The Engineered Lysin, CF-370, is Active Against Antibiotic-Resistant Gram-Negative Pathogens In vitro and Synergizes with Meropenem in Experimental Pseudomonas aeruginosa Pneumonia.

BACKGROUND: Lysins (cell wall hydrolases) targeting Gram-negative organisms require engineering to permeabilize the outer membrane and access subjacent peptidoglycan to facilitate killing. In the current study, the potential clinical utility for engineered lysin, CF-370, was examined in vitro and in vivo against Gram-negative pathogens important in human infections. METHODS: MICs and bactericidal activity were determined using standard methods. An in vivo proof-of-concept efficacy study was conducted using a rabbit acute pneumonia model caused by Pseudomonas aeruginosa. RESULTS: CF-370 exhibited potent antimicrobial activity, with MIC50/90 values (in µg/mL) for: P. aeruginosa, 1/2; Acinetobacter baumannii, 1/1; Escherichia coli, 0.25/1; Klebsiella pneumoniae, 2/4; Enterobacter cloacae 1/4; and Stenotrophomonas maltophilia 2/8. CF-370 furthermore demonstrated: i) bactericidal activity; (ii) activity in serum; iii) a low propensity for resistance; iv) anti-biofilm activity; and v) synergy with antibiotics. In the pneumonia model, CF-370 alone decreased bacterial densities in lungs, kidneys and spleen vs. vehicle control, and demonstrated significantly increased efficacy when combined with meropenem (vs either agent alone). CONCLUSIONS: CF-370 is the first engineered lysin described with potent broad spectrum in vitro activity against multiple clinically-relevant Gram-negative pathogens, as well as potent in vivo efficacy in an animal model of severe invasive multi-system infection.

Exebacase in Addition to Standard-of-Care Antibiotics for Staphylococcus aureus Bloodstream Infections and Right-Sided Infective Endocarditis: A Phase 3, Superiority-Design, Placebo-Controlled, Randomized Clinical Trial (DISRUPT).

BACKGROUND: Novel treatments are needed for Staphylococcus aureus bacteremia, particularly for methicillin-resistant S. aureus (MRSA). Exebacase is a first-in-class antistaphylococcal lysin that is rapidly bactericidal and synergizes with antibiotics. METHODS: In Direct Lysis of Staph Aureus Resistant Pathogen Trial of Exebacase (DISRUPT), a superiority-design phase 3 study, patients with S. aureus bacteremia/endocarditis were randomly assigned to receive a single dose of intravenous exebacase or placebo in addition to standard-of-care antibiotics. The primary efficacy outcome was clinical response at day 14 in the MRSA population. RESULTS: A total of 259 patients were randomized before the study was stopped for futility based on the recommendation of the unblinded Data Safety Monitoring Board. Clinical response rates at day 14 in the MRSA population (n = 97) were 50.0% (exebacase + antibiotics; 32/64) versus 60.6% (antibiotics alone; 20/33) (P = .392). Overall, rates of adverse events were similar across groups. No adverse events of hypersensitivity related to exebacase were reported. CONCLUSIONS: Exebacase + antibiotics failed to improve clinical response at day 14 in patients with MRSA bacteremia/endocarditis. This result was unexpected based on phase 2 data that established proof-of-concept for exebacase + antibiotics in patients with MRSA bacteremia/endocarditis. In the antibiotics-alone group, the clinical response rate was higher than that seen in phase 2. Heterogeneity within the study population and a relatively small sample size in either the phase 2 or phase 3 studies may have increased the probability of imbalances in the multiple components of day 14 clinical outcome. This study provides lessons for future superiority studies in S. aureus bacteremia/endocarditis. Clinical Trials Registration.NCT04160468.

Activity of Exebacase (CF-301) against Biofilms Formed by Staphylococcus epidermidis Strains Isolated from Prosthetic Joint Infections.

Staphylococcus epidermidis is one of the main pathogens responsible for bone and joint infections, especially those involving prosthetic materials, due to its ability to form biofilms. In these cases, biofilm formation, combined with increased antimicrobial resistance, often results in therapeutic failures. In this context, the development of innovative therapies active against S. epidermidis is a priority. The aim of this study was to evaluate the in vitro activity of the lysin exebacase (CF-301) against biofilms formed by 19 S. epidermidis clinical strains isolated from prosthetic joint infections (PJI). We determined the biomass and the remaining viable bacteria inside biofilms after 24 h of exposure to exebacase. Exebacase activity was compared to that of rifampicin, vancomycin, and daptomycin. The use of exebacase in addition to antibiotics was also assessed. Exebacase displayed (i) a significant anti-biomass activity on S. epidermidis biofilms at concentrations ≥5 mg/L (mean decrease up to 66% at 150 mg/L), (ii) significant bactericidal activity on biofilms at concentrations ≥50 mg/L (mean decrease up to 1.7 log CFU at 150 mg/L), (iii) synergistic effects when used in addition to rifampicin, vancomycin, or daptomycin. The extent of these activities varied by isolate. Exebacase can be considered a promising therapy in addition to rifampicin, vancomycin, or daptomycin in the context of PJI. Further in vitro studies are needed to understand its mechanism of action on S. epidermidis biofilms and in vivo investigations are required to confirm these data.

Exebacase Is Active In Vitro in Pulmonary Surfactant and Is Efficacious Alone and Synergistic with Daptomycin in a Mouse Model of Lethal Staphylococcus aureus Lung Infection.

Exebacase (CF-301) is a novel antistaphylococcal lysin (cell wall hydrolase) in phase 3 of clinical development for the treatment of Staphylococcus aureus bacteremia, including right-sided endocarditis, used in addition to standard-of-care antibiotics. In the current study, the potential for exebacase to treat S. aureus pneumonia was explored in vitro using bovine pulmonary surfactant (Survanta) and in vivo using a lethal murine pneumonia model. Exebacase was active against a set of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains, with an MIC90 of 2 µg/ml (n = 18 strains), in the presence of a surfactant concentration (7.5%) inhibitory to the antistaphylococcal antibiotic daptomycin, which is inactive in pulmonary environments due to specific inhibition by surfactant. In a rigorous test of the ability of exebacase to synergize with antistaphylococcal antibiotics, exebacase synergized with daptomycin in the presence of surfactant in vitro, resulting in daptomycin MIC reductions of up to 64-fold against 9 MRSA and 9 MSSA strains. Exebacase was also observed to facilitate the binding of daptomycin to S. aureus and the elimination of biofilm-like structures formed in the presence of surfactant. Exebacase (5 mg/kg of body weight 1 time every 24 h [q24h], administered intravenously for 3 days) was efficacious in a murine model of staphylococcal pneumonia, resulting in 50% survival, compared to 0% survival with the vehicle control; exebacase in addition to daptomycin (50 mg/kg q24h for 3 days) resulted in 70% survival, compared to 0% survival in the daptomycin-alone control group. Overall, exebacase is active in pulmonary environments and may be appropriate for development as a treatment for staphylococcal pneumonia.

Development of a Broth Microdilution Method for Exebacase Susceptibility Testing.

Exebacase (CF-301) belongs to a new class of protein-based antibacterial agents, known as lysins (peptidoglycan hydrolases). Exebacase, a novel lysin with antistaphylococcal activity, is in phase 3 of clinical development. To advance into the clinic, it was necessary to develop an accurate and reproducible method for exebacase MIC determination. The Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) method using cation-adjusted Mueller-Hinton broth (CAMHB) produced trailing MIC endpoints, and exebacase activity was diminished when frozen BMD panels were used. A modified BMD method was developed using CAMHB supplemented with 25% horse serum and 0.5 mM dl-dithiothreitol (CAMHB-HSD). Preliminary quality control (QC) ranges for Staphylococcus aureus ATCC 29213 of 0.25 to 1 µg/ml and for Enterococcus faecalis ATCC 29212 of 16 to 64 µg/ml were determined based on the results of a CLSI M23-defined MIC QC tier 1 study. These preliminary QC ranges validated the MIC data generated from a systematic study testing a discrete S. aureus strain collection using CAMHB-HSD to investigate the impact of parameters known to influence susceptibility test results and to evaluate the exebacase MIC distribution against clinical S. aureus isolates. Presentation of these data led to the CLSI Subcommittee on Antimicrobial Susceptibility Testing (AST) approval of the use of CAMHB-HSD to determine exebacase susceptibility and commencement of a multilaboratory (tier 2) QC study. Use of a standard BMD method and concomitant QC testing provides confidence in the assessment of test performance to generate accurate and reproducible susceptibility data during antibacterial drug development.

Exebacase in Addition to Daptomycin against MRSA.

Exebacase is a lysin (cell wall hydrolase) with direct lytic activity against Staphylococcus aureus including methicillin-resistant S. aureus (MRSA). Time-kill analysis experiments illustrated bactericidal activity of exebacase-daptomycin against MRSA strains MW2 and 494. Furthermore, exebacase in addition to daptomycin (10, 6, and 4 mg/kg/day) in a two-compartment ex vivo pharmacokinetic/pharmacodynamic simulated endocardial vegetation model with humanized doses resulted in reductions of 6.01, 4.99, and 2.81 log10 CFU/g (from initial inoculum) against MRSA strain MW2.

Determination of MIC Quality Control Parameters for Exebacase, a Novel Lysin with Antistaphylococcal Activity.

Exebacase (CF-301), a novel, antistaphylococcal lysin (cell wall hydrolase) is the first agent of this class to enter late-stage clinical development (phase 3, NCT04160468) for the treatment of Staphylococcus aureus bacteremia, including right-sided endocarditis. A multilaboratory Clinical and Laboratory Standards Institute (CLSI) M23-defined tier 2 quality control (QC) study was conducted to establish exebacase QC ranges for a new reference broth microdilution method. S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were selected as reference QC strains. Broth microdilution MIC QC ranges for exebacase spanned 4 log2 dilutions and contained 99.2% of the MIC results generated for the two reference strains. The QC ranges for exebacase were defined as 0.25 to 2 µg/ml and 8 to 64 µg/ml against S. aureus ATCC 29213 and E. faecalis ATCC 29212, respectively, and were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing. These QC ranges established for use with the reference broth microdilution method developed for exebacase susceptibility testing will ensure the test performance and accuracy of results generated during clinical development.

Efficacy assessment of lysin CF-296 in addition to daptomycin or vancomycin against Staphylococcus aureus in the murine thigh infection model.

OBJECTIVES: CF-296 is a lysin in pre-clinical development for the treatment of MSSA and MRSA infections, used in addition to standard-of-care (SOC) antibiotics. We evaluated the efficacy of CF-296 alone and in addition to daptomycin or vancomycin against Staphylococcus aureus in the neutropenic mouse thigh infection model. METHODS: Eight isolates (one MSSA and seven MRSA) were studied. Mice were administered five CF-296 monotherapy doses ranging from 0.5 to 50 mg/kg intravenously. To assess adjunctive therapy, mice received sub-therapeutic daptomycin alone, sub-therapeutic vancomycin alone, or the five CF-296 doses in addition to either daptomycin or vancomycin. RESULTS: Relative to starting inoculum (5.80 ± 0.31 log10 cfu/thigh), bacterial density in vehicle controls increased by +2.49 ± 0.98 across all eight strains. Relative to 24 h controls, a dose-response in bacterial killing (range -0.22 ± 0.87 to -2.01 ± 1.71 log10 cfu/thigh) was observed with increasing CF-296 monotherapy against the eight isolates. Daptomycin and vancomycin resulted in -1.36 ± 0.77 and -1.37 ± 1.01 log10 cfu/thigh bacteria reduction, respectively, relative to 24 h controls. Escalating CF-296 exposures (0.5-50 mg/kg) in addition to daptomycin resulted in an enhanced dose-response, ranging from bacterial killing of -0.69 to -2.13 log10 cfu/thigh, relative to daptomycin alone. Similarly, in addition to vancomycin, escalating CF-296 exposures resulted in bacterial reduction ranging from -1.37 to -2.29 log10 cfu/thigh, relative to vancomycin alone. CONCLUSIONS: Relative to SOC antibiotics (daptomycin or vancomycin), addition of CF-296 resulted in robust and enhanced antibacterial dose-response, achieving ≥1 log10 cfu/thigh decrease across most doses, highlighting a potential role for CF-296 adjunctive therapy against MSSA and MRSA isolates.

Exebacase Demonstrates In Vitro Synergy with a Broad Range of Antibiotics against both Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus.

In vitro synergy between an antimicrobial protein lysin (cell wall hydrolase) called exebacase and each of 12 different antibiotics was examined against Staphylococcus aureus isolates using a nonstandard medium approved for exebacase susceptibility testing by the Clinical and Laboratory Standards Institute. In the checkerboard assay format, fractional inhibitory concentration index values of ≤0.5, consistent with synergy, were observed for the majority of interactions tested. Synergy was further confirmed in time-kill assays.

Synergistic Activity of Exebacase (CF-301) in Addition to Daptomycin against Staphylococcus aureus in a Neutropenic Murine Thigh Infection Model.

We evaluated the efficacy of escalating doses of exebacase administered with subtherapeutic daptomycin exposures against 8 Staphylococcus aureus isolates in a neutropenic murine thigh infection model. Daptomycin alone resulted in mean growth of 0.39 ± 1.19 log10 CFU/thigh. When administered with daptomycin, exebacase resulted in a mean log10 CFU/thigh reduction of -1.03 ± 0.72 (range, -0.77 ± 0.98 to -1.20 ± 0.59) across evaluated doses (15 to 90 mg/kg), indicative of potential in vivo synergy.

Effect of the Lysin Exebacase on Cardiac Vegetation Progression in a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Endocarditis as Determined by Echocardiography.

Methicillin-resistant Staphylococcus aureus (MRSA) poses significant therapeutic challenges related to its frequency in clinical infections, innate virulence properties, and propensity for multiantibiotic resistance. MRSA is among the most common causes of endovascular infections, including infective endocarditis (IE). Our objective was to employ transthoracic echocardiography (TTE) to evaluate the effect of exebacase, a novel direct lytic agent, in experimental aortic valve MRSA IE. TTE was utilized to evaluate the in vivo effect of exebacase on MRSA-infected vegetation progression when combined with daptomycin (versus daptomycin alone). Primary intravegetation outcomes were maximum size, weights at sacrifice, and MRSA counts at infection baseline versus after 4 days of daptomycin treatment (alone or in addition to exebacase administered once on treatment day 1). A single dose of exebacase in addition to daptomycin cleared significantly more intravegetation MRSA than daptomycin alone. This was associated with a statistical trend toward reduced maximum vegetation size in the exebacase plus daptomycin versus the daptomycin alone therapy groups (P = 0.07). Also, mean vegetation weights in the exebacase-treated group were significantly lower than those of the daptomycin alone group (P < 0.0001). Maximum vegetation size by TTE correlated with vegetation weight (P = 0.005). In addition, intravegetation MRSA counts in the combination group were significantly lower than those of untreated controls (P < 0.0001) and the daptomycin alone group (P < 0.0001). This study suggests that exebacase has a salutary impact on MRSA-infected vegetation progression when combined with daptomycin, especially in terms of vegetation MRSA burden, size, and weight. Moreover, TTE appears to be an efficient noninvasive tool to assess therapeutic efficacies in experimental MRSA IE.

Postantibiotic and Sub-MIC Effects of Exebacase (Lysin CF-301) Enhance Antimicrobial Activity against Staphylococcus aureus.

CF-301 (exebacase) is a recombinantly produced bacteriophage-derived lysin (cell wall hydrolase) and is the first agent of this class to enter clinical development in the United States for treating bacteremia including endocarditis due to Staphylococcus aureus Whereas rapid bactericidal activity is the hallmark in vitro and in vivo response to CF-301 at exposures higher than the MIC, prolonged antimicrobial activity, mediated by cell wall damage, is predicted at concentrations less than the MIC. In the current study, a series of in vitro pharmacodynamic parameters, including the postantibiotic effect (PAE), postantibiotic sub-MIC effect (PA-SME), and sub-MIC effect (SME), were studied to determine how short-duration and sub-MIC CF-301 exposures affect the growth of surviving staphylococci and extend its antimicrobial activity. Mean PAE, PA-SME, and SME values up to 4.8, 9.3, and 9.8 h, respectively, were observed against 14 staphylococcal strains tested in human serum; growth delays were extended by 6 h in the presence of daptomycin. Exposures to CF-301 at sub-MIC levels as low as 0.001× to 0.01× MIC (∼1 to 10 ng/ml) resulted in aberrant cell wall ultrastructure, increased membrane permeability, dissipation of membrane potential, and inhibition of virulence phenotypes, including agglutination and biofilm formation. A mouse thigh infection model designed to study the PAE was used to confirm our findings and demonstrate in vivo growth delays of ≥19.3 h. Our findings suggest that at CF-301 concentrations less than the MIC during therapeutic use, sustained reductions in bacterial fitness and virulence may substantially enhance efficacy.

Lysocins: Bioengineered Antimicrobials That Deliver Lysins across the Outer Membrane of Gram-Negative Bacteria.

The prevalence of multidrug-resistant Pseudomonas aeruginosa has stimulated development of alternative therapeutics. Bacteriophage peptidoglycan hydrolases, termed lysins, represent an emerging antimicrobial option for targeting Gram-positive bacteria. However, lysins against Gram-negatives are generally deterred by the outer membrane and their inability to work in serum. One solution involves exploiting evolved delivery systems used by colicin-like bacteriocins (e.g., S-type pyocins of P. aeruginosa) to translocate through the outer membrane. Following surface receptor binding, colicin-like bacteriocins form Tol- or TonB-dependent translocons to actively import bactericidal domains through outer membrane protein channels. With this understanding, we developed lysocins, which are bioengineered lysin-bacteriocin fusion molecules capable of periplasmic import. In our proof-of-concept studies, components from the P. aeruginosa bacteriocin pyocin S2 (PyS2) responsible for surface receptor binding and outer membrane translocation were fused to the GN4 lysin to generate the PyS2-GN4 lysocin. PyS2-GN4 delivered the GN4 lysin to the periplasm to induce peptidoglycan cleavage and log-fold killing of P. aeruginosa with minimal endotoxin release. While displaying narrow-spectrum antipseudomonal activity in human serum, PyS2-GN4 also efficiently disrupted biofilms, outperformed standard-of-care antibiotics, exhibited no cytotoxicity toward eukaryotic cells, and protected mice from P. aeruginosa challenge in a bacteremia model. In addition to targeting P. aeruginosa, lysocins can be constructed to target other prominent Gram-negative bacterial pathogens.

Antimicrobial Activity of Exebacase (Lysin CF-301) against the Most Common Causes of Infective Endocarditis.

Exebacase, a recombinantly produced lysin (cell wall hydrolase), and comparator antibiotics were tested by the broth microdilution method against strain sets of Staphylococcus and Streptococcus spp., which are the most common causes of infective endocarditis in humans. Exebacase was active against all Staphylococcus spp. tested, including S. aureus and coagulase-negative staphylococci (MIC50/90, 0.5/1 µg/ml). Activity against Streptococcus spp. was variable, with S. pyogenes, S. agalactiae, and S. dysgalactiae (MIC50/90, 1/2 µg/ml) among the most susceptible.

The Antistaphylococcal Lysin, CF-301, Activates Key Host Factors in Human Blood To Potentiate Methicillin-Resistant Staphylococcus aureus Bacteriolysis.

Bacteriophage-derived lysins are cell-wall-hydrolytic enzymes that represent a potential new class of antibacterial therapeutics in development to address burgeoning antimicrobial resistance. CF-301, the lead compound in this class, is in clinical development as an adjunctive treatment to potentially improve clinical cure rates of Staphylococcus aureus bacteremia and infective endocarditis (IE) when used in addition to antibiotics. In order to profile the activity of CF-301 in a clinically relevant milieu, we assessed its in vitro activity in human blood versus in a conventional testing medium (cation-adjusted Mueller-Hinton broth [caMHB]). CF-301 exhibited substantially greater potency (32 to ≥100-fold) in human blood versus caMHB in three standard microbiologic testing formats (e.g., broth dilution MICs, checkerboard synergy, and time-kill assays). We demonstrated that CF-301 acted synergistically with two key human blood factors, human serum lysozyme (HuLYZ) and human serum albumin (HSA), which normally have no nascent antistaphylococcal activity, against a prototypic methicillin-resistant S. aureus (MRSA) strain (MW2). Similar in vitro enhancement of CF-301 activity was also observed in rabbit, horse, and dog (but not rat or mouse) blood. Two well-established MRSA IE models in rabbit and rat were used to validate these findings in vivo by demonstrating comparable synergistic efficacy with standard-of-care anti-MRSA antibiotics at >100-fold lower lysin doses in the rabbit than in the rat model. The unique properties of CF-301 that enable bactericidal potentiation of antimicrobial activity via activation of "latent" host factors in human blood may have important therapeutic implications for durable improvements in clinical outcomes of serious antibiotic-resistant staphylococcal infections.

Exebacase in Addition to Daptomycin Is More Active than Daptomycin or Exebacase Alone in Methicillin-Resistant Staphylococcus aureus Osteomyelitis in Rats.

Bacteriophage-derived lysins are being developed as anti-infective agents. In an acute osteomyelitis methicillin-resistant Staphylococcus aureus (MRSA) model, rats receiving no treatment or treatment with daptomycin, exebacase (CF-301), or daptomycin plus exebacase had means of 5.13, 4.09, 4.65, and 3.57 log10 CFU/gram of bone, respectively. All treated animals had fewer bacteria than did untreated animals (P ≤ 0.0001), with daptomycin plus exebacase being more active than daptomycin (P = 0.0042) or exebacase (P < 0.001) alone.

The PlyB Endolysin of Bacteriophage vB_BanS_Bcp1 Exhibits Broad-Spectrum Bactericidal Activity against Bacillus cereus Sensu Lato Isolates.

Lytic bacteriophages (or phages) drive bacterial mortality by elaborating exquisite abilities to bind, breach, and destroy bacterial cell membranes and subjugate critical bacterial cell functions. These antimicrobial activities make phages ideal candidates to serve as, or provide sources of, biological control measures for bacterial pathogens. In this study, we isolated the Myoviridae phage vB_BanS_Bcp1 (here referred to as Bcp1) from landfill soil, using a Bacillus anthracis host. The antimicrobial activities of both Bcp1 and its encoded endolysin, PlyB, were examined across different B. cereussensu lato group species, including B. cereussensu stricto, Bacillus thuringiensis, and Bacillus anthracis, with pathogenic potential in humans and multiple different uses in biotechnological applications. The Bcp1 phage infected only a subset (11 to 66%) of each B. cereussensu lato species group tested. In contrast, functional analysis of purified PlyB revealed a potent bacteriolytic activity against all B. cereussensu lato isolates tested (n = 79). PlyB was, furthermore, active across broad temperature, pH, and salt ranges, refractory to the development of resistance, bactericidal as a single agent, and synergistic with a second endolysin, PlyG. To confirm the potential for PlyB as an antimicrobial agent, we demonstrated the efficacy of a single intravenous treatment with PlyB alone or combination with PlyG in a murine model of lethal B. anthracis infection. Overall, our findings show exciting potential for the Bcp1 bacteriophage and the PlyB endolysin as potential new additions to the antimicrobial armamentarium.IMPORTANCE Organisms of the Bacillus cereussensu lato lineage are ubiquitous in the environment and are responsible for toxin-mediated infections ranging from severe food poisoning (B. cereussensu stricto) to anthrax (Bacillus anthracis). The increasing incidence of many of these infections, combined with the specter of antibiotic resistance, has created a need for novel antimicrobials with potent activity, including bacteriophages (or phages) and phage-encoded products (i.e., endolysins). In this study, we describe a broadly infective phage, Bcp1, and its encoded endolysin, PlyB, which exhibited a rapidly bacteriolytic effect against all B. cereussensu lato isolates tested with no evidence of evolving resistance. Importantly, PlyB was highly efficacious in a mouse model of lethal bacteremia with B. anthracis Both the Bcp1 phage and the PlyB endolysin represent novel mechanisms of action compared to antibiotics, with potential applications to address the evolving problem of antimicrobial resistance.

Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent.

Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of ≤0.25 µg/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 µg/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.