May 2000
Volume 41, Issue 6
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Immunology and Microbiology  |   May 2000
Lysostaphin Treatment of Methicillin-Resistant Staphylococcus aureus Keratitis in the Rabbit
Author Affiliations
  • Joseph J. Dajcs
    From the Department of Microbiology, Immunology, and Parasitology, LSU Medical Center in New Orleans; and
  • Emma B. H. Hume
    From the Department of Microbiology, Immunology, and Parasitology, LSU Medical Center in New Orleans; and
  • Judy M. Moreau
    From the Department of Microbiology, Immunology, and Parasitology, LSU Medical Center in New Orleans; and
  • Armando R. Caballero
    From the Department of Microbiology, Immunology, and Parasitology, LSU Medical Center in New Orleans; and
    Department of Microbiology and the Louisiana State University Eye Center, New Orleans, Louisiana.
  • Bennetta M. Cannon
    From the Department of Microbiology, Immunology, and Parasitology, LSU Medical Center in New Orleans; and
  • Richard J. O’Callaghan
    From the Department of Microbiology, Immunology, and Parasitology, LSU Medical Center in New Orleans; and
    Department of Microbiology and the Louisiana State University Eye Center, New Orleans, Louisiana.
Investigative Ophthalmology & Visual Science May 2000, Vol.41, 1432-1437. doi:
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      Joseph J. Dajcs, Emma B. H. Hume, Judy M. Moreau, Armando R. Caballero, Bennetta M. Cannon, Richard J. O’Callaghan; Lysostaphin Treatment of Methicillin-Resistant Staphylococcus aureus Keratitis in the Rabbit. Invest. Ophthalmol. Vis. Sci. 2000;41(6):1432-1437.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Abstract

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 ≤ 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 ≤ 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.

In most populations of the United States, Staphylococcus aureus is a leading cause of bacterial keratitis, especially among individuals with a previously compromised cornea. 1 2 3 4 Patients with epithelial trauma caused by contact lens wear or foreign bodies are more susceptible to Staphylococcus keratitis. 5 6 Staphylococcus adheres to protein deposits on contact lenses, permitting bacteria to come in contact with the corneal surface. 7 8 Tissue damage during Staphylococcus keratitis results from the action of bacterial products 9 and from the host inflammatory response to infection. 10 Staphylococcus keratitis can result in irreversible corneal scarring, resulting in loss of visual acuity or in blindness. 5  
Topical cefazolin, often used in combination with an aminoglycoside, or a fluoroquinolone (ciprofloxacin or ofloxacin) are the antibiotics most often prescribed for treating Staphylococcus keratitis. 5 11 12 13 Topical antibiotic drops are applied as frequently as every 15 to 30 minutes for 48 hours or longer. Methicillin-resistant Staphylococcus aureus (MRSA) strains have been treated successfully with ciprofloxacin 14 ; however, during the 1990s the susceptibility of MRSA strains to fluoroquinolones declined rapidly. Less than half of the current MRSA isolates remain susceptible to ciprofloxacin or ofloxacin. 15 16 17 18 19 The increasing incidence of fluoroquinolone-resistant MRSA strains has resulted in the more frequent use of vancomycin therapy for most MRSA infections. 15 16 20 21 22 23 However, vancomycin is a slow-acting antibiotic that has significant side effects. 20 24 Also of great concern is the recent emergence of rare mutant MRSA strains not susceptible to vancomycin. 25 26 27 28 Furthermore, there is concern that plasmid-borne vancomycin resistance will be transferred from Enterococcus faecalis to MRSA strains, creating multiple strains with resistance to essentially all available antibiotics. Vancomycin resistance has been conjugally transferred under laboratory conditions from E. faecalis to MRSA. 29 Hence the search for new antimicrobial agents is essential. 
Lysostaphin, a zinc metalloproteinase extracted from Staphylococcus simulans, can lyse S. aureus by disrupting its peptidoglycan layer. 30 31 The gene for lysostaphin has been successfully cloned and expressed in Bacillus sphaericus and Escherichia coli. 32 The major substrate for lysostaphin is the staphylococcal cell wall, 33 specifically the pentaglycine bridge found in the cell wall of S. aureus. 34 35 36 Lysostaphin has a molecular weight of 27 kDa 34 and contains one molecule of zinc per mole of protein. 36 The enzyme is destroyed by pepsin or trypsin and inhibited by Hg2+, Cu2+, and Zn2+ ions. 37  
The use of lysostaphin for chemotherapy was proposed over 30 years ago. 35 37 Lysostaphin purified from S. simulans was found to be effective in treating experimental staphylococcal infections in various nonocular animal models 35 38 and was once used systemically in a human neutropenic patient to treat staphylococcal abscesses. 39 Lysostaphin was also shown to be effective in reducing the nasal carriage of S. aureus in humans. 34 40 41  
Lysostaphin is now being reexamined as an antibacterial therapy because antibiotic resistance has become prevalent for many S. aureus strains. 42 43 44 Lysostaphin, to date, has not been described in the therapy of ocular infections or in the treatment of experimental ocular infections. 
Materials and Methods
Bacteria
MRSA strain 301 used in these studies was isolated from a human corneal ulcer and was previously analyzed in a rabbit keratitis model. 24 S. aureus strain ISP546, a methicillin-sensitive (MSSA) strain, is deficient in the accessory global regulator (agr) 45 46 and does not produce α-toxin, which is the main toxin associated with virulence and mediates corneal erosions. 9  
Minimal Inhibitory Concentration
Minimal inhibitory concentrations of lysostaphin and vancomycin were determined by the tube-broth dilution method using Mueller–Hinton broth (Difco, Detroit, MI) supplemented with 5% sodium chloride. 9 Approximately 105 colony-forming units (CFU)/ml of S. aureus were added to doubling dilutions of antibiotic and incubated at 35°C for 24 hours. The MIC was designated as the lowest concentration that inhibited growth of Staphylococcus as determined by the lack of turbidity. The MIC90 is the lowest concentration that inhibited 90% of the strains tested. 
Lysostaphin Inhibition Assay
MRSA 301 was grown for 24 hours in tryptic soy broth (TSB; Difco) at 37°C and washed three times in sterile Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.5). Approximately 109 CFU of MRSA 301 were resuspended in Tris-buffered saline with 0, 0.5, 1, 2.5, 5, 10, 50, 100, or 200 mM ZnCl. Lysostaphin (100 μg/ml) was added to the bacteria resuspended in buffered saline with ZnCl. After incubation at 37°C for 1 hour, the optical density was measured at a wavelength of 620 nm. The change in optical density (from 0.21 to 0.00 OD) between bacterial suspensions with lysostaphin and those without lysostaphin was determined as a measure of bacterial cell lysis, resulting from the action of lysostaphin. 30 47 The reduced change in optical density of samples with ZnCl measured the inhibition of lysostaphin activity. ZnCl at concentrations of 100 mM or higher inhibited approximately 94% of the lysostaphin activity. 
Rabbits
New Zealand White rabbits (2.0–3.0 kg) were treated and maintained in accordance with the tenets of the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research and in strict accordance with the institutional guidelines and The Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80-23). All rabbits were anesthetized by subcutaneous injection of a 1:5 mixture of xylazine (100 mg/ml, Rompum; Miles Laboratories, Shawnee, KS) and ketamine hydrochloride (100 mg/ml, Ketaset; Bristol Laboratories, Syracuse, NY). Proparacaine hydrochloride (0.5% Alcaine; Alcon Laboratories, Fort Worth, TX) was topically applied to each eye before intrastromal injection. 
Injection Model
MRSA 301 and ISP546 were grown to log phase in TSB (Difco) at 37°C and then diluted in TSB to approximately 10,000 CFU/ml for injection into corneas. Each cornea was intrastromally injected with 10μ l containing approximately 100 CFU as previously described. 11  
Antibiotic Preparation
Lysostaphin (Sigma, St. Louis, MO) was dissolved in sterile deionized water to a concentration of 2.8 mg/ml (0.28%). Vancomycin (Vancoled; Lederle Pharmaceuticals, Carolina, Puerto Rico) was dissolved in sterile deionized water and further diluted 1:4 in artificial tears (Tears Naturale Free; Alcon, Humacao, Puerto Rico) to a final concentration of 50 mg/ml (5.0%), the concentration recommended for clinical use. The pH of the vancomycin solution was adjusted to 6.5 with HCl before diluting in artificial tears. All antibiotics were prepared immediately before use and kept at 0 to 4°C. 
Treatment Schedule
Rabbits were topically treated for 5 hours postinfection with a single topical drop (45 μl) applied every 30 minutes. The treatment schedules were from 4 to 9 or 10 to 15 hours postinfection. Rabbits were killed 1 hour after the last treatment. Rabbits were randomly divided into three groups: group 1 received 0.28% lysostaphin, group 2 received 5.0% vancomycin, and group 3 was untreated. 
Bacterial Quantification
Corneas were prepared for bacterial quantification as previously described. 11 Briefly, corneas were removed aseptically, dissected, and homogenized in sterile buffered saline using a tissue homogenizer (Tekmar, Cincinnati, OH). Aliquots of corneal homogenates were serially diluted in buffered saline, plated in triplicate on tryptic soy agar plates (TSA; Difco), and incubated for 24 hours at 37°C. Saline solutions were buffered with either sodium phosphate (100 mM) or with Tris (50 mM; Sigma) supplemented with zinc chloride (200 mM). The number of viable S. aureus per cornea was expressed as base 10 logarithms. 
Slit Lamp Examinations
Slit lamp examinations (SLE) of rabbit eyes were performed using a Topcon biomicroscope (Koaku Kikai K.K., Tokyo, Japan) by two masked observers. Each of seven ocular parameters (injection, chemosis, corneal infiltrate, corneal edema, fibrin in the anterior chamber, hypopyon formation, and iritis) were graded on a scale of 0 to 4. The parameter grades were totaled to produce a single SLE score ranging from 0 (normal eye) to a theoretical maximum of 28, as previously described. 11 Corneal erosions were detected using fluoroscein (Fluor-I-Strip A.T.; Everst Laboratories, Philadelphia, PA), diameters were measured, and values were expressed in millimeters. 
Statistical Analysis
Data were analyzed using the Statistical Analysis System (Cary, NC) program for personal computers. For CFU determinations, analysis of variance and Student’s t-tests between least-squared means from each group showing statistical variances were performed. For SLE scores, nonparametric one-way analysis of variance (Kruskal–Wallis test) and Wilcoxon’s test were used for comparison among groups. P values ≤ 0.05 were considered significant. 
Results
Susceptibility of Strains
MICs of lysostaphin were determined for 34 strains of MRSA and 12 strains of MSSA (Table 1) . The MIC90 for MRSA were 2-fold lower than that of MSSA. All groups had an equivalent MIC90 for vancomycin. The MIC90 concentration of lysostaphin was 19.5-fold lower than the MIC90 concentration of vancomycin for MRSA and 39-fold lower than the MIC90 of vancomycin for MSSA. 
Treatment of Experimental MRSA Keratitis
With early therapy (4–9 hours postinfection), lysostaphin sterilized all corneas, whereas untreated corneas contained 6.52 ± 0.10 log CFU/cornea (P ≤ 0.0001; Fig. 1A ). No eyes treated with vancomycin were sterile, and these eyes had significantly more CFU per cornea than eyes treated with lysostaphin (P = 0.005; 2.30 ± 0.85 log CFU vs. 0.0 log CFU per cornea, respectively). 
When therapy was begun later (10–15 hours postinfection), lysostaphin reduced the CFU/cornea to 0.85 ± 0.46 log CFU compared to 6.59 ± 0.12 log CFU/cornea in the untreated eyes (P ≤ 0.0001; Fig. 1B ). In contrast, the number of log CFU per cornea in the vancomycin-treated group was not significantly different from the untreated group (5.83 ± 0.16; P = 0.1364). Lysostaphin therapy late in infection reduced the CFU/cornea approximately 100,000-fold more efficiently than vancomycin therapy. 
Treatment of Experimental Staphylococcus Keratitis without a Corneal Defect
To determine the ability of lysostaphin to penetrate the intact cornea, S. aureus strain ISP546, an agr-deficient mutant lacking the ability to cause corneal epithelial erosion was used. 45 46 When therapy of ISP546 infections began at 10 to 15 hours postinfection, lysostaphin penetrated the intact corneal epithelium and significantly reduced the CFU/cornea to 0.58 ± 0.34 log CFU/cornea compared with 5.94 ± 0.24 log CFU/cornea of untreated eyes (P ≤ 0.0001; Fig. 2 ). Vancomycin treatment of ISP546 keratitis resulted in a value not significantly different from the untreated eyes (5.41 ± 0.11 log CFU/cornea; P = 0.3677). Unlike infections with MRSA 301, no erosions were detectable by SLE with fluoroscein during the course of ISP546 infection. 
Evidence of In Situ Killing of S. aureus
To confirm that lysostaphin was penetrating the cornea, an experiment was performed in which the corneas were placed into an inhibitor of lysostaphin (ZnCl) immediately upon harvesting. The ZnCl (200 mM) inhibited potential lysostaphin-mediated killing of Staphylococcus during corneal homogenization and culturing. Lysostaphin was administered 4 to 9 hours postinfection, and the presence of ZnCl did not alter the experimental results compared with previous experiments in which ZnCl was not used. Lysostaphin sterilized 75% of treated corneas (0.33 ± 0.33 log CFU/cornea) compared with the untreated control group where no corneas were sterile (6.48 ± 0.10 log CFU/cornea; P ≤ 0.0001). 
In another experiment, rabbit eyes were infected with MRSA 301 and treated with 0.28% solution of lysostaphin every 30 minutes for 5 hours (4 to 9 hours postinfection). These treated eyes were found to develop only limited pathology through 25 hours postinfection (Fig. 3) , and these changes diminished with time such that by 36 hours postinfection the lysostaphin-treated eyes had SLE scores of less than 5, indicating minimal evidence of infection. These lysostaphin-treated rabbit eyes were observed for 6 additional days, with the SLE score reaching a value of 0 by day 2. There was no recurrent infection in these eyes over the next 4 days. This is in contrast with untreated rabbits infected with MRSA 301 whose SLE scores exceeded values of 18 by 25 hours postinfection and who had to be killed. 
Discussion
The present study demonstrated that lysostaphin is a highly effective therapy for experimental keratitis caused by S. aureus. Lysostaphin appears to be more effective than any other drug tested in the treatment of experimental S. aureus keratitis. 48 49 Early therapy with lysostaphin sterilized the infected corneas, including those infected with a MRSA strain. Lysostaphin also effectively killed a low-virulence strain of S. aureus, indicating that its effectiveness in the rabbit eye was not dependent on extensive erosion of the corneal epithelium. Lysostaphin had MIC values for multi–drug-resistant S. aureus that were similar to that of methicillin-sensitive S. aureus. Lysostaphin had MIC90 values 19.5- and 39-fold lower than those of vancomycin for MRSA and MSSA, respectively. 
The reduction in bacterial CFU in corneas treated with lysostaphin was shown to be due to in situ killing of bacteria and not the action of residual lysostaphin action in corneal homogenates. Furthermore, infected eyes treated with lysostaphin were observed over an extended period (7 days) and found to become free of discernable pathologic changes. 
Lysostaphin was an effective therapy during the late phases of Staphylococcus infection when bacterial replication was minimal. This finding agrees with the in vitro studies showing that lysostaphin’s activity is lethal to S. aureus, regardless of their metabolic state. 35 The lysing of actively multiplying, resting, or dead Staphylococcus is an unusual trait among antibiotics, and such activity evidences the potential of lysostaphin as an ocular antimicrobial therapy. 36 Tobramycin is the only other antimicrobial agent found to maintain its effectiveness in both the early and late phases of experimental Staphylococcus keratitis. 48 Tobramycin, however, has far less potency than lysostaphin and is not effective against MRSA strains. 48  
The ability of lysostaphin to penetrate the cornea could be related to its enzymatic activity. Lysostaphin has weak, but significant, proteolytic activity on mammalian tissue. 50 Lysostaphin was shown to be effective in degrading elastin, which has a high glycine content. 50 This proteolytic action could augment lysostaphin’s penetration through the epithelial barrier of the cornea. Such proteolytic action could be particularly important in understanding the effectiveness of lysostaphin in eyes infected with methicillin-sensitive strains of low virulence. The methicillin-sensitive strain was chosen for analysis because it fails to produce any visible defects in the corneal epithelium. The in vivo susceptibility of the low-virulence strain to lysostaphin illustrates the ability of the enzyme to penetrate the cornea. 
Previous studies have shown vancomycin to induce conjunctival inflammation and corneal edema. 20 24 Dissolving vancomycin in artificial tears was shown to significantly reduce, but not eliminate, this irritation. 20 Lysostaphin, however, did not show any irritation, as graded by slit lamp examination. Further studies of ocular lysostaphin administration are needed to determine whether any adverse effects are induced by repeat topical application of this enzyme. Because lysostaphin is a bacterial protein of 27 kDa, it has the potential to induce immunologic reactions. 
Lysostaphin has been investigated periodically over the past 30 years as a therapy for humans 39 and as an experimental systemic therapy in an animal model of infection (i.e., endocarditis). 42 Lysostaphin has been applied safely and effectively to human nasal passages of Staphylococcus carriers. 34 40 41 Although rechallenge of most subjects with a second intranasal application of lysostaphin was accomplished without reaction, 34 further study of the immune response has not yet been performed. The systemic use of lysostaphin in the past has not been encouraged because of the immunogenicity from the previously impure protein. However, the current availability of recombinant lysostaphin may provide an opportunity for a single, continuous, brief course of therapy. 39  
 
Table 1.
 
Determination of the MIC90 of Lysostaphin for S. aureus
Table 1.
 
Determination of the MIC90 of Lysostaphin for S. aureus
Strain Vancomycin (μg/ml)* Lysostaphin (μg/ml)* Vancomycin to Lysostaphin Ratio
S. aureus
Methicillin-sensitive, † 1.2 0.031 39
(0.6104–1.2207) (0.0078–0.0625)
Methicillin-resistant, † 1.2207 0.0625 19.5
(0.3052–2.4414) (0.0039–2.5)
Figure 1.
 
Lysostaphin treatment of rabbit eyes infected with MRSA 301. Rabbit corneas were infected with approximately 100 CFU of MRSA 301 and treated with vancomycin (50 mg/ml) or lysostaphin (2.8 mg/ml). (A) Rabbit eyes were treated every 30 minutes from 4 to 9 hours postinfection. (B) Rabbit eyes were treated every 30 minutes from 10 to 15 hours postinfection. All rabbits were killed 1 hour after treatment, and corneas were removed aseptically, dissected, and homogenized in sterile phosphate-buffered saline. Aliquots of corneal homogenates were serially diluted, plated in triplicate on tryptic soy agar plates, and incubated for 24 hours at 37°C. The number of viable S. aureus per cornea was expressed as base 10 logarithms ± SEM.
Figure 1.
 
Lysostaphin treatment of rabbit eyes infected with MRSA 301. Rabbit corneas were infected with approximately 100 CFU of MRSA 301 and treated with vancomycin (50 mg/ml) or lysostaphin (2.8 mg/ml). (A) Rabbit eyes were treated every 30 minutes from 4 to 9 hours postinfection. (B) Rabbit eyes were treated every 30 minutes from 10 to 15 hours postinfection. All rabbits were killed 1 hour after treatment, and corneas were removed aseptically, dissected, and homogenized in sterile phosphate-buffered saline. Aliquots of corneal homogenates were serially diluted, plated in triplicate on tryptic soy agar plates, and incubated for 24 hours at 37°C. The number of viable S. aureus per cornea was expressed as base 10 logarithms ± SEM.
Figure 2.
 
Lysostaphin treatment of rabbit eyes infected with S. aureus ISP546, a mutant strain unable to cause epithelial defects. Rabbit corneas were infected with approximately 100 CFU of S. aureus strain ISP546 and treated with vancomycin (50 mg/ml) or lysostaphin (2.8 mg/ml) every 30 minutes from 10 to 15 hours postinfection. Rabbits were killed 1 hour after treatment, and corneas were removed aseptically, dissected, and homogenized in sterile phosphate-buffered saline. Aliquots of corneal homogenates were serially diluted, plated in triplicate on tryptic soy agar plates, and incubated for 24 hours at 37°C. The number of viable S. aureus per cornea was expressed as base 10 logarithms ± SEM.
Figure 2.
 
Lysostaphin treatment of rabbit eyes infected with S. aureus ISP546, a mutant strain unable to cause epithelial defects. Rabbit corneas were infected with approximately 100 CFU of S. aureus strain ISP546 and treated with vancomycin (50 mg/ml) or lysostaphin (2.8 mg/ml) every 30 minutes from 10 to 15 hours postinfection. Rabbits were killed 1 hour after treatment, and corneas were removed aseptically, dissected, and homogenized in sterile phosphate-buffered saline. Aliquots of corneal homogenates were serially diluted, plated in triplicate on tryptic soy agar plates, and incubated for 24 hours at 37°C. The number of viable S. aureus per cornea was expressed as base 10 logarithms ± SEM.
Figure 3.
 
Extended analysis of lysostaphin effectiveness for experimental Staphylococcus keratitis. Rabbit corneas were infected with approximately 100 CFU of MRSA 301 and treated with lysostaphin (2.8 mg/ml) every 30 minutes from 4 to 9 hours postinfection. Rabbits were slit lamp examined every 5 hours for 30 hours and subsequently examined daily for 7 days. Control rabbits were killed at 25 hours postinfection.
Figure 3.
 
Extended analysis of lysostaphin effectiveness for experimental Staphylococcus keratitis. Rabbit corneas were infected with approximately 100 CFU of MRSA 301 and treated with lysostaphin (2.8 mg/ml) every 30 minutes from 4 to 9 hours postinfection. Rabbits were slit lamp examined every 5 hours for 30 hours and subsequently examined daily for 7 days. Control rabbits were killed at 25 hours postinfection.
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Figure 1.
 
Lysostaphin treatment of rabbit eyes infected with MRSA 301. Rabbit corneas were infected with approximately 100 CFU of MRSA 301 and treated with vancomycin (50 mg/ml) or lysostaphin (2.8 mg/ml). (A) Rabbit eyes were treated every 30 minutes from 4 to 9 hours postinfection. (B) Rabbit eyes were treated every 30 minutes from 10 to 15 hours postinfection. All rabbits were killed 1 hour after treatment, and corneas were removed aseptically, dissected, and homogenized in sterile phosphate-buffered saline. Aliquots of corneal homogenates were serially diluted, plated in triplicate on tryptic soy agar plates, and incubated for 24 hours at 37°C. The number of viable S. aureus per cornea was expressed as base 10 logarithms ± SEM.
Figure 1.
 
Lysostaphin treatment of rabbit eyes infected with MRSA 301. Rabbit corneas were infected with approximately 100 CFU of MRSA 301 and treated with vancomycin (50 mg/ml) or lysostaphin (2.8 mg/ml). (A) Rabbit eyes were treated every 30 minutes from 4 to 9 hours postinfection. (B) Rabbit eyes were treated every 30 minutes from 10 to 15 hours postinfection. All rabbits were killed 1 hour after treatment, and corneas were removed aseptically, dissected, and homogenized in sterile phosphate-buffered saline. Aliquots of corneal homogenates were serially diluted, plated in triplicate on tryptic soy agar plates, and incubated for 24 hours at 37°C. The number of viable S. aureus per cornea was expressed as base 10 logarithms ± SEM.
Figure 2.
 
Lysostaphin treatment of rabbit eyes infected with S. aureus ISP546, a mutant strain unable to cause epithelial defects. Rabbit corneas were infected with approximately 100 CFU of S. aureus strain ISP546 and treated with vancomycin (50 mg/ml) or lysostaphin (2.8 mg/ml) every 30 minutes from 10 to 15 hours postinfection. Rabbits were killed 1 hour after treatment, and corneas were removed aseptically, dissected, and homogenized in sterile phosphate-buffered saline. Aliquots of corneal homogenates were serially diluted, plated in triplicate on tryptic soy agar plates, and incubated for 24 hours at 37°C. The number of viable S. aureus per cornea was expressed as base 10 logarithms ± SEM.
Figure 2.
 
Lysostaphin treatment of rabbit eyes infected with S. aureus ISP546, a mutant strain unable to cause epithelial defects. Rabbit corneas were infected with approximately 100 CFU of S. aureus strain ISP546 and treated with vancomycin (50 mg/ml) or lysostaphin (2.8 mg/ml) every 30 minutes from 10 to 15 hours postinfection. Rabbits were killed 1 hour after treatment, and corneas were removed aseptically, dissected, and homogenized in sterile phosphate-buffered saline. Aliquots of corneal homogenates were serially diluted, plated in triplicate on tryptic soy agar plates, and incubated for 24 hours at 37°C. The number of viable S. aureus per cornea was expressed as base 10 logarithms ± SEM.
Figure 3.
 
Extended analysis of lysostaphin effectiveness for experimental Staphylococcus keratitis. Rabbit corneas were infected with approximately 100 CFU of MRSA 301 and treated with lysostaphin (2.8 mg/ml) every 30 minutes from 4 to 9 hours postinfection. Rabbits were slit lamp examined every 5 hours for 30 hours and subsequently examined daily for 7 days. Control rabbits were killed at 25 hours postinfection.
Figure 3.
 
Extended analysis of lysostaphin effectiveness for experimental Staphylococcus keratitis. Rabbit corneas were infected with approximately 100 CFU of MRSA 301 and treated with lysostaphin (2.8 mg/ml) every 30 minutes from 4 to 9 hours postinfection. Rabbits were slit lamp examined every 5 hours for 30 hours and subsequently examined daily for 7 days. Control rabbits were killed at 25 hours postinfection.
Table 1.
 
Determination of the MIC90 of Lysostaphin for S. aureus
Table 1.
 
Determination of the MIC90 of Lysostaphin for S. aureus
Strain Vancomycin (μg/ml)* Lysostaphin (μg/ml)* Vancomycin to Lysostaphin Ratio
S. aureus
Methicillin-sensitive, † 1.2 0.031 39
(0.6104–1.2207) (0.0078–0.0625)
Methicillin-resistant, † 1.2207 0.0625 19.5
(0.3052–2.4414) (0.0039–2.5)
Copyright 2000 The Association for Research in Vision and Ophthalmology, Inc.
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