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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 (fC max ) 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 MIC 50/90 . 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 fC max against MRSA and MSSA at standard inoculum density, though oritavancin exhibited the most rapid bactericidal activity. Oritavancin and daptomycin at their respective fC max 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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In this study, the impact of inoculum density on the growth inhibitory and killing activities of oritavancin and comparators (vancomycin, daptomycin and linezolid) in vitro against four Staphylococcus aureus strains at clinically relevant drug concentrations was studied. Broth microdilution and time-kill assays were performed using a standard inoculum [ca. 10(5)colony-forming units (CFU)/mL as per Clinical and Laboratory Standards Institute (CLSI) guidelines] and a high inoculum (ca. 10(7)CFU/mL). Whereas minimal inhibitory concentrations (MICs) of comparators were 2-8-fold higher when tested at high inoculum, oritavancin MICs were 16-fold higher for all strains at the high inoculum relative to the standard inoculum. However, in time-kill assays, when tested at its fC(min) [trough concentration of free (non-protein-bound) drug] and fC(max) (peak concentration of non-protein-bound drug), oritavancin retained its bactericidal activity against a vancomycin-susceptible, meticillin-susceptible S. aureus (VS-MSSA) strain and a vancomycin-susceptible, meticillin-resistant S. aureus (VS-MRSA) strain both at standard and high inocula. At its fC(max), oritavancin was bactericidal at standard inoculum but not at high inoculum against two vancomycin-intermediate S. aureus (VISA) strains. Against both VISA strains at standard inoculum, oritavancin at its fC(min) reduced cell density by between 2 and 3 log (bacteriostatic), predicting that it will retain activity against certain VISA infections. However, oritavancin had no substantial growth inhibitory effect against either VISA strain at high inoculum, suggesting that in rare VISA infections with an anticipated high bacterial burden such as endocarditis, alternative oritavancin dosing strategies, including combinations with other agents, may be explored., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

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.

Human serum albumin (HSA) did not affect oritavancin MICs against non-vancomycin-intermediate Staphylococcus aureus (non-VISA) strains. In time-kill assays, oritavancin bactericidal activity in the presence of HSA was significantly more rapid than comparators against non-VISA strains. HSA increased oritavancin MICs by 4-fold for VISA strains, reflective of reduced oritavancin activity in time-kill assays with HSA.

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.

The primary sigma factor of Staphylococcus aureus, sigma(SA), regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with sigma(SA) both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of sigma(SA) that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-sigma factor may occlude the -35 element recognition domain of sigma(SA). As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of sigma(SA) to DNA and sigma(SA)-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.

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 mug/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.

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.

A 9-laboratory M23-A2 quality control (QC) study was performed to evaluate reproducibility of oritavancin MICs against reference strains of Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae using broth microdilution assays in the presence of polysorbate 80. Polysorbate 80 has previously been shown to be required for accurate measurement of oritavancin broth microdilution MICs. Greater than 95% of replicate results (n = 270/organism) fell within the following QC ranges (in micrograms per milliliter): S. aureus ATCC 29213, 0.015 to 0.12; E. faecalis ATCC 29212, 0.008 to 0.03; and S. pneumoniae ATCC 49619, 0.001 to 0.004. Oritavancin MIC QC ranges were, thus, narrow and reproducible. Parameters affecting testing results in the presence of polysorbate 80 were also evaluated. Oritavancin MICs were equivalent to or within 1 doubling dilution of those obtained under standard Clinical and Laboratory Standards Institute testing conditions, regardless of incubation time (18, 24, or 48 h), Ca(2+) concentration, pH, or frozen panel storage time (up to 6 months).

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.

The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibacterial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics approach in providing tools to inhibit the bacterial RNA polymerase.

Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

Purpose: To assess the in vitro activity of ceftaroline and a panel of comparator agents against isolates of Gram-positive bacteria, including Staphylococcus aureus, Streptococcus pneumoniae, β-hemolytic streptococci, and coagulase-negative staphylococci (CoNS) from blood collected in Africa and Middle East (AfME), Asia Pacific (APAC), Europe, Latin America (LATAM), and North America from 2017 to 2020 as a part of the Antimicrobial Testing Leadership and Surveillance (ATLAS) program.Methods: Susceptibility and minimum inhibitory concentration were determined using broth microdilution for all antimicrobial agents by a central reference laboratory according to the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines.Results: Ceftaroline showed good activity (susceptibility ≥ 89.8%, MIC90 0.008– 2 mg/L) against all Gram-positive isolates tested. All isolates of methicillin-susceptible S. aureus, penicillin-susceptible S. pneumoniae, S. agalactiae, S. dysgalactiae, and S. pyogenes were susceptible to ceftaroline (MIC90 0.008– 0.25 mg/L). Ceftaroline susceptibility for MRSA isolates was 89.8% globally (MIC90 2 mg/L). Among the comparator agents, all isolates were susceptible to vancomycin, except S. epidermis (susceptibility, 99.9%). Among other agents, daptomycin, linezolid, and tigecycline showed potent activity (susceptibility ≥ 97.9%, MIC90 0.03– 2 mg/L) against all isolates tested.Conclusion: Ceftaroline showed potent in vitro activity against global bloodstream isolates of Gram-positive bacteria collected between 2017 and 2020. Monitoring and surveillance of global as well as regional longitudinal trends of resistance rates among Gram-positive isolates causing bloodstream infections are important to limit the spread of AMR, establish stewardship measures, and manage and appropriately treat infections. [ABSTRACT FROM AUTHOR]