Charles L. Balzli

Chemical Inhibition of Alpha-Toxin, a Key Corneal Virulence Factor of Staphylococcus aureus

PURPOSE alpha-Toxin mediates extreme corneal damage during Staphylococcus aureus keratitis. Chemical inhibition of this toxin was sought to provide relief from toxin-mediated pathology. METHODS Inhibition of alpha-toxin by phosphate-buffered saline (PBS), 0.1% methyl-beta-cyclodextrin (CD), or CD plus cholesterol (0.1%, CD-cholesterol) was assayed by hemolysis of rabbit erythrocytes. Pathologic changes in rabbit corneas injected with 12 hemolytic units of alpha-toxin suspended in PBS, 1% CD, or 1% CD-cholesterol were compared over time. Rabbit corneas injected with 10(2) colony forming units (CFU) of S. aureus were treated from 7 to 13 hours postinfection (PI) with a total of 15 drops of CD-cholesterol, CD, or PBS. Slit lamp examination (SLE) and measurement of erosions were performed at 13 hours PI and bacteria were quantified at 14 hours PI. RESULTS Toxin-mediated lysis of erythrocytes was inhibited up to 16,000-fold in the presence of CD-cholesterol compared with CD or PBS. Eyes injected with alpha-toxin mixed with CD-cholesterol had, at 7 hours postinjection, significantly smaller erosions than eyes injected with alpha-toxin in PBS or alpha-toxin mixed with CD (P = 0.0090 and P = 0.0035, respectively). Eyes infected with S. aureus and treated with CD-cholesterol had significantly lower SLE scores than eyes treated with CD or PBS (P <or= 0.0103 and P <or= 0.0017, respectively); however, there were no differences in the number of bacteria present (P >or= 0.0648). CONCLUSIONS CD-cholesterol is a potent inhibitor of alpha-toxin activity in vitro and an effective means to arrest corneal damage during S. aureus keratitis.

Effectiveness of a new tobramycin (0.3 % ) and dexamethasone (0.05 % ) formulation in the treatment of experimental Pseudomonas keratitis

ABSTRACT Objective: To quantitatively determine, in a Pseudomonas keratitis model, the anti-inflammatory and bactericidal properties of a new formulation of tobramycin (0.3 % ) and dexamethasone (0.05 % ) that utilizes a xanthan gum vehicle. Research methods: In a randomized and masked fashion, rabbit corneas (n ≥ 16 eyes per group) were intrastromally injected with 103 colony-forming units (CFU) of P. aeruginosa. Eyes were untreated or were administered a single drop every 15 min between 16 and 17 h postinfection (PI) and then a single drop every 30 min between 17 and 22 h PI, a total of 15 drops of either 0.1 % dexamethasone and 0.3 % tobramycin (TobraDex*; Tdex) or a new formulation 0.3 % tobramycin and 0.05 % dexamethasone with xanthan gum (TobraDex ST†; ST). Slit lamp examination scores (SLE ± SEM) were derived from grading seven parameters at 22 h PI. Rabbits were sacrificed at 23 h PI and the log CFU ± SEM per cornea was determined. Results: Untreated eyes had SLE scores of 11.11 ± 0.43 and had log CFU of 7.27 ± 0.06. Eyes treated with Tdex, as compared to the untreated eyes, had significantly lower SLE scores (7.39 ± 0.21, p < 0.0001) and significantly fewer bacteria (6.32 ± 0.29 log CFU, p = 0.0213). Eyes treated with ST had a SLE score (6.56 ± 0.19) that was significantly lower than both the untreated eyes (p < 0.0001) and the eyes treated with Tdex (p = 0.0124). Furthermore, eyes treated with ST had significantly fewer log CFU (5.78 ± 0.30) than untreated eyes (p = 0.0001) or eyes treated with Tdex (p = 0.0434). Conclusions: The ST formulation with xanthan gum demonstrated statistically superior anti-inflammatory and bactericidal properties as compared to Tdex. Limitations: Variations in inoculation procedures produced limited eye-to-eye differences in the infection.

Surfactant Protein D in Pseudomonas aeruginosa Keratitis

Purpose: To determine the importance of surfactant protein D in Pseudomonas keratitis. Methods: The surfactant D status of wild-type and surfactant D-deficient Black Swiss mice was confirmed by PCR reactions and immunoblot assay. Mouse corneas were infected with one of three strains of P. aeruginosa. At 1, 2, 3, and 6 days postinfection, eyes were scored by slit-lamp examination and bacteria per cornea quantified. Results: Infected wild-type mice had slit-lamp scores on 3 and 6 days postinfection that were significantly lower than those of surfactant D-deficient mice (p ≤ .0185) and only wild-type mice recovered from the infection. On day 6 postinfection, the number of bacteria per cornea was found to be significantly lower in wild-type mice as compared to surfactant D-deficient mice (p ≤ .0233). Conclusions: Surfactant D is important to the ocular innate host defense against Pseudomonas keratitis.

Penetration and effectiveness of prophylactic fluoroquinolones in experimental methicillin-sensitive or methicillin-resistant Staphylococcus aureus anterior chamber infections

PURPOSE: To determine the effectiveness of moxifloxacin and besifloxacin prophylactic therapy for experimental Staphylococcus aureus infections originating in the rabbit anterior chamber. SETTING: Microbiology Department, University of Mississippi Medical Center, Jackson, Mississippi, USA. DESIGN: Experimental study. METHODS: Minimum inhibitory concentrations (MICs) of moxifloxacin 0.5% and besifloxacin 0.6% for methicillin‐sensitive Staphylococcus aureus (MSSA) and methicillin‐resistant Staphylococcus aureus (MRSA) strains were determined. Eyes were treated with moxifloxacin, a moxifloxacin alternative formulation 0.5%, or besifloxacin (45 μL) 30 minutes or 60 minutes before anterior chamber infection (106 colony‐forming units [CFUs]). Aqueous humor was removed 30 minutes after infection for quantification of antibiotic and bacteria. RESULTS: The MIC for both organisms was 0.06 μg/mL for moxifloxacin and 0.03 μg/mL for besifloxacin. In MSSA infections, the untreated eyes contained 5.18 log CFU/mL, which was similar to besifloxacin‐treated eyes with either treatment (P≥.1091). Eyes treated with moxifloxacin or moxifloxacin alternative formulation contained significantly fewer CFUs than untreated controls or besifloxacin‐treated eyes with either treatment (P≤.0020). The aqueous humor in eyes treated with moxifloxacin or moxifloxacin alternative formulation contained significantly more drug than besifloxacin‐treated eyes at both prophylactic time points (P≤.0012). In MRSA infections, the untreated eyes contained 4.91 log CFU/mL, which was similar to besifloxacin‐treated eyes with either treatment (P≥.5830). Eyes treated with moxifloxacin or moxifloxacin alternative formulation contained significantly fewer CFUs than untreated controls or besifloxacin‐treated eyes at both prophylactic time points (P≤.0008). CONCLUSIONS: Moxifloxacin had greater in vivo effectiveness against MSSA and MRSA than besifloxacin. The aqueous antibiotic concentrations suggest limited penetration by besifloxacin, accounting for its lack of effectiveness. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Diverse Virulence of Staphylococcus aureus Strains for the Conjunctiva

Purpose: To determine the virulence of Staphylococcus aureus strains in the rabbit conjunctiva. Methods: Three strains of methicillin-sensitive S. aureus (8325-4, Newman, and UMCR1) and two strains of methicillin-resistant S. aureus (70490 and MW2) were analyzed. Rabbit bulbar conjunctivas (n ≥ 6 per group) were injected with 105 colony forming units (CFU) in 10 µl. Eyes were photographed and analyzed for pathology at 20 hr postinfection (PI) using slit lamp examination (SLE) to measure five parameters on a scale from 0 (normal) to 4 (severe): injection, chemosis, iritis, corneal edema, and pinpoint conjunctival hemorrhages. The parameter grades were added to produce a SLE score. Bacteria were enumerated and histopathological analysis was done at 20 hr PI. Myeloperoxidase assays were performed on conjunctival swabs (n ≥ 3 per strain) at 0 and 20 hr PI. Results: Conjunctivas injected with 8325-4 or Newman had SLE scores of 1.67 ± 0.12 and 0.81 ± 0.16, respectively. Strain 70490 produced an average SLE score of 2.94 ± 0.47, whereas MW2 produced a score of 5.04 ± 0.73. UMCR1 produced severe conjunctivitis having a SLE score of 13.25 ± 0.80. Only strain UMCR1 grew in the conjunctiva showing a 2.7 log increase in CFU; all other strains remained near the inoculated numbers or decreased as much as 1.85 logs. Myeloperoxidase activity was greatest in the tear film of UMCR1 infected eyes with over one million PMN present at 20 hr PI. Conclusions: Only one S. aureus strain, UMCR1, was able to cause a reproducible severe conjunctivitis. This conjunctival infection could be used to test new antimicrobials and to help understand the pathogenesis of conjunctivitis, especially in terms of overcoming the host defenses.

A Highly Virulent Staphylococcus aureus: Rabbit Anterior Chamber Infection, Characterization, and Genetic Analysis

PURPOSE To describe and characterize a Staphylococcus aureus strain with unique virulence that overcomes host defenses of the rabbit anterior chamber and mimics clinical cases of postcataract surgery endophthalmitis. METHODS Nine isolates of S. aureus were tested to determine their viability in the rabbit anterior chamber. Growth of UMCR1 in the anterior chamber was established and expressed as log colony-forming units per milliliter of aqueous humor. Pathologic changes produced by UMCR1 were documented by photographs, slit lamp examination, histopathologic analysis, and quantification of neutrophils. UMCR1 was characterized by antibiotic susceptibility, biochemical tests, ribotyping, genome restriction mapping, and multilocus sequence typing (MLST). RESULTS UMCR1 was the only S. aureus strain that grew within the anterior chamber, reaching log 6.97 ± 0.18 CFU/mL by 16 hours after infection. Pathologic changes included conjunctival injection, chemosis, corneal edema, severe iritis, fibrin accumulation, and a 193-fold increase in neutrophils by 16 hours after infection. UMCR1 was only resistant to sulfamethoxazole and, like other S. aureus isolates, polymyxin B. UMCR1 also had biochemical reactions and a ribotype pattern typical of S. aureus. The genomic reconstruction analysis of UMCR1 was most similar to strains MW2 and MSSA476. MLST revealed a 1 in 3198 nucleotide difference between UMCR1 and strains MW2 and MSSA476. CONCLUSIONS This study describes a unique S. aureus strain that overcomes host defenses and replicates in the anterior chamber. The survival and growth of this organism could be used for studies of S. aureus pathogenesis, host defenses, and effectiveness of antibiotics within the anterior chamber.

Identification and Potency of Cyclodextrin-Lipid Inhibitors of Staphylococcus aureus α-Toxin

Purpose: Staphylococcus aureus, a leading cause of bacterial keratitis, secretes α-toxin, a cytotoxin active on the corneal epithelium. This study describes the production and testing of chemical inhibitors of α-toxin action. Methods: Purified α-toxin was titered by its ability to lyse rabbit erythrocytes in buffered saline (PBS). To prepare potential toxin inhibitors, each of 18 lipids was incorporated into a complex with methyl-β-cyclodextrin (MβCD) or hydroxypropyl-β-cyclodextrin (HPβCD). Serial dilutions of each lipid-cyclodextrin (CD-lipid) complex were mixed with α-toxin prior to the addition of rabbit erythrocytes. Select CD-lipid complexes were mixed with 12 hemolytic units (HU) α-toxin and injected into the rabbit corneal stroma so the resulting corneal erosions could be measured at 4 and 8 hours post-injection (PI). Eyes injected with toxin alone, MβCD, or HPβCD alone served as controls. Results: Neither form of CD alone inhibited α-toxin. Of the 36 complexes prepared, 6 lipid-CD complexes were found to inhibit >100 HU of α-toxin. Four lipid complexes able to inhibit >200 HU of α-toxin were tested in toxin-injected corneas; at 4 and 8 hours PI, the complexes of cholesterol or lanosterol with MβCD and squalene or desmosterol with HPβCD caused a significant reduction in the corneal erosion size as compared to eyes injected with α-toxin alone (P ≤ 0.05). Conclusions: Specific lipid inclusion complexes with either MβCD or HPβCD demonstrated a significant inhibition of α-toxin in both in vitro and in vivo assays. Changes in either the cyclodextrin or lipid of a complex affected the inhibitory activity.

Fluoroquinolone therapy in a rabbit model of post-LASIK methicillin-resistant Staphylococcus aureus keratitis

PURPOSE: To develop a rabbit model of post‐laser in situ keratomileusis (LASIK) methicillin‐resistant Staphylococcus aureus (MRSA) keratitis for studying fluoroquinolone prophylaxis and treatment. SETTING: Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi, USA. METHODS: An MRSA keratitis isolate (5 μL, 500 colony forming units [CFU]) was inoculated underneath a corneal flap. Bacterial growth and pathology were determined by quantitative cultures (CFU) and slitlamp examination, respectively. The effectiveness of commercial moxifloxacin and gatifloxacin formulations was compared in 3 regimens: prophylaxis (4 drops before inoculation), early therapy (single drop hourly from 4 to 9 hours postinfection), and late therapy (single drop hourly from 10 to 15 hours postinfection). Zones of bacterial inhibition to known in vivo antibiotic concentrations were determined. RESULTS: Bacteria grew to a maximum of approximately 106 CFU/cornea within 10 hours postinfection. The slitlamp examination scores showed pathologic changes beginning 10 hours postinfection and progressed throughout the infection. For prophylaxis, eyes treated with moxifloxacin had significantly fewer CFU than gatifloxacin‐treated eyes or untreated controls (both P≤.0001). During early treatment, the antibiotics were equally effective in reducing CFU relative to untreated controls (P≤.0001). In late treatment, gatifloxacin and moxifloxacin caused significant reductions in CFU relative to untreated controls (P≤.0007 and P≤.0001, respectively). Moxifloxacin produced zones of bacterial inhibition significantly larger than those produced by gatifloxacin. CONCLUSIONS: Methicillin‐resistant S aureus inoculation beneath a rabbit corneal flap produced an infection that was useful for quantitative microbiological studies. A significant advantage in using moxifloxacin relative to gatifloxacin was observed in prophylaxis of keratitis (P = .0001).

Sustained anti-staphylococcal effect of lysostaphin in the rabbit aqueous humor.

Purpose: Staphylococcus aureus is an important cause of ocular infections including endophthalmitis. The purpose of this study was to determine the ability of a relatively large molecule, such as lysostaphin, to remain in the rabbit aqueous humor for extended periods while retaining its bactericidal activity. Methods: Lysostaphin, gatifloxacin, or Tris-buffered saline (TBS) was injected into the rabbit anterior chamber. Aqueous humor was sampled at 0.5, 1, 2, 3, and 4 days after injection, and then assayed for bactericidal activity against S. aureus. The anterior chamber of treated eyes was also challenged 2 days after treatment by an infection of S. aureus. The surviving bacteria were quantified to determine bactericidal effectiveness in the anterior chamber. The aqueous humor of lysostaphin injected eyes was assayed by western blot for the presence of the molecule 2 days post-injection. Results: The bactericidal activity of lysostaphin was confirmed by lysis of S. aureus and sensitivity to zinc. Eyes injected with lysostaphin showed no adverse reactions. Aqueous humor of gatifloxacin injected eyes demonstrated no greater effectiveness than that of TBS injected eyes in vitro at any time point assayed, whereas lysostaphin injected eyes retained potent bactericidal activity for at least 3 days. In an in vivo challenge, the anterior chamber of lysostaphin injected eyes retained significant bactericidal activity for at least 2 days after treatment, whereas gatifloxacin injected eyes demonstrated no significant difference from those injected with TBS. Western blot analysis demonstrated the presence of lysostaphin in the aqueous humor 2 days post-injection. Conclusions: Lysostaphin demonstrated the ability to remain in the aqueous humor for days while maintaining its bactericidal activity, an indication that a high molecular weight antimicrobial can provide prolonged prophylactic protection of the anterior chamber.

Staphylococcus aureus infection of the rabbit cornea following topical administration.

Purpose: To determine the ability of diverse S. aureus strains to infect the rabbit cornea following topical inoculation, with special emphasis on a strain of unusual virulence. Materials and Methods: S. aureus strains (5 × 105 colony forming units; CFU) were topically applied onto scarified rabbit corneas or 100 CFU were intrastromally injected into rabbit corneas. Eyes were scored by slit lamp examination (SLE) and corneas were cultured to determine the log CFU. Polymorphonuclear leukocytes (PMN) were quantified by myeloperoxidase assays and corneas underwent histopathological analysis. Hemolysin titers of S. aureus strains were determined and S. aureus interactions with rabbit tears or human corneal epithelial cells were investigated. Results: All strains injected into the cornea produced high SLE scores and multi-log increases in CFU. Following topical inoculation, four strains produced low SLE scores with no bacterial replication. One strain (UMCR1) topically infected the cornea, causing high SLE scores, extensive PMN infiltration, and multi-log increases in CFU. Histopathologic analysis demonstrated a PMN influx into the UMCR1-infected cornea, destruction of the corneal epithelium, and severe edema. Strain UMCR1 did not demonstrate a high hemolysin titer or resistance to the bactericidal activity of rabbit tears, but did invade human corneal epithelial cells with relatively high efficiency. Conclusions: One S. aureus strain demonstrated the ability to topically infect the rabbit cornea. This strain was previously found to be unique in its ability to infect the anterior chamber and conjunctiva, suggesting that a key mechanism may be employed to overcome the host defenses of these three ocular sites.