Identification of pathogenic factors potentially involved in Staphylococcus aureus keratitis using proteomics.

Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis.

The role of CXC chemokine receptor 2 in Staphylococcus aureus keratitis.

Staphylococcus aureus is a leading cause of corneal infection. CXC receptor 2 binding chemokines have been implicated in the pathogenesis of Pseudomonas aeruginosa keratitis. The role of this receptor in immune responses during Staphylococcus keratitis remains to be fully understood. Corneas of CXC receptor 2 knockout and wild-type mice (Cmkar -/- & Cmkar +/+) were scratched and 1 × 10(8) cfu/ml of strain Staph 38 applied. Twenty-four hours post-infection, mice were sacrificed and eyes harvested for enumeration of bacteria and measurement of myeloperoxidase levels. Production of inflammatory mediators, cellular adhesion molecules and chemokines in response to infection were investigated by ELISA, and PCR. 24 h after challenge with S. aureus, Cmkar -/- mice had developed a more severe response with a 50-fold higher bacterial load than WT mice. PMNs failed to penetrate the corneas of Cmkar -/- mice. However, concentrations of KC, MIP-2, IL-1ß and IL-6 were significantly elevated (6-13 fold) in Cmkar-/- mice. The concentration of LTB4 was decreased (2 fold). Cmkar-/- mice failed to upregulate mRNA for VCAM-1 or PECAM-1 in response to infection, but had constitutively higher levels of ICAM-1. A lack of CXC receptor 2 lead to an inability to control bacterial numbers as a result of failure of PMNs to penetrate the cornea to the site of infection, even when chemokines were more highly produced. These results imply that CXCR2-mediated signaling through upregulation of adhesion molecules is essential to margination of PMNs in this infection model.

In vivo performance of melimine as an antimicrobial coating for contact lenses in models of CLARE and CLPU.

PURPOSE: One strategy to minimize bacteria-associated adverse responses such as microbial keratitis, contact lens-induced acute red eye (CLARE), and contact lens induced peripheral ulcers (CLPUs) that occur with contact lens wear is the development of an antimicrobial or antiadhesive contact lens. Cationic peptides represent a novel approach for the development of antimicrobial lenses. METHODS: A novel cationic peptide, melimine, was covalently incorporated into silicone hydrogel lenses. Confirmation tests to determine the presence of peptide and anti-microbial activity were performed. Cationic lenses were then tested for their ability to prevent CLPU in the Staphylococcus aureus rabbit model and CLARE in the Pseudomonas aeruginosa guinea pig model. RESULTS: In the rabbit model of CLPU, melimine-coated lenses resulted in significant reductions in ocular symptom scores and in the extent of corneal infiltration (P < 0.05). Evaluation of the performance of melimine lenses in the CLARE model showed significant improvement in all ocular response parameters measured, including the percentage of eyes with corneal infiltrates, compared with those observed in the eyes fitted with the control lens (P < or = 0.05). CONCLUSIONS: Cationic coating of contact lenses with the peptide melimine may represent a novel method of prevention of bacterial growth on contact lenses and consequently result in reduction of the incidence and severity of adverse responses due to Gram-positive and -negative bacteria during lens wear.

Soft contact lens disinfection solution efficacy: clinical Fusarium isolates vs. ATCC 36031.

PURPOSE: To compare the disinfecting efficacy of five soft contact lens multipurpose disinfection solutions (MPDS) against Fusarium solani clinical isolates and the ISO standard ATCC 36031 strain. METHODS: Three commercially available and two recalled MPDS were tested using the ISO/CD 14,729 stand-alone test for contact lens care products against 10 ocular isolates of F. solani and the ATCC 36031 strain. The effect of filtering the fungal suspension before incubating in MPDS was also tested. An average log reduction in colony forming units at the manufacturer's minimum recommended disinfection time was determined and compared with criteria for stand-alone disinfection products for each MPDS against each strain. RESULTS: No difference between filtered and unfiltered fungal suspensions was observed for the ISO standard, whereas in one MPDS the representative clinical isolate showed significantly increased resistance when unfiltered. All but one solution met the stand-alone criteria of 1.0-log reduction of colony forming units against the recommended ISO standard strain ATCC 36031. However, there was wide variation in the ability of MPDS to meet the ISO disinfection criteria when tested against clinical isolates. Among the commercially available MPDS, the two polyquaternium-based solutions showed a higher disinfecting efficacy than the biguanide-based solution. The two recalled solutions showed a lower disinfecting efficacy than the polyquaternium-based solutions. Further, the clinical isolates were significantly more resistant to disinfection than was the recommended ISO strain. CONCLUSIONS: The effect of filtering the fungal suspension to remove hyphae seems to be relevant in the clinical isolate tested, but not in the ISO strain. Clinical isolates were significantly more resistant to disinfection than the recommended ISO strain in the presence of both the commercially available and the recalled MPDS. The use of clinical isolates in stand-alone disinfection testing is indicated. Because there were significant differences in increased resistance exhibited by clinical isolates and in a mixed (unfiltered) culture the use of a single laboratory strain may be insufficient to provide assurance that the disinfection solution will be effective against clinical isolates.

Staphylococcus aureus ocular isolates from symptomatic adverse events: antibiotic resistance and similarity of bacteria causing adverse events.

BACKGROUND: Staphylococcus is the leading cause of microbial keratitis. Staphylococcus aureus isolated from ocular infections with resistance to a wide range of antibiotics, including the commonly prescribed fluoroquinolones, is emerging. The aim of this study was to determine the current antibiotic susceptibilities of ocular S. aureus isolates and also determine whether isolates from different adverse events or those with similar antimicrobial susceptibilities are related. METHODS: A collection of 55 S. aureus strains from ocular adverse events were analysed for antibiotic susceptibility using disc diffusion (CDS method) and typed using PCR-ribotyping and pulsed-field gel electrophoresis (PFGE). RESULTS: S. aureus isolated from symptomatic ocular adverse events in the USA exhibited greater resistance to antibiotics than did those isolated from symptomatic ocular adverse events in Australia or India (p<0.05). A larger proportion of ulcerative keratitis isolates was found to be resistant to antibiotics than isolates from conjunctivitis. PFGE analysis separated related isolates determined by ribotype, on the basis of the adverse event caused by the isolate. Isolates were related within geographical regions and adverse event types. CONCLUSIONS: Similar isolates within a geographical location cause adverse events but there is a genetic difference between isolates causing corneal adverse events and those causing conjunctivitis. Isolates from corneal adverse events were more resistant to antibiotics, with those from the USA exhibiting the greatest resistance. This suggests that virulence may correlate with increased resistance to antibiotics.

Efficacy of contact lens multipurpose solutions against serratia marcescens.

PURPOSE: To compare the susceptibilities of clinical isolates of Serratia marcescens and the standard ISO ATCC 13880 strain to five contact lens multipurpose disinfection solutions (MPDSs). METHODS: Five commercially available MPDSs, containing either a polymeric biguanide or polyquaternium, were tested using ISO/CD 14729 stand-alone test for contact lens care products against four ocular isolates of S. marcescens and the strain ATCC 13880. An average log reduction in bacterial numbers at the manufacturer's minimum recommended disinfection time was determined and compared with the criteria for stand-alone disinfection products for each MPDS against each bacterial strain. RESULTS: All the MPDSs tested met the stand-alone criteria of 3-log reduction of viable bacteria against the ATCC strain of S. marcescens. However, there was more variability in their ability to meet disinfection criteria when tested against the clinical isolates. Two of the clinical isolates were significantly more resistant to disinfection than was the recommended ISO strain (p < or = 0.034). Two of the polyquaternium-1-based disinfection solutions (solutions D and E, p < or = 0.005) were less effective overall than the other MPDSs against S. marcescens. CONCLUSIONS: The importance of strain selection for the testing of MPDSs is indicated, and the use of a single laboratory strain may be insufficient to provide assurance that the disinfection solution will be effective against clinical isolates. Furthermore, clinical isolates of S. marcescens may show increased resistance to disinfection with polyquaternium.

Effects of topical administration of 12-methyl tetradecanoic acid (12-MTA) on the development of corneal angiogenesis.

Corneal vascularisation is a potentially devastating occurrence that can cause blindness. Currently, treatments for this condition are limited. In these studies, we have investigated a novel inhibitor of angiogenesis, 12-methyl tetradecanoic acid (12-MTA), to treat corneal vascularisation in mouse models of corneal alkali injury and corneal Pseudomonas aeruginosa infection. The effectiveness of 12-MTA was compared to treatment with dexamethasone. 12-MTA was found to be at least as effective as dexamethasone in reducing the angiogenesis that occurs following alkali injury or P. aeruginosa infection of the cornea. The major effect of both 12-MTA and dexamethasone in these models was to reduce the linear incursion of new blood vessels into the central cornea. A significantly better result was obtained at 14 days post-alkali injury when treatment was not delayed. A major advantage of treatment of alkali injury with 12-MTA compared to that with dexamethasone was the finding that there was a 5-fold less level of PMN infiltration and no persistent epithelial defects in corneas treated with 12-MTA compared to 50% of those treated with dexamethasone. Our studies indicate that 12-MTA may provide clinically significant advantages over conventional steroids for the treatment of vessel growth in the cornea.

A protective role for IL-6 in staphylococcal microbial keratitis.

PURPOSE: To determine whether interleukin-6 (IL-6) plays a protective role in Staphylococcus aureus keratitis in a gene knockout (gko) mouse model and to determine whether IL-6 may be used as a therapy to modulate host responses and control bacterial infection, thereby reducing scarring. METHODS: The eyes of IL-6 gko mice and wild-type mice were challenged topically with S. aureus and examined at 24 hours after infection. Keratitis was examined clinically and histologically. Bacterial and polymorphonuclear leukocytes (PMNs) were enumerated, and cytokine and chemokine levels were determined by ELISA. Exogenous IL-6 was administered to both IL-6 gko and wild-type mice, and clinical parameters were determined. RESULTS: IL-6 gko mice showed more severe disease, with increased bacterial counts and PMNs, than did wild-type mice. Changes in levels of chemokines and cytokines were also observed. Administration of exogenous IL-6 resulted in an improved outcome in IL-6 gko mice, with a threefold reduction in bacterial load. CONCLUSIONS: The data suggest an important regulatory role for IL-6 in modulating excessive inflammatory responses and in controlling bacterial proliferation. IL-6 may play a role in the priming and activation of neutrophils. It could represent a broad-spectrum therapy to improve outcomes in patients who have these potentially blinding infections.

A Staphylococcus aureus mouse keratitis topical infection model: cytokine balance in different strains of mice.

Staphylococcus is a leading cause of the potentially blinding disease microbial keratitis. Even with the use of antibiotic therapy, the host inflammatory response continues to damage the cornea, which may lead to blindness. Manipulation of the host response may help improve patient outcome from this devastating disease. We aim to understand the contribution of the host response to Staphylococcus aureus infection. A S. aureus keratitis mouse model was developed in both C57BL/6 and BALB/c mice using two different strains of S. aureus (8325-4 and Staph 38). Twenty-four hours postinfection, mice were killed and eyes were harvested for enumeration of bacteria, polymorphonuclear leucocytes, chemokines and cytokines. The laboratory strain 8325-4 was not as virulent as the clinical isolate Staph 38. In vitro data showed a 250-fold increase in invasion of human corneal epithelial cells by Staph 38 compared to 8325-4. BALB/c mice were susceptible to S. aureus infection whereas C57BL/6 mice were resistant. The resistant C57BL/6 mice were polarized towards a Th2 response, which may be protective for these mice. IL-4, IL-10 and IL-6 were elevated significantly in C57BL/6 mice infected with Staph 38 (P < 0.05). Macrophage inflammatory peptide (MIP)-2 was also significantly elevated in C57BL/6 mice (P < 0.001). The susceptible BALB/c mice had a muted cytokine response, which suggests that S. aureus might be 'walled off' during infection and might avoid host defences. IL-4, IL-10 and IL-6 cytokines may be protective during Gram-positive corneal infection and therefore may be useful for adjunct therapies in the treatment of this disease.

Secretory phospholipase A2 deposition on contact lenses and its effect on bacterial adhesion.

PURPOSE: Secretory phospholipase A2 (sPLA2) is a potent antibacterial enzyme in tears and has been found to kill Staphylococcus aureus rapidly in vitro. The purpose was to determine whether sPLA2 deposition is associated with contact lens (CL) type, if sPLA2 remains active on CLs, and if this has an effect on bacterial adhesion. METHODS: Ionic (etafilcon A) and nonionic (Polymacon) high-water, soft CLs were used. CLs were worn for 6 hours (daily wear, n = 39) or 6 nights on an extended-wear schedule (n = 25). Tears were collected from patients and worn contact lenses were removed and protein and active enzymes extracted for estimation of their levels. The number of S. aureus adhering to sPLA2-soaked CLs in vitro was also quantified. RESULTS: There was no significant difference in the concentration of sPLA2 in tears between groups of daily CL wearers. Significantly less sPLA2 was recovered from Polymacon CLs for both daily and extended wear compared with etafilcon A CLs (daily wear: 3 vs. 5 ng/lens; extended wear: 3 vs. 6 ng/lens; P < 0.05). sPLA2 activity correlated with protein amounts from lenses. Relatively less active sPLA2 was recovered from Polymacon contact lenses. sPLA2 reduced adhesion of Staphylococcus to contact lenses in vitro. CONCLUSIONS: Etafilcon A CLs absorb more active sPLA2 than Polymacon CLs, which increases with length of CL wear. The sequestering of sPLA2 onto CLs did not affect amounts of the enzyme in tears. sPLA2 adsorbed to a CL can reduce the viable Staphylococcus adhering to the CL, which may protect the eye from colonization by this pathogen.

Evasion of cellular ocular defenses by contact lens isolates of Serratia marcescens.

PURPOSE: Contact lens contamination by Serratia marcescens can lead to the development of microbial keratitis and contact lens-induced acute red eye, particularly during overnight contact lens wear. The aim of this study was to investigate the interaction of S. marcescens with polymorphonuclear leukocytes (PMNs), which occur in closed-eye tears. METHODS: The respiratory burst of PMNs, when incubated with S. marcescens, was measured using chemiluminescence. Phagocytosis of planktonic and biofilm-grown bacteria by PMNs was also determined. RESULTS: Some strains of S. marcescens did not induce a respiratory burst from PMNs and resisted phagocytosis regardless of opsonization. Phagocytic resistance and growth in the presence of PMNs was markedly increased when bacteria were grown as a biofilm on a contact lens. CONCLUSIONS: The ability of S. marcescens to resist phagocytosis and grow to high levels in the presence of PMNs, particularly when grown as a biofilm on contact lenses, may be a mechanism by which this bacterium can survive the ocular host defense system, grow, and cause keratitis or other adverse responses.

Effectiveness of mupirocin and polymyxin B in experimental Staphylococcus aureus, Pseudomonas aeruginosa, and Serratia marcescens keratitis.

PURPOSE: The purpose of this study was to determine the effectiveness of mupirocin and polymyxin B, alone and in combination, in vitro and in vivo using rabbit models of, and keratitis. METHODS: Rabbit eyes were intrastromally injected with 1,000 colony-forming units (CFUs) of or or 100 CFUs of Rabbits were then treated with 2.7 mg/mL mupirocin, 10,000 U/mL polymyxin B, a mupirocin:polymyxin B combination, or 0.3% ciprofloxacin. Vehicle and untreated controls were also included. Treatment schedules depended on the strain injected. The number of CFUs was determined for all eyes after treatment. RESULTS: The mupirocin:polymyxin B combination was effective for all three genera both in vitro and in vivo. For keratitis, the mupirocin:polymyxin B combination was more effective than either drug alone and significantly reduced the log number of bacteria in the cornea by more than 3 logs compared with the vehicle or untreated controls (p

Corneal pathogenesis of Staphylococcus aureus strain Newman.

PURPOSE: To determine the pathogenic role of gamma- and alpha-toxin in a rabbit model of Staphylococcus aureus keratitis. METHODS: S. aureus strains Newman (expressing gamma-toxin), Newman Delta(hlg) (deficient in gamma-toxin), Newman Delta(hlg)/pCU1 hlg(+) (chromosomal gamma-toxin-deficient mutant rescued by a plasmid encoding gamma-toxin), and Newman Delta(hla) (alpha-toxin-deficient) were intrastromally injected into rabbit corneas. Eyes were scored by slit lamp examination (SLE), and bacterial colony-forming units (CFU) per cornea were determined at 15, 20, and 25 hours after infection. Histologic examination of corneas was performed. Rabbits were immunized against alpha-toxin and subsequently challenged with S. aureus strain Newman. Western blot analyses of culture supernatants were performed to detect alpha-toxin production. RESULTS: All strains grew equivalently, producing approximately 7 log CFU per cornea at 25 hours after infection. SLE scores at 20 and 25 hours after infection revealed that strains Newman Delta(hlg) and Newman Delta(hla), although virulent, caused significantly less ocular damage and inflammation than their parent or the gamma-toxin genetically rescued strain (P