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Purpose/aim: Klebsiella pneumoniae causes a blinding infection called endogenous endophthalmitis. The role of innate immune recognition of K. pneumoniae in the eye during infection is not known. We hypothesized that intraocular recognition of K. pneumoniae was mediated by Toll-like receptor (TLR)-4 and may be dependent on MagA-regulated hypermucoviscosity., Materials and Methods: Experimental endophthalmitis was induced in C57BL/6J or TLR4(-/-) mice by intravitreal injection of 100 CFU of wild type or ΔmagA K. pneumoniae. Infection and inflammation were quantified by determining viable K. pneumoniae per eye, retinal responses via electroretinography, myeloperoxidase activity of infiltrating neutrophils and the proinflammatory cytokine and chemokine response., Results: C57BL/6J and TLR4(-/-) mice could not control intraocular wild-type K. pneumoniae growth. TLR4(-/-) mice were less able than C57BL/6J to control the intraocular growth of ΔmagA K. pneumoniae. Retinal function testing suggested that infection with ΔmagA K. pneumoniae resulted in less retinal function loss. There was a TLR4-dependent delay in initial neutrophil recruitment, regardless of the infecting organism. The proinflammatory cytokine/chemokine data supported these results. These findings were not due to an inability of TLR4(-/-) neutrophils to recognize or kill K. pneumoniae., Conclusions: These studies suggest that TLR4 is important in the early intraocular recognition and host response to K. pneumoniae. However, the role of MagA in TLR4-mediated intraocular recognition and subsequent inflammation is less clear.

Rapid vision loss and explosive inflammation are devastating consequences of Bacillus endophthalmitis that have not been well defined. We therefore analyzed the evolution of intraocular inflammation and loss of retinal architecture and function during experimental Bacillus endophthalmitis. Mice were intravitreally injected with 100 CFU of B. cereus, and eyes were analyzed for bacterial growth, retinal function, architectural changes and retinal cellular stress, inflammatory cytokines, and infiltrating cells. Retinal electrophysiologic and structural changes began as early as 4 to 6 hr postinfection. Significant declines in retinal function paralleled the loss of retinal architecture. Glial fibrillary acidic protein (GFAP) was detected in retina, indicating potential stress. Polymorphonuclear leukocyte (PMN) infiltration into the vitreous began as early as 4 hr postinfection, coinciding with a significant increase in TNF-alpha in the eye. These results indicated that acute inflammation and detrimental architectural and electrophysiologic changes in the retina began earlier than once thought, suggesting that therapeutic intervention should be given at the earliest possible time to avoid vision loss during Bacillus endophthalmitis.

Bacillus causes one of the most rapidly blinding intraocular infections: endophthalmitis. In this study, Bacillus spp. were isolated from ocular infection cases, taxonomically characterized by riboprint analysis, and screened for the presence of putative virulence factors. The ability of these isolates to kill retinal and corneal cells was examined, as were antibiotic susceptibility profiles. The majority of isolates belonged to the B. cereus taxonomic group of microorganisms and were identified as B. cereus (53%) or B. thuringiensis (26%). Toxins were identified in most B. thuringiensis and B. cereus isolates. Most B. cereus and B. thuringiensis killed corneal and retinal cells within 6 h. All isolates were susceptible to most antibiotics tested, with quinolones and vancomycin being the most potent. These findings represent the first report of B. thuringiensis as an important ocular pathogen, demonstrates the potential ocular toxicity of B. cereus and B. thuringiensis isolates, and identifies antibiotics whose efficacy against Bacillus were superior to those used clinically.

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.

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.

To determine the efficacy of a fluoroquinolone antibiotic in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) keratitis, topical administration of 0.3% ciprofloxacin was compared with topical 5.0% vancomycin or 5.0% cefazolin in experimental infections in the rabbit eye. The infections were established by intrastromal injection of 100 colony forming units (CFU) of MRSA, which resulted in greater than 10(6) CFU per cornea by 12 hr postinfection. Chemotherapy (one drop every 15 min) was given from 4-9, 10-15, or 10-20 hr postinfection. Early therapy (4-9 hr postinfection) with ciprofloxacin rendered all eyes free of bacteria; ciprofloxacin was significantly more effective than vancomycin or cefazolin. When treatment was initiated 6 hr later (10-15 hr postinfection), no corneas became free of bacteria, but ciprofloxacin was again more effective than vancomycin or cefazolin. Bacterial killing by ciprofloxacin after treatment from 10-20 hr postinfection was also significantly greater than that of vancomycin. Overall, the results show that ciprofloxacin is effective in killing methicillin-resistant staphylococcus aureus, and is most effective when applied during the very early stages of infection.