureus (MRSA) strains over 72 h. The area under the bacterial-kill curve (AUBKC) was used to assess the antibacterial effect of each dosing regimen at 24 h (AUBKC0-24), 48 h (AUBKC0-48), and 72 h (AUBKC0-72). The rapid bactericidal activities of oritavancin and daptomycin contributed to lower AUBKC0-24s for the three MRSA strains than with vancomycin (P < 0.05, as determined by analysis of variance [ANOVA]). Oritavancin exposure also resulted in a lower AUBKC0-48 and AUBKC0-72 against one MRSA strain and a lower AUBKC0-48 for another strain than did vancomycin exposure (P < 0.05). Furthermore, daptomycin exposure resulted in a lower AUBKC0-48 and AUBKC0-72 for one of the MRSA isolates than did vancomycin exposure (P < 0.05). Lower AUBKC0-24s for two of the MRSA strains (P < 0.05) were obtained with oritavancin exposure than with daptomycin. Thus, the antibacterial effect from the single-dose regimen of oritavancin is as effective as that from either once-daily dosing with daptomycin or twice-daily dosing with vancomycin against the MRSA isolates tested in an in vitro pharmacokinetic/pharmacodynamic model over 72 h. These results provide further justification to assess the single 1,200-mg dose of oritavancin for treatment of acute bacterial skin and skin structure infections.

Assessment by Time-Kill Methodology of the Synergistic Effects of Oritavancin in Combination with Other Antimicrobial Agents against Staphylococcus aureus

ABSTRACT Oritavancin is a semisynthetic lipoglycopeptide in clinical development for serious gram-positive infections. This study describes the synergistic activity of oritavancin in combination with gentamicin, linezolid, moxifloxacin, or rifampin in time-kill studies against methicillin-susceptible, vancomycin-intermediate, and vancomycin-resistant Staphylococcus aureus.

Ultrastructural Effects of Oritavancin on Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococcus

ABSTRACT The ultrastructural effects of the lipoglycopeptide oritavancin on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) were examined by transmission electron microscopy. Oritavancin but not vancomycin induced aberrant septum formation and loss of staining of nascent septal cross walls in MRSA. Septal distortions were also observed in VRE exposed to oritavancin.

Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in Staphylococcus aureus

Bacteriophages have evolved specific mechanisms that redirect bacterial metabolic pathways to the bacteriophage reproduction cycle. In this study, we characterized the bactericidal mechanism of two polypeptides from bacteriophages Twort and G1 that target the DNA sliding clamp of Staphylococcus aureus. The DNA sliding clamp, which tethers DNA polymerase to its template and thereby confers processivity upon the enzyme, was found to be essential for the viability of S. aureus. Expression of polypeptides TwortORF168 and G1ORF240 in S. aureus selectively inhibited DNA replication which in turn resulted in cell death. Both polypeptides specifically inhibited the S. aureus DNA replicase that was reconstituted in vitro but not the corresponding replicase of Streptococcus pyogenes. We demonstrated that inhibition of DNA synthesis is multifaceted and occurs via binding the DNA sliding clamp: TwortORF168 and G1ORF240 bound tightly to the DNA sliding clamp and prevented both its loading onto DNA and its interaction with DNA polymerase C. These results elucidate the impact of bacteriophage polypeptide expression upon DNA replication in the growing cell.

Assessment of Oritavancin Serum Protein Binding across Species

ABSTRACT Biophysical methods to study the binding of oritavancin, a lipoglycopeptide, to serum protein are confounded by nonspecific drug adsorption to labware surfaces. We assessed oritavancin binding to serum from mouse, rat, dog, and human by a microbiological growth-based method under conditions that allow near-quantitative drug recovery. Protein binding was similar across species, ranging from 81.9% in human serum to 87.1% in dog serum. These estimates support the translation of oritavancin exposure from nonclinical studies to humans.

Comparative in vitro activity of oritavancin and other agents against methicillin-susceptible and methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) infections constitute a threat to the public health due to their prevalence and associated mortality and morbidity. Several agents have been recently approved to treat MRSA skin infections including lipoglycopeptides (dalbavancin, oritavancin, and telavancin), ceftaroline, and tedizolid. This study compared the MIC, minimum bactericidal concentration (MBC), and time-kill of these agents alongside daptomycin, linezolid, and vancomycin against MRSA (n=15); meropenem, cefazolin, and nafcillin were also included against methicillin-susceptible S. aureus (MSSA [n=12]). MIC and MBC testing was conducted in accordance with Clinical and Laboratory Standards Institute guidelines, and time-kills were evaluated at multiples of the MIC and the free-drug maximum plasma concentration (fCmax) at both standard and high inoculum densities for a subset of MRSA (n=2) and MSSA (n=2). MRSA and MSSA were highly susceptible to all agents, with the lipoglycopeptides having the most potent activity by MIC50/90. All agents excluding tedizolid and linezolid were bactericidal by MBC for MRSA and MSSA, though dalbavancin and telavancin exhibited strain-specific bactericidal activity for MRSA. All agents excluding tedizolid and linezolid were bactericidal by time-kill at their respective fCmax against MRSA and MSSA at standard inoculum density, though oritavancin exhibited the most rapid bactericidal activity. Oritavancin and daptomycin at their respective fCmax maintained similar kill curves at high inoculum density. In contrast, the killing observed with other agents was typically reduced or slowed at high inoculum density. These data demonstrate the rapid bactericidal activity of oritavancin and daptomycin against S. aureus relative to other MRSA agents regardless of bacterial burden.