Aihua Tang

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.

Properties of PASP: A Pseudomonas Protease Capable of Mediating Corneal Erosions

PURPOSE To analyze PASP in terms of its gene distribution and expression, its corneal pathologic effects, its enzymatic properties, and the protectiveness of the immune response to this protease. METHODS Twenty-five strains of P. aeruginosa were analyzed for the PASP gene and secreted protein by PCR and Western blot analysis, respectively. Active recombinant (r)PASP (10 microg/20 microL) or heat-inactivated rPASP was intrastromally injected into rabbit corneas. Pathologic changes were monitored by slit lamp examination (SLE) and histopathology. Purified rPASP was assayed for cleavage of collagens and susceptibility to TLCK. Rabbit antibody to rPASP was produced and tested for enzyme inactivation, and actively immunized rabbits were challenged by intrastromal injection of active rPASP (5 microg). RESULTS All 25 strains of P. aeruginosa analyzed were positive for the PASP gene and protein. SLE scores of eyes injected with active rPASP were significantly higher than control eyes at all postinjection times (PI; P <or= 0.004). Histopathologic studies documented the destruction of the corneal epithelial layer and portions of the corneal stroma at 9 hours PI, and polymorphonuclear (PMN) leukocyte infiltration into the cornea by 24 hours after active rPASP injection. PASP cleaved type I and IV collagens and was susceptible to TLCK inhibition. PASP was present in the cytoplasm and periplasm, but only secreted PASP was enzymatically active. A high antibody titer (ELISA titer >or= 10,000) was produced, but this antibody did not protect against active rPASP challenge. CONCLUSIONS PASP is a commonly produced Pseudomonas protease that can cleave collagens and cause corneal erosions.

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.

Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

Abstract Purpose: To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. Methods: A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. Results: LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). Conclusions: The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

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).

Pseudomonas aeruginosa proteolytically alters the interleukin 22-dependent lung mucosal defense.

ABSTRACT The IL-22 signaling pathway is critical for regulating mucosal defense and limiting bacterial dissemination. IL-22 is unusual among interleukins because it does not directly regulate the function of conventional immune cells, but instead targets cells at outer body barriers, such as respiratory epithelial cells. Consequently, IL-22 signaling participates in the maintenance of the lung mucosal barrier by controlling cell proliferation and tissue repair, and enhancing the production of specific chemokines and anti-microbial peptides. Pseudomonas aeruginosa is a major pathogen of ventilator-associated pneumonia and causes considerable lung tissue damage. A feature underlying the pathogenicity of this bacterium is its capacity to persist and develop in the host, particularly in the clinical context of nosocomial lung infections. We aimed to investigate the ability of P. auruginosa to disrupt immune-epithelial cells cross-talk. We found that P. aeruginosa escapes the host mucosal defenses by degrading IL-22, leading to severe inhibition of IL-22-mediated immune responses. We demonstrated in vitro that, protease IV, a type 2 secretion system-dependent serine protease, is responsible for the degradation of IL-22 by P. aeruginosa. Moreover, the major anti-proteases molecules present in the lungs were unable to inhibit protease IV enzymatic activity. In addition, tracheal aspirates of patients infected by P. aeruginosa contain protease IV activity which further results in IL-22 degradation. This so far undescribed cleavage of IL-22 by a bacterial protease is likely to be an immune-evasion strategy that contributes to P. aeruginosa-triggered respiratory infections.