Fosfomycin-Daptomycin and Other Fosfomycin Combinations as Alternative Therapies in Experimental Foreign-Body Infection by Methicillin-Resistant Staphylococcus aureus

The efficacy of daptomycin, imipenem, or rifampin with fosfomycin was evaluated and compared with that of daptomycin-rifampin in a tissue cage model infection caused by methicillin-resistant Staphylococcus aureus (MRSA). Strain HUSA 304 was used. The study yielded the following results for MICs (in μg/ml): fosfomycin, 4; daptomycin, 1; imipenem, 0.25; and rifampin, 0.03. The study yielded the following results for minimum bactericidal concentration (MBC) (in μg/ml): fosfomycin, 8; daptomycin, 4; imipenem, 32; and rifampin, 0.5. Daptomycin-rifampin was confirmed as the most effective therapy against MRSA foreign-body infections. Fosfomycin combinations with high doses of daptomycin and rifampin were efficacious alternative therapies in this setting. Fosfomycin-imipenem was relatively ineffective and did not protect against resistance.     

High Activity of Fosfomycin and Rifampin against Methicillin-Resistant Staphylococcus aureus Biofilm In Vitro and in an Experimental Foreign-Body Infection Model

Increasing antimicrobial resistance reduces treatment options for implant-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA). We evaluated the activity of fosfomycin alone and in combination with vancomycin, daptomycin, rifampin, and tigecycline against MRSA (ATCC 43300) in a foreign-body (implantable cage) infection model. The MICs of the individual agents were as follows: fosfomycin, 1 μg/ml; daptomycin, 0.125 μg/ml; vancomycin, 1 μg/ml; rifampin, 0.04 μg/ml; and tigecycline, 0.125 μg/ml. Microcalorimetry showed synergistic activity of fosfomycin and rifampin at subinhibitory concentrations against planktonic and biofilm MRSA. In time-kill curves, fosfomycin exhibited time-dependent activity against MRSA with a reduction of 2.5 log₁₀ CFU/ml at 128 × the MIC. In the animal model, planktonic bacteria in cage fluid were reduced by <1 log₁₀ CFU/ml with fosfomycin and tigecycline, 1.7 log₁₀ with daptomycin, 2.2 log₁₀ with fosfomycin-tigecycline and fosfomycin-vancomycin, 3.8 log₁₀ with fosfomycin-daptomycin, and >6.0 log₁₀ with daptomycin-rifampin and fosfomycin-rifampin. Daptomycin-rifampin cured 67% of cage-associated infections and fosfomycin-rifampin cured 83%, whereas all single drugs (fosfomycin, daptomycin, and tigecycline) and rifampin-free fosfomycin combinations showed no cure of MRSA cage-associated infections. No emergence of fosfomycin resistance was observed in animals; however, a 4-fold increase in fosfomycin MIC (from 2 to 16 μg/ml) occurred in the fosfomycin-vancomycin group. In summary, the highest eradication of MRSA cage-associated infections was achieved with fosfomycin in combination with rifampin (83%). Fosfomycin may be used in combination with rifampin against MRSA implant-associated infections, but it cannot replace rifampin as an antibiofilm agent.     

Comparative Efficacies of Cloxacillin-Daptomycin and the Standard Cloxacillin-Rifampin Therapies against an Experimental Foreign-Body Infection by Methicillin-Susceptible Staphylococcus aureus

We compared the efficacies of daptomycin (doses equivalent to 8 to 10 mg/kg of body weight/day in humans) and cloxacillin alone with those of cloxacillin-rifampin and cloxacillin-daptomycin combinations, using a tissue cage methicillin-susceptible Staphylococcus aureus (MSSA) infection model. Monotherapies were less effective than combinations (P < 0.05), and daptomycin resistance emerged. Cloxacillin-daptomycin proved as effective as cloxacillin-rifampin and prevented the appearance of resistance; this combination may be an alternative anti-MSSA therapy, which may offer greater benefits in the early treatment of prosthetic joint infections (PJI).     

In Vivo Efficacy of Daptomycin against Methicillin-Resistant Staphylococcus aureus in a Mouse Model of Hematogenous Pulmonary Infection

Daptomycin is inactivated by pulmonary surfactant, but its effectiveness in hematogenous pulmonary infection has been poorly studied. The potential therapeutic application was evaluated in a methicillin-resistant Staphylococcus aureus (MRSA) hematogenous pulmonary infection mouse model. Compared with control results, daptomycin improved survival (P < 0.001) and decreased the number of abscesses and bacteria in the lungs (P < 0.01). Daptomycin may be an effective therapeutic option for MRSA hematogenous pulmonary infection.     

Comparison of Tigecycline and Vancomycin for Treatment of Experimental Foreign-Body Infection Due to Methicillin-Resistant Staphylococcus aureus

Twice-daily 7-day regimens of tigecycline (7 mg/kg) and vancomycin (50 mg/kg) were compared in a rat tissue cage model of chronic foreign-body infection due to methicillin (meticillin)-resistant Staphylococcus aureus strain MRGR3. Subcutaneously administered tigecycline reached levels in tissue cage fluid that were nearly equivalent or slightly superior to the antibiotic MIC (0.5 μg/ml) for strain MRGR3. After 7 days, equivalent, significant reductions in bacterial counts were recorded for tigecycline-treated and vancomycin-treated rats, compared with those for untreated animals.Antimicrobial therapy for foreign-body infections due to Staphylococcus aureus is challenging (38), in particular for multidrug-resistant hospital-associated and community-acquired isolates of methicillin (meticillin)-resistant S. aureus (MRSA) (3, 12, 15, 16). Tigecycline is a novel injectable glycylcycline broad-spectrum antibiotic that demonstrates excellent in vitro and in vivo activity against MRSA and other multiresistant organisms (9, 11, 22, 28, 32) and can overcome both major tetracycline resistance mechanisms, namely ribosomal protection (10, 23) and efflux (4, 27). Tigecycline has shown good activity in various animal models of serious MRSA infections (21, 39, 40), as well as against biofilm-embedded bacteria (14, 26).We previously used a rat tissue cage model of S. aureus chronic foreign-body infections for evaluating a number of antimicrobial agents, namely vancomycin (17), teicoplanin (31), imipenem (30), ceftobiprole (37), daptomycin (29, 35), and several fluoroquinolones (2, 17, 36). This study reports the activity of tigecycline compared to that of the reference anti-MRSA agent vancomycin in a tissue cage model of MRSA chronic foreign-body infection.(This study was presented in part at the 18th European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, Spain, April 2008.)MRSA strain MRGR3, whose properties were previously described (2, 5, 17, 29-31, 36, 37), was used for in vitro and in vivo studies. Strain MRGR3 is resistant to methicillin, gentamicin, erythromycin, tetracycline, and chloramphenicol (17).MICs of freshly prepared (1, 13, 25) tigecycline (Wyeth Research, Collegeville, PA) or vancomycin (Vancocin; Teva Pharma AG, Switzerland) for MRSA strain MRGR3 or quality control S. aureus ATCC 29213 were determined by broth macrodilution in cation-adjusted Mueller-Hinton broth (CAMHB), according to Clinical and Laboratory Standards Institute guidelines (7).The animal protocol used for evaluating the in vivo activities of tigecycline and vancomycin was previously described in detail (17, 37) and approved by the Ethics Committee of the Faculty of Medicine, University of Geneva, and the Veterinary Office of the State of Geneva. Three weeks after subcutaneous implantation of four tissue cages per animal in anesthetized Wistar rats (37), tissue cage fluids were checked for sterility (17).Pilot pharmacokinetic studies were performed using groups of noninfected rats to find an adequate dosing regimen of tigecycline for therapy of tissue cage infections as described previously (37). Tigecycline levels in cage fluids (and blood) were estimated by a microbiological assay (21), with a detection limit of 0.25 μg/ml. To account for protein binding, all plasma or tissue cage fluid samples were diluted with 1 volume of phosphate-buffered saline and assayed in duplicate, with reference to duplicate standard concentrations (0.25 to 8 μg/ml) of tigecycline, in phosphate-buffered saline supplemented with 50% plasma or pooled tissue cage fluids, respectively.Each tissue cage was chronically infected by inoculating 5 × 10⁵ CFU of log-phase MRGR3 (37). Two weeks later, all rats whose cage fluids contained ≥10⁵ CFU/ml received twice-daily doses (by the subcutaneous route for 7 days) of tigecycline (7 mg/kg), vancomycin (50 mg/kg), or no antibiotic (control group). Differences in CFU counts of cage fluid quantitative cultures, performed at day 1 (before treatment) and day 8 (12 h after the last injection of either tigecycline or vancomycin), were expressed as the change in number of log₁₀ CFU/ml (37) and evaluated by one-way analysis of variance and post-analysis of variance pairwise comparisons between individual groups via the Tukey HSD test (http://faculty.vassar.edu/lowry/VassarStats.html), using P values of <0.05 with two-tailed significance levels.Tigecycline resistance was screened by plating 10-fold-diluted cage fluids (100 μl) onto MH agar supplemented with 2 μg/ml tigecycline. No single colony grew on tigecycline-supplemented plates inoculated with 10⁸ CFU of in vitro-grown cultures of strain MRGR3.The MIC of tigecycline in CAMHB for MRSA strain MRGR3 was 0.5 μg/ml, namely at the upper limit of susceptibility breakpoints (7), and was unaffected by supplementation of CAMHB with 50% tissue cage fluid (data not shown). Since tigecycline did not produce a 3-log₁₀ reduction in the number of MRGR3 CFU/ml, it was not considered bactericidal. Nevertheless, supra-MIC levels (1, 2, and 4 μg/ml) of tigecycline produced a 2- to 3-log₁₀ decrease in the number of MRGR3 CFU/ml at 24 h. The vancomycin MIC and minimal bactericidal concentration for strain MRGR3 were 1 and 2 μg/ml, respectively (17).Average tigecycline levels, scored for tissue cage fluids (n = 6) from 0 to 12 h after subcutaneous administration, remained quite constant over time, showing ≤3-fold variations between results at different time points and moderate animal-to-animal differences (Fig. ​(Fig.1).1). A 7-mg/kg twice-daily regimen yielded cage fluid levels of 0.39 to 0.70 μg/ml tigecycline at day 4 and 0.33 to 1.01 μg/ml at day 7, such results thus being nearly equivalent or slightly superior to the antibiotic MIC for MRGR3. Tigecycline plasma levels at 2 h on day 4 were 1.87 ± 0.66 μg/ml, in agreement with other reports (8, 21). A 14-mg/kg twice-daily regimen led to plasma and tissue cage fluid tigecycline levels ca. twofold higher than the 7-mg/kg regimen (Fig. ​(Fig.1).1). Average peak and trough cage fluid levels of vancomycin were previously determined (17) as 12 and 2 μg/ml at 4 and 12 h, respectively.Open in a separate windowFIG. 1.Pharmacokinetic levels of tigecycline in tissue cage fluids of rats on day 4 (open symbols) or day 7 (closed symbols) of therapy every 12 h with 7 mg/kg (○) or 14 mg/kg (▵) of tigecycline. Each value is the mean result of six determinations.At day 1, mean bacterial counts for MRGR3-infected cages were not significantly different (P = 0.65) in controls (6.85 ± 0.19 log₁₀ CFU/ml; n = 28), tigecycline-treated rats (6.92 ± 0.13 log₁₀ CFU/ml; n = 29), or vancomycin-treated rats (6.70 ± 0.18 log₁₀ CFU/ml; n = 27). At day 8, significant (P < 0.01 versus controls) reductions were recorded in bacterial counts in cage fluids of both tigecycline-treated (−0.62 ± 0.17 CFU/ml; n = 29) and vancomycin-treated (−0.76 ± 0.18 log₁₀ CFU/ml; n = 27) rats, whereas the bacterial counts for controls slightly increased (+0.18 ± 0.19 log₁₀ CFU/ml; n = 28) (Fig. ​(Fig.2).2). The reductions in CFU counts for vancomycin-treated and tigecycline-treated rats were not significantly different. Finally, no MRGR3 isolate showing increased tigecycline MIC was observed in any posttherapy cage fluid sample (n = 29). The lack of emergence of MRGR3 derivates with diminished susceptibility to tigecycline is consistent with the difficulty in selecting laboratory-derived, tigecycline-resistant mutants of S. aureus (18), and it contrasts with the emergence of resistant subpopulations during low-dose daptomycin therapy of S. aureus-infected tissue cages (35).Open in a separate windowFIG. 2.Decrease in viable counts of MRSA MRGR3 in tissue cage fluids of rats treated for 7 days with tigecycline or vancomycin.Several studies performed with the rat tissue cage model demonstrated the low initial in vivo response of foreign-body-associated chronic MRSA infections (2, 5, 6, 17, 20, 29-31, 35-37). A much greater reduction of viable MRSA counts in cage fluids requires longer periods of antibiotic therapy (5), as found in clinical situations with foreign-body infections (38). Major pharmacokinetic properties of tigecycline, observed in human and animal studies, are very low plasma levels, long half-lives, and high volumes of distribution indicating extensive tigecycline distribution into the tissues (8, 11, 19, 28, 32, 40). In line with previous observations that showed a requirement for active, preferentially bactericidal, antibiotic levels for obtaining significant reductions of CFU counts in MRSA-infected cage fluids (29, 37), we selected for therapy a twice-daily 7-mg/kg regimen yielding cage fluid tigecycline levels above the MIC for strain MRGR3 for >50% of the dosing interval (32, 33), while minimizing the occurrence of side effects previously observed with higher-dose regimens (39). Our regimen is similar to those required for activity in other animal models of hard-to-treat S. aureus infections, such as endocarditis or osteomyelitis (21, 39), although its relevance to human therapy is not fully defined (32). In addition, the incomplete in vitro killing activity of tigecycline, namely a <3-log₁₀ reduction in number of MRGR3 CFU at 24 h, prevents a pharmacodynamic analysis of tigecycline in vivo activity more detailed than those of previously evaluated bactericidal antibiotics in MRSA-infected cages (29, 37). We can also speculate that other properties of tigecycline, namely its in vivo activity against intracellular, slowly growing, or biofilm-forming bacteria, might significantly contribute to tigecycline activity in MRSA-infected cages (34). Indeed, high intracellular levels of tigecycline were shown to accumulate in human polymorphonuclear neutrophils and prevent growth of phagocytized bacteria (24). Further studies are needed to elucidate the mechanisms of tigecycline activity against hard-to-treat MRSA infections.     

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.     

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.     

Efficacy of Daptomycin in Implant-Associated Infection Due to Methicillin-Resistant Staphylococcus aureus: Importance of Combination with Rifampin

Limited treatment options are available for implant-associated infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). We compared the activity of daptomycin (alone and with rifampin [rifampicin]) with the activities of other antimicrobial regimens against MRSA ATCC 43300 in the guinea pig foreign-body infection model. The daptomycin MIC and the minimum bactericidal concentration in logarithmic phase and stationary growth phase of MRSA were 0.625, 0.625, and 20 μg/ml, respectively. In time-kill studies, daptomycin showed rapid and concentration-dependent killing of MRSA in stationary growth phase. At concentrations above 20 μg/ml, daptomycin reduced the counts by >3 log₁₀ CFU/ml in 2 to 4 h. In sterile cage fluid, daptomycin peak concentrations of 23.1, 46.3, and 53.7 μg/ml were reached 4 to 6 h after the administration of single intraperitoneal doses of 20, 30, and 40 mg/kg of body weight, respectively. In treatment studies, daptomycin alone reduced the planktonic MRSA counts by 0.3 log₁₀ CFU/ml, whereas in combination with rifampin, a reduction in the counts of >6 log₁₀ CFU/ml was observed. Vancomycin and daptomycin (at both doses) were unable to cure any cage-associated infection when they were given as monotherapy, whereas rifampin alone cured the infections in 33% of the cages. In combination with rifampin, daptomycin showed cure rates of 25% (at 20 mg/kg) and 67% (at 30 mg/kg), vancomycin showed a cure rate of 8%, linezolid showed a cure rate of 0%, and levofloxacin showed a cure rate of 58%. In addition, daptomycin at a high dose (30 mg/kg) completely prevented the emergence of rifampin resistance in planktonic and adherent MRSA cells. Daptomycin at a high dose, corresponding to 6 mg/kg in humans, in combination with rifampin showed the highest activity against planktonic and adherent MRSA. Daptomycin plus rifampin is a promising treatment option for implant-associated MRSA infections.Implants are increasingly used in modern medicine to replace a compromised biological function or missing anatomical structure. Periprosthetic infections represent a devastating complication, causing high rates of morbidity and consuming considerable health care resources. Implant-associated infections are caused by microorganisms growing adherent to the device surface and embedded in an extracellular polymeric matrix, a complex three-dimensional structure called a microbial biofilm (8). Bacterial communities in biofilms cause persistent infection due to increased resistance to antibiotics and the immune system and the difficulty with eradicating them from the implant (6).Staphylococcus aureus is one of the leading pathogens causing implant-associated infections. Successful treatment requires the use of bactericidal drugs acting on surface-adhering microorganisms, which predominantly exist in the stationary growth phase. Previous in vitro, experimental, and clinical studies demonstrated that rifampin (rifampicin)-containing antimicrobial regimens were able to eradicate staphylococcal biofilms and cure implant-associated infections (23, 25). Quinolones are often used in combination with rifampin in order to prevent the emergence of rifampin resistance (4, 19, 21). However, methicillin (meticillin)-resistant S. aureus (MRSA) strains are often resistant to quinolones. In addition, MRSA strains were recently shown to have decreased susceptibility to vancomycin, reducing the efficacy of this drug. Therefore, alternative drugs for use in combination with rifampin against implant-associated infections are needed (12, 20).Daptomycin is a negatively charged cyclic lipopeptide with bactericidal activity against gram-positive organisms, including MRSA (17). The drug inserts into the bacterial cytoplasmic membrane in a calcium-dependent fashion, leading to rapid cell death without lysis, and causing only minimal inflammation (15). Daptomycin has been well tolerated in healthy volunteers dosed with up to 12 mg/kg of body weight intravenously for 14 days (2). Only limited data on the use of daptomycin in combination with rifampin against staphylococcal implant-associated infections are available.In this study, we investigated the activity of daptomycin against MRSA ATCC 43300 in vitro. In addition, we evaluated the activity of daptomycin in combination with rifampin in a cage-associated infection model in guinea pigs and compared the efficacy of the treatment with the efficacies of three other antibiotics commonly used against MRSA, vancomycin, linezolid, and levofloxacin (alone and in combination with rifampin).(Part of the results of the present study were presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 24 to 29 October 2008 [abstr. B-1000].)     

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.     

Efficacy of Linezolid in Treatment of Experimental Endocarditis Caused by Methicillin-Resistant Staphylococcus aureus

The efficacies of orally (p.o.) dosed linezolid and intravenously (i.v.) dosed vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) in rabbits with experimental aortic-valve endocarditis were investigated. After endocarditis was established with a recent clinical MRSA isolate, rabbits were dosed for 5 days with linezolid (p.o., three times a day) at either 25, 50, or 75 mg/kg of body weight or vancomycin (i.v., twice a day) at 25 mg/kg. The 25-mg/kg linezolid group had a high mortality rate and bacterial counts in the valve vegetations that were not different from those of the controls. Linezolid dosed p.o. at 50 and 75 mg/kg and i.v. vancomycin produced statistically significant reductions in bacterial counts compared to those of the untreated controls. The reduced bacterial counts and culture-negative valve rates for the animals treated with linezolid at 75 mg/kg were similar to those for the vancomycin-treated animals. Concentrations of linezolid in plasma were determined at several points in the dosing regimen. These results suggest that the efficacy of linezolid in this infection model is related to trough levels in plasma that remain above the MIC for this microorganism. At the ineffective dose of linezolid (25 mg/kg) the concentration at sacrifice was 0.045 times the MIC, whereas the concentrations of linezolid in plasma in the 50- and 75-mg/kg groups were 2 and 5 times the MIC at sacrifice, respectively. The results from this experimental model suggest that the oxazolidinone linezolid may be effective for the treatment of serious staphylococcal infections when resistance to other antimicrobials is present.     

Comparative Efficacy of Trovafloxacin in Experimental Endocarditis Caused by Ciprofloxacin-Sensitive, Methicillin-Resistant Staphylococcus aureus

The new fluoroquinolone trovafloxacin was tested against a ciprofloxacin-sensitive, methicillin-resistant Staphylococcus aureus strain in the rabbit model of endocarditis. Trovafloxacin was more effective than vancomycin (CFU/g of vegetation, 2.65 ± 1.87 versus 4.54 ± 2.80 [mean ± standard deviation]; P < 0.05) or ampicillin-sulbactam plus rifampin (4.9 ± 1.1 CFU/g). The addition of ampicillin-sulbactam to trovafloxacin tended to reduce titers further.     

Efficacy of Trovafloxacin against Experimental Staphylococcus aureus Endocarditis

Trovafloxacin is a new fluoronaphthyridone chemically and functionally related to members of the fluoroquinolone class of antimicrobial agents. The in vivo efficacy of the drug was compared with that of vancomycin by using the rabbit model of left-sided endocarditis. Rabbits infected with either a nafcillin-susceptible or -resistant test strain were treated with trovafloxacin (13.3 mg/kg of body weight every 12 h) or vancomycin (25 mg/kg of body weight every 8 h) for 4 days. In comparison with untreated controls, both antimicrobial agents effectively cleared bacteremia and significantly reduced bacterial counts in vegetations and tissues of animals infected with either test strain. No resistance to trovafloxacin emerged in test strains during therapy. We conclude that in this model trovafloxacin is as efficacious as vancomycin is and may serve as a viable alternative to vancomycin for use in humans with similar infections.Trovafloxacin (CP-99,219-27) is a fluoronaphthyridone antimicrobial agent related to fluoroquinolones such as ciprofloxacin and norfloxacin (2). It has potent activity against a broad range of bacterial species, including Staphylococcus aureus (4, 5, 15, 17, 22). The antibacterial activity of trovafloxacin versus S. aureus surpasses that of ciprofloxacin, and trovafloxacin may maintain clinically relevant activity against ciprofloxacin-resistant strains (1).The in vivo activity of trovafloxacin against selected pathogens has been assessed by using animal models, and the drug has shown therapeutic efficacy comparable to that of standard forms of therapy (3, 6, 13, 21). However, the efficacy of trovafloxacin in treating serious S. aureus infections has not been determined. In order to address this issue, we compared the therapeutic activities of trovafloxacin and vancomycin by using the rabbit model of left-sided S. aureus endocarditis. This model affords a severe test of antimicrobial activity in a serious systemic 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.     

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.     

Nine-Hospital Study Comparing Broth Microdilution and Etest Method Results for Vancomycin and Daptomycin against Methicillin-Resistant Staphylococcus aureus

Vancomycin and daptomycin MIC results for 1,800 randomly selected oxacillin (methicillin [meticillin])-resistant Staphylococcus aureus (MRSA) bloodstream isolates from nine U.S. hospitals (collected from 2002 to 2006) were determined by a reference broth microdilution (BMD) method using frozen-form panels with precise incremental dilutions and by the Etest technique. The Etest provided vancomycin and daptomycin MIC results that were consistently higher (0.5 to 1.5 log₂ dilution steps) than those provided by the reference BMD method. The dominant MRSA population (91.2% of MRSA isolates) would be categorized as vancomycin nonsusceptible by the MIC results from the Etest method if the susceptibility breakpoint was adjusted downward to ≤1 μg/ml, as suggested by clinical outcome studies.Vancomycin is still used extensively for the treatment of oxacillin (methicillin [meticillin])-resistant Staphylococcus aureus (MRSA) bacteremia, as well as other less serious MRSA infections. However, vancomycin treatment failure is not uncommon, even when the MRSA strains are fully susceptible to vancomycin according to the criterion (breakpoint MIC, ≤2 μg/ml) used by the Clinical and Laboratory Standards Institute (CLSI). A reduction in the efficacy of vancomycin against MRSA strains for which vancomycin MICs are elevated (to 1 to 2 μg/ml) has been widely reported, suggesting that modest elevations in MICs may explain some suboptimal clinical outcomes (10, 13).The vast majority of clinical laboratories use automated systems and a distinct minority use the disk diffusion method as the routine susceptibility testing technique. However, the disk diffusion method and some automated systems do not accurately detect vancomycin-intermediate S. aureus (7). In addition, there have been a growing number of reports showing discrepancies between in vitro susceptibility test results for vancomycin and clinical outcomes of MRSA infections treated with this glycopeptide (6, 11). Thus, many laboratories are being requested to assess exact MICs by reference or alternative methods, such as that of Etest (AB Biodisk, Solna, Sweden).Although daptomycin remains very active against S. aureus, an association between reduced susceptibility to vancomycin and reduced susceptibility to daptomycin has been reported by some investigators (1). S. aureus isolates classified as daptomycin nonsusceptible according to MICs (≥2 μg/ml) are still rare, and no definitive resistance mechanism has been identified. However, genetic mutations and increases in daptomycin MICs after prolonged vancomycin and/or daptomycin treatment of infections associated with septic arthritis, osteomyelitis, septic thrombophlebitis, and endocarditis, especially in the presence of intravenous catheters and other prosthetic devices, have been reported previously (1). Thus, microbiology laboratories may be required to perform proper daptomycin susceptibility testing. Since a daptomycin disk diffusion test has not been developed, daptomycin susceptibility has to be evaluated by reference methods (2, 3) or the Etest method. In the present study, we evaluated the correlation between vancomycin and daptomycin MICs obtained by the Etest technique and the CLSI reference broth microdilution (BMD) method.(This study was presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy-46th Infectious Diseases Society of America meeting, Washington, DC, 25 to 28 October 2008.)A total of 1,800 MRSA strains from nine hospitals, one in each of the U.S. census regions, were tested. Each medical center contributed 200 MRSA strains collected from patients with bloodstream infections from 2002 through 2006 (target, 40 strains per year per center). The participant centers were as follows: Tufts Medical Center, Boston, MA; New York Hospital Queens, New York, NY; Ochsner Clinic Foundation, New Orleans, LA; University of Colorado Denver, Aurora, CO; University of Nebraska Medical Center, Omaha, NE; University of Washington, Seattle, WA; University of Alabama at Birmingham, Birmingham, AL; Wayne State University, Detroit, MI; and the Medical University of South Carolina, Charleston, SC.MICs of daptomycin and vancomycin were determined by the reference BMD method (using frozen-form panels), with the appropriate medium variation (the addition of 50 mg/liter of calcium) for testing daptomycin (2). Thirty-six precise incremental dilutions of vancomycin between 64 and 0.06 μg/ml (64, 32, 16, 8, 4, 3, 2.5, 2, 1.875, 1.75, 1.625, 1.5, 1.375, 1.25, 1.125, 1, 0.938, 0.875, 0.813, 0.75, 0.688, 0.625, 0.563, 0.5, 0.469, 0.438, 0.406, 0.375, 0.344, 0.313, 0.281, 0.25, 0.188, 0.12, 0.094, and 0.06 μg/ml) were tested, and 20 dilutions of daptomycin between 16 and 0.06 μg/ml (16, 8, 4, 2, 1.5, 1, 0.875, 0.75, 0.625, 0.5, 0.438, 0.375, 0.313, 0.25, 0.219, 0.188, 0.156, 0.12, 0.094, and 0.06 μg/ml) were tested. Isolates were also evaluated by Etest for susceptibilities to daptomycin and vancomycin according to the recommendations of the Etest manufacturer (AB Biodisk). To compare the results obtained with the BMD method and those obtained with the Etest technique, BMD results were rounded up to the next dilution provided for the Etest method. Resistance to oxacillin was confirmed by the reference BMD method (dilution range tested, 0.5 to 4 μg/ml) and cefoxitin susceptibilities tested by disk diffusion (2, 3). Quality control strains S. aureus ATCC 25923 and Enterococcus faecalis ATCC 29212 were evaluated concurrently with every set of tests.MICs of vancomycin obtained by the Etest method were consistently higher (+0.5 to 1.5 log₂ dilutions) than those obtained by the BMD method (Fig. ​(Fig.1a).1a). For strains from all centers combined, the overall vancomycin MIC mode determined by the BMD method was 0.75 μg/ml (corresponding to 75.3% of values, including those rounded up from measured 0.563-, 0.625-, and 0.688-μg/ml MICs) and 96.9% of strains exhibited vancomycin MIC results of ≤1 μg/ml. In contrast, when tested by the Etest, MICs for 58.3 and 32.1% of MRSA strains were 1.5 and 2 μg/ml, respectively (Fig. ​(Fig.1a1a).Open in a separate windowFIG. 1.Scattergrams showing the correlations between vancomycin (a) and daptomycin (b) MICs obtained by the BMD method and the Etest. Solid lines indicate CLSI susceptibility breakpoints, and broken lines indicate complete agreement between results from the two methods.For all strains except one, the Etest yielded higher vancomycin MICs than the reference method. Among 1,628 strains for which the Etest MIC was 1.5 μg/ml (1,050 strains) or 2 μg/ml (578 strains), only 44 (2.7%) had BMD MIC results of >1 μg/ml. Furthermore, among 13 strains considered to be nonsusceptible (intermediate) to vancomycin (MIC, 3 or 4 μg/ml) according to the Etest results, only 1 had a vancomycin MIC result of >2 μg/ml by the reference method, demonstrating a positive predictive value of only 7.7%.Etest MICs of daptomycin were also slightly elevated (+0.5 to 1 log₂ dilution) compared to BMD MICs (Fig. ​(Fig.1b).1b). When tested by the BMD method, the daptomycin MIC mode was 0.19 μg/ml (corresponding to 55.4% of values, including MICs of 0.156 and 0.188 μg/ml) and 92.9% of strains exhibited daptomycin MIC results of ≤0.25 μg/ml. In contrast, when tested by the Etest, the MIC mode was 0.38 μg/ml (corresponding to 37.2% of values), which is 1 doubling dilution higher than that obtained by the BMD method. Among nine strains considered to be daptomycin nonsusceptible according to the Etest results, only two had a MIC result of >1 μg/ml when tested by the reference BMD method, demonstrating a positive predictive value of 22.2% (Fig. ​(Fig.1b1b).Although the emergence of vancomycin-intermediate S. aureus strains and that of vancomycin-resistant S. aureus strains are reasons for concern, these organisms still are extremely rare (12). Nevertheless, a number of studies have demonstrated increased clinical failure with MRSA isolates for which vancomycin MICs are increased (>1 μg/ml) but still within the CLSI-defined susceptibility range (≤2 μg/ml) (3, 11, 13). In addition, there are reports of the increase of vancomycin MICs over time (designated “MIC creep”) at individual institutions, although this trend has not been validated by large, multi-institutional studies (14). Thus, it is becoming clear to physicians that an “S” result for vancomycin is not sufficient to appropriately guide therapy and that the clinical laboratory should provide accurate and reliable MICs.Since most clinical laboratories use automated systems to perform susceptibility testing and these systems do not provide a precise vancomycin MIC, many laboratories are using alternative methods for testing vancomycin in selected cases (6). The Etest method is an attractive option for alternative vancomycin testing since it is easy to perform and cost-effective for testing only one drug-bug combination. However, the results of the present study clearly show that the Etest provides vancomycin and daptomycin MIC results consistently higher (by 0.5 to 1.5 log₂ dilutions) than those provided by precisely performed reference BMD tests. Furthermore, the fact that the higher vancomycin MIC results provided by the Etest appear to be more reliable in predicting vancomycin treatment responses (6) indicates that the vancomycin susceptibility breakpoint should be reevaluated.The susceptibility testing method used may have a large impact on physician decisions for vancomycin and daptomycin treatment of MRSA infections. According to the information presented here, the dominant MRSA population (91.2% of strains) would be categorized as vancomycin nonsusceptible by the Etest method if the susceptibility breakpoint was adjusted to ≤1 μg/ml, as suggested by some published clinical outcome studies on recognizing isolates for which vancomycin MICs are 2 μg/ml (4, 5, 8-10, 14). Daptomycin MICs were also affected by the method used, but to a lesser degree (MICs were found to be 0.5 to 1 dilution step higher by the Etest method than by the reference method, with 0.4% false-nonsusceptible results), and may also suffer from the perception of declining daptomycin potency.     

Vancomycin-Rifampin Combination Therapy Has Enhanced Efficacy against an Experimental Staphylococcus aureus Prosthetic Joint Infection

Treatment of prosthetic joint infections often involves a two-stage exchange, with implant removal and antibiotic spacer placement followed by systemic antibiotic therapy and delayed reimplantation. However, if antibiotic therapy can be improved, one-stage exchange or implant retention may be more feasible, thereby decreasing morbidity and preserving function. In this study, a mouse model of prosthetic joint infection was used in which Staphylococcus aureus was inoculated into a knee joint containing a surgically placed metallic implant extending from the femur. This model was used to evaluate whether combination therapy of vancomycin plus rifampin has increased efficacy compared with vancomycin alone against these infections. On postoperative day 7, vancomycin with or without rifampin was administered for 6 weeks with implant retention. In vivo bioluminescence imaging, ex vivo CFU enumeration, X-ray imaging, and histologic analysis were carried out. We found that there was a marked therapeutic benefit when vancomycin was combined with rifampin compared with vancomycin alone. Taken together, our results suggest that the mouse model used could serve as a valuable in vivo preclinical model system to evaluate and compare efficacies of antibiotics and combinatory therapy for prosthetic joint infections before more extensive studies are carried out in human subjects.