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Fimbrolide-Coated Antimicrobial Lenses: Their In Vitro and In Vivo Effects

ZHU, HUA PhD; KUMAR, AJAY PhD; OZKAN, JEROME BOptom; BANDARA, RANI BSc; DING, AIDONG MSc; PERERA, INDRANI MPhil; STEINBERG, PETER PhD; KUMAR, NARESH PhD; LAO, WILLIAM PhD; GRIESSER, STEFANI S. BSc; BRITCHER, LEANNE PhD; GRIESSER, HANS J. PhD; WILLCOX, MARK D.P. PhD

Author Information
Optometry and Vision Science: May 2008 - Volume 85 - Issue 5 - p 292-300
doi: 10.1097/OPX.0b013e31816bea0f

Abstract

In developed countries contact lens wear and particularly overnight wear of soft lenses is the main risk factor for development of microbial keratitis (MK) and this overrides all other risk factors in otherwise healthy eyes. Contact lens associated MK is of great concern because of the large numbers of lens wearers worldwide and because of the potential for poor visual outcome. Annualized incidence rates of MK vary in studies, although overall they are in good agreement, with rates in extended wear hydroxyethyl methacrylate (HEMA)-based lenses being around 19/10,000 wearers per year.1 This is approximately five times the rate if HEMA-lenses are worn on a daily wear schedule.1 The rate for extended wear of silicone-hydrogel lenses is very similar to that of HEMA-based lenses.1 Thus, it appears that the advent of highly oxygen permeable lenses has had no appreciable impact on rates of MK, although the silicone-hydrogel lenses are generally worn for longer periods of time (i.e., up to 30 day/nights, rather than 7 to 14 day/nights with HEMA-based lenses).

For contact lens-induced MK, the most common cause of infection remains the gram negative bacterium Pseudomonas aeruginosa. P. aeruginosa accounts for around 40 to 70% of isolates from MK in either HEMA-based lenses or silicone-based lenses.2 Other bacterial types commonly isolated include Staphylococcus aureus and Serratia marcescens. Acanthamoeba sp. can cause MK, and the events are almost always associated with contact lens wear.3 Members of the same bacterial types that cause MK can also cause other non-infectious keratitis events such as contact lens induced acute red eye (CLARE) and contact lens induced peripheral ulcers (CLPU).4,5 Therefore, there is a clear need for the development of contact lenses that can minimize the risk of developing these bacterial or amoebal adverse events.

Several possible antimicrobial lens coatings have been proposed. Silver coatings or silver within the lens matrix confers antibacterial properties to the lens.6–8 Other metals, such as gallium, zinc, and copper, may also act to reduce bacterial adhesion.9,10 Chitosan has been investigated as a tethered antimicrobial agent.11 Cationic peptides and selenium have also been added to the surface of lenses to reduce bacterial adhesion.12–14

A novel way of preventing bacterial adhesion to surfaces is to use substances that interfere with bacterial signaling systems. Many bacteria use a system known as quorum sensing to regulate gene expression. One of the most studied bacterial systems is that of P. aeruginosa. P. aeruginosa has two hierarchically organized quorum sensing systems termed the Las and Rhl systems, along with several other systems that interact with these two.15,16 The Las system is composed of LasR, a transcription regulator and LasI, a synthase that produces the quorum sensing molecule N-3-oxo-dodecanoyl-l-homoserine lactone (3O-C12-HSL). The Rhl similarly has RhlR and RhlI [which catalyzes the production of N-butyryl HSL (C4-HSL) and N-hexanoyl HSL (C6-HSL)]. These systems are termed quorum sensing as they regulate gene expression only when a sufficient quantity of the HSL is present in the bacterial cytoplasm. Levels of HSLs become sufficient once there is a large population of bacteria in a defined space such that their concentration reaches a threshold. Both systems regulate the production of several virulence traits in P. aeruginosa; the Las system regulates the production of lasB (elastase), lasA (staphylolysin), aprA (alkaline protease), toxA (exotoxin A), hcnABC (hydrogen-cyanide synthase), and lasI itself15,17–24 and the Rhl system regulates the production of rhlAB (rhamnolipid synthesis genes), rhlI, lasB (elastase), rpoS (the stationary phase sigma factor), lecA (type-1 lectin), lecB (type-II lectin), hcnABC (hydrogen-cyanide synthase), and genes involved in pyocyanin production.22,25–28 The two systems may also be involved in the initiation and stabilization of the biofilm mode of growth, i.e., attachment and subsequent growth of bacteria once bound to a surface (animate or inanimate; for review see Ref.29).

Fimbrolides (also known as furanones), initially isolated from the marine alga Delissia pulchra, have been shown to interfere specifically with the Las and Rhl quorum sensing systems of P. aeruginosa.30 Fimbrolides coated onto surfaces have been shown to reduce adhesion of P. aeruginosa.31 Furthermore, the fimbrolides/furanones can also interfere with other signaling systems in gram positive bacteria.32–34 Furanones, even when covalently bound to polymers, can control adhesion of Staphylococcus epidermidis although the mechanism of action of covalently bound furanones is not known.35,36 Thus, fimbrolides represent a potentially broad-spectrum antimicrobial technology. The current research assessed the ability of a fimbrolides, once covalently bound to a contact lens surface, to prevent the adhesion of a variety of bacteria and Acanthamoeba to contact lenses. We also assessed the ability of these lenses to be worn safely on the eye using an animal model and human volunteers.

MATERIALS AND METHODS

Strains and Culture Conditions

P. aeruginosa strain 6294, isolated from a MK patient,37,38S. aureus strain 31, isolated from case of contact lens associated peripheral ulceration39,40 and S. marcescens ATCC04135 isolated from MK were used in bacterial adhesion assays. Strains were inoculated into 10 ml of tryptone soya broth (TSB) and incubated at 37°C overnight. After centrifugation at 3000 rpm for 10 min, bacterial cells were washed once in phosphate buffered saline (PBS), and resuspended in 1/1000 TSB/PBS (or 1/50 TSB/PBS for S. aureus) to an OD660nm of 0.1 (1 × 108 colony forming units (CFU)/ml). The bacterial cell suspensions were then serially diluted (1/10) to 103 CFU/ml and used in adhesion assays.

Acanthamoeba castellani ATCC 30234 trophozoites were used in this study. Cryo-preserved Acanthamoeba cells were inoculated into 25 ml of peptone-yeast extract-glucose (PYG) culture media and incubated at 32°C for 7 to 10 days to obtain motile trophozoites. The culture was then collected and subcultured into 200 ml of PYG and incubated for 3 to 4 days to obtain trophozoites. After incubation, a sterile cell scraper was used to gently detach the trophozoites adhered to the base of the flask. The trophozoites were then centrifuged for 12 min at 1500 rpm to collect the cells. After centrifugation, trophozoites were resuspended in approximately 5 ml of PYG. The trophozoites cells were enumerated using a hemocytometer after staining with Trypan Blue. The final inoculum was then adjusted to 1.0 × 105 track forming units/ml either by dilution or centrifugation/concentration.

Chemical Attachment of the Fimbrolide to a Contact Lens Surface

Lotrafilcon A contact lenses (Base Curve 8.6, diameter 13.8 mm; Ciba Vision Inc) were coated with fimbrolide. First, individual lenses were plasma coated with a thin (∼20 nm thickness) polymeric coating fabricated by the gas plasma (glow discharge) polymerization of propanal to obtain an aldehyde functionalized surface similar to procedures reported previously.41,42 Poly (allylamine) (MW 70 kDa) was then covalently grafted from buffered aqueous solution onto the surface aldehyde groups via reductive amination, using sodium cyanoborohydride as the reduction agent. These amine surfaces were then used for the grafting of an acrylate derivative of the fimbrolide by a Michael-type addition reaction (Fig. 1). Briefly, plasma plus poly(allylamine)-coated lenses were soaked overnight with agitation in 1.5 mg/ml fimbrolide-acrylate solution (in 50% ethylene glycol, 10% ethanol, and 40% Milli Q water). The lenses were removed from the reaction mixture and washed three times in 10 ml of fresh solution (50% ethylene glycol, 10% ethanol, and 40% Milli Q water). The lenses were then washed three times with Milli Q water and once with PBS. Lenses were then autoclaved (at 121°C for 20 min) in 2 ml of PBS in a vial. Evaluation of the final lens wash in Milli Q water by liquid chromatography followed by mass spectrometry failed to find any free fimbrolide (data not shown). Thus, the lenses were truly coated with the fimbrolide and were not delivering free fimbrolide in in vitro or in vivo experiments.

FIGURE 1.
FIGURE 1.:
Scheme for chemical attachment of fimbrolide to lens surface. NH2, amine groups attached onto lens surface by grafting poly(allylamine) onto an aldehyde plasma coating.

Adhesion Assay

Fimbrolide-coated or uncoated control lenses (i.e., commercially available Lotrafilcon A lenses) were included in each experiment. Lenses were washed once with 1 ml PBS before the assay to eliminate any remaining lens packaging solution. The lenses were transferred into 1 ml of bacterial/amoebal suspension (prepared above) in 24 well tissue culture plates and incubated at 37°C for 24 h. After washing three times in 1 ml PBS, the contact lenses were stirred rapidly in 2 ml of PBS containing a small stirring bar. For bacterial strains, following log serial dilution in PBS, 3 × 50 μl of each dilution were plated on a nutrient agar plate for the bacterial counts. After incubation at 37°C overnight, CFU on the plate were counted and converted to CFU/lens by multiplying with the appropriate dilution factor. The bacterial adhesion on test lenses was compared with that on the control lenses, and the percentage reduction of bacterial adhesion was calculated accordingly. For amoeba, the detached trophozoites were transferred into an Eppendof tube and centrifuged for 12 min at 1500 rpm. After centrifugation the cell pellet was resuspended in the 200 μl PBS. The number of trophozoites in the suspension was measured using a hemocytometer (as above). The amoeba adhesion on test lenses was compared with that on the control lenses, and the percentage reduction of Acanthamoeba adhesion was calculated accordingly. Three lenses each from test and control group were included in each experiment and at least two repeats of experiment were applied to each test strain (total six lenses per microbe).

Cytotoxicity Tests

There have been reports on associated cytotoxicity of furanones, chemically related but structurally distinct from those used in the current study.43,44 Therefore the fimbrolide type used in the current study, once chemically attached to a contact lens, was assessed for cytotoxicity in two standard assays.

Cell Culture and Growth Inhibition (Based on ISO 10993).

Fimbrolide-coated or control contact lenses were extracted in saline at a ratio of eight lenses per 2.5 ml by autoclaving for a period of 1 h at 121°C. The saline was removed and diluted 1:3 with 1× Eagle’s Minimal Essential Medium (EMEM). Murine fibroblast L929 cells were grown for 24 h in 1× EMEM. The medium was then aspirated and replaced with medium containing sample solution (25% effective dilution, 1:3). The cell monolayer was then cultured for a further 48 h at 37°C. The cells were removed from the plates by trypsin digestion and cells numbers were counted. Controls were used; negative control was 1× EMEM, positive controls were 4, 5, and 7.5% ethanol in 1× EMEM. Cells numbers were compared with those in the controls and any reduction of cell numbers compared to the negative control in excess of 30% was considered evidence of significant cytotoxicity.

MEM Elution Assay.

Murine L929 cells were grown to near confluency in plastic Petri dishes, the medium was aspirated and replaced with a small volume of fresh medium. Fimbrolide-coated or control materials [latex (positive control) and silastic (negative control), or no materials] were placed directly on the cell monolayer. After incubation for 24 h at 37°C, cells were stained with a vital stain (Trypan Blue). Cytotoxicity assessed using bright field and phase-contrast microscopy. Cytotoxic responses were graded according to a standard key, which quantifies the zonal extent of cell damage (0: no cell damage zone around or under sample; 1: cell damage zone limited to area under sample; 2: zone extends <0.5 cm beyond sample; 3: zone extends 0.5 to 1.0 beyond sample; 4: zone extends >1.0 cm beyond sample). A reactivity grade above 1 is indicative of a significant cytotoxic response under the conditions of this assay.

Lens Parameter Measurements

Five fimbrolide-coated human lenses were randomly picked for metrology testing using standard techniques. Briefly, the central thickness of each lens was measured using a low force tactile probe (<20 mN). The procedure was repeated five times. The lens diameter was measured in a wet cell using a Nikon profile projector with horizontal x-y table and digital position readout. The procedure was repeated to obtain a total of four diameter readings. Base curve equivalent was a calculated value corresponding to the average curvature of the back surface and was calculated using center thickness measurements and edge thickness. Refractive index was measured using a Model WY1A Abbé Refractometer. The refractometer was calibrated using a standard glass block (Edmund Scientific). After removal from PBS, each lens was placed concave up on the refractometer platform and the cover closed to flatten it, and a reading taken. The lens was then immediately returned to PBS. Three independent readings were taken for each lens, and the average value was recorded.

Animal Model for Assessing the Safety of Fimbrolide-Coated Lenses Worn on an Extended Wear Schedule

Lotrafilcon A contact lenses designed specially to fit the guinea pig cornea were used in this study and were coated with fimbrolide as described above, or remained uncoated (but with a plasma coating equivalent to that of commercially available human lenses). Institute of Medical and Veterinary Science Guinea Pigs were purchased from Institute of Medical and Veterinary Science, Veterinary Service Division, Gilles Plains, SA. All guinea pigs were housed in accordance with National Institutes of Health guidelines and institutional ethics committee clearance was obtained before animal experiments. Guinea pigs were 6 to 9 months old at the start of the experiments.

Ten guinea pigs were used in this study. All the guinea pigs underwent a detailed ocular examination using a slit lamp (Zeiss, Germany) before lens insertion. Lenses including eight fimbrolide-coated lenses (see above) and two process control lenses (i.e., plasma polymer plus polyallylamine only) were inserted on eyes of guinea pigs chosen at random while the contralateral eyes received normal plasma-treated lotrafilcon A lenses. The lenses were left on the eyes for a period of 4 weeks. The eyes of the guinea pigs were monitored daily for the presence of lenses in both eyes. Lost lenses were replaced as and when discovered. Ocular slit lamp evaluation was conducted following 1 day, 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks of continuous wear of test and control lenses. The measurements during slit lamp examination included cornea epithelial staining (0 to 4 grades, 0.1 steps), corneal infiltration (0 to 4 grades, 0.1 steps), limbal conjunctiva redness (0 to 4 grades, 0.1 steps), conjunctival chemosis (0 to 4 grades, 0.1 steps), and discharge (0 to 4 grades, 0.1 steps). Total adverse response scores were calculated by adding all the grades of ocular measurement together. The single observer was masked regarding which lens was in the animals eyes.

Assessing the Safety of Fimbrolide-Coated Lenses in Human Volunteers

A double-masked, contralateral, randomized, controlled study was conducted using fimbrolide-coated and marketed lotrafilcon A lenses (CIBA Vision). Lenses were randomly assigned to be worn in either eye of each of 10 subjects. The duration of wear was 20 to 22 h, including 8 h of eye closure (sleep). After an ocular slit lamp examination was conducted at baseline, lenses were inserted and then the eyes were evaluated immediately after insertion (10-min wear), at 6 h after insertion, before sleep and upon waking from sleep.

Slit lamp evaluation, conducted by a single masked observer, included measurements of lens wettability (0 to 4 grade; 0.5 steps), amount of front or back surface deposits (0 to 4 grade; 0.5 steps), number of mucin balls, lens centration of eye (mm from center on x and y axis), primary gaze lens movement (mm), lens tightness [using finger push up; grade 1(tight) − 100(loose)], overall fit assessment (grade 0 to 4; 0.1 steps), bulbar, palbebral, and limbal conjunctival redness (grade 0 to 4; 0.1 steps), corneal and conjunctival staining (after fluorescein instillation and viewing using Wratten filter; grade 0 to 4; 0.1 steps), and conjunctival indentation (grade 0 to 4; 0.1 steps). Subjects were asked to rate the lens comfort based on overall impression of ocular comfort, ocular dryness, lens edge awareness, and lens awareness at the time of the examination (1 = very uncomfortable, dry or aware; 10 = very comfortable, not dry, unaware). Subjects were also asked for any preference for either lens (right or left eye, or neither eye preferred).

Data Analysis

Differences in adhesion of microbes to control and furanone-coated lenses were tested after converting adhesion data into logarithm using Mann-Whitney U test and data are presented as medians and 25th and 75th percentile ranges. Contact lens parameters were analyzed using Student’s t-test. Animal ocular measurements of test and control lens wearing eyes were compared using paired t-test. Human clinical and subjective ratings were summarized using descriptive statistics. Differences between lens types were determined at each visit using paired t-test or Wilcoxon rank sum test based on the type of variable. Lens preference was reported as frequency and percentage for each preference category. Initially the proportion of subjects with “no-preference” was compared with those who had a preference. Following this, specific lens preference was analyzed after excluding the “no-preference” category. Preference was tested for equality using a binomial test with a test proportion of 0.5. A p value of ≤0.05 was considered statistically significant.

RESULTS

Bacterial and Amoebal Adhesion to Lenses

Fig. 2 shows the reduction of the adhesion of the microbes to lenses coated with fimbrolides compared with the control lenses. All strains/types tested showed significant reduction in adhesion, giving reductions of 67% for P. aeruginosa (p = 0.000), 87% for S. marcescens (p = 0.01), 92% for S. aureus (p = 0.000), and 70% for Acanthamoeba trophozoites (p = 0.004). Thus, the fimbrolide-coated lenses were antiadhesive/antimicrobial.

FIGURE 2.
FIGURE 2.:
Reduction in adhesion of microbes on lotrafilcon A lenses coated with fimbrolide. Data are presented as box plots showing median, 25th and 75th percentile ranges. All experiments were repeated at least three times. Data in gray shade are for control lenses and data in white for furanone-coated lenses.

Toxicity Profile of the Fimbrolide-Coated Lenses

The cell-growth inhibition assay was a valid assay as controls performed as expected [7% ethanol gave a reduction in cell growth of 87% compared to the negative (media) control]. Fimbrolide-coated lenses only showed 4% inhibition of growth. In the direct contact assay, the fimbrolide-coated lenses behaved in the same way as normal commercially available lotrafilcon A lenses, showing only a small annulus of dead cells under the contact area (grade 1). The positive and negative controls behaved as expected. The fimbrolide-coated lenses were thus not toxic in either cytotoxicity assay.

Lens Parameter Measurements

These tests were performed on the human lenses only. The commercially available lotrafilcon A lenses (with power of −1.00) had a lens diameter of 13.85 mm, a central thickness of 72 μm and a calculated base curve of 8.6 mm. The refractive index of the commercial lenses was 1.4272. Similarly, the fimbrolide-coated lenses (with power of −1.00) had a lens diameter of 13.85 mm, a central thickness of 71 μm, a calculated base curve of 8.6 mm and a refractive index of 1.4249 ± 0.0003. Although the refractive index of the lenses was slightly reduced, none of the measures for the fimbrolide-coated lenses were statistically different to those of the commercially available lenses.

Animal Model Lens Wear

A few animals lost lenses during wear which were replaced at the time of examination (but the total lens wearing time for animal did not exceed 28 days). At 72 h of lens wear, two lenses, one test and one control were lost from both eyes of one animal, these lenses were replaced within 24 h of noticing loss of lenses. Two test and two control lenses were lost during the 2nd week of lens wear. These lenses were replaced within 24 h of loss. One more animal was discontinued from the study in week 3 as it was repeatedly losing lenses and the data for this animal was not included in any data analysis. Another animal was discontinued from the study because of the presence of corneal infiltrates in the control eye at day 14. The remaining Guinea Pigs wore the lenses safely for the whole of the 28 days. The changes that occurred to the Guinea Pigs conjunctivas and corneas are shown in Figs. 3 and 4, respectively. There was no statistically significant difference between fimbrolide-coated and control lenses for any of the variables shown in these figures at any time point. Also, eyes wearing process-control lenses (i.e., lotrafilcon A lenses plasma coated with plasma polymer plus polyallylamine but not the fimbrolide) also behaved the same as the control (i.e., plasma coated as per human commercially available lenses) in all of these variables over the 28 day time frame.

FIGURE 3.
FIGURE 3.:
Conjunctival responses of the Guinea Pig during wear of fimbrolide-coated and control lotrafilcon A lenses. Standard deviations of data were between 20 and 40% of the mean values for redness responses. For chemosis and discharge, standard deviations were greater because of the infrequency of developing these responses during lens wear. Mean of data from 10 animals.
FIGURE 4.
FIGURE 4.:
Corneal staining responses to wear of fimbrolide-coated and control lenses on Guinea Pig eyes. Standard deviations of data were between 35 and 80% of the mean values. Mean data from 10 animals.

There were two events of corneal infiltration that occurred only in the control lens wearing eyes. At day 14, one control lens wearing eye but not the contralateral fimbrolide-lens wearing eye was found to have extensive corneal infiltration and redness. Lens wear was discontinued for this animal and the condition resolved without any treatment within 5 days. On day 28, one more control lens wearing eye was found to have corneal infiltrates and again the fimbrolide-coated lens wearing eye was found to be within normal limits for conjunctival responses and had no corneal staining or infiltration. Fig. 5 shows the adverse response scores (a summation of all adverse response scores calculated from sum of corneal and conjunctival variables and infiltrate grades). With the exception of the increase in score caused by the infiltrates outlined above, there were no differences between adverse responses scores for either lens type. Overall, the conjunctival and corneal responses during lens wear were graded as mild (i.e., below 2 in 0 to 4 grading scale).

FIGURE 5.
FIGURE 5.:
Adverse response scores during wear of fimbrolide-coated and control lenses by Guinea Pigs. Adverse response scores calculated from sum of corneal and conjunctival variables in Figs. 3 and 4 plus infiltrate grade. Data are presented as box plots showing median, 25th and 75th percentile ranges. Data in gray shade are for control lenses and data in white for furanone-coated lenses.

Human Lens Wear

Lens surface characteristics were graded on a 0 to 4 scale in 0.5 unit steps. There were no differences in lens wettability, amount of front or back surface deposits, or number of mucin balls between fimbrolide-coated and control lenses when the subjects were examined after lens insertion, 6 h of open eye wear and immediately following eye opening after sleep. The only variable that approached significance was for back surface debris where there was a trend for the fimbrolide-coated lenses to have more back surface debris on awaking (p = 0.066). However, the grade of back surface debris was mild (1.0 ± 0.4 vs. 0.6 ± 0.3).

For lens fitting characteristics (x or y centration, primary gaze movement, lens tightness), there were no significant differences between fimbrolide-coated and control lenses. The bulbar or limbal redness responses (after 6 h of lens wear, before sleep and after sleep) were not significantly different from each other and were graded overall as 2.3 ± 0.1 vs. 2.3 ± 0.1 for bulbar redness and 2.1 ± 0.2 vs. 2.0 ± 0.2 for limbal redness for fimbrolide-coated vs. control, respectively (Fig. 6). There were also no significant differences between the corneal or conjunctival staining responses during wear of either lens type. For conjunctival indentation, the only significant difference was a slight increase in temporal conjunctival indentation on awaking with the fimbrolide-coated lenses (0.4 ± 0.5 vs. 0.2 ± 0.3, p = 0.039).

FIGURE 6.
FIGURE 6.:
Conjunctival and limbal redness response to fimbrolide-coated and control lenses on the human eye for 24 h [including immediately after sleep (24 h time point)]. Data are mean results from 10 human volunteers.

Both control and fimbrolide-treated lenses were generally comfortable (Fig. 7) and were within the range seen within this clinic for the silicone hydrogel lens type. Although the fimbrolide-coated lenses generally were slightly less comfortable, had slightly increased dryness, lens edge and lens awareness, the only significant difference was that the subjects noted slightly more lens awareness for the fimbrolide-coated lenses on awaking (p = 0.037). Overall, the subjects also preferred the control lenses to the fimbrolide-coated lenses, although the differences were not statistically significant (Fig. 8).

FIGURE 7.
FIGURE 7.:
Comfort during wear of furanone-coated and control lenses. Data are presented as box plots showing median, 25th and 75th percentile ranges.
FIGURE 8.
FIGURE 8.:
Preferences of the lens wearers for the lenses after insertion (A), after 6 h of lens wear (B), and upon awaking (C). Data is % of subjects in each category.

DISCUSSION

This study has demonstrated that fimbrolides can reduce the adhesion of bacteria and Acanthamoeba to lenses after covalent attachment of the fimbrolides to the lens surface. In addition, the fimbrolide-coated lenses were able to be worn safely by human volunteers and in an animal model.

The reported effects of fimbrolides on bacteria have largely examined their interactions in solution, i.e., when the fimbrolides are free in solution. A report by Hume et al.35 demonstrated that the use of fimbrolides, when chemically linked into polystyrene blocks, inhibited the adhesion of Staphylococcus epidermidis and adverse responses during implantation of fimbrolide-coated catheters in sheep. The current study is the first to demonstrate that covalently bound fimbrolides can also prevent adhesion of P. aeruginosa, S. aureus, S. marcescens, and Acanthamoeba to contact lenses. This is significant as these bacterial types and the amoeba are associated with most cases of MK caused by wearing contact lenses.2,3,45,46 The molecular mechanism(s) that underlie this inhibition of adhesion by attached fimbrolides is not known. In solution, fimbrolides interact with quorum sensing transcription regulators in P. aeruginosa47 and this regulator resides within the bacterial cytoplasm. The nature of the interactions between fimbrolides and other bacteria is not known, nor is the nature of the interaction of the fimbrolides with Acanthamoeba. The fimbrolide-coated lenses were extensively washed in water before use in these experiments, thus making any observed effects because of free fimbrolides remaining adsorbed to the lenses very unlikely. For the covalently bound fimbrolides to cause an effect there must be some form of interaction with the surface of the bacteria. Interestingly, in Agrobacterium tumifaciens the transcriptional regulator TraR (homologue of P. aeruginosa LasR and RhlR) binds to the cytoplasmic membrane of the bacterium.48 Similarly, in Vibrio harveyi there is a membrane associated receptor for the AI-2 quorum sensing molecule (reviewed in Ref. 49). Further work is needed to understand the molecular basis of the inhibition of adhesion mediated by fimbrolides once chemically bound to surfaces. As fimbrolides do not kill bacteria, but simply switch off characteristics such as biofilm formation, we believe the potential for bacteria to develop resistance to fimbrolides is low, and no fimbrolide-resistant bacteria have been isolated from the marine environment where Delisea pulchra is found (unpublished data). However, monitoring ocular bacteria for the develop of resistance should certainly be undertaken in larger scale clinical trials.

The level of adhesion inhibition was between 67 and 92% and was significant. The supposition being tested when examining the ability of antibacterial surfaces to prevent bacterial adhesion is that a reduction in adhesion would lead to reductions in adverse responses caused as a result of bacterial adhesion. There is no generally accepted level for this reduction on contact lens surfaces as there are no antibacterial contact lenses available that have been tested to determine whether they do indeed reduce adverse responses from occurring. However, it is possible to set some minimal level of adhesion, based on numbers of bacteria required to initiate adverse responses in animal models. Around 23,000 P. aeruginosa on a contact lens are required to produce CLARE-like response in 50% of Guinea Pigs eyes.2 Halving that number leads to reduction in response rate from 50 to 33% and by around 10,000 bacteria on a lens there is no CLARE-like response. Thus, for P. aeruginosa adhesion, for every 50% reduction in adhesion there is an approximate reduction in CLARE-like responses from 50 to 10%. In a rabbit model of CLPU,50 there is also a response to numbers of bacteria on a contact lens. In this case, the number of adherent S. aureus on a lens is related to size of the epithelial defect.2 To halve the ulcer size, the numbers of bacteria need to be reduce by approximately 35% (i.e., an epithelial defect of 6 mm is produced by approximately 6000 bacteria on the lens and an epithelial defect of 3 mm is produced by approximately 4000 bacteria on a lens). As we do not know the numbers of bacteria that initially colonize a contact lens that go on to cause adverse responses in humans, it seems appropriate to set as a minimum a 50% reduction in adhesion for an antibacterial surface as being likely to reduce adverse response rates. We believe it is unlikely that large numbers of bacteria initially adhere to a lens surface. It is more likely that smaller numbers of bacteria adhere initially and then accumulate to numbers that can cause adverse responses.

The fimbrolide lenses passed preclinical cytotoxicity tests and were considered to be safe for use in animal model and, assuming no safety issues in the animal model, in human eyes. This study is also the first to report on the safety of an antibacterial contact lens in preclinical and clinical testing. A previous study has shown that selenium-coated lenses, in a rabbit lens wear model, can be worn safely for up to 4 weeks,14 but there was no subsequent clinical evaluation of safety in humans. In the present study, the fimbrolide-coated lenses performed almost identically to the control lotrafilcon A lenses in either the Guinea Pig model or in human volunteers. The only exception was in subjective responses in the human volunteers, where, on occasion, the fimbrolide-coated lenses apparently caused more lens awareness and the Lotrafilcon A lenses were preferred over the fimbrolide-coated lenses after overnight wear. However, it is difficult to definitely link these responses, and responses such as increased debris behind lenses or lens indentation of the conjunctiva, to the fimbrolides per se as the fimbrolide coating process involved several additional laboratory steps that could also have affected the comfort/preference responses.

In conclusion, this study has demonstrated the efficacy of fimbrolide-coated lenses in vitro to control bacterial and amoebal adhesion, as well as their safety during a small scale clinical trial. Whether these lens will produce a reduction in adverse responses during wear, especially extended wear, is a possibility but requires more extensive clinical trials. Given the frequency of CLARE and CLPU responses (around 5 to 20% of wearers per year), prospective clinical trials of antimicrobial lenses designed to measure reductions in these responses are possible. However, given the relative infrequency of MK (i.e., frank infection; 1/500 people per year), the ability of antimicrobial lenses to control or reduce this response is only likely to be determined in postmarket studies.

ACKNOWLEDGMENTS

This study was partly funded by the Institute for Eye Research and Biosignal Ltd, and a project grant from the NHMRC.

Mark Willcox

Institute for Eye Research

University of New South Wales

Rupert Myers Building

Sydney, NSW 2052

Australia

e-mail: m.willcox@ier.org.au

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Keywords:

contact lenses; adverse events; antimicrobial surface; clinical study; Pseudomonas

© 2008 American Academy of Optometry