October 2016
Volume 57, Issue 13
Open Access
Cornea  |   October 2016
Melimine-Coated Antimicrobial Contact Lenses Reduce Microbial Keratitis in an Animal Model
Author Affiliations & Notes
  • Debarun Dutta
    School of Optometry and Vision Science, University of New South Wales, Sydney, Australia
  • Ajay K. Vijay
    School of Optometry and Vision Science, University of New South Wales, Sydney, Australia
  • Naresh Kumar
    School of Chemistry, University of New South Wales, Sydney, Australia
  • Mark D. P. Willcox
    School of Optometry and Vision Science, University of New South Wales, Sydney, Australia
  • Correspondence: Debarun Dutta, School of Optometry and Vision Science, Level 3, North Wing - Rupert Myers Building, Gate 14, Barker Street, The University of New South Wales, Sydney NSW 2052, Australia; debarun.dutta@unsw.edu.au
Investigative Ophthalmology & Visual Science October 2016, Vol.57, 5616-5624. doi:10.1167/iovs.16-19882
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      Debarun Dutta, Ajay K. Vijay, Naresh Kumar, Mark D. P. Willcox; Melimine-Coated Antimicrobial Contact Lenses Reduce Microbial Keratitis in an Animal Model. Invest. Ophthalmol. Vis. Sci. 2016;57(13):5616-5624. doi: 10.1167/iovs.16-19882.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose: To determine the ability of antimicrobial peptide melimine-coated contact lenses to reduce the incidence of microbial keratitis (MK) in a rabbit model of contact lens wear.

Methods: In vitro antimicrobial activity of melimine-coated contact lenses was determined against Pseudomonas aeruginosa by viable count and a radiolabeled assay. The amount of lipopolysaccharide (LPS) associated with bacteria bound to melimine-coated and control lenses was determined. Ocular swabs from rabbit eyes were collected for assessment of ocular microflora. A rabbit model for MK was developed that used overnight wear of contact lenses colonized by P. aeruginosa in the absence of a corneal scratch. During lens wear, detailed ocular examinations were performed, and the incidence of MK was investigated. Bacteria associated with worn lenses and infected corneas were determined by viable plate count.

Results: Inhibition in viable and total P. aeruginosa adhesion by melimine-coated contact lenses was 3.1 log10 and 0.4 log10, respectively. After colonization, the amount of LPS on lenses was approximately the same with or without melimine. Gram-positive bacteria were found in all the ocular swabs followed by fungus (42%). Melimine-coated lens wear was protective and significantly (odds ratio 10.12; P = 0.012) reduced the incidence of P. aeruginosa–driven MK in the rabbit model. The antimicrobial lenses were associated with significantly (P < 0.001) lower ocular scores, indicating improved ocular signs compared with controls.

Conclusions: This study showed that contaminated contact lenses can produce MK without corneal epithelial defect in an animal model. Melimine-coated contact lenses reduced the incidence of MK associated with P. aeruginosa in vivo. Development of MK requires viable bacteria adherent to contact lenses, and bacterial debris adherent at the lens surface did not cause keratitis.

Microbial keratitis (MK) is a serious ocular condition that has the potential to cause blindness.1 Risk factors for MK include ocular surface trauma, ocular surface disease, contact lens wear, and hypoxia.25 Trauma and ocular surface diseases remain the major risk factors for MK in developing countries,6 whereas contact lens wear is the predominant risk factor for MK in developed countries.2,7 Depending on the study design and location, contact lens wear now accounts for approximately 12% to 66% of all MK events.815 The annualized incidence of MK ranges between 9.3 and 20.9 during overnight wear of lenses and 2.2 and 3.5 per 10,000 wearers during daily wear of lenses.1618 With the introduction of high oxygen–permeable silicone hydrogel contact lenses in 1998, it was anticipated that these materials might be able to reduce the incidence rate of MK. However, subsequent studies confirmed that these materials had little effect on absolute and relative risk of MK.19 Although various microorganisms can cause MK, Pseudomonas aeruginosa has been repeatedly reported to be the predominant causative organism.20,21 
Sankaridurg et al.1 detailed that the possible sequence of events leading to contact lens–related MK include microbial adhesion to contact lens followed by adhesion to damaged corneal epithelium, then replication and release of toxins and polymorphonuclear leukocyte (PMN) infiltration. The PMNs and bacteria release enzymes and toxins that lead to collagenolysis of the stroma and suppuration. Because bacterial adhesion to contact lenses is an essential step in the development of MK, it is believed that if adhesion to contact lenses was controlled, the incidence of MK may be reduced.22,23 There have been various attempts to create antimicrobial contact lenses that can reduce the adhesion of pathogenic microorganisms. Antimicrobials, such as silver,24 nonsteroidal anti-inflammatory drugs,2527 selenium,28 quorum-sensing inhibitors,29 poly(ethylene glycol),30,31 phosphorylcholine,32 and antimicrobial peptides, have been examined in vitro.33 
Antimicrobial peptides (AMPs) are a class of antimicrobial agents that are active against a wide spectrum of microorganisms, such as bacteria, viruses, fungi, protozoa, and parasites, including antibiotic-resistant bacteria.34,35 Melimine is a synthetic peptide consisting of 29 amino acid residues derived from melittin and protamine.36 Melimine has high activity, which exceeds that of a mixture of the two parent peptides.37 Earlier studies have shown that melimine-coated contact lenses can reduce P. aeruginosa adhesion to contact lenses33,35 and can reduce the incidence and severity of noninfectious keratitis in a Guinea pig model.38 Melimine is not cytotoxic to mammalian cells in vitro, or animals' eyes, and can be worn safely by humans.35,39 
This study aimed (1) to determine whether there was a significant amount of dead bacteria on melimine-coated contact lenses and whether this could lead to the development of keratitis, and (2) to develop a rabbit model for MK and determine whether melimine-coated contact lenses could reduce the incidence of MK induced by P. aeruginosa
Methods
Melimine (T-L-I-S-W-I-K-N-K-R-K-Q-R-P-R-V-S-R-R-R-R-R-R-G-G-R-R-R-R) was synthesized by conventional solid-phase peptide synthesis protocols and was obtained from American Peptide Company (Sunnyvale, CA, USA; >95% purity). Melimine was covalently attached to etafilcon A (Johnson & Johnson Vision Care, Inc., Jacksonville, FL, USA; base curve: 8.7 mm, diameter: 14.0 mm, power: −3.00 Diopter) lenses as described earlier.35 Briefly, melimine was diluted in sterile PBS (NaCl 8 g L−1, KCl 0.2 g L−1, Na2HPO4 1.15 g L−1, KH2PO4 0.2 g L−1, pH 7.4). Contact lenses were washed in PBS, then placed in 0.1 mol L−1 sodium acetate buffer, pH 5.0, containing 2 mg mL−1 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) for 15 minutes at 25°C. Lenses were washed three times in PBS and then resuspended in 3 mg mL−1 melimine in PBS and incubated for 2 hours at 37°C with gentle shaking. Subsequently, lenses were washed three times in sterile PBS, and then resuspended in 2 mL 10% wt/vol NaCl overnight followed by soaking in PBS for 2 hours and autoclaving (121°C, 15 minutes). Etafilcon A lenses were used because they are commercially available and contain carboxylic acid groups (provided by the incorporation of methacrylic acid during manufacture) that facilitates covalent binding of amine groups on peptides. 
In Vitro Bacterial Adhesion
Pseudomonas aeruginosa strain 6294 (isolated from a case of MK) was grown overnight in tryptone soya broth (TSB; Oxoid, Basingstoke, UK). Pseudomonas aeruginosa 6294 is an invasive strain and the minimum inhibitory concentration for melimine is 250 μL mL−1.4043 The adhesion conditions of P. aeruginosa 6294 with contact lenses have reported previously.35,44 Briefly, 1 mL P. aeruginosa (1.0 × 106 CFU mL−1) was added to uncoated control or melimine-coated lenses prewashed in PBS, and incubated for 18 hours with shaking (120 rpm). Then, contact lenses were washed three times with PBS to remove nonadherent bacterial cells and stirred rapidly in 2 mL fresh PBS containing a small magnetic stirring bar. This PBS was serially diluted (1/10) in Dey Engley neutralizing broth (Becton, Dickson and Company, Sparks, MD, USA), and a 3 × 50-μL aliquot of each dilution was transferred to tryptone soya agar (Oxoid, Basingstoke, UK) containing Tween 80 and lecithin for recovery of bacterial cells. After 24-hour incubation at 37°C, viable microorganisms were enumerated as CFU per contact lens. Measurements were performed in triplicate on a minimum of three separate occasions. 
To determine the effect of melimine-coated lenses on the total number (viable + nonviable cells) of bacterial adherent cells, P. aeruginosa 6294 was grown overnight with 20 μL 3H-uridine (0.02 mCi; PerkinElmer, Glen Waverley, Australia) in TSB, then centrifuged, washed, resuspended in PBS44 and incubated with lenses as described for viable cell adhesion. Subsequently, 0.5 mL of the bottle contents was added with 4.5 mL Opti-Fluor scintillation cocktail (Packard Instrument Co., Downers Grove, IL, USA) to 6 mL Pony H-I vials (PerkinElmer, Turku, Finland). The vials were vortexed and then placed in a β scintillation counter (Wallac 1400 DSA; PerkinElmer, Turku, Finland) to estimate radioactivity associated with the lenses. The disintegrations per minute from each lens were converted to number of bacterial cells mm−1 of lens surface area based on a standard calibration curve of known numbers of radiolabeled bacteria. Triplicates of each lens type (with and without) were included in each adhesion experiment and the experiment was repeated a minimum of three times. 
Detection of Endotoxin
Melimine-coated and uncoated contact lenses were incubated overnight with P. aeruginosa 6294 following the methods described for viable cell adhesion. Following incubation, lenses were washed to remove loosely bound bacteria and stirred rapidly in 2 mL pyrogen-free cell culture grade water (PromoCell GMBH, Heidelberg, Germany). This solution was diluted 100 times in a pyrogen-free container (Kinesis Australia, Redland Bay, Australia) and endotoxin associated with both the melimine-coated and uncoated contact lenses was determined by Limulus amoebocyte lysate (LAL) assay (Lonza, Walkersville, MD, USA). In brief, 50 μL LAL reagent was added to 50 μL × 100 diluted samples in a 96-well endotoxin-free plate (CELLSTAR, Greiner bio-one, Frickenhausen, Germany); 100 μL prewarmed (37 ± 1°C) chromogenic substrate was added after 10 minutes and 100 μL stop reagent (acetic acid, 25% vol/vol glacial acetic acid; Ajax Finechem Pty Ltd., Taren Point, NSW, Australia) added after 16 minutes. During this time, the side of a 96-well plate was gently tapped to facilitate mixing. Endotoxin units (EU) per lens were calculated after measuring the absorbance on a SpectrafluorPlus microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at 405 to 410 nm and converting the absorbance into EU by reference to a standard curve. Standard curves were plotted for each experiment from four standard endotoxin solutions that were prepared from standard Escherichia coli endotoxin (Lonza) of known potency and following manufacturer's guidelines. Endotoxin units per lens were calculated. Measurements were performed in triplicate. 
Animal Model for Assessing the Incidence of MK With Melimine-Coated Contact Lenses
This was a prospective, controlled study conducted to determine whether melimine-coated contact lenses could reduce the incidence of MK caused by adherent P. aeruginosa in a rabbit model compared with uncoated control lenses. All animals were treated strictly in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and before study commencement, approval from the University of New South Wales Animal Care and Ethics Committee was obtained. A total of 27 conventionally reared New Zealand white rabbits (Nanowie Animal Facility, Bellbrae, VIC, Australia) were allocated for this study. They were acclimatized for at least 1 week before the study start. Only rabbits in good general health, and having eyes free of clinically significant ocular irritation or staining were used in the study. The nictitating membrane was not removed from the rabbits' eyes. 
Aseptic swabs from the right eye upper bulbar conjunctiva and lower lid margins of seven rabbits were taken using sterile calcium alginate swabs (Puritan, Guilford, ME, USA) to determine the rabbit normal ocular microbiota. The swabs were then placed in a vial containing 2 mL sterile PBS and vortexed for 30 seconds. Aliquots of the saline (400 μL) were inoculated onto three chocolate agar plates and one Sabouraud's dextrose agar plate. One of the three chocolate agar plates was incubated at 35°C in 5% CO2 (2 days), one in aerobic conditions (2 days), and one under anaerobic conditions (4 days). The Sabouraud's dextrose agar plate was incubated at 25°C for 7 days to culture for yeasts and fungi. Microbial growth on the various media was enumerated and the number of CFUs of each distinct colony type per swab was calculated.45 Representative bacterial colonies from each plate were Gram stained and identified using Analytical Profile Index (Biomerieux, Grenoble, France) commercially available kits. 
Pseudomonas aeruginosa strain 6294 was grown overnight in TSB at 37°C and then washed three times in PBS after centrifugation at 1157g at 18°C for 10 minutes. Pseudomonas aeruginosa was resuspended in 10 times diluted TSB in PBS to an OD660nm of 0.1 (1.0 × 108 CFU mL−1). Noncoated control and melimine-coated lenses were washed in PBS and transferred to 1 mL bacterial suspension in wells of 24-well tissue culture plates, and incubated for 18 hours with shaking (120 rpm). Incubated contact lenses were washed once with PBS to remove nonadherent bacterial cells before inserting into the rabbit eyes. 
Rabbits wore either melimine-coated or control contact lenses with or without adherent P. aeruginosa in their left eyes for 24 hours, while the right eye served as a nonlens control. Before lens insertion, lenses were examined for particulate matter, physical damage, and inversion. Rabbits were reexamined after 6 hours of lens insertion to monitor retention of lens and in case of any lens loss, the respective type of lens with or without adherent bacteria was reinserted. Rabbits were administered subcutaneous 0.01 mg kg−1 buprenorphine. Contact lens fit was assessed using slit-lamp biomicroscopy, including lens centration, corneal coverage, and tightness. All rabbits had a detailed ocular examination and photographs taken by a single trained optometrist. Slit-lamp biomicroscopy was performed using a Nikon photographic slit lamp (Nikon FS-3V; Tokyo, Japan), which provided up to ×32 magnification. Detailed anterior segment examinations were carried out, including sodium fluorescein (Chauvin Pharmaceuticals Ltd., Surry, UK) staining. Wratten #12 filter (Bausch & Lomb, Rochester, NY, USA) was used in conjunction with cobalt blue filter to excite fluorescence. Photographs were taken and then graded, using the CCLRU grading scale,46 by a single trained masked optometrist who recorded details of corneal and conjunctival parameters (Table 1). Ocular examinations were performed at baseline and immediately after lens removal at 24 hours and at 48 hours (after 24 hours of no lens wear). The ocular variables assessed and scores given during each examination are listed in Table 1. All rabbits were closely monitored during lens wear for any indication of stress by examining their movements, alertness, gait, behavior, vocalizations, and respiration. After 24 hours, lenses from rabbits' left eyes were aseptically collected in 2 mL sterile PBS and processed to determine viable bacterial count. If MK developed, the animal was immediately euthanized and the left cornea was collected to determine viable bacterial count. All rabbits wore contact lenses for up to three occasions if MK did not develop. If MK developed, the rabbits were euthanized. A minimum of 2 weeks of wash out was provided between each lens wear for rabbits to recover from any effects of lens wear. 
Table 1
 
Scoring of the Rabbit Ocular Response During Contact Lens Wear
Table 1
 
Scoring of the Rabbit Ocular Response During Contact Lens Wear
After removal from eyes, worn and unworn melimine-coated or control contact lenses were stirred rapidly in 2 mL PBS containing a small magnetic stirring bar to remove bacterial cells. Following log serial dilutions in PBS, 3 × 50 μL of each dilution was plated on nutrient agar (NA; Oxoid, Basingstoke, UK). After 24 hours of incubation at 37°C, viable bacteria were enumerated as CFU lens−1. Corneal buttons collected from infected corneas were homogenized (IKA tissue homogenizer; Wilmington, NC, USA) in 4 mL PBS, serially diluted in PBS, and 3 × 50 μL of each dilution was plated on NA. Following 24 hours of incubation at 37°C, viable bacteria were enumerated as CFU cornea−1
Data Analysis
Data were analyzed using Excel (Office; Microsoft, Redmond, WA, USA) and Statistical Package for the Social Sciences software (SPSS) for Windows, version 21.0 (SPSS, Inc., Chicago, IL, USA). The in vitro bacterial adhesion data were log10 (x + 1) transformed before data analysis, where x is the adherent bacteria in CFU lens−1. Differences in microbial adhesion and lens-associated endotoxin were analyzed using independent 2-sample t-test. During the animal study, percent lens retention was calculated as ([actual wear time for duration of study]/[total possible wear time for duration of study]) × 100. Data obtained from corneal evaluations in the MK model study were examined for differences using a Wilcoxon sign ranked test for corneal scores and a χ2 test for corneal infiltrates with MK. All statistical tests were two sided; odds ratio and relative risk for development of MK were determined with test lenses compared with control. Analytical manipulation of the data, such as the sum or frequency of scores, was calculated where appropriate. 
Results
Figure 1 shows total and viable adhesion of P. aeruginosa (at the initial inoculum of 1 × 106 CFU mL−1) to melimine-coated and control lenses. Melimine-coated lenses produced 3.1 ± 0.6 log10 inhibition in viable adhesion 18 hours after incubation with bacteria. Compared with viable adhesion, there was substantially higher total adhesion by P. aeruginosa on melimine-coated lenses (P < 0.05) in the same time frame. Melimine-coated lenses demonstrated a small but statistically significant 0.4 log10 inhibition in total adhesion for P. aeruginosa (P < 0.05) 18 hours after incubation with bacteria. Melimine-coated contact lenses adsorbed 110 ± 11 EU lens−1 of endotoxin (Fig. 2) over an 18-hour incubation period. Control lenses after adhesion of P. aeruginosa had a slightly but not statistically (P = 0.14) higher endotoxin level of 143 ± 32 EU lens−1 over the same time frame. 
Figure 1
 
Number of total and viable P. aeruginosa 6294 adherent to melimine-coated and control contact lenses following 18 hours of incubation. *Significantly higher total bacterial adhesion than viable bacterial adhesion to melimine-coated lenses. #Significantly lower total bacterial adhesion to melimine-coated lenses than uncoated control lenses.
Figure 1
 
Number of total and viable P. aeruginosa 6294 adherent to melimine-coated and control contact lenses following 18 hours of incubation. *Significantly higher total bacterial adhesion than viable bacterial adhesion to melimine-coated lenses. #Significantly lower total bacterial adhesion to melimine-coated lenses than uncoated control lenses.
Figure 2
 
Amount of endotoxin associated with control and melimine-coated lenses after incubation with P. aeruginosa for 18 hours.
Figure 2
 
Amount of endotoxin associated with control and melimine-coated lenses after incubation with P. aeruginosa for 18 hours.
Table 2 details the percentage of rabbit eyes that were colonized with microorganisms and the type of microorganisms. Twenty-eight percent of lower lid and 57% of upper bulbar conjunctiva were sterile. Gram-positive bacteria were found in all the other ocular swabs. Fungus was found in 42% of both lower lid and upper bulbar conjunctiva. Moraxella urethralis was the only gram-negative bacteria that was isolated, from 14% of rabbit lower lid and upper bulbar conjunctiva. 
Table 2
 
Types of Microorganisms Isolated From Rabbit Eyes Before Contact Lens Wear
Table 2
 
Types of Microorganisms Isolated From Rabbit Eyes Before Contact Lens Wear
Table 3 demonstrates the demographics of contact lens wear for rabbits, including number of rabbits that retained contact lenses during this study. A total of 22 and 19 eyes were exposed to control and melimine-coated P. aeruginosa preincubated contact lenses, respectively, and 59% and 58% of eyes retained lenses after 24 hours of wear, respectively (Table 3). Twenty-two and 13 eyes were exposed to sterile control and melimine-coated lenses, and lens retention was 95% and 92%, respectively. All the animals included in the study maintained good health and no abnormal behavior was observed during the study. All the lenses had acceptable contact lens fit indicated by good centration, 360° corneal coverage, and optimal lens tightness. Sixty-nine percent of rabbit eyes that wore P. aeruginosacolonized control lenses for 24 hours developed MK compared with 18% for melimine-coated lenses (P = 0.012). A score for the response of the rabbit eyes was generated from measurement of parameters, including conjunctival redness, conjunctival edema, discharge, and focal and diffuse infiltrates (summarized as “ocular score”). Table 4 summarizes these findings from the eyes that retained lenses for 24 hours, indicating that the ocular scores associated with P. aeruginosacolonized melimine-coated contact lenses were significantly (P < 0.05) lower than P. aeruginosacolonized control lenses. The relative risk of developing MK during melimine-coated lens wear was 0.26 (95% confidence interval [CI] 0.71–0.96; P = 0.044) and the odds ratio was 10.12 (95% CI 1.46–69.93; P = 0.018) when compared with control lens wear, indicating the melimine-coated lens wear was protective against developing MK. 
Table 3
 
Number of Rabbits Wearing Contact Lenses and That Developed MK
Table 3
 
Number of Rabbits Wearing Contact Lenses and That Developed MK
Table 4
 
Ocular Responses Following Wear of Contact Lenses Preincubated With P. aeruginosa in Rabbits
Table 4
 
Ocular Responses Following Wear of Contact Lenses Preincubated With P. aeruginosa in Rabbits
Figure 3 details the ocular response scores for rabbit eyes wearing lenses with and without adherent P. aeruginosa for both control and melimine-coated lenses. When lenses without adherent P. aeruginosa were worn, there were no significant differences between melimine-coated and control lenses (P > 0.05) for any ocular variables, including total ocular score. Pseudomonas aeruginosacolonized melimine-coated lenses showed similar scores compared with counterparts without adherent bacteria (P > 0.5) except for the total scores (Fig. 3; P = 0.03). For P. aeruginosacolonized control lenses, the mean total score was 14.3 ± 8.4, whereas P. aeruginosacolonized melimine-coated lenses had mean total scores of 4.2 ± 2.1 (P = 0.001). 
Figure 3
 
Ocular responses of rabbit eyes that retained contact lenses for 24 hours.
Figure 3
 
Ocular responses of rabbit eyes that retained contact lenses for 24 hours.
Figure 4 is a bubble chart that details the total ocular scores of rabbit eyes following wear of P. aeruginosacolonized control and melimine-coated lenses after 24 hours and 48 hours. The size of the bubbles indicates the number of rabbit eyes with that score. For instance, after 48 hours of wear of P. aeruginosacolonized melimine-coated lenses, nine rabbits showed total ocular scores of 1.0 as represented by a large bubble, whereas only two rabbits showed an ocular score of 1.0 with P. aeruginosacolonized control lenses after 48 hours, and that is represented by a small bubble. The black bubbles indicate eyes that developed MK during lens wear. After 24 hours, three rabbits that wore P. aeruginosacolonized control lenses had total ocular scores of more than 5, whereas only one rabbit wearing P. aeruginosacolonized melimine-coated lenses had a similar score; other rabbits had lower scores in P. aeruginosacolonized melimine-coated lenses. After 48 hours, nine rabbit eyes wearing P. aeruginosacolonized control lenses showed ocular scores of 10 or more, indicating a severe adverse event, compared with only one rabbit eye wearing P. aeruginosacolonized melimine-coated lenses. 
Figure 4
 
Total ocular scores following wear of P. aeruginosa adherent to contact lenses after 24 and 48 hours. Total ocular scores of 10 or more were regarded as a significant adverse event. Bubble size indicates the number of animals with same ocular score, and the smallest bubbles seen represent data from one rabbit. Black bubbles indicate incidence of microbial keratitis.
Figure 4
 
Total ocular scores following wear of P. aeruginosa adherent to contact lenses after 24 and 48 hours. Total ocular scores of 10 or more were regarded as a significant adverse event. Bubble size indicates the number of animals with same ocular score, and the smallest bubbles seen represent data from one rabbit. Black bubbles indicate incidence of microbial keratitis.
Figure 5 illustrates representative appearances of rabbit eyes seen with slit-lamp biomicroscopy. Figure 5A shows a healthy eye with normal redness and other ocular parameters following wear of sterile control lenses, whereas Figure 5B shows fluorescein staining with full-thickness loss of epithelium (black arrow) observed following 24 hours of wear of a P. aeruginosacolonized control lens. Figure 5C shows conjunctival redness (arrow) observed following 24 hours of wear of P. aeruginosacolonized control contact lens, and Figure 5D shows a case of MK observed during wear of a P. aeruginosacolonized control contact lens. The MK was characterized by a large, dense, irregular infiltrate (white arrow) with diffuse conjunctival swelling and hyperemia (white arrow). 
Figure 5
 
Representative images of slit-lamp findings of rabbit eyes captured at ×7.5 magnification. (A) Healthy rabbit eye; (B) fluorescein staining of full-thickness loss of epithelium; (C) conjunctival hyperemia with mild discharge; (D) microbial keratitis.
Figure 5
 
Representative images of slit-lamp findings of rabbit eyes captured at ×7.5 magnification. (A) Healthy rabbit eye; (B) fluorescein staining of full-thickness loss of epithelium; (C) conjunctival hyperemia with mild discharge; (D) microbial keratitis.
Contact lenses analyzed before insertion into rabbit eyes showed that after incubation with 1 × 108 CFU mL−1 the number of bacterial cells on control lenses was 7.3 ± 0.5 log10 CFU lens−1, whereas on the melimine-coated lenses, there was 6.3 ± 0.4 log10 CFU lens−1 and this difference was statistically significant (P < 0.001). After lens wear, the number of P. aeruginosa cultured from control lenses was 5.4 ± 0.4 log10 CFU lens−1 and from melimine-coated lenses was 4.8 ± 0.2 CFU log10 lens−1 (P < 0.001). The corneas of eyes that developed MK were processed to determine the number of P. aeruginosa associated with each eye. Corneas that had been exposed to control or melimine-coated lens wear were associated with 6.8 ± 0.4 log10 or 6.8 ± 0.8 log10 cultivable P. aeruginosa, respectively (P > 0.05). 
Discussion
This study for the first time has demonstrated that melimine-coated contact lenses can reduce the incidence of MK associated with P. aeruginosa infection in an animal model. The animal model developed was an acute MK model in rabbit eyes wearing bacteria-colonized contact lenses without any epithelial scratch or defect. Bacterial debris was associated with melimine-coated contact lenses, but this debris did not result in MK and caused only a low grade of ocular response during the 24 hours of lens wear. 
Frequency of MK in eyes that wore lenses for 24 hours with control lens wear was 69%, whereas when wearing melimine-coated contact lenses for 24 hours, the frequency of MK was only 18%. Furthermore, the ocular response to melimine-coated lens wear in the presence of adherent P. aeruginosa was also significantly reduced. These results are similar to the results obtained in a Guinea pig model in which melimine-coated lenses were able to reduce the incidence and severity of noninfectious keratitis.38 Previous models of MK with P. aeruginosa have either used injection of viable bacteria into the cornea of rabbits,47 or used rats that had worn lenses for a continuous period of 72 hours before wearing contact lenses soaked in 1 × 1011 Pseudomonas mL−1 followed by topical application of 1 × 1010 P. aeruginosa 19660 bacterial cells, and an additional topical application of the same concentration for up to 3 days for MK to be obtained.48 Another model used rats wearing contact lenses with 106 or 105 P. aeruginosa PAO1 (green florescent protein-producing mutant) adherent to the lenses and lens wear for up to 10 days before MK developed.49 Our rabbit model is a more acute model of contact lens–induced MK. We previously reported that contact lens–induced MK in an acute Guinea pig model was dependent on prior scratching of the cornea,50 much like is the case in a nonlens-wearing mouse model.51 The reason why rabbits can produce an acute MK in the absence of corneal damage during lens wear, and why not all rabbits are affected, requires further investigation, but there was no evidence of differences in corneal and conjunctival staining or conjunctival redness for those animals that developed MK before lens wear. 
Melimine-coated contact lenses significantly reduced adhesion of viable bacteria in agreement with our earlier results.33,35 However, when the number of total bacterial cells adherent to lenses was assessed using uptake and incorporation of radiolabeled uridine, there was no apparent reduction in total bacterial adhesion. Bacterial growth and uptake of 3H-uridine during bacterial growth and incorporation into bacterial RNA is an established method of tracking adhesion.52 An earlier report using confocal microscopy and LIVE/DEAD staining35 showed significantly reduced numbers of dead bacteria on melimine-coated lenses. Interaction of melimine causes rapid disruption of bacterial cytoplasmic membranes resulting in cell death.53 The lack of visible intact dead bacterial cells35 (or at least to levels comparable to the amount of radioactive RNA present on lenses in the current study) might indicate that the bacteria are totally destroyed on adhesion to bound-melimine, such that no cell form can be seen with microscopy but cellular debris (i.e., RNA) is associated with the lens surface. RNA is a negatively charged molecule (polyanion)54 that may bind to the positively charged melimine surface. The presence of endotoxin on the surfaces of lenses is further evidence of bacterial debris on the lens surface. Endotoxins are released when gram-negative bacteria are killed.55 
Given the presence of bacterial debris (endotoxin and RNA, perhaps other components) on the melimine-coated lens surfaces, it is intriguing that the melimine-coated lenses after interaction with P. aeruginosa did not result in the production of a large ocular inflammatory response. Lipopolysaccharide or bacterial RNA and DNA on melimine-coated contact lenses could be recognized by Toll-like receptor (TLR)4 or TLR9, which are expressed by polarized corneal epithelial cells, bulbar conjunctiva, and stromal fibroblasts.56 It has been suggested that ocular surface epithelial cells may initiate an immunosilent condition for TLR-mediated innate immunity to inhibit unwanted inflammatory responses to nonpathogenic microbiota.57 Ocular surface epithelial cells are regularly exposed to these bacteria and thus may have a low propensity for TLR-mediated inflammation.57 Also, the polarity of ocular surface cells,56 and consequent sequestering of TLRs away from the surface of the eye, also may prevent a healthy nonwounded ocular surface from interacting with the cellular debris. We have previously shown in a rabbit model of noninfectious keratitis for the human condition known as contact lens–induced peripheral ulcer, that dead staphylococci are not able to provoke an ocular surface inflammatory response during lens wear for 24 hours.58 It may be that a time longer than 24 hours of exposure to melimine-coated lenses with associated bacterial debris is required for an ocular surface inflammatory response to occur, and we are currently investigating this possibility. 
As the aim of developing melimine-coated contact lenses is to use them to reduce contact lens–associated keratitis in humans; it is worthwhile considering the possible effect of the bacterial debris on the human ocular surface. During the study, we found that commensal ocular microbiota of the rabbits was similar to earlier reports59 and healthy human ocular microbiota,60 which is predominantly composed of gram-positive bacteria, especially Staphylococcus sp. There was no evidence of cultivable P. aeruginosa as part of the normal ocular microbiota of rabbit eyes, and so we assume that all these bacteria cultured after contact lens wear were from the colonized contact lenses. Although the number of pathogenic bacteria that initially colonize a contact lens before the onset of MK is not known, it is unlikely that it is at the level used in the current study (1.0 × 108 CFU mL−1). It is perhaps more likely that much lower numbers of bacteria initially colonize a contact lens, and then grow to levels that initiate the infection. Over the course of 13 nights of wear of etafilcon A contact lenses, the numbers of bacteria that could be cultured from lenses usually ranged between 10 and 30 CFU lens−1.61 During daily wear of contact lenses, lenses are stored in contact lens cases, but reports on the number of viable bacteria in a lens case rarely exceeds 1 × 105 CFUs per lens case well.62 Therefore, if lower numbers of bacteria adhere initially, melimine coatings would likely greatly reduce the likelihood of them growing to large numbers on the lens surface. This would mean that it might take several days for debris to accumulate on lenses. Reusable contact lenses are commonly disposed of on a monthly or 2-weekly basis.63 We are currently running a clinical trial of melimine-coated contact lenses worn on a 2-weekly disposable extended wear schedule to examine the production of noninfectious keratitis compared with noncoated lenses, and this will definitively examine whether melimine coatings are associated with reductions, no change, or even increases in adverse events. 
The finding that melimine coatings can reduce the adhesion of P. aeruginosa to contact lenses is confirmation of earlier studies that showed that melimine coatings can reduce microbial adhesion to a variety of biomaterials, including contact lenses in vitro.35,64,65 The reduction of the viable numbers of P. aeruginosa after wear is similar to previous findings with a Guinea pig model of noninfectious keratitis.38 This reduction in viable numbers of adherent bacteria is likely the reason for the reduced incidence of MK and severity of ocular responses in the absence of MK. When MK did occur in the presence of melimine-coated lenses, the numbers of bacteria that were cultured from the cornea were the same as the numbers in non–melimine-coated eyes, indicating that if viable bacteria are released from melimine-coated lenses they still have the capacity to cause disease. Thus, it is possible, given the data described herein and elsewhere,38 that melimine-coated lenses will reduce the incidence of both MK and noninfectious keratitis, but will not completely eliminate the disease. 
In this study, some rabbits lost lenses during the 24 hours of wear of lenses with adherent P. aeruginosa, and this rate of lens loss was higher than when rabbits wore lenses in the absence of adherent P. aeruginosa (Table 3).39 This may indicate that the rabbits experienced increased discomfort in the presence of the adherent bacteria and so actively removed the lenses from their eyes. Melimine-coated lenses also have high activity against bacteria other than P. aeruginosa that are implicated in MK.35 Although it is likely, further work is needed to confirm its capacity in reducing such MK in vivo. This study was a proof-of-principal test of the effectiveness of antimicrobial contact lenses in a model of MK; as such, we did not consider the feasibility to manufacture melimine-coated lenses as a commercial product or its potential cost for large-scale production. It may be that melimine or other antimicrobial coatings are eventually brought to market, and the likelihood of this happening will depend on their antimicrobial efficacy as well as ease and cost of manufacture. 
In summary, this study for the first time has shown that melimine-coated antimicrobial contact lenses have the capacity to significantly reduce the incidence of MK associated with P. aeruginosa contamination. Development of MK requires viable bacteria present at the ocular surface, and bacterial debris adherent to the lens, such as endotoxin, does not cause MK and results in only minor ocular changes. 
Acknowledgments
The authors thank Praveen Yerramothu, School of Optometry and Vision Science, The University of New South Wales, Australia, for helping with animal handling. 
The corresponding author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and there is no discrepancy from the study as planned. 
Disclosure: D. Dutta, None; A.K. Vijay, None; N. Kumar, None; M.D.P. Willcox, P 
References
Sankaridurg PR, Holden B, Jalbert I. Adverse events and infections: which ones and how many? In: Sweeney D, ed. Silicone Hydrogels: Continuous-Wear Contact Lenses. Sydney Australia: Butterworth-Heinemann; 2004: 217–274.
Dart JK. Predisposing factors in microbial keratitis: the significance of contact lens wear. Br J Ophthalmol. 1988; 72: 926–930. [CrossRef] [PubMed]
Dart JK, Stapleton F, Minassian D. Contact lenses and other risk factors in microbial keratitis. Lancet. 1991; 338: 650–653. [CrossRef] [PubMed]
Solomon OD, Loff H, Perla B, et al. Testing hypotheses for risk factors for contact lens-associated infectious keratitis in an animal model. CLAO J. 1994; 20: 109–113. [PubMed]
Ong HS, Corbett MC. Corneal infections in the 21st century. Postgrad Med J. 2015; 91: 565–571. [CrossRef] [PubMed]
Ormerod LD, Smith RE. Contact lens-associated microbial keratitis. Arch Ophthalmol. 1986; 104: 79–83. [CrossRef] [PubMed]
Szczotka-Flynn LB, Pearlman E, Ghannoum M. Microbial contamination of contact lenses, lens care solutions and their accessories: a literature review. Eye Contact Lens. 2010; 36: 116–129. [CrossRef] [PubMed]
Bourcier T, Thomas F, Borderie V, Chaumeil C, Laroche L. Bacterial keratitis: predisposing factors, clinical and microbiological review of 300 cases. Br J Ophthalmol. 2003; 87: 834–838. [CrossRef] [PubMed]
Fong CF, Tseng CH, Hu FR, Wang IJ, Chen WL, Hou YC. Clinical characteristics of microbial keratitis in a university hospital in Taiwan. Am J Ophthalmol. 2004; 137: 329–336. [CrossRef] [PubMed]
Keay L, Edwards K, Naduvilath T, et al. Microbial keratitis predisposing factors and morbidity. Ophthalmology. 2006; 113: 109–116. [CrossRef] [PubMed]
Mela EK, Giannelou IP, Koliopoulos JX, Gartaganis SP. Ulcerative keratitis in contact lens wearers. Eye Contact Lens. 2003; 29: 207–209. [CrossRef] [PubMed]
Wong T, Ormonde S, Gamble G, McGhee CN. Severe infective keratitis leading to hospital admission in New Zealand. Br J Ophthalmol. 2003; 87: 1103–1108. [CrossRef] [PubMed]
Gebauer A, McGhee CN, Crawford GJ. Severe microbial keratitis in temperate and tropical Western Australia. Eye. 1996; 10: 575–580. [CrossRef] [PubMed]
Rattanatam T, Heng WJ, Rapuano CJ, Laibson PR, Cohen EJ. Trends in contact lens-related corneal ulcers. Cornea. 2001; 20: 290–294. [CrossRef] [PubMed]
Baier RE. Comments on cell adhesion to biomaterial surfaces: conflicts and concerns. J Biomed Mater Res. 1982; 16: 173–175. [CrossRef] [PubMed]
Lam DS, Houang E, Fan DS, Lyon D, Seal D, Wong E. Incidence and risk factors for microbial keratitis in Hong Kong: comparison with Europe and North America. Eye. 2002; 16: 608–618. [CrossRef] [PubMed]
Poggio EC, Glynn RJ, Schein OD, et al. The incidence of ulcerative keratitis among users of daily-wear and extended-wear soft contact lenses. N Engl J Med. 1989; 321: 779–783. [CrossRef] [PubMed]
Cheng KH, Leung SL, Hoekman HW, et al. Incidence of contact-lens-associated microbial keratitis and its related morbidity. Lancet. 1999; 354: 181–185. [CrossRef] [PubMed]
Stapleton F, Keay L, Edwards K, Holden B. The epidemiology of microbial keratitis with silicone hydrogel contact lenses. Eye Contact Lens. 2013; 39: 79–85. [CrossRef] [PubMed]
Green M, Apel A, Stapleton F. Risk factors and causative organisms in microbial keratitis. Cornea. 2008; 27: 22–27. [CrossRef] [PubMed]
Konda N, Motukupally SR, Garg P, Sharma S, Ali MH, Willcox MD. Microbial analyses of contact lens-associated microbial keratitis. Optom Vis Sci. 2014; 91: 47–53. [CrossRef] [PubMed]
Dutta D, Willcox MD. Antimicrobial contact lenses and lens cases: a review. Eye Contact Lens. 2014; 40: 312–324. [CrossRef] [PubMed]
Dutta D, Cole N, Willcox M. Factors influencing bacterial adhesion to contact lenses. Mol Vis. 2012; 18: 14–21. [PubMed]
Willcox MD, Hume EB, Vijay AK, Petcavich R. Ability of silver-impregnated contact lenses to control microbial growth colonisation. J Optom. 2010; 3: 143–148. [CrossRef]
Muller E, Takeda S, Shiro H, Goldmann D, Pier GB. Occurrence of capsular polysaccharide adhesin among clinical isolates of coagulase-negative Staphylococci. J Infect Dis. 1993; 168: 1211–1218. [CrossRef] [PubMed]
Arciola CR, Montanaro L, Caramazza R, Sassoli V, Cavedagna D. Inhibition of bacterial adherence to a high-water-content polymer by a water-soluble, nonsteroidal, anti-inflammatory drug. J Biomed Mater Res. 1998; 42: 1–5. [CrossRef] [PubMed]
Bandara BMK, Sankaridurg PR, Willcox MDP. Non-steroidal anti-inflammatory agents decrease bacterial colonisation of contact lenses and prevent adhesion to human corneal epithelial cells. Curr Eye Res. 2004; 29: 245–251. [CrossRef] [PubMed]
Mathews SM, Spallholz JE, Grimson MJ, Dubielzig RR, Gray T, Reid TW. Prevention of bacterial colonization of contact lenses with covalently attached selenium and effects on the rabbit cornea. Cornea. 2006; 25: 806–814. [CrossRef] [PubMed]
Zhu H, Kumar A, Ozkan J, et al. Fimbrolide-coated antimicrobial lenses: their in vitro and in vivo effects. Optom Vis Sci. 2008; 85: 292–300. [CrossRef] [PubMed]
Kingshott P, Thissen H, Griesser HJ. Effects of cloud-point grafting, chain length and density of PEG layers on competitive adsorption of ocular proteins. Biomaterials. 2002; 23: 2043–2056. [CrossRef] [PubMed]
Kingshott P, Wei J, Bagge-Ravn D, Gadegaard N, Gram L. Covalent attachment of poly(ethylene glycol) to surfaces, critical for reducing bacterial adhesion. Langmuir. 2003; 19: 6912–6921. [CrossRef]
Selan L, Palma S, Scoarughi G, et al. Phosphorylcholine impairs susceptibility to biofilm formation of hydrogel contact lenses. Am J Ophthalmol. 2009; 147: 134–139. [CrossRef] [PubMed]
Willcox MDP, Hume EBH, Aliwarga Y, Kumar N, Cole N. A novel cationic-peptide coating for the prevention of microbial colonization on contact lenses. J Appl Microbiol. 2008; 105: 1817–1825. [CrossRef] [PubMed]
Nguyen LT, Haney EF, Vogel HJ. The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol. 2011; 29: 464–472. [CrossRef] [PubMed]
Dutta D, Cole N, Kumar N, Willcox MD. Broad spectrum antimicrobial activity of melimine covalently bound to contact lenses. Invest Ophthalmol Vis Sci. 2013; 54: 175–182. [CrossRef] [PubMed]
Willcox MD, Hume E, Aliwarga Y, Kumar N, Cole N. A novel cationic-peptide coating for the prevention of microbial colonization on contact lenses. J Appl Microbiol. 2008; 105: 1817–1825. [CrossRef] [PubMed]
Aliwarga Y, Hume EBH, Lan J, Willcox MD. Antimicrobial peptides: a potential role in ocular therapy. Clin Exp Ophthalmol. 2001; 29: 157–160. [CrossRef] [PubMed]
Cole N, Hume EB, Vijay AK, Sankaridurg P, Kumar N, Willcox MD. In vivo performance of melimine as an antimicrobial coating for contact lenses in models of CLARE and CLPU. Invest Ophthalmol Vis Sci. 2010; 51: 390–395. [CrossRef] [PubMed]
Dutta D, Ozkan J, Willcox MD. Biocompatibility of antimicrobial melimine lenses: rabbit and human studies. Optom Vis Sci. 2014; 91: 570–581. [CrossRef] [PubMed]
Fleiszig SM, Evans DJ, Do N, Vallas V, Shin S, Mostov KE. Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity. Infect Immun. 1997; 65: 2861–2867. [PubMed]
Sawa T, Ohara M, Kurahashi K, et al. In vitro cellular toxicity predicts Pseudomonas aeruginosa virulence in lung infections. Infect Immun. 1998; 66: 3242–3249. [PubMed]
Bandara M, Sankaridurg P, Zhu H, Hume E, Willcox M. Effect of salicylic acid on the membrane proteome and virulence of Pseudomonas aeruginosa. Invest Ophthalmol Vis Sci. 2016; 57: 1213–1220. [CrossRef] [PubMed]
Dutta D, Kumar N, Willcox M. Antimicrobial activity of four cationic peptides immobilised to poly-hydroxyethylmethacrylate. Biofouling. 2016; 32: 429–438. [CrossRef] [PubMed]
Dutta D, Willcox MD. A laboratory assessment of factors that affect bacterial adhesion to contact lenses. Biology. 2013; 2: 1269–1281. [CrossRef]
Willcox MD, Harmis N, Holden B. Bacterial populations on high-Dk silicone hydrogel contact lenses: effect of length of wear in asymptomatic patients. Clin Exp Optom. 2002; 85: 172–175. [CrossRef] [PubMed]
Terry RL, Schnider CM, Holden BA, et al. CCLRU standards for success of daily and extended wear contact lenses. Optom Vis Sci. 1993; 70: 234–243. [CrossRef] [PubMed]
Caballero A, Thibodeaux B, Marquart M, Traidej M, O'Callaghan R. Pseudomonas keratitis: protease IV gene conservation, distribution and production relative to virulence and other Pseudomonas proteases. Invest Ophthalmol Vis Sci. 2004; 45: 522–530. [CrossRef] [PubMed]
Szliter EA, Barrett RP, Gabriel MM, Zhang Y, Hazlett LD. Pseudomonas aeruginosa-induced inflammation in the rat extended-wear contact lens model. Eye Contact Lens. 2006; 32: 12–18. [CrossRef] [PubMed]
Tam C, Mun JJ, Evans DJ, Fleiszig SM. The impact of inoculation parameters on the pathogenesis of contact lens-related infectious keratitis. Invest Ophthalmol Vis Sci. 2010; 51: 3100–3106. [CrossRef] [PubMed]
Willcox MD, Sankaridurg PL, Zhu H, et al. Inflammation and Infection and the effects of the closed eye. In: Sweeney D, ed. Silicone Hydrogels: Continuous-Wear Contact Lenses. Sydney, Australia: Butterworth-Heinemann; 2004: 90–125.
Hazlett LD, Rosen DD, Berk RS. Age-related susceptibility to Pseudomonas aeruginosa ocular infections in mice. Infect Immun. 1978; 20: 25–29. [PubMed]
Yamauchi K, Miwa S. Erythrocyte metabolism and enzyme chemistry in aplastic anemia. Nihon Rinsho. 1978; 36: 2707–2719. [PubMed]
Rasul R, Cole N, Balasubramanian D, Chen R, Kumar N, Willcox MD. Interaction of the antimicrobial peptide melimine with bacterial membranes. Int J Antimicrob Agents. 2010; 35: 566–572. [CrossRef] [PubMed]
Lee JC, Gutell RR. Diversity of base-pair conformations and their occurrence in rRNA structure and RNA structural motifs. J Mol Biol. 2004; 344: 1225–1249. [CrossRef] [PubMed]
Elsbach P. Mechanisms of disposal of bacterial lipopolysaccharides by animal hosts. Microbes Infect. 2000; 2: 1171–1180. [CrossRef] [PubMed]
Chang JH, McCluskey PJ, Wakefield D. Toll-like receptors in ocular immunity and the immunopathogenesis of inflammatory eye disease. Br J Ophthalmol. 2006; 90: 103–108. [CrossRef] [PubMed]
Ueta M, Nochi T, Jang MH, et al. Intracellularly expressed TLR2s and TLR4s contribution to an immunosilent environment at the ocular mucosal epithelium. J Immunol. 2004; 173: 3337–3347. [CrossRef] [PubMed]
Wu P, Stapleton F, Willcox MD. The causes of and cures for contact lens-induced peripheral ulcer. Eye Contact Lens. 2003; 29: 63–66. [CrossRef]
Okuda H, Campbell LH. Conjunctival bacterial flora of the clinically normal New Zealand white rabbit. Lab Anim Sci. 1974; 24: 831–833. [PubMed]
Ueta M, Kinoshita S. Innate immunity of the ocular surface. Brain Res Bull. 2010; 81: 219–228. [CrossRef] [PubMed]
Sweeney DF, Stapleton F, Leitch C, Taylor J, Holden BA, Willcox MD. Microbial colonization of soft contact lenses over time. Optom Vis Sci. 2001; 78: 100–105. [CrossRef] [PubMed]
Willcox MD, Carnt N, Diec J, et al. Contact lens case contamination during daily wear of silicone hydrogels. Optom Vis Sci. 2010; 87: 456–464. [CrossRef] [PubMed]
Efron N, Nichols JJ, Woods CA, Morgan PB. Trends in US contact lens prescribing 2002 to 2014. Optom Vis Sci. 2015; 92: 758–767. [CrossRef] [PubMed]
Chen R, Willcox MD, Ho KK, Smyth D, Kumar N. Antimicrobial peptide melimine coating for titanium and its in vivo antibacterial activity in rodent subcutaneous infection models. Biomaterials. 2016; 85: 142–151. [CrossRef] [PubMed]
Willcox MD, Hume E, Aliwarga Y, Kumar N, Cole N. A novel cationic-peptide coating for the prevention of microbial colonization on contact lenses. J Appl Microbiol. 2008; 105: 1817–1825. [CrossRef] [PubMed]
Figure 1
 
Number of total and viable P. aeruginosa 6294 adherent to melimine-coated and control contact lenses following 18 hours of incubation. *Significantly higher total bacterial adhesion than viable bacterial adhesion to melimine-coated lenses. #Significantly lower total bacterial adhesion to melimine-coated lenses than uncoated control lenses.
Figure 1
 
Number of total and viable P. aeruginosa 6294 adherent to melimine-coated and control contact lenses following 18 hours of incubation. *Significantly higher total bacterial adhesion than viable bacterial adhesion to melimine-coated lenses. #Significantly lower total bacterial adhesion to melimine-coated lenses than uncoated control lenses.
Figure 2
 
Amount of endotoxin associated with control and melimine-coated lenses after incubation with P. aeruginosa for 18 hours.
Figure 2
 
Amount of endotoxin associated with control and melimine-coated lenses after incubation with P. aeruginosa for 18 hours.
Figure 3
 
Ocular responses of rabbit eyes that retained contact lenses for 24 hours.
Figure 3
 
Ocular responses of rabbit eyes that retained contact lenses for 24 hours.
Figure 4
 
Total ocular scores following wear of P. aeruginosa adherent to contact lenses after 24 and 48 hours. Total ocular scores of 10 or more were regarded as a significant adverse event. Bubble size indicates the number of animals with same ocular score, and the smallest bubbles seen represent data from one rabbit. Black bubbles indicate incidence of microbial keratitis.
Figure 4
 
Total ocular scores following wear of P. aeruginosa adherent to contact lenses after 24 and 48 hours. Total ocular scores of 10 or more were regarded as a significant adverse event. Bubble size indicates the number of animals with same ocular score, and the smallest bubbles seen represent data from one rabbit. Black bubbles indicate incidence of microbial keratitis.
Figure 5
 
Representative images of slit-lamp findings of rabbit eyes captured at ×7.5 magnification. (A) Healthy rabbit eye; (B) fluorescein staining of full-thickness loss of epithelium; (C) conjunctival hyperemia with mild discharge; (D) microbial keratitis.
Figure 5
 
Representative images of slit-lamp findings of rabbit eyes captured at ×7.5 magnification. (A) Healthy rabbit eye; (B) fluorescein staining of full-thickness loss of epithelium; (C) conjunctival hyperemia with mild discharge; (D) microbial keratitis.
Table 1
 
Scoring of the Rabbit Ocular Response During Contact Lens Wear
Table 1
 
Scoring of the Rabbit Ocular Response During Contact Lens Wear
Table 2
 
Types of Microorganisms Isolated From Rabbit Eyes Before Contact Lens Wear
Table 2
 
Types of Microorganisms Isolated From Rabbit Eyes Before Contact Lens Wear
Table 3
 
Number of Rabbits Wearing Contact Lenses and That Developed MK
Table 3
 
Number of Rabbits Wearing Contact Lenses and That Developed MK
Table 4
 
Ocular Responses Following Wear of Contact Lenses Preincubated With P. aeruginosa in Rabbits
Table 4
 
Ocular Responses Following Wear of Contact Lenses Preincubated With P. aeruginosa in Rabbits
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