Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing.

Orthopedic implant infections are a significant clinical problem, with current therapies limited to surgical debridement and systemic antibiotic regimens. Lysostaphin is a bacteriolytic enzyme with high antistaphylococcal activity. We engineered a lysostaphin-delivering injectable PEG hydrogel to treat Staphylococcus aureus infections in bone fractures. The injectable hydrogel formulation adheres to exposed tissue and fracture surfaces, ensuring efficient, local delivery of lysostaphin. Lysostaphin encapsulation within this synthetic hydrogel maintained enzyme stability and activity. Lysostaphin-delivering hydrogels exhibited enhanced antibiofilm activity compared with soluble lysostaphin. Lysostaphin-delivering hydrogels eradicated S. aureus infection and outperformed prophylactic antibiotic and soluble lysostaphin therapy in a murine model of femur fracture. Analysis of the local inflammatory response to infections treated with lysostaphin-delivering hydrogels revealed indistinguishable differences in cytokine secretion profiles compared with uninfected fractures, demonstrating clearance of bacteria and associated inflammation. Importantly, infected fractures treated with lysostaphin-delivering hydrogels fully healed by 5 wk with bone formation and mechanical properties equivalent to those of uninfected fractures, whereas fractures treated without the hydrogel carrier were equivalent to untreated infections. Finally, lysostaphin-delivering hydrogels eliminate methicillin-resistant S. aureus infections, supporting this therapy as an alternative to antibiotics. These results indicate that lysostaphin-delivering hydrogels effectively eliminate orthopedic S. aureus infections while simultaneously supporting fracture repair.

Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis.

AIMS: The biofilm produced by Staphylococcus aureus isolates involved in clinical or subclinical bovine mastitis and the activity of nisin and lysostaphin against the preformed biofilm produced by these strains were investigated. METHODS AND RESULTS: Eighteen strains were tested and all produced biofilm. Eight strains with distinct biofilm composition were selected for the antimicrobial activity assays. The minimal inhibitory concentration of each bacteriocin was determined against the planktonic cells and ranged from 15·6 to 500 µg ml(-1) for nisin, and from 3·9 to 50 µg ml(-1) , for lysostaphin. Lysostaphin treatment (0·4 µg ml(-1) ) for 4 h caused a strong Staph. aureus 4181 biofilm detachment and death of the majority of the sessile cells, while nisin treatment (100 µg ml(-1) ) for the same time caused only a great reduction in cell viability. Additionally, combination of both bacteriocins for 4 h resulted in significant death of the sessile cells but no biofilm detachment. CONCLUSIONS: The treatment with lysostaphin alone or in combination with nisin was effective in killing most biofilm sessile cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The action of lysostaphin, either alone or in combination with nisin, against established staphylococcal biofilm may represent an alternative to bovine mastitis control. However, the duration of the treatment should be considered for its application so that the best effectiveness can be achieved.

Evaluation of a lysostaphin-fusion protein as a dry-cow therapy for Staphylococcus aureus mastitis in dairy cattle.

This study evaluated the efficacy of a recombinant lysostaphin fused to a protein transduction domain (rLYS-PTD) as a dry-cow therapy for the treatment of experimentally induced chronic, subclinical Staphylococcus aureus mastitis. Twenty-two Holstein dairy cows were experimentally infected with Staph. aureus in a single pair of diagonal mammary quarters approximately 45d before dry off. Staphylococcus aureus-infected mammary quarters of cows were randomly assigned to 1 of 2 treatment groups at dry off: (1) 279mg of rLYS-PTD in 50mL of vehicle (n=11 cows; 22 quarters) or (2) 50mL of vehicle solution (n=11 cows; 22 quarters) by intramammary infusion. All cows were followed for 30d postpartum to determine cure rates using bacteriologic culture, somatic cell counts, and clinical mastitis scores. No cures were recorded in either the treatment or control groups. Milk somatic cell count, bacterial colony counts, and mastitis scores did not significantly differ between treatment groups. In conclusion, rLYS-PTD was not an effective dry-cow therapeutic for chronic, subclinical Staph. aureus mastitis at the tested dose and formulation.

Bioluminescence and 19F magnetic resonance imaging visualize the efficacy of lysostaphin alone and in combination with oxacillin against Staphylococcus aureus in murine thigh and catheter-associated infection models.

Staphylococci are the leading cause of hospital-acquired infections worldwide. Increasingly, they resist antibiotic treatment owing to the development of multiple antibiotic resistance mechanisms in most strains. Therefore, the activity and efficacy of recombinant lysostaphin as a drug against this pathogen have been evaluated. Lysostaphin exerts high levels of activity against antibiotic-resistant strains of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). The therapeutic value of lysostaphin has been analyzed in two different clinically relevant in vivo models, a catheter-associated infection model and a thigh infection model. We infected mice with luciferase-expressing S. aureus Xen 29, and the efficacies of lysostaphin, vancomycin, oxacillin, and combined lysostaphin-oxacillin were investigated by determining numbers of CFU, detecting bioluminescent signals, and measuring the accumulation of perfluorocarbon emulsion at the site of infection by (19)F magnetic resonance imaging. Lysostaphin treatment significantly reduced the bacterial burden in infected thigh muscles and, after systemic spreading from the catheter, in inner organs. The efficiency of lysostaphin treatment was even more pronounced in combinatorial therapy with oxacillin. These results suggest that recombinant lysostaphin may have potential as an anti-S. aureus drug worthy of further clinical development. In addition, both imaging technologies demonstrated efficacy patterns similar to that of CFU determination, although they proved to be less sensitive. Nonetheless, they served as powerful tools to provide additional information about the course and gravity of infection in a noninvasive manner, possibly allowing a reduction in the number of animals needed for research evaluation of new antibiotics in future studies.

Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands.

Staphylococci cause bovine mastitis, with Staphylococcus aureus being responsible for the majority of the mastitis-based losses to the dairy industry (up to $2 billion/annum). Treatment is primarily with antibiotics, which are often ineffective and potentially contribute to resistance development. Bacteriophage endolysins (peptidoglycan hydrolases) present a promising source of alternative antimicrobials. Here we evaluated two fusion proteins consisting of the streptococcal λSA2 endolysin endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin (λSA2-E-Lyso-SH3b) or the staphylococcal phage K endolysin, LysK (λSA2-E-LysK-SH3b). We demonstrate killing of 16 different S. aureus mastitis isolates, including penicillin-resistant strains, by both constructs. At 100 µg/ml in processed cow milk, λSA2-E-Lyso-SH3b and λSA2-E-LysK-SH3b reduced the S. aureus bacterial load by 3 and 1 log units within 3 h, respectively, compared to a buffer control. In contrast to λSA2-E-Lyso-SH3b, however, λSA2-E-LysK-SH3b permitted regrowth of the pathogen after 1 h. In a mouse model of mastitis, infusion of 25 µg of λSA2-E-Lyso-SH3b or λSA2-E-LysK-SH3b into mammary glands reduced S. aureus CFU by 0.63 or 0.81 log units, compared to >2 log for lysostaphin. Both chimeras were synergistic with lysostaphin against S. aureus in plate lysis checkerboard assays. When tested in combination in mice, λSA2-E-LysK-SH3b and lysostaphin (12.5 µg each/gland) caused a 3.36-log decrease in CFU. Furthermore, most protein treatments reduced gland wet weights and intramammary tumor necrosis factor alpha (TNF-α) concentrations, which serve as indicators of inflammation. Overall, our animal model results demonstrate the potential of fusion peptidoglycan hydrolases as antimicrobials for the treatment of S. aureus-induced mastitis.

Evaluation of the antimicrobial activity of lysostaphin-coated hernia repair meshes.

Bacterial infections by antibiotic-resistant Staphylococcus aureus strains are among the most common postoperative complications in surgical hernia repair with synthetic mesh. Surface coating of medical devices/implants using antibacterial peptides and enzymes has recently emerged as a potentially effective method for preventing infections. The objective of this study was to evaluate the in vitro antimicrobial activity of hernia repair meshes coated by the antimicrobial enzyme lysostaphin at different initial concentrations. Lysostaphin was adsorbed on pieces of polypropylene (Ultrapro) mesh with binding yields of ∼10 to 40% at different coating concentrations of between 10 and 500 µg/ml. Leaching of enzyme from the surface of all the samples was studied in 2% (wt/vol) bovine serum albumin in phosphate-buffered saline buffer at 37°C, and it was found that less than 3% of adsorbed enzyme desorbed from the surface after 24 h of incubation. Studies of antibacterial activity against a cell suspension of S. aureus were performed using turbidity assay and demonstrated that the small amount of enzyme leaching from the mesh surface contributes to the lytic activity of the lysostaphin-coated samples. Colony counting data from the broth count (model for bacteria in wound fluid) and wash count (model for colonized bacteria) for the enzyme-coated samples showed significantly decreased numbers of CFU compared to uncoated samples (P < 0.05). A pilot in vivo study showed a dose-dependent efficacy of lysostaphin-coated meshes in a rat model of S. aureus infection. The antimicrobial activity of the lysostaphin-coated meshes suggests that such enzyme-leaching surfaces could be efficient at actively resisting initial bacterial adhesion and preventing subsequent colonization of hernia repair meshes.

Clinical efficacy of intravaginal recombinant lysostaphin administration on endometritis in sows.

Recombinant lysostaphin has been used for the treatment of cow endometritis and mastitis in China. To our knowledge, no scientific effort has been made to evaluate the efficacy of lysostaphin in sows with clinical endometritis. Lysostaphin, loaded in effervescent tablets that were completely foamed and dissolved within 30 min in the presence of water or body fluids and released active lysostaphin, were administered vaginally on endometritis sows in this study. The clinical recovery, bacterial clearance and reproductive performance of sows with endometritis were investigated. We found that the 400U dosage (400U lysostaphin/pill/time, repeat once on the third day, a total of two times, with 10% oxytetracycline uterine injection as a control) is the most effective treatment. Staphylococcus aureus was the most prevalent finding (34%, n = 188), followed by Streptococcus (32%, n = 181), Escherichia coli (19%, n = 104) and other bacilli (15%, n = 83) before treatment by drugs. Administration of lysostaphin resulted in an extremely significant (p < .01) reduction in S. aureus (0.18 ± 0.25 from 4.57 ± 0.33) and Streptococcus (0.11 ± 0.14 from 3.88 ± 0.29), as well as a significant (p < .05) reduction in E. coli (0.55 ± 0.42 from 3.11 ± 0.14). Mixed infections (83%) were predominant before treatment, in contrast to single infections (61%) after treatment. Large-scale trials were conducted to verify the clinical efficacy of lysostaphin on sow endometritis. The average cure rate of 400u lysostaphin on sow endometritis(82.5%) was higher than the antibiotic group(72.17%). In addition, our results revealed that intravaginal administration of lysostaphin had no adverse effect on the reproductive performance of sows. Thus, this lysostaphin has potential application value as a new method alternative to antibiotics to treat endometritis in sows.

Globally deimmunized lysostaphin evades human immune surveillance and enables highly efficacious repeat dosing.

There is a critical need for novel therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant pathogens, and lysins are among the vanguard of innovative antibiotics under development. Unfortunately, lysins' own microbial origins can elicit detrimental antidrug antibodies (ADAs) that undermine efficacy and threaten patient safety. To create an enhanced anti-MRSA lysin, a novel variant of lysostaphin was engineered by T cell epitope deletion. This "deimmunized" lysostaphin dampened human T cell activation, mitigated ADA responses in human HLA transgenic mice, and enabled safe and efficacious repeated dosing during a 6-week longitudinal infection study. Furthermore, the deimmunized lysostaphin evaded established anti-wild-type immunity, thereby providing significant anti-MRSA protection for animals that were immune experienced to the wild-type enzyme. Last, the enzyme synergized with daptomycin to clear a stringent model of MRSA endocarditis. By mitigating T cell-driven antidrug immunity, deimmunized lysostaphin may enable safe, repeated dosing to treat refractory MRSA infections.

Lysostaphin eradicates established Staphylococcus aureus biofilms in jugular vein catheterized mice.

OBJECTIVES: Staphylococcus aureus infections associated with indwelling devices can be very difficult to treat due to the recalcitrant nature of bacterial biofilms to conventional antibiotics. Lysostaphin has been shown to clear S. aureus biofilms in vitro, and in this study we determined whether lysostaphin could also eradicate established S. aureus biofilms on implanted jugular vein catheters in mice. METHODS: Jugular vein catheterized mice (four to six per group) challenged with S. aureus developed multiorgan infection and biofilm infections on the catheters. The infected mice with established biofilms received various doses of recombinant lysostaphin through the catheters, administered up to three times daily for up to 4 days. Some mice also received lysostaphin combined with nafcillin. Following treatment, mice were sacrificed and cfu on the catheter and in the liver and heart were determined. In another set of experiments, implanted jugular vein catheters in mice were pre-instilled with lysostaphin to determine whether this pre-treatment would protect the mice from biofilm infection. RESULTS AND CONCLUSIONS: Lysostaphin administered at 15 mg/kg in combination with 50 mg/kg nafcillin three times per day for 4 days eradicated established S. aureus, including methicillin-resistant S. aureus, biofilms from implanted catheters and sterilized heart and liver infections of S. aureus-infected mice. Furthermore, a single pre-instillation of 10 mg/kg lysostaphin in catheters completely protected catheterized mice from a subsequent biofilm infection. These results demonstrate that lysostaphin is an effective treatment as well as prophylaxis for S. aureus biofilms on indwelling catheters.

The bactericidal effect of lysostaphin coupled with liposomal vancomycin as a dual combating system applied directly on methicillin-resistant Staphylococcus aureus infected skin wounds in mice.

BACKGROUND AND AIM: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of surgical infection, and its resistance to numerous conventional antibiotics makes treatment difficult. Although vancomycin is often an effective agent for the initial therapy of MRSA, clinical failure sometimes occurs. Therefore, there is an urgent need to develop better therapies. Here, we prepared some vancomycin-loaded nanoliposomes coupled with anti-staphylococcal protein (lysostaphin) and evaluated their in vitro and in vivo efficacy as a topical MRSA therapy. METHODS: Vancomycin was encapsulated in liposomes, and the coupling of lysostaphin with the surface of liposomes was carried out through cyanuric functional groups. The bactericidal efficacies and a full characterization were evaluated. To define different nanoliposomal-bacterium interactions and their bactericidal effect, flow cytometry was employed. Finally, in vivo, the topical antibacterial activity of each formulation was measured against surgical wound MRSA infection in a mouse model. RESULTS: High encapsulation and conjugation efficiency were achieved for all formulations. All the formulations showed a significant reduction in bacterial counts (p<0.05). The targeted liposomes more effectively suppress bacterial infection in vitro and in vivo relative to equivalent doses of untargeted vancomycin liposome. The flow cytometry results confirmed liposome-bacterium interactions, which increased during the incubation time. The maximum binding rate and the bactericidal effect were significantly higher in targeted liposomes (p<0.05) compared with control liposomes. CONCLUSION: Our data suggest a novel nano-vehicle (lysostaphin-conjugated coupled liposomal vancomycin) which could be used as a great topical antimicrobial construct for treatment of MRSA skin infections.

Lysostaphin and BMP-2 co-delivery reduces S. aureus infection and regenerates critical-sized segmental bone defects.

Staphylococcus aureus is the most common pathogen associated with bacterial infections in orthopedic procedures. Infections often lead to implant failure and subsequent removal, motivating the development of bifunctional materials that both promote repair and prevent failure due to infection. Lysostaphin is an anti-staphylococcal enzyme resulting in bacterial lysis and biofilm reduction. Lysostaphin use is limited by the lack of effective delivery methods to provide sustained, high doses of enzyme to infection sites. We engineered a BMP-2-loaded lysostaphin-delivering hydrogel that simultaneously prevents S. aureus infection and repairs nonhealing segmental bone defects in the murine radius. Lysostaphin-delivering hydrogels eradicated S. aureus infection and resulted in mechanically competent bone. Cytokine and immune cell profiling demonstrated that lysostaphin-delivering hydrogels restored the local inflammatory environment to that of a sterile injury. These results show that BMP-2-loaded lysostaphin-delivering hydrogel therapy effectively eliminates S. aureus infection while simultaneously regenerating functional bone resulting in defect healing.

Lysostaphin: an antistaphylococcal agent.

Lysostaphin is a zinc metalloenzyme which has a specific lytic action against Staphylococcus aureus. Lysostaphin has activities of three enzymes namely, glycylglycine endopeptidase, endo-beta-N-acetyl glucosamidase and N-acteyl muramyl-L-alanine amidase. Glycylglycine endopeptidase specifically cleaves the glycine-glycine bonds, unique to the interpeptide cross-bridge of the S. aureus cell wall. Due to its unique specificity, lysostaphin could have high potential in the treatment of antibiotic-resistant staphylococcal infections. This review article presents a current understanding of the lysostaphin and its applications in therapeutic agent as a treatment against antibiotic-resistant S. aureus and methicillin-resistant S. aureus (MRSA) infections, either alone or in combination with other antibiotics.

Staphylococcus biofilm components as targets for vaccines and drugs.

Staphylococci have become the most common cause of nosocomial infections, especially in patients with predisposing factors such as indwelling or implanted foreign polymer bodies. The pathogenesis of foreign-body associated infections with S.aureus and S. epidermidis is mainly related to the ability of these bacteria to form thick, adherent multilayered biofilms. In a biofilm, staphylococci are protected against antibiotic treatment and attack from the immune system, thus making eradication of the infections problematic. This necessitates the discovery of novel prophylactic and therapeutic strategies to treat these infections. In this review, we provide an overview of staphylococcal biofilm components and discuss new possible approaches to controlling these persistent biofilm-dwelling bacteria.

The effectiveness of lysostaphin therapy for experimental coagulase-negative Staphylococcus endophthalmitis.

PURPOSE: To quantitatively determine the effectiveness of lysostaphin therapy for experimental endophthalmitis mediated by coagulase-negative Staphylococcus species, the leading cause of postsurgical endophthalmitis. METHODS: Minimal inhibitory concentrations (MIC) of lysostaphin were determined for 54 isolates representing the following species: S. epidermidis, S. warneri, S. haemolyticus, S. cohnii, S. simulans, and S. capitis. The effectiveness of lysostaphin therapy was tested in an experimental model of endophthalmitis by intravitreally injecting log phase bacteria (100 colony-forming units; cfu) into rabbit eyes (n = 3 eyes per group). At 8 hr postinfection (PI), lysostaphin (250 microg) was injected intravitreally, and the number of cfu/ml of vitreous was determined at 24 hr PI. RESULTS: Average MIC for S. epidermidis was 0.7 microg/ml for 90% of the 33 strains tested. Six methicillin-resistant strains of S. epidermidis (MRSE) had an average MIC of 0.74 micro g/ml. All other species had MIC values of =1.1 microg/ml, except for one strain of S. capitis (MIC = 15.6 microg/ml) and one S. haemolyticus (MIC = 1.41 microg/ml). At 24 hr PI, all untreated eyes had between 5.7 and 8.08 log cfu/ml vitreous humor. Treatment with lysostaphin significantly reduced the cfu/ml as compared with untreated eyes for 13 strains tested in vivo (p = 0.020), but not for two S. haemolyticus strains (p = 0.13), two MRSE strains (p = 0.544), or one S. cohnii strain (p = 0.1366). Treatment with lysostaphin reduced the cfu/ml of methicillin-sensitive S. epidermidis strains by 6 logs; for S. warneri, there was a 2 log reduction; and for the other species a 4 log reduction in cfu/ml relative to untreated eyes. CONCLUSIONS: Lysostaphin was mostly effective in treating coagulase-negative staphylococcal experimental endophthalmitis.

Lysostaphin treatment of methicillin-resistant Staphylococcus aureus keratitis in the rabbit.

PURPOSE: To determine the efficacy of lysostaphin treatment of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a rabbit model. METHODS: The sensitivity to lysostaphin and vancomycin were compared for 34 MRSA and 12 methicillin-sensitive strains. Methicillin-resistant S. aureus strain 301 (MRSA 301) or a methicillin-sensitive strain of low virulence, ISP546, was intrastromally injected into rabbit corneas. Rabbit eyes were treated topically every 30 minutes from 4 to 9 or 10 to 15 hours postinfection with 0.28% lysostaphin or 5.0% vancomycin. Rabbits were killed and corneas were excised and cultured to determine the number of colony forming units (CFU) per cornea. RESULTS: Ninety percent minimal inhibitory concentrations were at least 19-fold lower for lysostaphin than for vancomycin. With early therapy (4 -9 hours postinfection) lysostaphin sterilized all MRSA 301-infected corneas, whereas untreated corneas contained 6.52 log CFU/cornea (P < or = 0.0001). Corneas infected with MRSA 301 and treated similarly with vancomycin retained 2.3 +/-0.85 log CFU/cornea, and none were sterile. When therapy was begun later (10-15 hours postinfection) the residual bacteria in lysostaphin-treated eyes were significantly less numerous than in vancomycin-treated eyes (0.58 +/- 0.34 vs. 5.83 +/- 0.16 log CFU/cornea, respectively; P < or = 0.0001). Three experiments were performed to demonstrate that lysostaphin penetrated the cornea to kill bacteria in vivo; lysostaphin-treated eyes were found to recover from infection, bacteria that did not cause epithelial defects (ISP546) were susceptible to lysostaphin, and inhibition of lysostaphin when harvesting corneas did not alter the observed therapeutic values of lysostaphin. CONCLUSIONS: Lysostaphin is very effective in treating keratitis mediated by methicillin-sensitive or methicillin-resistant S. aureus.

Immunity to lysostaphin and its therapeutic value for ocular MRSA infections in the rabbit.

PURPOSE: To determine the effects of immunization against lysostaphin on the bactericidal action of lysostaphin in ocular tissue and the possible induction of allergic reactions. METHODS: Rabbits were immunized against lysostaphin by subcutaneous, intranasal, or topical routes. Anti-lysostaphin antibody titers were determined by ELISA and by neutralization of lysostaphin. Methicillin-resistant Staphylococcus aureus was intrastromally or intravitreously injected into rabbit eyes. Eyes were treated either topically with drops of lysostaphin (0.3%) or with a single intravitreous injection (0.1 mL) of lysostaphin (0.1%). At the time of death, corneas or vitreous humors were cultured to determine the number of colony forming units (CFU). RESULTS: Rabbits in keratitis experiments that were immunized subcutaneously, intranasally, or topically had serum antibody titers of 10,240, 187, and 1,867, respectively, and neutralization titers of 8 or less. In both normal and immunized rabbits with keratitis, lysostaphin significantly reduced the log CFU to less than 1 log, whereas the untreated eyes contained more than 10(6) CFU/cornea (P < or = 0.0001). Rabbits that were subcutaneously or topically immunized for endophthalmitis experiments had serum antibody titers of 1636 or 137, respectively, and neutralization titers of 2 or less. A single intravitreous injection of lysostaphin (0.1%) sterilized all eyes of immunized and nonimmune rabbits with endophthalmitis. No adverse effects were observed with the administration of lysostaphin to either normal or immunized rabbit eyes. CONCLUSIONS: Lysostaphin treatment of immunized rabbits was effective in treating S. aureus-infected eyes, despite the presence of anti-lysostaphin antibody. No adverse reactions were produced by administration of lysostaphin to immunized rabbits.

Lysostaphin is effective in treating methicillin-resistant Staphylococcus aureus endophthalmitis in the rabbit.

PURPOSE: To determine the effectiveness of lysostaphin treatment of experimental endophthalmitis caused by methicillin-resistant Staphylococcus aureus (MRSA). METHODS: In one experiment, rabbits were injected in the mid-vitreous with 50 or 200 CFU of S. aureus; untreated groups and groups injected intra-vitreally at 8 or 24 hours postinfection with vehicle or lysostaphin (0.1 mg/ml) were compared in terms of CFU/ml vitreous at 24 or 48 hours postinfection. Histopathology of untreated and treated eyes was also compared. To quantify the potency of lysostaphin, additional rabbits were injected with 50 CFU of S. aureus and untreated eyes and eyes treated at 8 hours with 0.001, 0.01 or 0.05 mg/ml were compared in terms of CFU/ml vitreous at 24 hours postinfection. RESULTS: Vitreous of untreated eyes or vehicle-treated eyes injected with 50 or 200 CFU of S. aureus contained 5-10 million CFU/ml at 24 or 48 hours postinfection. All eyes treated with lysostaphin at 8 hours postinfection had less than 1 log CFU/ml in the vitreous (P >or= 0.0001). Similarly, eyes treated with lysostaphin at 24 hours postinfection had approximately 1 log of CFU/ml at 48 hours postinfection. None of the untreated eyes were sterile and 88% or 50% of the eyes treated at 8 or 24 hours postinfection, respectively, were sterile. Eyes treated with lysostaphin at 8, but not 24, hours postinfection had less pronounced pathologic changes than the untreated eyes (P = 0.002). A significant reduction in the CFU/ml vitreous at 24 hours postinfection was obtained by treating infected eyes at 8 hours postinfection with lysostaphin at concentrations of >or=0.001 mg/ml (P

Effects of lysostaphin on Staphylococcus aureus infections of the mouse mammary gland.

A 5 to 6 log10 reduction in the viable count of Staphylococcus aureus was produced in vitro with 10 micrograms lysostaphin ml-1 milk. Infusion of the lactating murine mammary gland with 10 micrograms lysostaphin, immediately following inoculation with 10(8) colony forming units of S aureus, resulted in a significant 2 to 3 log10 reduction in viable S aureus (P less than 0.02) within 30 minutes. Pre-infusion with 10 micrograms lysostaphin either immediately before or one hour before staphylococcal challenge reduced the recovery of S aureus by more than 6 log10 and greatly reduced pathological changes typical of S aureus mastitis. This clearly demonstrates that lysostaphin has considerable potential for the therapeutic or prophylactic control of staphylococcal mastitis.

[Topical use of lysostaphin for Staphylococcus aureus infection of burn wounds in mice].

The effect of lysostaphin on the burn wounds infected with Staphylococcus aureus was studied in mice. The results showed that the mortality and incidence of bacterial isolation in wounds were 17.2% and 8.3%, respectively, in mice treated by lysostaphin, and the figures were significantly lower than that treated by SD-Ag (40.6% and 100%) or base (44.1% and 94.7%). In lysostaphin group the bacterial count of subeschar tissue was 1325 cfu/g, compared with more than 10(9) cfu/g in both SD-Ag and base groups. The results demonstrate that lysostaphin has powerful killing effect on S. aureus, and may be used as atopical antimicrobial to control burn wound infection with S. aureus.

Lysostaphin-coated titan-implants preventing localized osteitis by Staphylococcus aureus in a mouse model.

The increasing incidence of implant-associated infections induced by Staphylococcus aureus (SA) in combination with growing resistance to conventional antibiotics requires novel therapeutic strategies. In the current study we present the first application of the biofilm-penetrating antimicrobial peptide lysostaphin in the context of bone infections. In a standardized implant-associated bone infection model in mice beta-irradiated lysostaphin-coated titanium plates were compared with uncoated plates. Coating of the implant was established with a poly(D,L)-lactide matrix (PDLLA) comprising lysostaphin formulated in a stabilizing and protecting solution (SPS). All mice were osteotomized and infected with a defined count of SA. Fractures were fixed with lysostaphin-coated locking plates. Plates uncoated or PDLLA-coated served as controls. All mice underwent debridement and lavage on Days 7, 14, 28 to determine the bacterial load and local immune reaction. Fracture healing was quantified by conventional radiography. On Day 7 bacterial growth in the lavages of mice with lysostaphin-coated plates showed a significantly lower count to the control groups. Moreover, in the lysostaphin-coated plate groups complete fracture healing were observed on Day 28. The fracture consolidation was accompanied by a diminished local immune reaction. However, control groups developed an osteitis with lysis or destruction of the bone and an evident local immune response. The presented approach of terminally sterilized lysostaphin-coated implants appears to be a promising therapeutic approach for low grade infection or as prophylactic strategy in high risk fracture care e.g. after severe open fractures.