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.

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.

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.

CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests.

Effective evaluations of antimicrobial susceptibility tests (ASTs) require robust study design. The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing has recognized that many published studies reporting the performance of commercial ASTs (cASTs) suffer from major design and/or analysis flaws, rendering the results difficult or impossible to interpret. This minireview outlines the current consensus of the Methods Development and Standardization Working Group of the CLSI Subcommittee on Antimicrobial Susceptibility Testing regarding best practices for systematic evaluation of the performance of an AST, including the analysis and presentation of essential data intended for publication.

Ceftaroline efficacy against high-MIC clinical Staphylococcus aureus isolates in an in vitro hollow-fibre infection model.

Objectives: The current CLSI and EUCAST clinical susceptible breakpoint for 600 mg q12h dosing of ceftaroline (active metabolite of ceftaroline fosamil) for Staphylococcus aureus is ≤1 mg/L. Efficacy data for S. aureus infections with ceftaroline MIC ≥2 mg/L are limited. This study was designed to generate in-depth pharmacokinetic/pharmacodynamics (PK/PD) understanding of S. aureus isolates inhibited by ≥ 2 mg/L ceftaroline using an in vitro hollow-fibre infection model (HFIM). Methods: The PK/PD target of ceftaroline was investigated against 12 diverse characterized clinical MRSA isolates with ceftaroline MICs of 2 or 4 mg/L using q8h dosing for 24 h. These isolates carried substitutions in the penicillin-binding domain (PBD) and/or the non-PBD. Additionally, PD responses of mutants with ceftaroline MICs ranging from 2 to 32 mg/L were evaluated against the mean 600 mg q8h human-simulated dose over 72 h. Results: The mean stasis, 1 log10-kill and 2 log10-kill PK/PD targets were 29%, 32% and 35% f T>MIC, respectively. In addition, these data suggest that the PK/PD target for MRSA is not impacted by the presence of substitutions in the non-PBD commonly found in isolates with ceftaroline MIC values of ≤ 2 mg/L. HFIM studies with 600 mg q8h dosing demonstrated a sustained long-term bacterial suppression for isolates with ceftaroline MICs of 2 and 4 mg/L. Conclusions: Overall, efficacy was demonstrated against a diverse collection of clinical isolates using HFIM indicating the utility of 600 mg ceftaroline fosamil for S. aureus isolates with MIC ≤4 mg/L using q8h dosing.

In Vitro Activity of Ceftaroline against Staphylococcus aureus Isolated in 2012 from Asia-Pacific Countries as Part of the AWARE Surveillance Program.

Ceftaroline, the active metabolite of the prodrug ceftaroline-fosamil, is an advanced-generation cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA). This investigation provides in vitro susceptibility data for ceftaroline against 1,971 S. aureus isolates collected in 2012 from seven countries (26 centers) in the Asia-Pacific region as part of the Assessing Worldwide Antimicrobial Resistance and Evaluation (AWARE) program. Broth microdilution as recommended by the CLSI was used to determine susceptibility. In all, 62% of the isolates studied were MRSA, and the ceftaroline MIC90 for all S. aureus isolates was 2 µg/ml (interpretive criteria: susceptible, ≤1 µg/ml). The overall ceftaroline susceptibility rate for S. aureus was 86.9%, with 100% of methicillin-sensitive S. aureus isolates and 78.8% of MRSA isolates susceptible to this agent. The highest percentages of ceftaroline-nonsusceptible MRSA isolates came from China (47.6%), all of which showed intermediate susceptibility, and Thailand (37.1%), where over half (52.8%) of isolates were resistant to ceftaroline (MIC, 4 µg/ml). Thirty-eight ceftaroline-nonsusceptible isolates (MIC values of 2 to 4 µg/ml) were selected for molecular characterization. Among the isolates analyzed, sequence type 5 (ST-5) was the most common sequence type encountered; however, all isolates analyzed from Thailand were ST-228. Penicillin-binding protein 2a (PBP2a) substitution patterns varied by country, but all isolates from Thailand had the Glu239Lys substitution, and 12 of these also carried an additional Glu447Lys substitution. Ceftaroline-fosamil is a useful addition to the antimicrobial agents that can be used to treat S. aureus infections. However, with the capability of this species to develop resistance to new agents, it is important to recognize and monitor regional differences in trends as they emerge.

In Vitro Activity of Ceftaroline against Staphylococcus aureus Isolates Collected in 2012 from Latin American Countries as Part of the AWARE Surveillance Program.

The in vitro activities of ceftaroline and comparators, using broth microdilution, were determined against 1,066 Staphylococcus aureus isolates from hospitalized patients. Seventeen medical centers from Latin American countries contributed isolates. Methicillin-resistant S. aureus (MRSA) percentages ranged from 46% (Brazil) to 62% (Argentina). All methicillin-susceptible S. aureus (MSSA) isolates were susceptible to ceftaroline. Ceftaroline activity against MRSA varied with MIC90s of 0.5 (Venezuela) to 2 (Brazil, Chile, and Colombia) µg/ml, which was the highest MIC value. ST-5 was the most common sequence type.

Molecular characterization of MRSA isolates bracketing the current EUCAST ceftaroline-susceptible breakpoint for Staphylococcus aureus: the role of PBP2a in the activity of ceftaroline.

OBJECTIVES: The objectives of this study were to characterize contemporary MRSA isolates and understand the prevalence and impact of sequence variability in PBP2a on ceftaroline susceptibility. METHODS: A total of 184 MRSA isolates collected from 28 countries were collected and characterized. RESULTS: WT PBP2a proteins were found in MRSA distributed evenly over the ceftaroline MIC range of 0.5-2 mg/L (n=56). PBP2a variations found in 124 isolates fell into two categories: (i) 12 isolates contained a substitution in the transpeptidase pocket located in the penicillin-binding domain and exhibited significantly decreased ceftaroline susceptibility (typically 8 mg/L); and (ii) isolates with substitutions in the non-penicillin-binding domain (nPBD) in a region proposed to be functionally important for cell wall biogenesis. The majority (71%) of isolates containing only nPBD variations were inhibited by 2 mg/L ceftaroline, 23% by ≤1 mg/L and 6% by 4 mg/L. These data suggest that the WT MRSA distribution extends beyond the current EUCAST and CLSI susceptible breakpoints and includes isolates inhibited by 2 mg/L ceftaroline. SCCmec type IV was the predominant type in the ceftaroline-susceptible population (68%), whereas it only represented 6% of the non-susceptible population. The variations of MLST lineages were fewer among the non-susceptible group. CONCLUSIONS: This study suggests that MRSA populations with a WT PBP2a and those with nPBD variations overlap significantly and that PBP2a sequence-independent factors contribute to ceftaroline susceptibility. Whereas characterization of isolates with a ceftaroline MIC of 2 mg/L enriched for isolates with nPBD variations, it was not a discrete population. In contrast, the rare isolates containing a substitution in the transpeptidase-binding pocket were readily differentiated.

The Lysin Exebacase Has a Low Propensity for Resistance Development in Staphylococcus aureus and Suppresses the Emergence of Resistance to Antistaphylococcal Antibiotics.

Exebacase (CF-301) belongs to a novel class of protein-based antibacterial agents, called lysins (peptidoglycan hydrolases). Exebacase exhibits potent antistaphylococcal activity and is the first lysin to initiate clinical trials in the United States. To support clinical development, the potential for resistance development to exebacase was assessed over 28 days of serial daily subculture in the presence of increasing concentrations of the lysin performed in its reference broth medium. Exebacase MICs remained unchanged over serial subculture for three replicates each of methicillin-susceptible Staphylococcus aureus (MSSA) strain ATCC 29213 and methicillin-resistant S. aureus (MRSA) strain MW2. For comparator antibiotics also tested, oxacillin MICs increased by 32-fold with ATCC 29213 and daptomycin and vancomycin MICs increased by 16- and 8-fold, respectively, with MW2. Serial passage was also used to examine the capacity of exebacase to suppress selection for increased oxacillin, daptomycin, and vancomycin MICs when used together in combination, wherein daily exposures to increasing concentrations of antibiotic were performed over 28 days with the added presence of fixed sub-MIC amounts of exebacase. Exebacase suppressed increases in antibiotic MICs over this period. These findings are consistent with a low propensity for resistance to exebacase and an added benefit of reducing the potential for development of antibiotic resistance. IMPORTANCE To guide development of an investigational new antibacterial drug, microbiological data are required to understand the potential for development of resistance to the drug in the target organism(s). Exebacase is a lysin (peptidoglycan hydrolase) that represents a novel antimicrobial modality based on degradation of the cell wall of Staphylococcus aureus. Exebacase resistance was examined here using an in vitro serial passage method that assesses the impact of daily exposures to increasing concentrations of exebacase over 28 days in medium approved for use in exebacase antimicrobial susceptibility testing (AST) by the Clinical and Laboratory Standards Institute (CLSI). No changes in susceptibility to exebacase were observed over the 28-day period for multiple replicates of two S. aureus strains, indicating a low propensity for resistance development. Interestingly, while high-level resistance to commonly used antistaphylococcal antibiotics was readily obtained using the same method, the added presence of exebacase acted to suppress antibiotic resistance development.

Rapid bacteriolysis of Staphylococcus aureus by lysin exebacase.

Lysins (peptidoglycan hydrolases) are promising new protein-based antimicrobial candidates under development to address rising antibiotic resistance encountered among pathogenic bacteria. Exebacase is an antistaphylococcal lysin and the first member of the lysin class to have entered clinical trials in the United States. In this study, the bacteriolytic activity of exebacase was characterized with time-kill assays, turbidity reduction assays, and microscopy. Three methicillin-susceptible Staphylococcus aureus and three methicillin-resistant S. aureus isolates were tested in time-kill assays over a range of concentrations from 0.25 to 8 × MIC. Exebacase demonstrated a concentration-dependent killing and showed bactericidal activity (≥3 log10 kill achieved relative to the starting inoculum) within 3 h at 1 × MIC against all strains tested. Dose-dependent lysis by exebacase was, furthermore, observed in the turbidity reduction assay, wherein decreases in initial OD600 of 50% were observed within ~15 min at concentrations as low as 4 µg/mL. Membrane dissolution, loss of cytoplasmic material, and lysis were confirmed by video and electron microscopy. The demonstrated rapid bacteriolytic effect of exebacase is an important distinguishing feature of this novel modality. IMPORTANCE To guide the development of an investigational new antibacterial entity, microbiological data are required to evaluate the killing kinetics against target organism(s). Exebacase is a lysin (peptidoglycan hydrolase) that represents a novel antimicrobial modality based on degradation of the cell wall of Staphylococcus aureus. Killing by exebacase was determined in multiple assay formats including time-kill assays, wherein reductions of viability of ≥3 log10 colony-forming units/mL were observed within 3 h for multiple different isolates tested, consistent with very rapid bactericidal activity. Rapid reductions in optical density were likewise observed in exebacase-treated cultures, which were visually consistent with microscopic observations of rapid lysis. Overall, exebacase provides a novel antimicrobial modality against S. aureus, characterized by a rapid cidal and lytic activity.

Determination of moxifloxacin anaerobic susceptibility breakpoints according to the Clinical and Laboratory Standards Institute guidelines.

A summary of the key data presented to Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards) in determination of moxifloxacin anaerobic breakpoints is presented. The breakpoint analysis required review of a variety of data, including bacteriologic and clinical outcomes by MIC of anaerobic isolates from prospective clinical trials in patients with complicated intra-abdominal infections, human and animal pharmacokinetic/pharmacodynamic (PK/PD) information and in vitro models, MIC distributions of indicated organisms, and animal model efficacy data for strains with MIC values around prospective breakpoints. The compilation of the various components of this breakpoint analysis supports the US Food and Drug Administration (FDA) and CLSI moxifloxacin anaerobic breakpoints of < or =2 mg/L (susceptible), 4 mg/L (intermediate), and > or =8 mg/L (resistant), and provides information to European investigators for interpretation of MICs prior to establishment of the European Committee on Antimicrobial Susceptibility Testing breakpoints.

Multi-center and multi-method evaluation of in vitro activities of ceftaroline against S. aureus.

This five-site study was performed to assess the reproducibility of ceftaroline MIC and disk results for Staphylococcus aureus. Three commercial broth microdilution, three gradient diffusion and ceftaroline 5µg disk diffusion methods were compared to a reference broth microdilution method against challenge isolates (n = 41) and isolates collected at four European sites (n = 30/site). For four MIC methods (Sensititre and three gradient diffusion methods), 99.0% of consolidated MIC results were within +/- 1 dilution of the reference MIC. Categorical agreement rates based on EUCAST breakpoints for the challenge isolates were 75.6-100% and for disk testing were 78.0-92.7%. There was no clear distinction between isolates with MIC results of 1 and 2mg/L with regard to variation in MIC or molecular genotyping results. The addition of an intermediate category for isolates with MIC results of 2mg/L would help to identify these isolates as borderline susceptible/non-susceptible isolates.

Fluoroquinolone resistance in Streptococcus pneumoniae: evidence that gyrA mutations arise at a lower rate and that mutation in gyrA or parC predisposes to further mutation.

Fluoroquinolones are being increasingly used for acute lower respiratory tract infection where Streptococcus pneumoniae is the most important bacterial pathogen. S. pneumoniae becomes resistant to quinolone antibiotics by mutations in a small section of the parC and gyrA genes. In this study, we investigated the mutation rates and spectrum of resistance when ciprofloxacin and gemifloxacin were the selective agents. When ciprofloxacin was the selective agent, parC mutants arose at a rate of 1.1 x 10(-9) mutations per cell division. There were two double mutants: parC + gyrA and parC + gyrB, and these mutations arose in as few as five generations. When gemifloxacin was the selective agent, all but one of the colonies growing on the x2 MIC plate had no mutations in gyrA or parC. The only mutation identified was in gyrA, and it appeared at a rate of 1.6 x 10(-11). When the gemifloxacin MIC of strains with mutations in parC was determined, there was no change from the susceptible parent. These data indicate that S. pneumoniae becomes resistant to gemifloxacin through mutation in gyrA rather than parC. Because gyrA mutations arise at a lower rate than parC mutations, it is likely that resistance to gemifloxacin will emerge more slowly than is seen with those quinolones that become resistant through an initial mutation in parC. The rate at which second-step mutants emerged was 1.3 x 10(-8) for parC Serine 79 Tyrosine and 7.2 x 10(-9) for gyrA Serine 81 Phenylalanine, 12 and 450 times higher, respectively, than for first-step rates, suggesting that mutation in either gene readies the genome for further mutation.

Examination of single and multiple mutations involved in resistance to quinolones in Staphylococcus aureus by a combination of PCR and denaturing high-performance liquid chromatography (DHPLC).

Detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that has been used to detect mutations in human DNA. We report on the first study to use this technique as a tool to detect mutations in genes encoding antibiotic resistance in bacteria. Three methicillin-sensitive and three methicillin-resistant clinical Staphylococcus aureus isolates, susceptible to ciprofloxacin (MIC Leu, Ser-112-->Pro, Glu-88-->Lys in GyrA, Glu-84-->Val, Ser-80-->Phe in GrlA, Pro-456-->Ser in GyrB and Glu-422-->Asp, Pro-451-->Ser, Asp-432-->Gly in GrlB. Mutations could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run) and reliable technique for characterizing antibiotic resistance in bacteria.