Correlation of Staphylococcus Epidermidis Phenotype and Its Corneal Virulence.

PURPOSE: S. epidermidis is an ocular pathogen and a leading cause of keratitis. It produces hemolysins and at least 3 proteases. The purpose of the present study is to compare the secretion of hemolysins and proteases between 28 ocular isolates and one non-ocular strain and to determine their relationship to ocular virulence in selected strains using a rabbit model of infection. MATERIALS AND METHODS: Culture supernatants were compared for protease production and hemolysis. Selected strains were injected into rabbit corneas and their virulence and pathology recorded. The major protease activity in a virulent strain was identified and the gene was cloned and expressed as a recombinant protein. The corneal toxicity of this protease was determined. Antibodies to the native protease were generated and tested for neutralizing activity in vivo and in vitro. The corneal pathology of the S. epidermidis protease was compared to the pathology of S. aureus V8 protease. RESULTS: Strains that exhibited the least protease activity in vitro caused significantly less ocular pathology in vivo (p ≤ 0.003). Strains that were hemolytic and secreted a major protease had numerically higher SLE scores. This protease was identified as the serine protease Esp. The recombinant Esp protease caused extensive pathology when injected into the corneal stroma (7.62 ± 0.33). Antibody generated against native Esp did not neutralize the activity of the protease in vivo or in vitro. The antibody reacted with Esp proteases secreted by other S. epidermidis strains. S. epidermidis Esp protease and its homologue in S. aureus caused similar ocular pathology when injected in the rabbit corneal stroma. CONCLUSION: Hemolysins and proteases seem to be important in corneal pathology caused by S. epidermidis infections. The Esp protease mediates significant corneal damage. S. epidermidis Esp and S. aureus V8 protease caused similar and extensive edema in rabbit corneas.

Staphylococcus aureus Superantigen-Like Protein SSL1: A Toxic Protease.

Staphylococcus aureus is a major cause of corneal infections that can cause reduced vision, even blindness. Secreted toxins cause tissue damage and inflammation resulting in scars that lead to vision loss. Identifying tissue damaging proteins is a prerequisite to limiting these harmful reactions. The present study characterized a previously unrecognized S. aureus toxin. This secreted toxin was purified from strain Newman ΔhlaΔhlg, the N-terminal sequence determined, the gene cloned, and the purified recombinant protein was tested in the rabbit cornea. The virulence of a toxin deletion mutant was compared to its parent and the mutant after gene restoration (rescue strain). The toxin (23 kDa) had an N-terminal sequence matching the Newman superantigen-like protein SSL1. An SSL1 homodimer (46 kDa) had proteolytic activity as demonstrated by zymography and cleavage of a synthetic substrate, collagens, and cytokines (IL-17A, IFN-γ, and IL-8); the protease was susceptible to serine protease inhibitors. As compared to the parent and rescue strains, the ssl1 mutant had significantly reduced virulence, but not reduced bacterial growth, in vivo. The ocular isolates tested had the ssl1 gene, with allele type 2 being the predominant type. SSL1 is a protease with corneal virulence and activity on host defense and structural proteins.

Mechanism of Pseudomonas aeruginosa Small Protease (PASP), a Corneal Virulence Factor.

Purpose: Pseudomonas aeruginosa is the leading cause of contact lens-associated bacterial keratitis. Secreted bacterial proteases have a key role in keratitis, including the P. aeruginosa small protease (PASP), a proven corneal virulence factor. We investigated the mechanism of PASP and its importance to corneal toxicity. Methods: PASP, a serine protease, was tested for activity on various substrates. The catalytic triad of PASP was sought by bioinformatic analysis and site-directed mutagenesis. All mutant constructs were expressed in a P. aeruginosa PASP-deficient strain; the resulting proteins were purified using ion-exchange, gel filtration, or affinity chromatography; and the proteolytic activity was assessed by gelatin zymography and a fluorometric assay. The purified PASP proteins with single amino acid changes were injected into rabbit corneas to determine their pathological effects. Results: PASP substrates were cleaved at arginine or lysine residues. Alanine substitution of PASP residues Asp-29, His-34, or Ser-47 eliminated protease activity, whereas PASP with substitution for Ser-59 (control) retained activity. Computer modeling and Western blot analysis indicated that formation of a catalytic triad required dimer formation, and zymography demonstrated the protease activity of the homodimer, but not the monomer. PASP with the Ser-47 mutation, but not with the control mutation, lacked corneal toxicity, indicating the importance of protease activity. Conclusions: PASP is a secreted serine protease that can cleave proteins at arginine or lysine residues and PASP activity requires dimer or larger aggregates to create a functional active site. Most importantly, proteolytic PASP molecules demonstrated highly significant toxicity for the rabbit cornea.

Pseudomonas aeruginosa Protease IV Exacerbates Pneumococcal Pneumonia and Systemic Disease.

Pneumonia is a pulmonary disease affecting people of all ages and is consistently a leading cause of childhood mortality and adult hospitalizations. Streptococcus pneumoniae and Pseudomonas aeruginosa are major lung pathogens commonly associated with community-acquired and nosocomial pneumonia. Additionally, mixed lung infections involving these bacterial pathogens are increasing in prevalence and are frequently more severe than single infections. The cooperative interactions of these two pathogens that impact pulmonary disease severity are understudied. A major secreted virulence factor of P. aeruginosa, protease IV (PIV), cleaves interleukin 22 (IL-22), a cytokine essential for maintaining innate mucosal defenses against extracellular pathogens. Here, we investigate the ability of PIV to augment the virulence of a pneumococcal strain with limited virulence, S. pneumoniae EF3030, in a C57BL/6 murine model of pneumonia. We demonstrate that pulmonary coinfection involving P. aeruginosa 103-29 and S. pneumoniae EF3030 results in pneumococcal bacteremia that is abrogated during pneumococcal coinfection with a PIV-deficient strain. Furthermore, intratracheal administration of exogenous PIV and EF3030 resulted in abundant immune cell infiltration into the lung with large abscess formation, as well as severe bacteremia leading to 100% mortality. Heat-inactivated PIV did not worsen pneumonia or reliably induce bacteremia, suggesting that the specific activity of PIV is required. Our studies also show that PIV depletes IL-22 in vivo Moreover, PIV-mediated enhancement of pneumonia and disease severity was dependent on the expression of pneumolysin (Ply), a prominent virulence factor of S. pneumoniae Altogether, we reveal that PIV and Ply additively potentiate pneumonia in a murine model of lung infection.IMPORTANCES. pneumoniae remains the leading cause of bacterial pneumonia despite widespread use of pneumococcal vaccines, forcing the necessity for appropriate treatment to control pneumococcal infections. Coinfections involving S. pneumoniae with other bacterial pathogens threaten antibiotic treatment strategies and disease outcomes. Currently, there is not an effective treatment for alveolar-capillary barrier dysfunction that precedes bacteremia. An understanding of the dynamics of host-pathogen interactions during single and mixed pulmonary infections could elucidate proper treatment strategies needed to prevent or reduce invasive disease. Antibiotic treatment decreases bacterial burden in the lung but also increases acute pathology due to cytotoxins released via antibiotic-induced bacterial lysis. Therefore, targeted therapeutics that inhibit or counteract the effects of bacterial proteases and toxins are needed in order to limit pathology and disease progression. This study identifies the cooperative effect of PIV and Ply, products of separate lung pathogens that additively alter the lung environment and facilitate invasive disease.

Reactions with Antisera and Pathological Effects of Staphylococcus aureus Gamma-Toxin in the Cornea.

PURPOSE: This study analyzed the toxicity of purified gamma-toxin from Staphylococcus aureus and the protectiveness of antisera to gamma-toxin in the rabbit cornea. MATERIALS AND METHODS: Gamma-toxin was purified from cultures of alpha-toxin deficient S. aureus strain Newman Δhla. Antisera to native gamma-toxin (Hlg) were produced in rabbits. These antisera and a commercial polyclonal antibody to recombinant HlgB (rHlgB) were analyzed for specificity and toxin neutralization. Heat-inactivated gamma-toxin, active gamma-toxin either alone or with antisera or with commercial antibody to rHlgB, was injected into the rabbit cornea to observe the pathological effects using slit lamp examination scoring (SLE) and histological analyses. RESULTS: Eyes with intrastromal injection of gamma-toxin developed SLE scores that were significantly higher than eyes injected with heat-inactivated gamma-toxin (p ≤ 0.003). Slit lamp and histological examination of eyes revealed that gamma-toxin injected into the cornea mediated conjunctival injection and chemosis, iritis, fibrin accumulation in the anterior chamber, and polymorphonuclear neutrophil infiltration of the cornea and iris. Also, eyes injected with gamma-toxin plus antisera to native whole gamma-toxin or HlgB, but not with commercial antibody to rHlgB, yielded significantly lower SLE scores than eyes injected with gamma-toxin alone (p ≤ 0.003). CONCLUSIONS: This study illustrates that S. aureus gamma-toxin is capable of causing significant corneal pathology. Furthermore, the use of polyclonal antisera specific for native gamma-toxin was found to inhibit the damaging effects of the toxin in the rabbit cornea.

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-ß-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.

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

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.

Pseudomonas aeruginosa small protease (PASP), a keratitis virulence factor.

PURPOSE: The virulence contribution of Pseudomonas aeruginosa small protease (PASP) during experimental keratitis was studied by comparing a PASP-deficient mutant with its parent and rescue strains. METHODS: The pasP gene of P. aeruginosa was replaced with the tetracycline resistance gene via allelic exchange. A plasmid carrying the pasP gene was introduced into the PASP-deficient mutant to construct a rescue strain. The PASP protein in the culture supernatants was determined by Western blot analysis. Corneal virulence was evaluated in rabbit and mouse keratitis models by slit lamp examination (SLE), bacterial enumeration, and/or histopathological analysis. Various host proteins and the rabbit tear film were analyzed for their susceptibility to PASP degradation. RESULTS: The PASP-deficient mutant produced a significantly lower mean SLE score when compared with the parent or rescue strain (P ≤ 0.03) at 29 hours postinfection (PI). All of the strains grew equally in the rabbit cornea (P = 0.971). Corneas infected with the PASP-deficient mutant showed moderate histopathology compared with those infected with the parent or rescue strain, which produced severe pathology inclusive of epithelial erosions, corneal edema, and neutrophil infiltration. In the mouse model, eyes inoculated with the PASP-deficient mutant had a significantly lower mean SLE score at 24 hours PI than the eyes inoculated with the parent or rescue strain (P ≤ 0.007). PASP was found to degrade complement C3, fibrinogen, antimicrobial peptide LL-37, and constituents of the tear film. CONCLUSIONS: PASP is a commonly secreted protease of P. aeruginosa that contributes significantly to the pathogenesis of keratitis.

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 × 10(5) 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 10(5) 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.

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 (10(6) 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.

The effectiveness of an improved combination therapy for experimental Staphylococcus aureus keratitis.

INTRODUCTION: antibiotic and steroid combination therapies, such as tobramycin with dexamethasone, are often used in ophthalmology to treat or prevent infection and inflammation. The purpose of this study was to use a model of Staphylococcus aureus keratitis to quantify and compare the effectiveness of a standard tobramycin and dexamethasone combined therapy, with each drug individually, and with a new formulation of the two drugs in a xanthan gum vehicle. METHODS: rabbit corneas were intrastromally injected with a methicillin-sensitive S. aureus (MSSA) or a methicillin-resistant S. aureus (MRSA) strain. Rabbit eyes were treated every hour from 10 to 15 hours postinfection (PI) with 0.1% dexamethasone, 0.3% tobramycin, 0.3% tobramycin with 0.1% dexamethasone, or 0.3% tobramycin with 0.05% dexamethasone in a xanthan gum vehicle (ST). Slit lamp examinations (SLE) were performed on infected eyes and pathology scored at 15 hours PI. At 16 hours PI, colony forming units (CFUs) per cornea were quantified. RESULTS: the CFUs in eyes treated with dexamethasone alone were similar to untreated control eyes for MSSA or MRSA infections. All other treatment groups had significantly less CFUs per cornea than untreated eyes. The eyes treated with the ST formulation had significantly fewer CFUs per cornea than all other treatment groups when infected with MSSA or MRSA. The SLE scores of MSSA or MRSA infected eyes treated with tobramycin alone were similar to untreated control eyes. All other treatment groups had significantly lower SLE scores than untreated controls eyes, but were not significantly different from each other. CONCLUSION: the results of this study demonstrated that the tobramycin and dexamethasone combination therapy with a xanthan gum vehicle has an improved bactericidal effectiveness compared to the commercially available formulation, and maintains a similar anti-inflammatory effect while containing half the amount of steroid.

Sustained anti-staphylococcal effect of lysostaphin in the rabbit aqueous humor.

PURPOSE: Staphylococcus aureus is an important cause of ocular infections including endophthalmitis. The purpose of this study was to determine the ability of a relatively large molecule, such as lysostaphin, to remain in the rabbit aqueous humor for extended periods while retaining its bactericidal activity. METHODS: Lysostaphin, gatifloxacin, or Tris-buffered saline (TBS) was injected into the rabbit anterior chamber. Aqueous humor was sampled at 0.5, 1, 2, 3, and 4 days after injection, and then assayed for bactericidal activity against S. aureus. The anterior chamber of treated eyes was also challenged 2 days after treatment by an infection of S. aureus. The surviving bacteria were quantified to determine bactericidal effectiveness in the anterior chamber. The aqueous humor of lysostaphin injected eyes was assayed by western blot for the presence of the molecule 2 days post-injection. RESULTS: The bactericidal activity of lysostaphin was confirmed by lysis of S. aureus and sensitivity to zinc. Eyes injected with lysostaphin showed no adverse reactions. Aqueous humor of gatifloxacin injected eyes demonstrated no greater effectiveness than that of TBS injected eyes in vitro at any time point assayed, whereas lysostaphin injected eyes retained potent bactericidal activity for at least 3 days. In an in vivo challenge, the anterior chamber of lysostaphin injected eyes retained significant bactericidal activity for at least 2 days after treatment, whereas gatifloxacin injected eyes demonstrated no significant difference from those injected with TBS. Western blot analysis demonstrated the presence of lysostaphin in the aqueous humor 2 days post-injection. CONCLUSIONS: Lysostaphin demonstrated the ability to remain in the aqueous humor for days while maintaining its bactericidal activity, an indication that a high molecular weight antimicrobial can provide prolonged prophylactic protection of the anterior chamber.

A highly virulent Staphylococcus aureus: rabbit anterior chamber infection, characterization, and genetic analysis.

PURPOSE: To describe and characterize a Staphylococcus aureus strain with unique virulence that overcomes host defenses of the rabbit anterior chamber and mimics clinical cases of postcataract surgery endophthalmitis. METHODS: Nine isolates of S. aureus were tested to determine their viability in the rabbit anterior chamber. Growth of UMCR1 in the anterior chamber was established and expressed as log colony-forming units per milliliter of aqueous humor. Pathologic changes produced by UMCR1 were documented by photographs, slit lamp examination, histopathologic analysis, and quantification of neutrophils. UMCR1 was characterized by antibiotic susceptibility, biochemical tests, ribotyping, genome restriction mapping, and multilocus sequence typing (MLST). RESULTS: UMCR1 was the only S. aureus strain that grew within the anterior chamber, reaching log 6.97 ± 0.18 CFU/mL by 16 hours after infection. Pathologic changes included conjunctival injection, chemosis, corneal edema, severe iritis, fibrin accumulation, and a 193-fold increase in neutrophils by 16 hours after infection. UMCR1 was only resistant to sulfamethoxazole and, like other S. aureus isolates, polymyxin B. UMCR1 also had biochemical reactions and a ribotype pattern typical of S. aureus. The genomic reconstruction analysis of UMCR1 was most similar to strains MW2 and MSSA476. MLST revealed a 1 in 3198 nucleotide difference between UMCR1 and strains MW2 and MSSA476. CONCLUSIONS: This study describes a unique S. aureus strain that overcomes host defenses and replicates in the anterior chamber. The survival and growth of this organism could be used for studies of S. aureus pathogenesis, host defenses, and effectiveness of antibiotics within the anterior chamber.

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= 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.

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= 0.0648). 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.

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 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. RESULTS: 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. 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).

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.

Age-related differences in rabbits during experimental Staphylococcus aureus keratitis.

PURPOSE: To analyze age-related changes in susceptibility to experimental Staphylococcus aureus keratitis and purified alpha-toxin in rabbits. METHODS: 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. RESULTS: 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. CONCLUSIONS: Corneas and erythrocytes of young rabbits, relative to aged rabbits, are significantly more susceptible to S. aureus keratitis and to alpha-toxin.