Slit Lamp Examination Scores Research Articles

Staphylococcus Alpha-Toxin Action on the Rabbit Iris: Toxic Effects and Their Inhibition

Purpose: Staphylococcus aureus infection of the anterior chamber can occur after cataract surgery, causing inflammation and extensive damage to the iris. Alpha-toxin, the most potent S. aureus corneal toxin, was tested as a possible mediator of damage to the iris, and alpha-toxin anti-serum and a chemical toxin inhibitor were tested as potential pathology-reducing agents.Methods: The hemolytic activity of alpha-toxin and its inhibition by a chemical inhibitor or anti-serum were quantified in vitro. Purified alpha-toxin, heat-inactivated toxin, or alpha-toxin plus normal serum, alpha-toxin anti-serum, or the chemical inhibitor, methyl-β-cyclodextrin-cholesterol (CD-cholesterol), was injected into the rabbit anterior chamber. Pathological changes were photographed, quantified by slit-lamp examination (SLE) scoring, and further documented by histopathological analysis.Results: At five hours post-injection, eyes injected with alpha-toxin or heat-inactivated toxin had a mean SLE score of 7.3 ± 0.59 or 0.84 ± 0.19, respectively. Active toxin caused moderate to severe iris edema, severe erosion of the iris, and mild to moderate fibrin accumulation in the anterior chamber. Alpha-toxin plus anti-serum or CD-cholesterol, in contrast to alpha-toxin alone, caused less iris edema and epithelium sloughing as well as significantly lower SLE scores than eyes receiving alpha-toxin alone (p ≤ 0.019).Conclusion: Alpha-toxin caused extensive iris damage and inflammation, and either anti-alpha-toxin anti-serum or CD-cholesterol was able to significantly reduce toxin-mediated damage and inflammation.

Moxifloxacin as Postoperative Prophylaxis forEnterococcus faecalis-Induced Endophthalmitis After Cataract Surgery in Aphakic Rabbits

The development of endophthalmitis after cataract surgery often results in significant vision loss. Inhibition of bacterial proliferation in the anterior chamber using antibiotic eye drops is important to prevent endophthalmitis after cataract surgery. We aimed to determine the sensitivity of fluoroquinolones against Enterococcus faecalis ocular isolates and the efficacy of fluoroquinolones to prevent E. faecalis-induced endophthalmitis in aphakic rabbits. The minimum inhibitory concentrations (MICs) of moxifloxacin (MFLX) and levofloxacin (LVFX) used in ophthalmic solutions for 13 E. faecalis isolates obtained from the conjunctival sac or endophthalmitis cases were determined. Eye drops containing MFLX (0.5%), LVFX (0.5%), or saline were administered to aphakic rabbits with endophthalmitis induced by E. faecalis. The eye drops were administered immediately after lensectomy and at 3 and 6 h after cataract surgery (early instillation group) or immediately after lensectomy and at 12 and 15 h after cataract surgery (delayed instillation group). Bacterial growth, electroretinography (ERG), and slit-lamp examination (SLE) were determined throughout the course of infection. In vitro susceptibility testing revealed that the MICs of MFLX for E. faecalis isolates were lower than those of LVFX. In the early ocular instillation groups, MFLX significantly improved SLE scores, ERG, and viable bacterial counts compared with LVFX and saline (all, P<0.05). There were no significant differences in any parameter between MFLX and saline in the delayed ocular instillation groups. Early ocular instillation of MFLX delays retinal damage compared with LVFX when used to treat E. faecalis-induced endophthalmitis after cataract surgery.

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.

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.

The Streptococcus pneumoniae Capsule Is Required for Full Virulence in Pneumococcal Endophthalmitis

To determine whether Streptococcus pneumoniae capsule was necessary for pathogenesis of pneumococcal endophthalmitis. An isogenic capsule-deficient strain was created using homologous recombination. New Zealand White rabbits were injected intravitreously with 10(2) colony-forming units (CFU) of the parent strain or the capsule mutant. Slit lamp examination (SLE), electroretinography, and myeloperoxidase activity were performed 24 and 48 hours postinfection (PI). Serial dilutions of vitreous were plated to quantitate CFU, eyes were extracted for histology, and host cytokine mRNA expression was determined. Eyes infected with the parent strain had significantly higher SLE scores than eyes infected with the capsule-deficient strain 24 and 48 hours PI (P < 0.001). CFU recovered from eyes infected with the capsule mutant were significantly fewer than CFU recovered from eyes infected with the parent strain 24 and 48 hours PI (P < 0.001). The parent strain caused a significantly greater decrease in retinal function and more retinal destruction than the mutant strain 48 hours PI (P = 0.026). Vitreal IL-1β, IL-6, and TNF-α were upregulated by both the parent and mutant strain 12 hours PI. By 48 hours PI, there was significantly more neutrophil infiltration in the vitreous infected with the parent strain. Endophthalmitis caused by the encapsulated strain is more damaging to retinal function and structural integrity. These findings indicate that capsule is an important virulence factor of S. pneumoniae endophthalmitis, in contrast to keratitis, suggesting that the anatomic host site in pneumococcal ocular infections is important.

Immunization with Pneumolysin Protects Against Both Retinal and Global Damage Caused byStreptococcus pneumoniaeEndophthalmitis

To determine whether immunization with pneumolysin (PLY) protects against pneumococcal endophthalmitis. New Zealand white rabbits were immunized with a mutant form of PLY that retains only 1% of its cytolytic activity until serum IgG titers were ≥51,200. For a negative control, rabbits were immunized with phosphate-buffered saline (mock). Each vitreous was injected with 10(2) colony-forming units of a clinical endophthalmitis isolate of Streptococcus pneumoniae. Severity of endophthalmitis was graded by slit lamp examination at 24 and 48 h postinfection (PI). Serial dilutions of vitreous were plated for bacterial colony-forming units quantitation, eyes were extracted for histology, and a whole blood survival assay was performed. Immunized rabbits had a significantly lower mean slit lamp examination score at 24 and 48 h PI when compared to mock immunized rabbits (P ≤ 0.002). There was not a significant difference in bacterial load in the vitreous at 24 or 48 h PI. Histological sections showed that retinas of mock immunized rabbits appeared to be destroyed, whereas those of PLY immunized rabbits remained largely intact. Damage spread to the aqueous humor, stroma, and conjunctiva of mock immunized rabbits by 48 h PI. Minimal damage was observed in the vitreous of PLY immunized rabbits and did not spread to other parts of the eye. Whole blood from immunized rabbits inhibited the growth of bacteria better than whole blood from mock immunized rabbits. Immunization with PLY helps protect the eye from damage caused by pneumococcal endophthalmitis.

Properties of PASP: APseudomonasProtease Capable of Mediating Corneal Erosions

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. 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). 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. PASP is a commonly produced Pseudomonas protease that can cleave collagens and cause corneal erosions.

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

alpha-Toxin mediates extreme corneal damage during Staphylococcus aureus keratitis. Chemical inhibition of this toxin was sought to provide relief from toxin-mediated pathology. 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. 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). CD-cholesterol is a potent inhibitor of alpha-toxin activity in vitro and an effective means to arrest corneal damage during S. aureus keratitis.

Development of a Streptococcus pneumoniae Keratitis Model in Mice

Background:Streptococcus pneumoniae is a common cause of bacterial keratitis, and models to examine the ocular pathogenesis of this bacterium would aid in efforts to treat pneumococcal keratitis. The aim of this study was to establish a murine model of pneumococcal keratitis. Methods: The corneas of A/J, BALB/c or C57BL/6 mice were scratched and topically infected with a clinical strain of S. pneumoniae. Slitlamp examination (SLE), enumeration of bacteria in the corneas and histology were performed. Results: Bacteria were recovered from the eyes of A/J mice on postinfection (PI) days 1 [1.96 ± 0.61 log₁₀ colony-forming units (CFU)] and 3 (1.41 ± 0.71 log₁₀ CFU). SLE scores were significantly higher in the infected A/J mice as compared to the BALB/c or C57BL/6 mice on PI day 3 (p < 0.0001) and steadily increased over time, reaching a maximal value of 3.00 ± 0.35 on PI day 10. Histopathology revealed stromal edema and the influx of polymorphonuclear leukocytes on PI days 7 and 10, and corneal disruption on PI day 7. Conclusions:S. pneumoniae keratitis was established in A/J mice, but not BALB/c or C57BL/6 mice.

A comparison of pneumolysin activity and concentration in vitro and in vivo in a rabbit endophthalmitis model

The purpose of this study was to determine whether the in vitro activity and concentration of Streptococcus pneumoniae pneumolysin correlated to the pathogenesis of S. pneumoniae endophthalmitis. Five S. pneumoniae clinical endophthalmitis strains were grown in media to similar optical densities (OD), and extracellular milieu was tested for pneumolysin activity by hemolysis of rabbit red blood cells. Pneumolysin concentration was determined using a sandwich ELISA. Rabbit vitreous was injected with 10(2) colony-forming units (CFU) of 1 of 2 different strains with low hemolytic activity (n = 10 and 12 for strains 4 and 5, respectively) or 1 of 3 different strains with high hemolytic activity (n = 12 per strain). Pathogenesis of endophthalmitis infection was graded by slit lamp examination (SLE) at 24 hours post-infection. Bacteria were recovered from infected vitreous and quantitated. The SLE scores of eyes infected with strains having high hemolytic activity were significantly higher than the scores of those infected with strains having low hemolytic activity (P < 0.05). Pneumolysin concentration in vitro, however, did not correlate with hemolysis or severity of endophthalmitis. Bacterial concentrations from the vitreous infected with 4 of the strains were not significantly different (P > 0.05). These data suggest that pneumolysin hemolytic activity in vitro directly correlates to the pathogenesis of S. pneumoniae endophthalmitis. The protein concentration of pneumolysin, however, is not a reliable indicator of pneumolysin activity.

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

ABSTRACTObjective: 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.

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

To develop a rabbit model of post-laser in situ keratomileusis (LASIK) methicillin-resistant Staphylococcus aureus (MRSA) keratitis for studying fluoroquinolone prophylaxis and treatment. Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi, USA. An MRSA keratitis isolate (5 microL, 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. Bacteria grew to a maximum of approximately 10(6) 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 < or = .0001). During early treatment, the antibiotics were equally effective in reducing CFU relative to untreated controls (P < or = .0001). In late treatment, gatifloxacin and moxifloxacin caused significant reductions in CFU relative to untreated controls (P < or = .0007 and P < or = .0001, respectively). Moxifloxacin produced zones of bacterial inhibition significantly larger than those produced by gatifloxacin. 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).

Age-Related Differences in Rabbits during ExperimentalStaphylococcus aureusKeratitis

To analyze age-related changes in susceptibility to experimental Staphylococcus aureus keratitis and purified alpha-toxin in rabbits. Intrastromal injection of S. aureus (100 colony-forming units [CFUs]) induced keratitis in young (6-8 weeks) and aged (approximately 30 months) New Zealand White rabbits. Bacteria and polymorphonuclear leukocytes (PMNs) per cornea were quantified. Purified alpha-toxin at 1, 10, 25, or 50 hemolytic units (HU) or heat-inactivated alpha-toxin was intrastromally injected into corneas, and pathologic changes were determined by slit lamp examination (SLE) and histopathologic analysis. alpha-Toxin hemolysis assays were performed using erythrocytes from young and aged rabbits. S. aureus keratitis produced significantly higher SLE scores in young rabbits than in aged rabbits at 15, 20, and 25 hours postinfection (PI; P < or = 0.001); aged rabbits essentially recovered from S. aureus keratitis by 7 days PI. At 25 hours PI, numbers of CFUs and PMNs in corneas of young and aged rabbits were equivalent (P > or = 0.6); the bacterial burden in aged rabbits declined by 5 logs per cornea from day 1 to day 7 PI. Intrastromal injection of > or =10 HU alpha-toxin also produced significantly more disease in young than in aged rabbit corneas (P < or = 0.05), whereas 1 HU or heat-inactivated toxin yielded negligible pathologic changes in either group. Hemolysis assays of erythrocytes from young rabbits demonstrated greater susceptibility to alpha-toxin compared with those from aged rabbits. Corneas and erythrocytes of young rabbits, relative to aged rabbits, are significantly more susceptible to S. aureus keratitis and to alpha-toxin.

Cholesterol as Treatment for Pneumococcal Keratitis: Cholesterol-Specific Inhibition of Pneumolysin in the Cornea

The purpose of this study was to determine whether cholesterol, the host cell receptor for pneumolysin of Streptococcus pneumoniae, could effectively treat pneumococcal keratitis. New Zealand White rabbits were intrastromally injected with 10(5) colony-forming units (CFUs) of S. pneumoniae D39. Corneas were treated with topical drops of 1% cholesterol every 2 hours beginning 25 hours after infection and were examined by slit lamp microscopy 24, 36, and 48 hours after infection. Rabbits were killed, and CFUs were recovered from the corneas after the final slit lamp examination (SLE). Minimal inhibitory concentration (MIC) assays of cholesterol against bacteria were performed. Specific inhibition of pneumolysin by cholesterol in the rabbit cornea was tested by intrastromal injection of pneumolysin with or without cholesterol and was compared with cholesterol inhibition of pneumolysin in vitro using hemolysis assays with rabbit erythrocytes. Corneas treated with cholesterol had significantly lower SLE scores 48 hours after infection than corneas treated with vehicle (P = 0.0015). Treated corneas also had significantly less log(10) CFUs than vehicle-treated corneas (P = 0.0006). Cholesterol at a 1% concentration was bactericidal to bacteria in vitro, and lower concentrations of cholesterol were partially inhibitory in a concentration-dependent manner. Cholesterol also specifically inhibited 1 mug pneumolysin in vivo and up to 50 ng pneumolysin in vitro. Topical cholesterol is an effective treatment for S. pneumoniae keratitis. Cholesterol not only inhibits pneumolysin, it is also bactericidal.

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 (10 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.

Effects of Toxin Production in a Murine Model ofStaphylococcus aureusKeratitis

To investigate the corneal virulence of toxin-deficient mutants of Staphylococcus aureus in young and aged mice in a topical inoculation model of keratitis. Corneas of young and aged A/J mice were scarified and topically inoculated with a log phase S. aureus parent strain (8325-4), an alpha-toxin-deficient mutant (DU1090), or an Agr-defective mutant (ISP546) deficient in production of multiple toxins or with purified alpha-toxin. Slit lamp examination (SLE) and histopathology were performed, and bacterial colony-forming units (CFU) and myeloperoxidase (MPO) activity were determined. The infection of young mice with the mutant strains demonstrated significantly lower SLE scores (P < or = 0.0001) and reduced histopathologic changes compared with infections with the parent bacterial strain. Either mutant strain of S. aureus produced SLE scores in aged mice through 9 days after infection (PI) that were significantly lower than those of aged mice similarly infected with the toxin-producing parent strain (P < or = 0.0001). Despite use of identical inocula, the CFU per eye were greater for the parent than the mutant strains from 1 to 5 days PI in the young mice (P < or = 0.0372) and from 1 to 3 days PI in the aged mice (P < or = 0.0018). MPO activities were at the maximum at day 1 PI and were similar overall for all infections. Administration of purified alpha-toxin caused greater gross and histopathologic changes in eyes of aged mice than in those of young mice. Bacterial toxins, and especially alpha-toxin, can mediate corneal disease in mice. Differences in severity of S. aureus keratitis in aged versus young mice correlates with their susceptibility to alpha-toxin.

Ocular Virulence of Capsule-DeficientStreptococcus pneumoniaein a Rabbit Keratitis Model

Determine the ocular virulence of noncapsular Streptococcus pneumoniae in a rabbit keratitis model. Mice were infected intraperitoneally with 10(5) colony-forming units (CFUs) of Avery's strain (capsular type 2) or strain R6 (a noncapsular derivative of type 2), and mortality was monitored daily. In addition, 10(5) CFU of each strain was injected into rabbit corneas. Bacterial loads in rabbit corneas were determined at 20 or 48 hours after infection. Slit lamp examination (SLE) of rabbit eyes was performed at 24, 36, and 48 hours after infection. Controls included corneas inoculated with bacterial suspension medium and UV-killed bacteria. One hundred percent mortality was observed in mice infected intraperitoneally with the encapsulated strain at 2 days after infection, whereas all mice infected with the nonencapsulated strain survived for 21 days. The nonencapsulated strain caused the same pathologic effects in the rabbit cornea as the encapsulated strain at 24, 36, and 48 hours after infection (P > or = 0.080). Control corneas showed no pathologic effects and had significantly lower SLE scores than corneas infected with live bacteria (P < or = 0.001). Mean bacteria log CFU +/- SEM recovered at 20 hours after infection were 7.069 +/- 0.094 for the encapsulated and 6.533 +/- 0.116 for the nonencapsulated strain (P = 0.001). Bacteria recovered from the corneas at 48 hours after infection were 6.712 +/- 0.349 and 1.807 +/- 0.462 for the encapsulated and nonencapsulated strains, respectively (P < 0.001). The S. pneumoniae noncapsular strain was as virulent in the rabbit cornea as was the encapsulated strain, but unlike the encapsulated strain, was avirulent in the mouse peritoneum.

Effectiveness of Topical Taurolidine versus Ciprofloxacin, Ofloxacin, and Fortified Cefazolin in a Rabbit Staphylococcus aureus Keratitis Model

Purpose. Taurolidine is a broad-spectrum, nonantibiotic antimicrobial agent, not previously tested against the common causes of bacterial keratitis. This study, employing an experimental rabbit model of Staphylococcus aureus keratitis, investigated the effectiveness of topical taurolidine in reducing the number of bacteria, and its effectiveness was compared with topical ciprofloxacin, ofloxacin, and 5% cefazolin.Methods. The right corneas of all rabbits were intrastromally injected with 100 colony-forming units of Staphylococcus aureus ATCC strain 25923. The animals were divided into the following seven groups: Group 1 (6 rabbits) received taurolidine, group 2 (6 rabbits) received ciprofloxacin, group 3 (6 rabbits) received ofloxacin, group 4 (6 rabbits) received cefazolin, group 5 (5 rabbits) received polyvinylpyrrolidone (vehicle), group 6 (4 rabbits) received sterile water, and group 7 (4 rabbits) was left untreated (control group). The eyes were topically treated every 30 min with the above-mentioned substances from 4 to 9 h postinjection. One hour after the last drop administration (at 10 h postinjection), signs of inflammation were scored in a masked fashion by slit-lamp examination. Then, their corneas were processed. The number of colony-forming units (cfu) per cornea in all eyes was also determined.Results. All antimicrobial (taurolidine, ciprofloxacin, ofloxacin, and cefazolin) treatments significantly reduced cfu numbers and slit-lamp examination scores compared with untreated eyes, eyes that received the vehicle, or eyes with sterile water (all p values < 0.05). Regarding cfu numbers, although taurolidine therapy was significantly less effective than ciprofloxacin or ofloxacin, there was no significant difference between taurolidine and cefazolin groups. However, taurolidine had similar clinical examination scores with the other antimicrobials, while it had lower scores than the vehicle, sterile water, or untreated eyes. Conclusions. The results obtained in this study suggest that topical taurolidine is an effective, novel ocular chemotherapeutic agent for the therapy of rabbit experimental Staphylococcus aureus keratitis. This drug may be a useful and promising ocular antimicrobial.

Susceptibility of aged mice to Staphylococcus aureus keratitis

Purpose. To determine the effect of age on the extent of pathogenesis of Staphylococcus keratitis in the mouse. Methods. Corneas of young and aged mice (BALB/c, A/J, and C57BL/6) were scarified and topically inoculated with S. aureus. Slit lamp examination (SLE) and histopathology were performed, and bacterial colony forming units and myeloperoxidase activity were determined. Results. SLE scores of infected eyes of aged mice were significantly higher at days 1 and 3 postinfection (PI) as compared to infected young mice. Histopathological changes observed in all aged mice were more severe than those in young mice. Young BALB/c and A/J mice demonstrated minimal signs of keratitis by day 3 PI, whereas aged mice of both strains demonstrated severe keratitis by day 3. Young C57BL/6 mice showed no clinical signs of keratitis, whereas aged C57BL/6 mice demonstrated moderate keratitis. Conclusions. Aged mice with S. aureus keratitis demonstrated increased pathology as compared to young mice.

A new topical model of Staphylococcus corneal infection in the mouse.

To establish, in the scarified mouse eye, a new model of Staphylococcus aureus keratitis suitable for studies of pathogenesis and host defense mechanisms. Corneas of three strains of mice (BALB/c, A/J, and C57BL/6) were scarified and inoculated with S. aureus strain 8325-4. Mice underwent slit lamp examination (SLE) at 1, 3, 5, 7, and 9 days after infection and were killed. Histopathologic analyses, determination of bacterial colony-forming units (CFU), and myeloperoxidase (MPO) activity assays were performed at each time point. S. aureus keratitis developed in both BALB/c and A/J strains of mice, but not in C57BL/6. The BALB/c and A/J strains demonstrated greater susceptibility to infection, as evidenced by significantly higher SLE scores and more viable bacteria per infected eye than in C57BL/6 mice at 5, 7, and 9 days after infection (P <or= 0.0001). Histopathologic analysis and MPO assays of infected A/J mice both revealed an influx of polymorphonuclear leukocytes (PMNs). Histology demonstrated presence of leukocytes in the aqueous humor, migration of PMNs into infected tissue, corneal erosion, and edema in the eyes of infected A/J mice. Whereas infected BALB/c mice demonstrated both PMN migration and corneal edema, eyes of infected C57BL/6 mice failed to show even mild histopathologic changes. These studies demonstrate the establishment of Staphylococcus keratitis in the mouse eye. This model should provide for a large range of future studies that are currently unavailable in the rabbit keratitis model, particularly those requiring a genetically altered host or specific immunologic reagents.