Pharmacokinetic-Pharmacodynamic Modeling of the In Vitro Activities of Oxazolidinone Antimicrobial Agents against Methicillin-Resistant Staphylococcus aureus

Linezolid is the first FDA-approved oxazolidinone with activity against clinically important gram-positive pathogens, including methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). RWJ-416457 is a new oxazolidinone with an antimicrobial spectrum similar to that of linezolid. The goal of the present study was to develop a general pharmacokinetic (PK)-pharmacodynamic (PD) model that allows the characterization and comparison of the in vitro activities of oxazolidinones, determined in time-kill curve experiments, against MRSA. The in vitro activities of RWJ-416457 and the first-in-class representative, linezolid, against MRSA OC2878 were determined in static and dynamic time-kill curve experiments over a wide range of concentrations: 0.125 to 8 μg/ml (MIC, 0.5 μg/ml) and 0.25 to 16 μg/ml (MIC, 1 μg/ml), respectively. After correction for drug degradation during the time-kill curve experiments, a two-subpopulation model was simultaneously fitted to all data in the NONMEM VI program. The robustness of the model and the precision of the parameter estimates were evaluated by internal model validation by nonparametric bootstrap analysis. A two-subpopulation model, consisting of a self-replicating, oxazolidinone-susceptible and a persistent, oxazolidinone-insusceptible pool of bacteria was appropriate for the characterization of the time-kill curve data. The PK-PD model identified was capable of accounting for saturation in growth, delays in the onsets of growth and drug-induced killing, as well as naturally occurring bacterial death. The simultaneous fit of the proposed indirect-response, maximum-effect model to the data resulted in concentrations that produced a half-maximum killing effect that were significantly (P < 0.05) lower for RWJ-416457 (0.41 μg/ml) than for linezolid (1.39 μg/ml). In combination with the appropriate PK data, the susceptibility-based two-subpopulation model identified may provide valuable guidance for the selection of oxazolidinone doses or dose regimens for use in clinical studies.Gram-positive pathogens are major causes of a wide range of infections, including skin and skin structure infections and life-threatening infections such as bacteremia, endocarditis, and pneumonia (18, 24). The treatment of these infections has become increasingly challenging due to the rapid development of resistance to the first-choice antibiotics. In many cases, only a few agents, such as vancomycin, retain activity against these multiresistant strains, despite its increased utilization. However, with the appearance of vancomycin-resistant strains, for example, vancomycin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, the once powerful antibiotic arsenal is becoming increasingly ineffective (3, 12). Therefore, it is critically important to develop new potent antibiotics or antibiotic classes that may successfully treat these infections.In 2000, the FDA approved the use of linezolid, the first representative of a novel class of antibiotics, the oxazolidinones. At the time of approval, linezolid was one of the few agents that showed activity against vancomycin-resistant strains (2, 5). However, resistance to linezolid was reported as early as 2002 (10, 32). In addition, toxic side effects such as reversible thrombocytopenia, neutropenia, and, rarely, neuropathy have occurred during prolonged use (4, 13, 16, 28). Due to these limitations, there is a definite opportunity to develop new oxazolidinones with optimized exposure-response relationships. RWJ-416457 is a new investigational oxazolidinone that is being developed as both an oral and an intravenous formulation for the treatment of infections caused by clinically important gram-positive bacteria. RWJ-416457 has a MIC that is frequently two- to fourfold lower than that of linezolid against multidrug-resistant gram-positive pathogenic bacteria, including methicillin (meticillin)-resistant S. aureus (MRSA), vancomycin-intermediate susceptible S. aureus, vancomycin-resistant S. aureus, vancomycin-resistant enterococci, and penicillin-resistant streptococci (8, 17). Although the MIC is routinely determined in clinical settings and has contributed much to the understanding of antibiotic dosing, it does not provide any information on the time course of bacterial growth or antibiotic-induced killing (21, 27). More detailed information can be obtained from the evaluation of growth and kill profiles over time (time-kill curves). A major strength of the time-kill curve approach is its capability of simulating the effect of changing concentrations on the antimicrobial outcome. Changing concentration time-kill curves can, subsequently, be used to evaluate the efficacies of antibiotics with different half-lives (t_1/2s). Once these experiments have been performed, a mathematical model can be simultaneously fitted to the data and the respective pharmacodynamic (PD) parameters can be calculated. These PD parameters can then be linked to in vivo pharmacokinetic (PK) information for prediction of the clinical outcome.The aims of this study were (i) to establish a general mathematical model that is appropriate for characterizing the in vitro PDs of oxazolidinones determined in static as well as dynamic time-kill curve experiments and (ii) to apply this model in order to compare the in vitro potencies of the investigational oxazolidinone RWJ-416457 and the first-in-class representative linezolid.     

Efficacy of LY333328 against Experimental Methicillin-Resistant Staphylococcus aureus Endocarditis

The in vivo efficacy of LY333328, a new glycopeptide antibiotic, was compared with that of vancomycin by using the rabbit model of left-sided methicillin-resistant Staphylococcus aureus endocarditis. Animals received LY333328 or vancomycin (25 mg/kg of body weight every 24 or 8 h, respectively) for 4 days. These drugs were equally effective in clearing bacteremia and in reducing bacterial counts in vegetations and tissues. We conclude that in this model, LY333328 was microbiologically effective and may be a therapeutic alternative to vancomycin.     

Ceftobiprole- and Ceftaroline-Resistant Methicillin-Resistant Staphylococcus aureus

The role of mecA mutations in conferring resistance to ceftobiprole and ceftaroline, cephalosporins with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, was determined with MRSA strains COL and SF8300. The SF8300 ceftaroline-passaged mutant carried a single mecA mutation, E447K (E-to-K change at position 447), and expressed low-level resistance. This mutation in COL conferred high-level resistance to ceftobiprole but only low-level resistance to ceftaroline. The COL ceftaroline-passaged mutant, which expressed high-level resistance to ceftobiprole and ceftaroline, had mutations in pbp2, pbp4, and gdpP but not mecA.     

Epigallocatechin Gallate Synergistically Enhances the Activity of Carbapenems against Methicillin-Resistant Staphylococcus aureus

Combinations of carbapenems and epigallocatechin gallate (EGCg; a main constituent of tea catechins) showed potent synergy against 24 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). MICs of imipenem in the presence of EGCg at 3.125, 6.25, 12.5, and 25 microg/ml, were restored to the susceptible breakpoint (< or =4 microg/ml) for 8, 38, 46, and 75% of the MRSA isolates, respectively. Similar results were also observed for combinations of panipenem or meropenem and EGCg. Therefore, the combinations may be worthy of further evaluation in vivo against MRSA infection.     

Role of Interleukin-12 in Protection against Pulmonary Infection with Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen associated with nosocomial pneumonia and is an increasing threat for severe community-acquired pneumonia. We have now investigated the role of interleukin-12 (IL-12) in protective immunity against lung infection with MRSA. The importance of IL-12 in protection from pulmonary MRSA infection was demonstrated by the finding that IL-12p35-deficient mice had a lower survival rate, higher bacterial burdens in lung and spleen, and decreased expression of interferon gamma (IFN-γ) in the lung compared to wild-type mice. These effects were completely reversed by replacement intranasal therapy with recombinant IL-12. Furthermore, exogenous IL-12 treatment of wild-type mice 24 h before pulmonary challenge with a lethal dose of MRSA significantly improved bacterial clearance and resulted in protection from death. The IL-12-treated mice had increased numbers of lung natural killer (NK) cells and neutrophils and higher levels of IFN-γ in the lung and serum compared to untreated mice. The major source of IL-12-driven IFN-γ expression in the lung was the NK cell, and the direct target of pulmonary IFN-γ was the lung macrophage, as shown using mice with a macrophage-specific defect in interferon gamma (IFN-γ) signaling (MIIG mice). Importantly, combination therapy with linezolid and IL-12 following intranasal MRSA infection significantly increased survival compared to that of mice receiving linezolid or IL-12 alone. These results indicate that IL-12-based immunotherapy may hold promise for treatment of MRSA pneumonia.     

Pharmacodynamic Profile of Tigecycline against Methicillin-Resistant Staphylococcus aureus in an Experimental Pneumonia Model

Tigecycline (TGC) is an extended-spectrum antibiotic with activity against Staphylococcus aureus, including methicillin (meticillin)-resistant S. aureus strains, which are well-recognized pathogens in nosocomial pneumonia. The objective of this study was to characterize the exposure-response relationship for TGC against S. aureus in an immunocompromised BALB/c murine pneumonia model. Six S. aureus isolates were studied, and the TGC MICs for those isolates ranged from 0.125 to 0.5 mg/liter. The pharmacokinetics (PK) of TGC in serum and bronchoalveolar lavage (BAL) fluid were evaluated, as was the level of protein binding of the compound in this murine species. Administration of TGC at 1.56 to 150 mg/kg of body weight/day in single or two to three divided doses was used in the efficacy studies. TGC displayed linear PK and had a mean half-life of 10.9 ± 2.5 h. Efficacy was highly correlated with the area under the free concentration-time curve (fAUC)/MIC (r² = 0.93). The 80% and 50% effective exposure indexes and the stasis exposure index were similar between the isolates (means ± standard deviations, 3.04 ± 1.12, 1.84 ± 1.3, and 1.9 ± 1.5, respectively). Maximal efficacy was predicted at a 2.85-log₁₀-CFU reduction. TGC appeared to accumulate in the interstitial space, as the ratios of the fAUC from 0 to 8 h of epithelial lining fluid to plasma were 7.02, 15.11, and 23.95 for doses of 12.5, 25, and 50 mg/kg, respectively. TGC was highly effective in this murine pneumonia model. In light of current MIC distributions, the fAUC/MIC targets that we defined against S. aureus are readily achievable in humans given conventional doses of TGC.Staphylococcus aureus has long been recognized as an important cause of infection, and the emergence of S. aureus strains with the methicillin (meticillin)-resistant phenotype (methicillin-resistant S. aureus [MRSA]) has further complicated management. Both community-acquired MRSA (CA-MRSA) and hospital-acquired MRSA (HA-MRSA) strains have been associated with severe and difficult-to-treat infections. While the most common site of staphylococcal infection is the skin and skin structures, the surveillance of 8,792 invasive MRSA cases in the United States showed that pneumonia is the second most common clinical manifestation of MRSA infection (13.3% overall; 14% of the strains were CA-MRSA and 28% were HA-MRSA) (9).Tigecycline (TGC) is a broad-spectrum glycylcycline with efficacy against gram-positive and gram-negative bacteria, including drug-resistant bacteria such as MRSA. The MIC₉₀ of TGC against methicillin-susceptible S. aureus (MSSA) and MRSA strains is reported to be ≤0.25 mg/liter (6). TGC is approved by the FDA for use for the treatment of complicated skin and skin structure infections and complicated intra-abdominal infections. Pneumonia is an important clinical manifestation of infection with drug-resistant bacteria; therefore, many in vivo and in vitro studies of TGC for the treatment of lower respiratory infection are ongoing (data available at http://www.clinicaltrialssearch.org/tigecycline_versus_imipenemcilastatin_for_the_treatment_of_subjects_with_nosocomial_pneumonia.html and http://www.medicalnewstoday.com/articles/53035.php).Previously, we demonstrated the efficacy of TGC against Acinetobacter spp. in a murine pneumonia model (10) and against S. aureus in a murine thigh infection model (3). We also found that TGC penetrated well into lung tissue, as displayed by high concentrations in bronchoalveolar (BAL) fluid (4). In the present study, we aimed to explore the exposure-response relationship for TGC against S. aureus in an immunocompromised BALB/c murine pneumonia model.     

Copper Complexation Screen Reveals Compounds with Potent Antibiotic Properties against Methicillin-Resistant Staphylococcus aureus

Macrophages take advantage of the antibacterial properties of copper ions in the killing of bacterial intruders. However, despite the importance of copper for innate immune functions, coordinated efforts to exploit copper ions for therapeutic interventions against bacterial infections are not yet in place. Here we report a novel high-throughput screening platform specifically developed for the discovery and characterization of compounds with copper-dependent antibacterial properties toward methicillin-resistant Staphylococcus aureus (MRSA). We detail how one of the identified compounds, glyoxal-bis(N4-methylthiosemicarbazone) (GTSM), exerts its potent strictly copper-dependent antibacterial properties on MRSA. Our data indicate that the activity of the GTSM-copper complex goes beyond the general antibacterial effects of accumulated copper ions and suggest that, in contrast to prevailing opinion, copper complexes can indeed exhibit species- and target-specific activities. Based on experimental evidence, we propose that copper ions impose structural changes upon binding to the otherwise inactive GTSM ligand and transfer antibacterial properties to the chelate. In turn, GTSM determines target specificity and utilizes a redox-sensitive release mechanism through which copper ions are deployed at or in close proximity to a putative target. According to our proof-of-concept screen, copper activation is not a rare event and even extends to already established drugs. Thus, copper-activated compounds could define a novel class of anti-MRSA agents that amplify copper-dependent innate immune functions of the host. To this end, we provide a blueprint for a high-throughput drug screening campaign which considers the antibacterial properties of copper ions at the host-pathogen interface.     

Ceftaroline-Fosamil Efficacy against Methicillin-Resistant Staphylococcus aureus in a Rabbit Prosthetic Joint Infection Model

Ceftaroline (CPT), the active metabolite of the prodrug ceftaroline-fosamil (CPT-F), demonstrates in vitro bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) and is effective in rabbit models of difficult-to-treat MRSA endocarditis and acute osteomyelitis. However, its in vivo efficacy in a prosthetic joint infection (PJI) model is unknown. Using a MRSA-infected knee PJI model in rabbits, the efficacies of CPT-F or vancomycin (VAN) alone and combined with rifampin (RIF) were compared. After each partial knee replacement with a silicone implant that fit into the tibial intramedullary canal was performed, 5 × 10⁷ MRSA CFU (MICs of 0.38, 0.006, and 1 mg/liter for CPT, RIF, and VAN, respectively) was injected into the knee. Infected animals were randomly assigned to receive no treatment (controls) or CPT-F (60 mg/kg of body weight intramuscularly [i.m.]), VAN (60 mg/kg i.m.), CPT-F plus RIF (10 mg/kg i.m.), or VAN plus RIF starting 7 days postinoculation and lasting for 7 days. Surviving bacteria in crushed tibias were counted 3 days after ending treatment. Although the in vivo mean log₁₀ CFU/g of CPT-treated (3.0 ± 0.9, n = 12) and VAN-treated (3.5 ± 1.1, n = 12) crushed bones was significantly lower than those of controls (5.6 ± 1.1, n = 14) (P < 0.001), neither treatment fully sterilized the bones (3/12 were sterile with each treatment). The mean log₁₀ CFU/g values for the antibiotics in combination with RIF were 1.9 ± 0.5 (12/14 were sterile) for CPT-F and 1.9 ± 0.5 (12/14 were sterile) for VAN. In this MRSA PJI model, the efficacies of CPT-F and VAN did not differ; thus, CPT appears to be a promising antimicrobial agent for the treatment of MRSA PJIs.     

Methicillin-Resistant Strains of Staphylococcus aureus Phage Type 92

Methicillin-resistant (Mec(r)) strains of Staphylococcus aureus received for phage typing from several hospitals in New York City were resistant to the international set of typing phages but susceptible to experimental phage 92. Subsequently, strains of type 92 were detected in two outbreaks with Mec(r) strains in two other locations in the United States. In all instances, type 92 was predominant among the Mec(r) strains isolated in each hospital. With the exception of one strain, the methicillin resistance of the Mec(r) strains investigated was homogeneous. In most instances, isolates from the same hospital were closely similar in their antibiotic resistance patterns. The strains isolated in New York City could be divided into three groups by the host range of their lysogenic phages and by antigenic structure. Transduction experiments indicated that the transfer of chromosomal tetracycline resistance from Mec(r) strains into a strain susceptible to several international typing phages renders the latter nontypable. However, the acceptor strain remains susceptible to experimental phages 92 and 88. Transduction of methicillin resistance had no effect on the phage susceptibility of the acceptor strain. It is possible that the presence of chromosomal tetracycline resistance is a determining factor in the phage susceptibility of Mec(r) strains isolated in New York City.     

Effects of Bacteriocins on Methicillin-Resistant Staphylococcus aureus Biofilm

Control of biofilms formed by microbial pathogens is an important subject for medical researchers, since the development of biofilms on foreign-body surfaces often causes biofilm-associated infections in patients with indwelling medical devices. The present study examined the effects of different kinds of bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by certain bacteria, on biofilms formed by a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). The activities and modes of action of three bacteriocins with different structures (nisin A, lacticin Q, and nukacin ISK-1) were evaluated. Vancomycin, a glycopeptide antibiotic used in the treatment of MRSA infections, showed bactericidal activity against planktonic cells but not against biofilm cells. Among the tested bacteriocins, nisin A showed the highest bactericidal activity against both planktonic cells and biofilm cells. Lacticin Q also showed bactericidal activity against both planktonic cells and biofilm cells, but its activity against biofilm cells was significantly lower than that of nisin A. Nukacin ISK-1 showed bacteriostatic activity against planktonic cells and did not show bactericidal activity against biofilm cells. Mode-of-action studies indicated that pore formation leading to ATP efflux is important for the bactericidal activity against biofilm cells. Our results suggest that bacteriocins that form stable pores on biofilm cells are highly potent for the treatment of MRSA biofilm infections.     

Prevalence of Chlorhexidine-Resistant Methicillin-Resistant Staphylococcus aureus following Prolonged Exposure

Chlorhexidine has been increasingly utilized in outpatient settings to control methicillin-resistant Staphylococcus aureus (MRSA) outbreaks and as a component of programs for MRSA decolonization and prevention of skin and soft-tissue infections (SSTIs). The objective of this study was to determine the prevalence of chlorhexidine resistance in clinical and colonizing MRSA isolates obtained in the context of a community-based cluster-randomized controlled trial for SSTI prevention, during which 10,030 soldiers were issued chlorhexidine for body washing. We obtained epidemiological data on study participants and performed molecular analysis of MRSA isolates, including PCR assays for determinants of chlorhexidine resistance and high-level mupirocin resistance and pulsed-field gel electrophoresis (PFGE). During the study period, May 2010 to January 2012, we identified 720 MRSA isolates, of which 615 (85.4%) were available for molecular analysis, i.e., 341 clinical and 274 colonizing isolates. Overall, only 10 (1.6%) of 615 isolates were chlorhexidine resistant, including three from the chlorhexidine group and seven from nonchlorhexidine groups (P > 0.99). Five (1.5%) of the 341 clinical isolates and five (1.8%) of the 274 colonizing isolates harbored chlorhexidine resistance genes, and four (40%) of the 10 possessed genetic determinants for mupirocin resistance. All chlorhexidine-resistant isolates were USA300. The overall prevalence of chlorhexidine resistance in MRSA isolates obtained from our study participants was low. We found no association between extended chlorhexidine use and the prevalence of chlorhexidine-resistant MRSA isolates; however, continued surveillance is warranted, as this agent continues to be utilized for infection control and prevention efforts.     

Correlation between Vancomycin MIC Values and Those of Other Agents against Gram-Positive Bacteria among Patients with Bloodstream Infections Caused by Methicillin-Resistant Staphylococcus aureus

An increase in the distribution of vancomycin MIC values among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates has been noted. It is postulated that the shift in vancomycin MIC values may be associated with a concurrent rise in the MIC values of other anti-MRSA agents. Scant data are available on the correlation between vancomycin MIC values and the MIC values of other anti-MRSA agents. This study examined the correlation between vancomycin MIC values and the MIC values of daptomycin, linezolid, tigecycline, and teicoplanin among 120 patients with bloodstream infections caused by MRSA at a tertiary care hospital between January 2005 and May 2007. For each included patient, the MIC values of the antibiotics under study were determined by the Etest method and were separated into the following two categories: day 1 (index) and post-day 1 (subsequent). For subsequent isolates, the MIC values for each antibiotic from the post-day 1 terminal isolate were used. Among the index isolates, there was a significant correlation (P value, <0.01) between the MIC values for vancomycin and daptomycin and between the MIC values for vancomycin and teicoplanin. The MIC values for daptomycin were significantly correlated with linezolid, tigecycline, and teicoplanin MIC values. Among the 48 patients with subsequent isolates, vancomycin MIC values were significantly correlated with MIC values for daptomycin, linezolid, and teicoplanin (ρ value of ≥0.38 for all comparisons). This study documented an association between vancomycin MIC values and the MIC values of other anti-MRSA antibiotics among patients with bloodstream infections caused by MRSA primarily treated with vancomycin.An increase in the distribution of vancomycin MIC values among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates has been noted in several recent reports (3, 11, 14). This shift is a concern because a growing number of studies have shown that patients with infections caused by MRSA with vancomycin MIC values at the higher end of the Clinical and Laboratory Standards Institute (CLSI) and Food and Drug Administration (FDA) susceptibility range are less responsive to vancomycin (3, 4, 5, 7, 12, 13). Clinicians must now consider using alternative therapies for such patients. However, it is unclear whether the observed shift in the distribution of vancomycin MIC values for MRSA is associated with similar shifts in the distribution of MIC values of other anti-MRSA agents.It is postulated that the shift in vancomycin MIC values may be associated with a concurrent rise in the MIC values of other anti-MRSA agents. Scant data are available on the correlation between vancomycin MIC values and the MIC values of other anti-MRSA agents. To date, analyses have been limited to the index MRSA isolate and have primarily focused on the correlation between vancomycin and daptomycin (6, 10, 11).This study examined the correlation between vancomycin MIC values and the MIC values of daptomycin, linezolid, tigecycline, and teicoplanin among patients with bloodstream infections caused by MRSA. Given the recent reports describing the emergence of resistance during therapy, our analyses included both the index (day 1) and subsequent (post-day 1) isolates.     

Pharmacodynamic Profile of GSK2140944 against Methicillin-Resistant Staphylococcus aureus in a Murine Lung Infection Model

GSK2140944 is a novel bacterial type II topoisomerase inhibitor with in vitro activity against key causative respiratory pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). We described the pharmacodynamics of GSK2140944 against MRSA in the neutropenic murine lung infection model. MICs of GSK2140944 were determined by broth microdilution. Plasma and epithelial lining fluid (ELF) pharmacokinetics were evaluated to allow determination of pulmonary distribution. Six MRSA isolates were tested. GSK2140944 doses of 1.56 to 400 mg/kg of body weight every 6 h (q6h) were utilized. Efficacy as the change in log₁₀ CFU at 24 h compared with 0 h controls and the area under the concentration-time curve for the free, unbound fraction of a drug (fAUC)/MIC required for various efficacy endpoints were determined. GSK2140944 MICs were 0.125 to 0.5 mg/liter against the six MRSA isolates. ELF penetration ratios ranged from 1.1 to 1.4. Observed maximal decreases were 1.1 to 3.1 log₁₀ CFU in neutropenic mice. The mean fAUC/MIC ratios required for stasis and 1-log-unit decreases were 59.3 ± 34.6 and 148.4 ± 83.3, respectively. GSK2140944 displayed in vitro and in vivo activity against MRSA. The pharmacodynamic profile of GSK2140944, as determined, supports its further development as a potential treatment option for pulmonary infections, including those caused by MRSA.     

Targeting Methicillin-Resistant Staphylococcus aureus with Short Salt-Resistant Synthetic Peptides

The seriousness of microbial resistance combined with the lack of new antimicrobials has increased interest in the development of antimicrobial peptides (AMPs) as novel therapeutics. In this study, we evaluated the antimicrobial activities of two short synthetic peptides, namely, RRIKA and RR. These peptides exhibited potent antimicrobial activity against Staphylococcus aureus, and their antimicrobial effects were significantly enhanced by addition of three amino acids in the C terminus, which consequently increased the amphipathicity, hydrophobicity, and net charge. Moreover, RRIKA and RR demonstrated a significant and rapid bactericidal effect against clinical and drug-resistant Staphylococcus isolates, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), linezolid-resistant S. aureus, and methicillin-resistant Staphylococcus epidermidis. In contrast to many natural AMPs, RRIKA and RR retained their activity in the presence of physiological concentrations of NaCl and MgCl₂. Both RRIKA and RR enhanced the killing of lysostaphin more than 1,000-fold and eradicated MRSA and VRSA isolates within 20 min. Furthermore, the peptides presented were superior in reducing adherent biofilms of S. aureus and S. epidermidis compared to results with conventional antibiotics. Our findings indicate that the staphylocidal effects of our peptides were through permeabilization of the bacterial membrane, leading to leakage of cytoplasmic contents and cell death. Furthermore, peptides were not toxic to HeLa cells at 4- to 8-fold their antimicrobial concentrations. The potent and salt-insensitive antimicrobial activities of these peptides present an attractive therapeutic candidate for treatment of multidrug-resistant S. aureus infections.     

N-Terminally Modified Linear and Branched Spermine Backbone Dipeptidomimetics against Planktonic and Sessile Methicillin-Resistant Staphylococcus aureus

Toward the discovery of useful therapeutic molecules, we report the design and synthesis of a focused library of new ultrashort N-terminally modified dipeptidomimetics, with or without modifications in the spermine backbone leading to linear (series 1) or branched (series 2) tryptophans, as antimicrobial agents. Eight peptidomimetics in the library showed good antibacterial activity (MICs of 1.77 to 14.2 μg/ml) against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis bacterial strains. Tryptophan fluorescence measurements on artificial bacterial or mammalian mimic membranes and assessment of the MRSA potential depolarization ability of the designed compounds revealed membrane interactions dependent on tryptophan positioning and N-terminal tagging. Among active peptidomimetics, compounds 1c and 1d were found to be nonhemolytic, displaying rapid bactericidal activity (at 4× MIC) against exponentially growing MRSA. Further, scanning electron microscopy of peptidomimetic 1c- and 1d-treated MRSA showed morphological changes with damage to cell walls, defining a membrane-active mode of action. Moreover, peptidomimetics 1c and 1d did not induce significant drug resistance in MRSA even after 17 passages. We also investigated the activity of these molecules against MRSA biofilms. At sub-MIC levels (∼2 to 4 μg/ml), both peptidomimetics inhibited biofilm formation. At concentrations higher than the MIC (35 to 140 μg/ml), peptidomimetics 1c and 1d significantly reduced the metabolic activity and biomass of mature (24-h) MRSA biofilms. These results were corroborated by confocal laser scanning microscopy (live/dead assay). The in vitro protease stability and lower cytotoxicity of peptidomimetics against peripheral blood mononuclear cells (PBMCs) support them being novel staphylocidal peptidomimetics. In conclusion, this study provides two peptidomimetics as potential leads for treatment of staphylococcal infections under planktonic and sessile conditions.     

Synergism between a Novel Chimeric Lysin and Oxacillin Protects against Infection by Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is the causative agent of several serious infectious diseases. The emergence of antibiotic-resistant S. aureus strains has resulted in significant treatment difficulties, intensifying the need for new antimicrobial agents. Toward this end, we have developed a novel chimeric bacteriophage (phage) lysin that is active against staphylococci, including methicillin-resistant S. aureus (MRSA). The chimeric lysin (called ClyS) was obtained by fusing the N-terminal catalytic domain of the S. aureus Twort phage lysin with the C-terminal cell wall-targeting domain from another S. aureus phage lysin (phiNM3), which displayed Staphylococcus-specific binding. ClyS was expressed in Escherichia coli, and the purified protein lysed MRSA, vancomycin-intermediate strains of S. aureus (VISA), and methicillin-sensitive (MSSA) strains of S. aureus in vitro. In a mouse nasal decolonization model, a 2-log reduction in the viability of MRSA cells was seen 1 h following a single treatment with ClyS. One intraperitoneal dose of ClyS also protected against death by MRSA in a mouse septicemia model. ClyS showed a typical pattern of synergistic interactions with both vancomycin and oxacillin in vitro. More importantly, ClyS and oxacillin at doses that were not protective individually protected synergistically against MRSA septic death in a mouse model. These results strongly support the development of ClyS as an attractive addition to the current treatment options of multidrug-resistant S. aureus infections and would allow for the reinstatement of antibiotics shelved because of mounting resistance.Staphylococcus aureus is an opportunistic pathogen inhabiting human skin and mucous membranes and is the causative agent of a variety of skin and soft-tissue infections as well as serious infections, such as pneumonia, meningitis, endocarditis, and osteomyelitis. S. aureus exotoxins also cause disease syndromes such as bullous impetigo, scalded skin syndrome, and toxic shock syndrome. While outbreaks of cutaneous infections in otherwise-healthy people may be managed well without antibiotics (34), in compromised individuals staphylococci are an important cause of life-threatening nosocomial infections, such as ventilator-acquired pneumonia (VAP) (20). The spread of methicillin-resistant S. aureus (MRSA) has been of critical concern to health care providers, and the further distribution of Panton-Valentine leukocidin (PVL) carrying community-acquired MRSA strains to the hospital (33) poses a threat that is even more serious than that of MRSA alone due to the striking pathogenicity of this toxin (2).Currently, 40 to 60% of nosocomial infections of S. aureus are resistant to oxacillin (27), and greater than 60% of the isolates are resistant to methicillin (13). Treating infections caused by drug-resistant S. aureus has become increasingly difficult and therefore is a major concern among health care professionals. To combat this challenge, the development of new and effective antibiotics belonging to different classes are being aggressively pursued. A number of new antimicrobial agents, such as linezolid, quinupristin-dalfopristin, daptomycin, telavancin, new glycopeptides, and ceftobiprole, have been introduced or are under clinical development (1). However, clinical isolates of MRSA with resistance to these new classes of antibiotics have been reported already (25, 38, 41). Consequently, there is an urgent need to develop novel therapeutic agents or antibiotic alternatives that are active against MRSA. As an option, current antibiotics to which MRSA strains are resistant may be resurrected as viable candidates in the treatment of MRSA when used in combination with other agents, offering a new dimension to a dwindling list of potential anti-infectives for this pathogen.The carriage of both methicillin-susceptible S. aureus (MSSA) and MRSA in the human anterior nares is the major reservoir for S. aureus infection. Studies have shown that roughly 80% of the population could be nasally colonized by S. aureus, and that colonization increases the risk factor for developing other more serious S. aureus infections (19). The elimination of nasal carriage in the community or in the hospital setting could reduce the risk of infection and slow the spread of drug-resistant S. aureus (19). Only one agent, intranasal mupirocin, has been approved in this indication. Mupirocin ointment must be applied twice daily to the anterior nares for 3 to 5 days. When used in this manner, mupirocin ointment has been shown to significantly reduce the risk of postoperative staphylococcal infection in patients who were S. aureus carriers (32). However, the value of mupirocin is compromised by a relatively high rate of resistance mutations (6) and by potential compliance issues associated with its method of use. Despite these issues, preoperative prophylaxis with mupirocin already is mandated for cardiovascular surgery (11) and also has been recommended for orthopedic surgery (43). Clearly a superior product for intranasal prophylaxis in at-risk patients would be valuable.Bacteriophage endolysins (lysins) are a new class of antimicrobial agents that are emerging as effective agents for the prevention and treatment of bacterial infections. Lysins are cell wall hydrolases that are produced during the infection cycle of double-stranded DNA bacteriophages, enabling the release of progeny virions. When applied exogenously, native or recombinant lysins are able to cleave the integral peptidoglycan bonds of susceptible Gram-positive bacteria, resulting in rapid cell lysis (30). Lysins have been developed against a number of Gram-positive pathogens, including Streptococcus pyogenes (30), Streptococcus pneumoniae (22), Bacillus anthracis (36), enterococci (46), group B streptococci (5), and Staphylococcus aureus (31, 35). The efficacy of most of these lysins has been demonstrated in in vivo models. Several unique characteristics of lysins make them attractive antibacterial candidates against Gram-positive pathogens. These include (i) rapid antibacterial activity both in vitro and in vivo; (ii) narrow lytic spectrum (species specific); (iii) strong receptor-binding affinity, typically in the nanomolar range; (iv) very low probability of developing resistance, since the binding epitopes on the bacteria are essential for viability; (v) safety; and (vi) relative ease of modification by genetic engineering (12).The development of a highly active S. aureus-specific lysin has been challenging either due to the lack of expression in a heterologous host, the insolubility of the expressed protein, or poor expression, except for the results of one report (to our knowledge) of a S. aureus-specific lysin from phage phiMR11 (35). To circumvent these issues, a few studies have reported the construction of truncated (16) or chimeric versions of lysins (26).Typically, lysins have two distinct functional domains consisting of an N-terminal catalytic domain for peptidoglycan hydrolysis and a C-terminal binding domain for the recognition of surface moieties on the bacterial cell walls. The catalytic domains are relatively conserved among lysins, and the activities can be classified into three basic groups based on peptidoglycan bond specificity: (i) glycosidases that hydrolyze linkages within the amino sugar moieties; (ii) endopeptidases that cleave the peptide moiety; and (iii) amidases that hydrolyze the amide bond connecting the glycan strand and stem peptide. The binding domains, however, are not conserved among lysins. Hence, the binding domain often imparts species specificity, because the binding targets, typically carbohydrates associated with the peptidoglycan, display species-specific distribution (V. A. Fischetti, personal communication). The modular architecture of lysins is an important feature with respect to their development as antimicrobial agents. This enables the creation of chimeric enzymes by swapping lysin domains and thereby altering binding specificity, enzymatic activity, or both (7, 10, 23, 37).In this paper, we describe the genetic engineering of a novel chimeric lysin constructed by fusing the catalytic domain of a Staphylococcus-specific phage lysin with a unique binding domain from a different Staphylococcus phage lysin. This engineered lysin, called ClyS (for chimeric lysin for staphylococci), was soluble and highly active, and it displayed rapid and specific lytic activity against susceptible and drug-resistant staphylococci. We demonstrate the protective activity of ClyS in in vivo colonization and septicemia models as well as its synergistic activity with oxacillin in in vitro and in vivo models. These results highlight the potential of ClyS as a novel therapeutic agent for the treatment of MRSA and other staphylococcal infections.