Staphylococcus aureus Metabolic Adaptations during the Transition from a Daptomycin Susceptibility Phenotype to a Daptomycin Nonsusceptibility Phenotype

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The success of S. aureus as a pathogen is due in part to its many virulence determinants and resistance to antimicrobials. In particular, methicillin-resistant S. aureus has emerged as a major cause of infections and led to increased use of the antibiotics vancomycin and daptomycin, which has increased the isolation of vancomycin-intermediate S. aureus and daptomycin-nonsusceptible S. aureus strains. The most common mechanism by which S. aureus acquires intermediate resistance to antibiotics is by adapting its physiology and metabolism to permit growth in the presence of these antibiotics, a process known as adaptive resistance. To better understand the physiological and metabolic changes associated with adaptive resistance, six daptomycin-susceptible and -nonsusceptible isogenic strain pairs were examined for changes in growth, competitive fitness, and metabolic alterations. Interestingly, daptomycin nonsusceptibility coincides with a slightly delayed transition to the postexponential growth phase and alterations in metabolism. Specifically, daptomycin-nonsusceptible strains have decreased tricarboxylic acid cycle activity, which correlates with increased synthesis of pyrimidines and purines and increased carbon flow to pathways associated with wall teichoic acid and peptidoglycan biosynthesis. Importantly, these data provided an opportunity to alter the daptomycin nonsusceptibility phenotype by manipulating bacterial metabolism, a first step in developing compounds that target metabolic pathways that can be used in combination with daptomycin to reduce treatment failures.     

Daptomycin In Vitro Activity against Methicillin-Resistant Staphylococcus aureus Is Enhanced by d-Cycloserine in a Mechanism Associated with a Decrease in Cell Surface Charge

The killing activity of daptomycin against an isogenic pair of daptomycin-susceptible and daptomycin-nonsusceptible (DNS) methicillin-resistant Staphylococcus aureus (MRSA) strains was enhanced by the addition of certain cell wall agents at 1× MIC. However, when high inocula of the DNS strain were used, no significant killing was observed in our experiments. Cytochrome c binding assays revealed d-cycloserine as the only agent associated with a reduction in the cell surface charge for both strains at the concentrations used.     

A Novel Point Mutation Promotes Growth Phase-Dependent Daptomycin Tolerance in Staphylococcus aureus

Recalcitrance of genetically susceptible bacteria to antibiotic killing is a hallmark of bacterial drug tolerance. This phenomenon is prevalent in biofilms, persisters, and also planktonic cells and is associated with chronic or relapsing infections with pathogens such as Staphylococcus aureus. Here we report the in vitro evolution of an S. aureus strain that exhibits a high degree of nonsusceptibility to daptomycin as a result of cyclic challenges with bactericidal concentrations of the drug. This phenotype was attributed to stationary growth phase-dependent drug tolerance and was clearly distinguished from resistance. The underlying genetic basis was revealed to be an adaptive point mutation in the putative inorganic phosphate (P_i) transporter gene pitA. Drug tolerance caused by this allele, termed pitA6, was abrogated when the upstream gene pitR was inactivated. Enhanced tolerance toward daptomycin, as well as the acyldepsipeptide antibiotic ADEP4 and various combinations of other drugs, was accompanied by elevated intracellular concentrations of P_i and polyphosphate, which may reversibly interfere with critical cellular functions. The evolved strain displayed increased rates of survival within human endothelial cells, demonstrating the correlation of intracellular persistence and drug tolerance. These findings will be useful for further investigations of S. aureus drug tolerance, toward the development of additional antipersister compounds and strategies.     

Heterogeneity of Genetic Pathways toward Daptomycin Nonsusceptibility in Staphylococcus aureus Determined by Adjunctive Antibiotics

Daptomycin is increasingly used in combination with other antibiotics to enhance antimicrobial efficacy and/or to mitigate the emergence of daptomycin nonsusceptibility (DNS). This study used a clinical methicillin-resistant Staphylococcus aureus (MRSA) strain in which DNS emerged upon therapy to examine the influence of antibiotic combinations on the development of mutations in specific genes (mprF, rpoBC, dltA, cls2, and yycFG) previously associated with DNS. Whole genomes of bacteria obtained following 28 days of in vitro exposure to daptomycin with or without adjunctive clarithromycin, linezolid, oxacillin, or trimethoprim-sulfamethoxazole were sequenced, and the sequences were compared to that of the progenitor isolate. The addition of oxacillin to medium containing daptomycin prevented the emergence of mprF mutation but did not prevent rpoBC mutation (P < 0.01). These isolates maintained susceptibility to daptomycin during the combined exposure (median MIC, 1 mg/liter). Daptomycin plus clarithromycin or linezolid resulted in low-level (1.5 to 8 mg/liter) and high-level (12 to 96 mg/liter) DNS, respectively, and did not prevent mprF mutation. However, these same combinations prevented rpoBC mutation. Daptomycin alone or combined with linezolid or trimethoprim-sulfamethoxazole resulted in high-level DNS and mutations in mprF plus rpoBC, cls2, and yycFG. Combining daptomycin with different antimicrobials alters the mutational space available for DNS development, thereby favoring the development of predictable collateral susceptibilities.     

Antimicrobial Activity of Ceftaroline Tested against Staphylococci with Reduced Susceptibility to Linezolid,Daptomycin, or Vancomycin from U.S. Hospitals, 2008 to 2011

Vancomycin, linezolid, and daptomycin are very active against staphylococci, but isolates with decreased susceptibility to these antimicrobial agents are isolated sporadically. A total of 19,350 Staphylococcus aureus isolates (51% methicillin resistant [MRSA]) and 3,270 coagulase-negative staphylococci (CoNS) were collected consecutively from 82 U.S. medical centers from January 2008 to December 2011 and tested for susceptibility against ceftaroline and comparator agents by the reference broth microdilution method. Among S. aureus strains, 14 isolates (0.07%) exhibited decreased susceptibility to linezolid (MIC, ≥8 μg/ml), 18 (0.09%) to daptomycin (MIC, ≥2 μg/ml), and 369 (1.9%) to vancomycin (MIC, ≥2 μg/ml; 368 isolates at 2 μg/ml and 1 at 4 μg/ml). Fifty-one (1.6%) CoNS were linezolid resistant (MIC, ≥8 μg/ml), and four (0.12%) were daptomycin nonsusceptible (MIC, ≥2 μg/ml). Ceftaroline was very active against S. aureus overall (MIC_50/90, 0.5/1 μg/ml; 98.5% susceptible), including MRSA (MIC_50/90, 0.5/1 μg/ml; 97.2% susceptible). All daptomycin-nonsusceptible and 85.7% of linezolid-resistant S. aureus isolates were susceptible to ceftaroline. Against S. aureus isolates with a vancomycin MIC of ≥2 μg/ml, 91.9, 96.2, and 98.9% were susceptible to ceftaroline, daptomycin, and linezolid, respectively. CoNS strains were susceptible to ceftaroline (MIC_50/90, 0.25/0.5 μg/ml; 99.1% inhibited at ≤1 μg/ml), including methicillin-resistant (MIC_50/90, 0.25/0.5 μg/ml), linezolid-resistant (MIC_50/90, 0.5/0.5 μg/ml), and daptomycin-nonsusceptible (4 isolates; MIC range, 0.03 to 0.12 μg/ml) strains. In conclusion, ceftaroline demonstrated potent in vitro activity against staphylococci with reduced susceptibility to linezolid, daptomycin, or vancomycin, and it may represent a valuable treatment option for infections caused by these multidrug-resistant staphylococci.     

Antimicrobial Salvage Therapy for Persistent Staphylococcal Bacteremia Using Daptomycin Plus Ceftaroline

Purpose

Guidelines recommend daptomycin combination therapy as an option for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia after vancomycin failure. Recent data suggest that combining daptomycin with a β-lactam may have unique benefits; however, there are very limited clinical data regarding the use of ceftaroline with daptomycin.

Methods

All 26 cases from the 10 medical centers in which ceftaroline plus daptomycin was used for treatment of documented refractory staphylococcal bacteremia from March 2011 to November 2012 were included. In vitro (synergy studies, binding assays, cathelicidin LL-37 killing assays), and in vivo (virulence assays using a murine subcutaneous infection model) studies examining the effects of ceftaroline with daptomycin were also performed.

Findings

Daptomycin plus ceftaroline was used in 26 cases of staphylococcal bacteremia (20 MRSA, 2 vancomycin-intermediate S aureus, 2 methicillin-susceptible S aureus [MSSA], 2 methicillin-resistant S epidermidis). Bacteremia persisted for a median of 10 days (range, 3–23 days) on previous antimicrobial therapy. After daptomycin plus ceftaroline was started, the median time to bacteremia clearance was 2 days (range, 1–6 days). In vitro studies showed ceftaroline synergy against MRSA and enhanced MRSA killing by cathelicidin LL-37 and neutrophils. Ceftaroline also induced daptomycin binding in MSSA and MRSA to a comparable degree as nafcillin. MRSA grown in subinhibitory concentrations of ceftaroline showed attenuated virulence in a murine subcutaneous infection model.

Implications

Ceftaroline plus daptomycin may be an option to hasten clearance of refractory staphylococcal bacteremia. Ceftaroline offers dual benefit via synergy with both daptomycin and sensitization to innate host defense peptide cathelicidin LL37, which could attenuate virulence of the pathogen.     

In Vitro Pharmacodynamics of Human Simulated Exposures of Ceftaroline and Daptomycin against MRSA,hVISA, and VISA with and without Prior Vancomycin Exposure

The effects of prior vancomycin exposure on ceftaroline and daptomycin therapy against methicillin-resistant Staphylococcus aureus (MRSA) have not been widely studied. Humanized free-drug exposures of vancomycin at 1 g every 12 h (q12h), ceftaroline at 600 mg q12h, and daptomycin at 10 mg/kg of body weight q24h were simulated in a 96-h in vitro pharmacodynamic model against three MRSA isolates, including one heteroresistant vancomycin-intermediate S. aureus (hVISA) isolate and one VISA isolate. A total of five regimens were tested: vancomycin, ceftaroline, and daptomycin alone for the entire 96 h, and then sequential therapy with vancomycin for 48 h followed by ceftaroline or daptomycin for 48 h. Microbiological responses were measured by the changes in log₁₀ CFU during 96 h from baseline. Control isolates grew to 9.16 ± 0.32, 9.13 ± 0.14, and 8.69 ± 0.28 log₁₀ CFU for MRSA, hVISA, and VISA, respectively. Vancomycin initially achieved ≥3 log₁₀ CFU reductions against the MRSA and hVISA isolates, followed by regrowth beginning at 48 h; minimal activity was observed against VISA. The change in 96-h log₁₀ CFU was largest for sequential therapy with vancomycin followed by ceftaroline (−5.22 ± 1.2, P = 0.010 versus ceftaroline) and for sequential therapy with vancomycin followed by ceftaroline (−3.60 ± 0.6, P = 0.037 versus daptomycin), compared with daptomycin (−2.24 ± 1.0), vancomycin (−1.40 ± 1.8), and sequential therapy with vancomycin followed by daptomycin (−1.32 ± 1.0, P > 0.5 for the last three regimens). Prior exposure of vancomycin at 1 g q12h reduced the initial microbiological response of daptomycin, particularly for hVISA and VISA isolates, but did not affect the response of ceftaroline. In the scenario of poor vancomycin response for high-inoculum MRSA infection, a ceftaroline-containing regimen may be preferred.     

Case-control study comparing de novo and daptomycin-exposed daptomycin-nonsusceptible Enterococcus infections

Understanding factors associated with de novo daptomycin-nonsusceptible Enterococcus (DNSE) infections will aid in better understanding the mechanisms of daptomycin nonsusceptibility. We conducted a case-control study to compare patients with DNSE infections who were daptomycin treatment naïve (n = 9) and those with DNSE infections who had exposure to daptomycin (n = 13). Less frequent exposure to antimicrobials, increased susceptibility to nitrofurantoin and gentamicin, and shorter duration of hospitalization were associated with de novo DNSE infection, suggesting a potential community reservoir.     

Identification of a Novel Clone,ST736, among Enterococcus faecium Clinical Isolates and Its Association with Daptomycin Nonsusceptibility

Resistance to daptomycin in enterococcal clinical isolates remains rare but is being increasingly reported in the United States and worldwide. There are limited data on the genetic relatedness and microbiological and clinical characteristics of daptomycin-nonsusceptible enterococcal clinical isolates. In this study, we assessed the population genetics of daptomycin-nonsusceptible Enterococcus faecium (DNSE) clinical isolates by multilocus sequence typing (MLST) and whole-genome sequencing analysis. Forty-two nonduplicate DNSE isolates and 43 randomly selected daptomycin-susceptible E. faecium isolates were included in the analysis. All E. faecium isolates were recovered from patients at a tertiary care medical center in suburban New York City from May 2009 through December 2013. The daptomycin MICs of the DNSE isolates ranged from 6 to >256 μg/ml. Three major clones of E. faecium (ST18, ST412, and ST736) were identified among these clinical isolates by MLST and whole-genome sequence-based analysis. A newly recognized clone, ST736, was seen in 32 of 42 (76.2%) DNSE isolates and in only 14 of 43 (32.6%) daptomycin-susceptible E. faecium isolates (P < 0.0001). This report provides evidence of the association between E. faecium clone ST736 and daptomycin nonsusceptibility. The identification and potential spread of this novel E. faecium clone and its association with daptomycin nonsusceptibility constitute a challenge for patient management and infection control at our medical center.     

Ceftaroline Restores Daptomycin Activity against Daptomycin-Nonsusceptible Vancomycin-Resistant Enterococcus faecium

Daptomycin-nonsusceptible vancomycin-resistant Enterococcus faecium (VRE) strains are a formidable emerging threat to patients with comorbidities, leaving few therapeutic options in cases of severe invasive infections. Using a previously characterized isogenic pair of VRE strains from the same patient differing in their daptomycin susceptibilities (Etest MICs of 0.38 mg/liter and 10 mg/liter), we examined the effect of ceftaroline, ceftriaxone, and ampicillin on membrane fluidity and susceptibility of VRE to surface binding and killing by daptomycin and human cathelicidin antimicrobial peptide LL37. Synergy was noted in vitro between daptomycin, ampicillin, and ceftaroline for the daptomycin-susceptible VRE strain, but only ceftaroline showed synergy against the daptomycin-nonsusceptible VRE strain (∼2 log₁₀ CFU reduction at 24 h). Ceftaroline cotreatment increased daptomycin surface binding with an associated increase in membrane fluidity and an increase in the net negative surface charge of the bacteria as evidenced by increased poly-l-lysine binding. Consistent with the observed biophysical changes, ceftaroline resulted in increased binding and killing of daptomycin-nonsusceptible VRE by human cathelicidin LL37. Using a pair of daptomycin-susceptible/nonsusceptible VRE strains, we noted that VRE is ceftaroline resistant, yet ceftaroline confers significant effects on growth rate as well as biophysical changes on the cell surface of VRE that can potentiate the activity of daptomycin and innate cationic host defense peptides, such as cathelicidin. Although limited to just 2 strains, these finding suggest that additional in vivo and in vitro studies need to be done to explore the possibility of using ceftaroline as adjunctive anti-VRE therapy.     

Ceftaroline Increases Membrane Binding and Enhances the Activity of Daptomycin against Daptomycin-Nonsusceptible Vancomycin-Intermediate Staphylococcus aureus in a Pharmacokinetic/Pharmacodynamic Model

New antimicrobial agents and novel combination therapies are needed to treat serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to daptomycin and vancomycin. The purpose of this study was to evaluate the combination of ceftaroline plus daptomycin or vancomycin in an in vitro pharmacokinetic/pharmacodynamic model. Simulations of ceftaroline-fosamil at 600 mg per kg of body weight every 8 h (q8h) (maximum free-drug concentration in serum [fC_max], 15.2 mg/liter; half-life [t_1/2], 2.3 h), daptomycin at 10 mg/kg/day (fC_max, 11.3 mg/liter; t_1/2, 8 h), vancomycin at 2 g q12h (fC_max, 30 mg/liter; t_1/2, 6 h), ceftaroline plus daptomycin, and ceftaroline plus vancomycin were evaluated against a clinical, isogenic MRSA strain pair: D592 (daptomycin susceptible and heterogeneous vancomycin intermediate) and D712 (daptomycin nonsusceptible and vancomycin intermediate) in a one-compartment in vitro pharmacokinetic/pharmacodynamic model over 96 h. Therapeutic enhancement of combinations was defined as ≥2 log₁₀ CFU/ml reduction over the most active single agent. The effect of ceftaroline on the membrane charge, cell wall thickness, susceptibility to killing by the human cathelicidin LL37, and daptomycin binding were evaluated. Therapeutic enhancement was observed with daptomycin plus ceftaroline in both strains and vancomycin plus ceftaroline against D592. Ceftaroline exposure enhanced daptomycin-induced depolarization (81.7% versus 72.3%; P = 0.03) and killing by cathelicidin LL37 (P < 0.01) and reduced cell wall thickness (P < 0.001). Fluorescence-labeled daptomycin was bound over 7-fold more in ceftaroline-exposed cells. Whole-genome sequencing and mutation analysis of these strains indicated that change in daptomycin susceptibility is related to an fmtC (mprF) mutation. The combination of daptomycin plus ceftaroline appears to be potent, with rapid and sustained bactericidal activity against both daptomycin-susceptible and -nonsusceptible strains of MRSA.     

Characterization of a daptomycin-nonsusceptible vancomycin-intermediate Staphylococcus aureus strain in a patient with endocarditis

We analyzed the emergence of daptomycin nonsusceptibility in a patient with persistent vancomycin-intermediate Staphylococcus aureus (VISA) bacteremia. The daptomycin-nonsusceptible VISA's cell wall demonstrated a reduction in muramic acid O-acetylation, a phenotypic parameter not previously reported for VISA; some isolates also contained a single point mutation in the mprF gene.     

Frequency and Distribution of Single-Nucleotide Polymorphisms within mprF in Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides

MprF is responsible for the lysinylation of phosphatidylglycerol (PG) to synthesize the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) are associated with a gain-in-function phenotype in terms of daptomycin resistance in Staphylococcus aureus. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation.) Using 22 daptomycin-susceptible (DAP^s)/daptomycin-resistant (DAP^r) clinical methicillin-resistant S. aureus (MRSA) strain pairs, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs, (ii) the relationships of the SNPs to both daptomycin resistance and cross-resistance to the prototypical endovascular host defense peptide (HDP) thrombin-induced platelet microbicidal protein (tPMP), and (iii) the impact of mprF SNPs on positive surface charge phenotype and modifications of membrane PL profiles. Most of the mprF SNPs identified in our DAP^r strains were clustered within the two MprF loci, (i) the central bifunctional domain and (ii) the C-terminal synthase domain. Moreover, we were able to correlate the presence and location of mprF SNPs in DAP^r strains with HDP cross-resistance, positive surface charge, and L-PG profiles. Although DAP^r strains with mprF SNPs in the bifunctional domain showed higher resistance to tPMPs than DAP^r strains with SNPs in the synthase domain, this relationship was not observed in positive surface charge assays. These results demonstrated that both charge-mediated and -unrelated mechanisms are involved in DAP resistance and HDP cross-resistance in S. aureus.     

Daptomycin versus Vancomycin in Treatment of Methicillin-Resistant Staphylococcus aureus Meningitis in an Experimental Rabbit Model

In this study, we aimed to compare the antibacterial activities of daptomycin and vancomycin in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) meningitis (induced by MRSA strain ATCC 43300) in an experimental rabbit meningitis model. After an 8-h period of treatment, bacterial counts decreased significantly in both treatment groups compared to the control group (P < 0.05). However, there was no statistically significant difference between treatment groups. Our results suggest that the antibacterial activity of daptomycin is similar to vancomycin for treatment in the experimental MRSA meningitis model in rabbits.     

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

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 (AUBKC_0-24), 48 h (AUBKC_0-48), and 72 h (AUBKC_0-72). The rapid bactericidal activities of oritavancin and daptomycin contributed to lower AUBKC_0-24s 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 AUBKC_0-48 and AUBKC_0-72 against one MRSA strain and a lower AUBKC_0-48 for another strain than did vancomycin exposure (P < 0.05). Furthermore, daptomycin exposure resulted in a lower AUBKC_0-48 and AUBKC_0-72 for one of the MRSA isolates than did vancomycin exposure (P < 0.05). Lower AUBKC_0-24s 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.     

Antimicrobial susceptibility of daptomycin and comparator agents tested against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci: trend analysis of a 6-year period in US medical centers (2005-2010)

We evaluated daptomycin activity trends against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE; MIC, ≥8 μg/mL) in a 6-year period (2005–2010) following US regulatory release for clinical use. Consecutive, unique patient strains of clinical significance were collected in 32 US medical centers and susceptibility tested in a central laboratory against daptomycin and various comparator agents by reference broth microdilution methods. A total of 22 858 S. aureus (12 181 [53.3%] MRSA), 4312 Enterococcus faecalis (195 [4.5%] VRE), and 2462 Enterococcus faecium (1867 [75.8%] VRE) were evaluated. Daptomycin susceptibility rates were 99.94%, 99.98%, and 99.68% for S. aureus, E. faecalis, and E. faecium, respectively. Among MRSA (daptomycin MIC_50/90, 0.25/0.5 μg/mL), only 13 (0.11%) daptomycin-non-susceptible (MIC, ≥2 μg/mL) isolates were observed with no MIC creep over the study interval. Daptomycin was very active against vancomycin-resistant E. faecalis (MIC_50/90, 1/2 μg/mL) and E. faecium (MIC_50/90, 2/2 μg/mL). Among VRE, only 4 daptomycin-non-susceptible isolates (all E. faecium) were detected. In conclusion, daptomycin demonstrated sustained activity against an extensive collection of clinical isolates of MRSA and VRE from numerous US medical centers over the last 6 monitored years.     

Bactericidal activity of bacteriophage endolysin HY-133 against Staphylococcus aureus in comparison to other antibiotics as determined by minimum bactericidal concentrations and time-kill analysis

Methicillin-resistant Staphylococcus aureus (MRSA) decolonization is expensive and time consuming, and new agents are necessary due to increasing resistance rates. The administration of bacteriophages or particularly their endolysins may offer an alternative treatment strategy and could provide a solution to overcome the selection pressure due to classical antibiotics. Here, the bactericidal activity was characterized for the recombinant chimeric bacteriophage endolysin HY-133 in comparison to other antimicrobials. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for 2 reference strains, 24 clinical MRSA and methicillin-susceptible S. aureus (MSSA) isolates, as well as 6 isolates with high-level mupirocin resistance. Additionally, HY-133 activity against bacteria in stationary or exponential growth phase was compared in 12 isolates. Time-kill curves were performed with 2 representative isolates to investigate the pharmacodynamics until 48-h incubation time. All experiments were performed in comparison to daptomycin and mupirocin. The MIC_50/90 and MBC_50/90 values were in the range 0.12–0.5?mg/L for all 3 growth conditions comparable to daptomycin with 0.5/0.5?mg/L, respectively. The MBC was almost always equal the MIC and without considerable differences between MSSA and MRSA. Time-kill curves revealed a rapid bactericidal effect of HY-133 within the first 2 h, similar to daptomycin. Even with low concentrations, the recombinant endolysin HY-133 was highly active against all tested MSSA and MRSA isolates including mupirocin-resistant isolates. The application of this alternative agent may offer a future strategy for MRSA/MSSA decolonization and, potentially, for treatment purposes.