Armando R. Caballero

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.

Quantitative comparison of fluoroquinolone therapies of experimental Gram-negative bacterial keratitis

Purpose. To determine the effectiveness of topically applied fluoroquinolones for experimental Pseudomonas or Serratia keratitis. Methods. Bacteria were injected intrastromally (10 3 colony forming units [CFU]). From 16 to 22 hours post-infection (PI), a single topical drop of moxifloxacin (Vigamox ®, 0.545%), levofloxacin (Quixin™, 0.5%), ofloxacin (Ocuflox ®, 0.3%) or ciprofloxacin (Ciloxan ®, 0.3%) was applied every 30 minutes. At 23 hours PI, corneas were cultured quantitatively. Results. For Pseudomonas keratitis, untreated eyes contained 7 log CFU/cornea and antibiotic-treated eyes demonstrated a = 5-log reduction in CFU/cornea (p = 0.0001). Moxifloxacin, levofloxacin, or ciprofloxacin therapies were not significantly different from each other (p = 0.67). For Serratia keratitis, untreated eyes contained 7 log CFU/cornea whereas treated eyes had a = 2-log reduction (p = 0.0001). Moxifloxacin therapy proved most effective (p = 0.001). Conclusions. Overall, moxifloxacinwas the most effective of the four fluoroquinolones in reducing CFU/cornea in the rabbit model of Gram-negative keratitis.

Corneal Virulence of Pseudomonas aeruginosa Elastase B and Alkaline Protease Produced by Pseudomonas putida

Purpose: To measure the specific virulence contributions of two Pseudomonas aeruginosa proteases, elastase B and alkaline protease, when expressed separately by Pseudomonas putida in a rabbit model of bacterial keratitis. Methods: P. putida KT2440 was transformed with plasmids that enabled the extracellular production of either elastase or alkaline protease. Protease expression was confirmed by zymography and immunoblotting. P. putida expressing elastase, alkaline protease, or vector alone was injected intrastromally (103 colony forming units [CFU]) into rabbit corneas (n = 6). Infected eyes were graded by slit-lamp examination (SLE) at 20, 24, 28, and 32 hr postinfection (PI). Rabbits were sacrificed at 33 hr PI, and the log CFU (±SEM) per cornea was determined. Results: SLE scores for eyes infected with P. putida producing elastase were significantly higher than those infected with vector alone at all time points (p ≤ 0.008). SLE scores for eyes infected with P. putida producing alkaline protease were not significantly higher than the control (p ≥ 0.1), but small erosions formed in 33% of corneas. At both 24 and 28 hr PI, the SLE scores for corneas infected with P. putida producing elastase were significantly higher than those infected with P. putida producing alkaline protease (p ≤ 0.002). Conclusions: Elastase production by P. putida caused significant increases in SLE scores whereas expression of alkaline protease caused limited corneal erosions. This suggests that the production of elastase during P. aeruginosa keratitis enhances ocular pathology, whereas alkaline protease production contributes to limited corneal erosion.

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.

Protection from Streptococcus pneumoniae Keratitis by Passive Immunization with Pneumolysin Antiserum

PURPOSE To determine whether passive immunization with pneumolysin antiserum can reduce corneal damage associated with pneumococcal keratitis. METHODS New Zealand White rabbits were intrastromally injected with Streptococcus pneumoniae and then passively immunized with control serum, antiserum against heat-inactivated pneumolysin (HI-PLY), or antiserum against cytotoxin-negative pneumolysin (psiPLY). Slit lamp examinations (SLEs) were performed at 24, 36, and 48 hours after infection. An additional four corneas from rabbits passively immunized with antiserum against psiPLY were examined up to 14 days after infection. Colony forming units (CFUs) were quantitated from corneas extracted at 20 and 48 hours after infection. Histopathology of rabbit eyes was performed at 48 hours after infection. RESULTS SLE scores at 36 and 48 hours after infection were significantly lower in rabbits passively immunized with HI-PLY antiserum than in control rabbits (P < or = 0.043). SLE scores at 24, 36, and 48 hours after infection were significantly lower in rabbits passively immunized with psiPLY antiserum than in control rabbits (P < or = 0.010). The corneas of passively immunized rabbits that were examined up to 14 days after infection exhibited a sequential decrease in keratitis, with an SLE score average of 2.000 +/- 1.586 at 14 days. CFUs recovered from infected corneas were not significantly different between each experimental group and the respective control group at 20 or 48 hours after infection (P > or = 0.335). Histologic sections showed more corneal edema and polymorphonuclear leukocyte (PMN) infiltration in control rabbits compared with passively immunized rabbits. CONCLUSIONS HI-PLY and psiPLY both elicit antibodies that provide passive protection against S. pneumoniae 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.

The effectiveness of tobramycin and Ocuflox® in a prophylaxis model of Staphylococcus keratitis

Purpose. To determine the effectiveness of prophylactic antibiotic treatment prior to intra-corneal infection with Staphylococcus aureus. Methods. One topical drop of Tobrex ® (0.3% tobramycin), tobramycin (0.3%) in the Tobrex ® vehicle with 0.05% dodecyl maltoside (DDM)/4.0% hydroxypropylmethycellulose (HPMC), Ocuflox ® (0.3% ofloxacin) or DDM/HPMC vehicle were applied to rabbit eyes at one or five hours prior to injection of bacteria. Approximately 500 colony-forming units (CFU) of S. aureus strain 8325-4 were injected into the corneal stroma. Rabbits were sacrificed five hours after infection and corneal homogenates were cultured to determine the number of colony forming units (CFU) per cornea. Results. Rabbits treated at five hours prior to infection with tobramycin-DDM/HPMC reduced the bacterial load by approximately 2.4 log CFU/cornea as compared to the untreated control (3.47 ± 0.98 vs. 5.71 ± 0.14 log CFU/cornea, respectively; P = 0.0010); however, Ocuflox ®, Tobrex ®, or DDM/HPMC vehicle did not significantly reduce the log CFU (P = 0.4837). Rabbits treated at 1 hour prior to infection with Ocuflox ® or tobramycin-DDM/HPMC had significantly reduced CFU/cornea (1.31 ± 0.86 and 0.48 ± 0.31 log CFU/cornea, respectively) as compared to the untreated group (5.71 ± 0.14 log CFU/cornea; P = 0.0001). Neither Tobrex ® nor the DDM/HPMC vehicle significantly reduced the CFU/cornea compared to the untreated group (P = 0.2312). Conclusions. This pre-treatment model of Staphylococcus keratitis quantitatively measured the prophylactic effectiveness of topical antibiotic formulations. An important finding was that a tobramycin-DDM/HPMC formulation was highly effective as a prophylactic medication.

Corneal virulence of LasA protease--deficient Pseudomonas aeruginosa PAO1.

Purpose. Pseudomonas aeruginosa PAO1 deficient in LasA protease was reported to be ocularly avirulent. However, the avirulence of this mutant could not attributed to the loss of LasA protease. The purpose of this study was to define the mechanism for such a mutants inability to cause corneal disease. Methods. A LasA protease–deficient mutant of P. aeruginosa PAO1 was constructed by allelic exchange. Virulence of this mutant in mouse and rabbit models of keratitis was assessed by scoring for ocular disease and quantitating viable bacteria from infected corneas. Adherence to scarified mouse corneal tissue was determined with an organ culture assay. Results. In the mouse eye, the LasA protease–deficient mutant was not virulent, despite being as adherent as its parent strain. Virulence of the mutant was also significantly reduced in the rabbit eye. Complementation with las A did not restore virulence in either model of infection. Neither the mutant nor the mutant complemented with las A grew well in ocular tissue. An analysis of the mutant showed that it was auxotrophic for leucine. Conclusion. These data show that the mutants avirulence in the eye is caused by poor growth in the ocular environment and not the loss of a functional las A gene.

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.