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Purpose: To study antibody production to Pseudomonas aeruginosa protease IV (PIV) for immunoassay development and to assess the possible role of antibody in arresting corneal damage., Methods: Rabbits were immunized with PIV, urea-soluble recombinant PIV (rPIV), or precipitated rPIV. Antibody was analyzed by ELISA and Western blotting. Antibody-mediated inhibition of PIV activity was tested by colorimetric assay and during keratitis by slit-lamp examination of infected eyes., Results: Antibody was not produced after PIV immunization but was induced by rPIV. Rabbits immunized first with soluble and then precipitated rPIV produced high titers (log(10)) to rPIV (4.28 +/- 0.09) and significantly higher titers to PIV (3.90 +/- 0.06) compared to the other immunized groups. Antibody to rPIV reacted with PIV, but neither neutralized enzyme activity in vitro nor protected infected rabbits in vivo., Conclusions: The present study demonstrates that PIV is a virulence factor which can escape a protective immune response.

Purpose: Staphylococcus aureus causes severe corneal infections that often result in corneal scarring and blindness. Presently, therapy often involves the use of a fluoroquinolone antibiotic. This study, employing an experimental rabbit model of Staphylococcus keratitis, compared the effectiveness of two commonly prescribed formulations of fluoroquinolones to an experimental formulation, ciprofloxacin with polystyrene sulfonate (ciprofloxacin-PSS). The ciprofloxacin-PSS formulation uses an ion exchange resin to aid in the delivery of drug to the cornea., Methods: Early (4-9 h postinfection, PI) and late (10-15 h PI) therapies were studied, employing 5 groups: ciprofloxacin-PSS, ciprofloxacin, ofloxacin, PSS vehicle. and untreated. Dosing regimens were: every 30 min, 60 min, or a single drop applied at 9 h PI. Eyes were observed by slit lamp examination (SLE) and bacterial colony forming units (CFU) per cornea were determined., Results: Early phase therapy with ciprofloxacin-PSS, ciprofloxacin, or ofloxacin administered every 30 or 60 min were equally effective (P > or = 0.2880), decreasing CFU per cornea by >5 log. Ciprofloxacin was significantly more active than ciprofloxacin-PSS or ofloxacin (P < or = 0.0410) when applied as a single drop. Late therapy with ciprofloxacin-PSS, ciprofloxacin, or ofloxacin administered every 30 or 60 min resulted in >3 log decrease in CFU per cornea relative to controls (P < or = 0.0001)., Conclusions: Topical treatment of experimental Staphylococcus keratitis with ciprofloxacin-PSS, ciprofloxacin, or ofloxacin was effective. The effectiveness of ciprofloxacin-PSS suggests that improved drug delivery systems employing an ion exchange resin could be useful in an ocular fluoroquinolone formulation.

Purpose: A Pseudomonas mutant deficient in protease IV has significantly reduced virulence in experimental keratitis. In the present study, the corneal toxicity of purified protease IV and its ability to augment the virulence of protease-IV-deficient bacteria were analyzed., Methods: The toxicity of purified protease IV was determined by intrastromally injecting the exoenzyme (20-200 ng) into the cornea. The effects of protease IV on the corneal virulence of the protease-IV-deficient strain, PA103-29::Tn9, were determined by injecting eyes with 1000 CFU of log phase bacteria plus either 200 ng active purified protease IV or 200 ng heat-inactivated protease IV. Changes in ocular disease, determined by slit-lamp examination, were measured at 3, 16, 22, and 27 hours after infection. Colony-forming units per cornea were quantified at 27 hours after infection., Results: Purified protease IV at doses from 50 to 200 ng induced epithelial defects within 3 hours of injection. Injection of 20 ng active protease IV or heat-inactivated protease IV (200 ng) had no effect on ocular tissue. Corneal virulence of the protease-IV-deficient strain was augmented by intrastromal injection with purified protease IV but not with heat-inactivated protease IV (P < or = 0.0001). Neither active nor heat-inactivated protease IV altered the growth of bacteria in the cornea (6 log units; P = 0.81)., Conclusions: The important role of protease IV in corneal virulence was demonstrated by direct toxicity and by its ability to significantly augment the virulence of protease-IV-deficient Pseudomonas.

Purpose: The role of exoproteins in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated in three animal models by assessing the relationship between corneal virulence and the activities of exotoxin A, elastase, alkaline protease, and an uncharacterized protease, protease IV., Methods: The four Pseudomonal strains tested included a prototype strain (ATCC 27853) producing exotoxin A, elastase, and alkaline protease; a parent strain (PA103) producing only exotoxin A and protease IV; a mutant (PA103-29) producing only protease IV; and a mutant (PA103-AP1) producing exotoxin A and having only approximately 5% of the protease IV activity of its parent. Corneal virulence was evaluated in the mouse scratch, rabbit scratch, and rabbit intrastromal models in terms of clinical signs (slit lamp examination, slit lamp examination), and viable bacteria., Results: Protease IV, the only protease produced by PA103 and PA103-29, was found to produce a unique band on zymograms (120 kDa) and to react distinctively with a synthetic substrate. Evidence for the role of protease IV in corneal virulence included two findings: PA103-29,which produced protease IV but not the other exoproteins, caused infections that were as severe as those caused by the prototype strain (ATCC 27853) in all three models (P>0.24); and PA103-AP1, the strain deficient in 95% of the parent protease IV activity, mediated infections characterized by slit lamp examination scores significantly lower than those of infections caused by the parent (PA103) or the prototype strain (ATCC 27853) in the rabbit and mouse scratch models (P<0.02)., Conclusions: Protease IV was found to be a novel Pseudomonas protease contributing to corneal virulence in rabbits and mice when infections were initiated at the corneal surface. Furthermore, production of protease IV in low quantities was sufficient for virulence when the topical stages of keratitis were bypassed by an intrastromal injection of Pseudomonas.

Ciprofloxacin is a fluoroquinolone antibiotic with broad spectrum bactericidal activity. A commercially available form of ciprofloxacin contains benzalkonium chloride (BAC) (0.006%) and EDTA (0.05%) as preservatives. Since BAC has been shown to cause adverse changes in corneal epithelial cells, a formulation of ciprofloxacin devoid of BAC and EDTA but with the same effectiveness would be valuable. We present here the results of experiments designed to assess the efficacy of a BAC-free and EDTA-free formulation of ciprofloxacin, Ciprofloxacin-polystyrene sulfonate (PSS), in experimental models of Pseudomonas aeruginosa and Staphylococcus aureus keratitis. Both formulations of ciprofloxacin sterilized corneas infected with P aeruginosa, and both formulations showed equal bactericidal activity for S aureus. Normal eyes treated with either formulation showed mild conjunctival irritation compared to untreated normal eyes. The bactericidal activities of both formulations of ciprofloxacin were excellent. Therefore, the Ciprofloxacin-PSS formulation could serve as an effective single drug therapy for ocular infections.

Our previous work has demonstrated the importance of pneumolysin in the virulence of S. pneumoniae in a rabbit intracorneal model. This was accomplished by showing that deletion of the gene encoding pneumolysin resulted in reduced virulence, whereas restoration of the wild-type gene resulted in restoration of the virulent phenotype. To assess the importance of a particular domain in the pneumolysin molecule, we have now constructed a strain which produces a pneumolysin molecule which is hemolytic but which bears a site-specific mutation in the domain known to be associated with the complement-activating properties of this molecule. Comparison of the virulence of this strain with that of a strain bearing the wild-type gene showed statistically significantly lower total slit lamp examination (SLE) scores at 12, 18, 24, and 36 h (particularly with respect to fibrin formation), but no difference at 48 h. Determination of colony forming units (CFU) in eyes infected with the two strains showed approximately 10(6) bacteria per cornea until 36 h. Between 36 and 48 h, the bacteria were almost completely cleared with very few bacteria recoverable at the later time point. The loss of virulence observed with this mutation in the complement-activation domain of pneumolysin, though less than that observed with the gene deletion mutant, suggests that complement activation by pneumolysin has a significant role in the pathology observed in this model of corneal infection.

Ciprofloxacin and prednisolone, but not an aminoglycoside and dexamethasone, were previously found to be effective in killing bacteria and reducing inflammation for the treatment of Pseudomonas keratitis. We investigated the therapeutic effectiveness of tobramycin/prednisolone and ciprofloxacin/dexamethasone in a rabbit model of experimental keratitis to increase our understanding of the effectiveness of antibiotic/steroid combinations. To our knowledge, this is the first analysis of the effectiveness of a combination of ciprofloxacin and dexamethasone for experimental keratitis. Two experiments were conducted. In the first experiment, 36 rabbits were divided into six groups: 1) untreated; 2) prednisolone acetate, 1.0%; 3) prednisolone phosphate, 1.0%; 4) tobramycin, 1.36%; 5) tobramycin plus prednisolone acetate; 6) tobramycin plus prednisolone phosphate. In the second experiment, 23 rabbits were divided into four groups: 1) untreated; 2) ciprofloxacin, 0.3%, plus dexamethasone alcohol, 0.1%; 3) ciprofloxacin; 4) dexamethasone alcohol. Topical antibiotic and/or steroid was given for 10 h, from 16 to 26 h postinfection, one drop every 15 min for the first hour and then every 30 min for the remaining 9 h. At 27 h postinfection, eyes were evaluated by slit lamp examination (SLE) and assayed for the presence of bacteria in terms of colony forming units (CFU) per cornea. Both prednisolone acetate and prednisolone phosphate reduced ocular inflammation (as determined by SLE), compared with no treatment (P < or = 0.036); the phosphate was more effective (P = 0.005). Tobramycin alone and in combination with prednisolone also significantly reduced SLE, compared with no treatment (P < or = 0.006). The bactericidal activity of tobramycin was not affected by either steroid formulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of staphylococcal keratitis includes tobramycin drops at repeated intervals, a prolonged therapy that is disruptive to the patient. To identify a regimen involving less frequent drug application, we compared the efficacy of fortified tobramycin (1.36%) administered by collagen shields or in topical drop form to rabbit corneas intrastromally infected with staphylococci. Eyes were treated with shields hydrated in and supplemented with fortified tobramycin drops (1.36%) applied every 1, 2, 5, or 10 h, from 10 to 20 h postinfection. For topical drop treatment alone, tobramycin was applied following the identical regimen. Untreated corneas contained 10(6) colony forming units. Shields supplemented with tobramycin drops applied every 1, 2, or 5 h sterilized 100% of the corneas. Shields supplemented with tobramycin drops applied at 10 h sterilized 58% of the corneas. Topical delivery of tobramycin every h sterilized all corneas; drops alone applied at longer intervals, such as 2, 5, or 10 h, sterilized 83%, 17%, and 0% of the corneas, respectively. Collagen shield delivery of tobramycin with supplemental topical drops can eradicate staphylococci in this model with less frequent dosing intervals than are required with topical therapy alone.

Purpose: To compare the chemotherapeutic efficacies of ciprofloxacin (0.3%) and fortified (1.36%) tobramycin for the treatment of methicillin-sensitive and methicillin-resistant Staphylococcus aureus keratitis during early and late stages of infection., Methods: Rabbit corneas were intrastromally injected with 10(2) colony-forming units (CFU) of methicillin-sensitive S. aureus (MSSA) or methicillin-resistant S. aureus (MRSA). Topical therapy was initiated at either 4 hours postinfection (early stage) or at 10 hours postinfection (late stage). Drops were administered every 15 minutes for 5 hours. Corneal bacterial counts and aqueous humor antibiotic concentrations were determined., Results: Early administration of ciprofloxacin sterilized all MSSA-infected corneas and 83% of MRSA-infected corneas. Late administration of ciprofloxacin reduced the numbers of viable MSSA and MRSA to 3.6 and 3.7 log10 CFU per cornea, respectively, but did not sterilize any corneas. Early administration of fortified (1.36%) tobramycin sterilized all MSSA-infected corneas but none of the MRSA-infected corneas. Late administration of tobramycin reduced the viable MSSA to very low numbers (0.5 and 0.0 log10, respectively) and sterilized 33% of MSSA-infected corneas, but had little effect on MRSA-infected corneas., Conclusions: Early in infection, ciprofloxacin was highly effective against MSSA and MRSA, whereas tobramycin was effective only against MSSA. During later stages of infection, tobramycin was more effective than ciprofloxacin against MSSA, and neither antibiotic was effective against MRSA. Thus, ciprofloxacin is limited by the time of application and tobramycin is limited by the resistance of the MRSA strain.

This study was conducted to determine the therapeutic efficacy of 3.0 mg/ml ciprofloxacin administered concurrently with one of two salts of prednisolone for the treatment of experimental pseudomonal keratitis. Rabbit corneas were injected intrastromally with Pseudomonas aeruginosa ATCC strain 27853. Sixteen hr after injection, rabbits were randomly divided into four treatment groups (3 rabbits, 6 eyes per group): 1) ciprofloxacin plus prednisolone acetate; 2) ciprofloxacin plus prednisolone phosphate; 3) ciprofloxacin only; 4) untreated. Signs of inflammation were graded in a masked fashion by slit lamp examination (SLE) and by estimating polymorphonuclear leukocyte (PMN) numbers in corneas 27 hr after injection. SLE scores and PMN numbers were significantly lower (P < 0.02) in eyes receiving either salt of prednisolone plus ciprofloxacin compared to the untreated controls. In contrast, SLE scores and PMN numbers were not significantly different in eyes treated with ciprofloxacin alone, compared to untreated controls (P > 0.13). No viable bacteria were recovered from any eye treated with ciprofloxacin (groups 1, 2, and 3). Ciprofloxacin concentrations in the aqueous humor of eyes in groups 1, 2, and 3 were greater than 15-fold higher than the MIC for P. aeruginosa 27853. These results suggest that either salt of prednisolone, when combined with ciprofloxacin, reduces ocular inflammation without affecting the antimicrobial efficacy of the antibiotic.

To study the role of the host inflammatory response in Pseudomonas aeruginosa keratitis, rabbits were made leukopenic with intravenous injections of cyclophosphamide and dexamethasone. Twenty-four hr later, keratitis was initiated in all rabbits with an intrastromal injection of 1,000 log phase P. aeruginosa strain 27853. Slit lamp examination of eyes showed that leukopenic rabbits had significantly less (P < 0.0001) ocular pathology at 16, 22, and 27 hr postinfection. The number of viable bacteria recovered from corneas of leukopenic rabbits was the same as the number recovered from nonleukopenic rabbits (P = 0.95). These results suggest that the host inflammatory response significantly contributes to the overall ocular pathology associated with P. aeruginosa keratitis, but does not influence the survival of the infecting organism in the cornea at the height of the infection.

We compared collagen shields hydrated in 1.36% tobramycin with topical 1.36% tobramycin for sustained treatment of experimental Pseudomonas keratitis in rabbits. Antibiotic therapy for a total of 24 hours was initiated 14 hours after an intrastromal injection of 10(3) logarithmic phase Pseudomonas aeruginosa. Seven groups were treated as follows: groups 1-3, collagen shields hydrated in tobramycin supplemented with topical 1.36% tobramycin drops at 4, 6, or 8 hour intervals; group 4, collagen shields hydrated in tobramycin without any further topical supplementation; group 5, topical tobramycin therapy, initially every half-hour for 4 hours, then hourly; group 6, collagen shields hydrated in balanced saline solution; and group 7, no treatment. Each group contained four eyes. The groups treated with collagen shields supplemented every 4 or 6 hours with topical tobramycin had significantly fewer colony forming units (CFU) than those receiving topical or collagen shield therapy alone (P < 0.001). The group treated with collagen shields hydrated in sterile saline had 10(7) CFU per cornea, which was not significantly different from the untreated group (P > 0.2). Collagen shields hydrated in tobramycin and supplemented with topical tobramycin were effective in sustained treatment of experimental Pseudomonas keratitis.