The tear film coats the hydrophobic surface of the epithelium and consists of three distinct layers: outermost lipid layer, the middle aqueous layer (containing electrolytes, proteins, enzymes, and other components), and the mucin layer.1,2 The lipid layer, which is primarily secreted by meibomian gland comprises two phases, a thin polar phase and a thick non-polar phase.3–5 The polar phase consists mainly of phospholipids,3,6 and the non-polar phase consists of cholesterol and wax esters.5 It is believed that the lipid layer prevents evaporation of the tear fluid and facilitates movement of the eyelid during blinking.6–8
Immediately after insertion in the eye, contact lenses absorb a variety of tear film components including lipids and proteins.9–13 These deposition cause contact lens spoilage, which is believed to be the important factor in clinical complications such as discomfort,14,15 reduced visual acuity,16,17 dryness,14,15,18 and bacterial contamination of the contact lens surface.19–27 The degree of tear film deposition on contact lenses is affected by lens wear basis,28–30 lens material,29,31,32 and types of lens care system used.29,33 Because of relative hydrophobicity, silicone hydrogel contact lenses have been shown to adsorb more lipid compared with conventional contact lenses. However, compared with conventional lenses they accumulate extremely low levels of protein.13,30,32,34
A number of studies have reported the presence of lipids in the deposits of contact lenses.9,10,29,31,32,35–37 Lipid deposits consist of oleic acid, oleic acid methyl ester, and cholesterol on worn contact lenses.32 Cholesterol has been shown to be one of the most frequently detected lipids on silicone hydrogel lenses.10,29,32 All silicone hydrogel lens types tested in vitro adsorbed significantly higher amount of cholesterol compared with phosphatidyl ethanolamine.38 In vivo, lens polymer and care solutions can affect the amount of cholesterol deposition onto silicone hydrogel contact lenses during wear.29
Several reports have shown that tear film protein deposits,19,21,23,25,39 lens material,19,40,41 duration of wear,19,26,42 and surface roughness43,44 can affect the ability of bacteria to bind to the contact lens surface. Pseudomonas aeruginosa adhesion was increased when albumin was coated on to etafilcon A or polymacon lenses.25 The adhesion of Staphylococcus aureus can be either increased or remained unchanged by adsorption of lysozyme to the lens surface.23,27 To date, few studies have examined the bacterial adhesion to silicone hydrogel contact lenses,41,45–47 and none of these reports have investigated the effect of lipid deposition on bacterial adhesion to contact lenses.
In this study, we analyzed in vivo cholesterol deposits on two types of silicone hydrogel lenses in combination with lens care solutions. We further examined the effect that cholesterol adsorption to contact lens had on bacterial adhesion by comparing the adhesion of strains of P. aeruginosa and S. aureus to clean contact lenses, and to contact lenses soaked in cholesterol solution in vitro. Results of this study will be valuable data indicating whether cholesterol as frequently detected tear component on silicone hydrogel contact lenses will influence the rate of bacterial contamination of the lens surface.
MATERIALS AND METHODS
Contact Lenses and Lens Care Solutions
The commercially available silicone hydrogel contact lenses used in this study were PureVision (Balafilcon A; Bausch and Lomb, Rochester, NY), a silicone hydrogel FDA group III ionic lens, which is surface treated by gas plasma and composed of a 36% water content silicone N-vinyl pyrrolidone polymer, and Acuvue Oasys (Senofilcon A; Johnson and Johnson Vision Care, Jacksonville, FL), an FDA group I non-ionic lens, which has no surface modification but contains polyvinyl pyrrolidone. The commercially available multipurpose solutions Opti-Free Replenish (containing polyquad and Aldox as preservatives, and Tetronic 1304 as a surfactant; Alcon, Fort Worth, TX), and Menicare Soft (Epica Cold) (containing polyhexanide as a disinfectant and macrogolglycerol hydroxystearate 60 as a detergent, Menicon Co Ltd., Japan) and AQuify (containing polyhexanide as a disinfectant and poloxamer 407 as surfactant, CIBA Vision, Atlanta, GA) were used for lens disinfection and cleaning.
Collection of Daily and Extended Wear Contact Lenses
Contact lenses were collected from a group of 22 participants in a randomized, open label, parallel group, and cross-over study (Australian New Zealand Clinical Trials Registry, ACTRN12608000557336), consisting of 1-month daily wear (DW) for each lens care solution and 1-month extended wear. Participants were randomly allocated to bilateral wear of either PureVision or Acuvue Oasys contact lenses for the duration of the study (3 months in total). Each group was randomly assigned to use either Opti-Free Replenish or Menicare Soft lens care solutions for the first month of DW, and then swapped the types of lens care solutions used for the second month of DW. Data for the effect of use of AQuify solution on amount of cholesterol deposited on test lens types were obtained from a previous data set that used the same clinical and laboratory techniques as outlined herein.29 For bacterial adhesion assay, contact lenses were collected from subjects who wore PureVision (n = 22) or Acuvue Oasys (n = 8) as DW in conjunction with AQuify. Lens replacement schedule was fortnightly for Acuvue Oasys and monthly for PureVision. The worn contact lenses were collected in 2-mL phosphate-buffered saline solution (PBS, pH 7.4) once they were removed from the patients' eyes, kept temporarily at 4°C, then transferred to −80°C before extraction of deposits. Latex non-powdered surgical gloves were applied to remove the contact lenses from participants' eyes to prevent contamination from the hands. All protocols for this part of the study were approved by the Institutional Human Ethics Review Committee and complied with the Declaration of Helsinki.
Determination of Cholesterol from Worn and Cholesterol-Soaked Contact Lenses
Cholesterol was purchased from Sigma Aldrich (Castle Hill, NSW, Australia). Aliquots were taken from stock (solubilized in methanol) to make cholesterol solutions in PBS at 37°C (at concentrations 40, 20, 10, 5, 2.5, 1.25 μg/mL).38 Five worn or four lenses soaked in cholesterol were pooled together for extraction. Lenses were blotted dry and placed into a glass vial containing 3 mL of extraction solvent methanol:chloroform (1:1, v/v) and incubated at ambient temperature overnight (18 h) with gentle shaking. The extraction solutions were then transferred to new glass vial and evaporated under a gentle stream of nitrogen gas and reconstituted in 80 μL of methanol:chloroform (1:1, v/v). Extracts were stored at −80°C for analysis. Extraction was repeated twice for worn contact lenses and three times for each type of lens soaked in each concentration of cholesterol.
Thin layer chromatography (TLC) analysis was adapted from those of Bontempo and Rapp.29,48 In brief, 20 μl of lipid extract or cholesterol standards were loaded onto TLC plate (highly purified silica gel plates incorporate a gypsum/polymer binder, Sigma Aldrich). The plate was run sequentially in hexane, benzene, and then hexane:ether:acetic acid (30:20:0.5). After drying, the plate was sprayed with 25% sulfuric acid and the color of the bands was developed by heating the plate at 100°C for 1 h. The image of separated bands on plate was scanned by using GS-800 calibrated densitometer (Bio-Rad Laboratories, Hercules, CA) and bands were analyzed and quantified by using Quantity One software (Bio-Rad Laboratories) based on the known concentrations of standards.
Adsorption of Cholesterol In Vitro to Contact Lenses
Four lenses of each type were cycled in various concentrations of cholesterol aiming to achieve the similar amount of deposition to worn lenses.29 Each lens was removed from the package and washed three times with 1 mL PBS for each wash in a 24-well plate. Each lens was soaked in 1 mL of PBS containing various concentrations of cholesterol (Table 2) for 24 h at 37°C as one cycle. Cycling was repeated as designated (Table 2) and fresh cholesterol solutions were used for each cycle. After cycling, lenses were removed from cholesterol solution and washed three times in 1 mL PBS for each wash. Lenses were then placed in 1.5 mL microfuge tubes and stored at −80°C immediately for further extraction and determination of cholesterol adsorption as described above.
Bacterial Adhesion to Cholesterol-Soaked Contact Lenses
P. aeruginosa strains 6294 (MK isolate), 6206 (MK isolate), GSU-3 (MK isolate), and S. aureus strains 031 (CLPU isolate), 038 (MK isolate), and ATCC 6538 (human isolate) were used in the adhesion experiment. Strains were grown in 10 mL minimal medium [1.0 g D-glucose, 7.0 g K2HPO4, 2.0 g KH2PO4, 0.5 g sodium citrate, 1.0 g (NH4)2SO4, and 0.1 g MgSO4 in 1 liter distilled H2O, pH 7.2]45 (for viable adhesion) or minimal medium containing 3H-uridine (0.2%, for total adhesion) overnight at 37°C. Bacterial cells were collected and washed by centrifugation and resuspended in sterile PBS to an optical density of 0.1 at 660 nm [108 colony forming unit (CFU)/mL], then 10-fold serially diluted in PBS to a final concentration of 107 CFU/mL. Worn and unworn lenses (n = 3 for each repeat) with or without cholesterol soaking (Table 2) were washed three times in PBS. Lenses were then transferred into each well of a 24-well tissue culture plate and inoculated with 1 mL of bacterial suspension at 37°C for 18 h. For recovery of viable cells, each lens was aseptically removed and washed three times, then placed in plastic vials containing 2 mL PBS and a sterile magnetic stirrer24 and vortexed for 1 min at maximum speed to detach adhered bacterial cells. The cell suspensions were serially (10-fold) diluted. Dilutions were plated onto nutrient agar and incubated at 37°C for 18 h. After incubation, numbers of CFU were counted. Total number of bacteria adhered on lens surfaces were estimated after measuring the radioactivity of lenses using a β scintillation counter (Wallac 1400 DSA) and converting the radioactivity into the numbers of cells by using a standard curve. Standard curves were plotted for each experiment from radioactivity measurements of known counts of the radiolabeled bacteria. Lenses without cholesterol treatment and immersed in PBS were included in the adhesion assay as controls. Triplicates of each lens type were included in each adhesion experiment and the experiment was repeated three times for each challenge strain to collect the data from a total of nine lenses for each group.
Effect of Cholesterol on Bacterial Growth
Cholesterol was examined for possible effects on bacterial growth. P. aeruginosa 6294 and S. aureus strains 031, 038, ATCC6538 were grown in minimal medium containing cholesterol (at concentrations 40, 20, 10, 5, 2.5, 1.25, and 0 μg/mL) overnight. Bacteria were then enumerated by viable count.49
One-way analysis of variance with post hoc multiple comparisons were adjusted using Benferroni to compare the amounts of cholesterol absorbed to lenses in vitro. Pilot experiments on unworn lenses indicated that a minimum of three lenses were needed to show a difference of 0.5 log with a 95% confidence and 80% power; therefore, we have performed three repeated measurements, and each measurement was accomplished with extract from four pooled lenses. Log transformed CFU/lens (for viable cells) or cells/lens (for total counts) data were compared with various concentrations of cholesterol-soaked and unsoaked lenses using Linear Mixed Model (a form of the General Linear Mixed Model) for each strain and lens type. Pilot experiments on unworn lenses indicated that for bacterial adhesion assay a minimum of seven lenses were needed in each arm to show a difference of 0.5 log with a 95% confidence and 80% power, and we used eight worn Acuvue Oasys, 22 worn PureVision, and total number of nine lenses for each group of cholesterol-soaked and unsoaked lenses. Linear mixed model was used to account for the replicates within a same group of samples. Post hoc multiple comparisons were adjusted using Bonferroni correction when compared with one control category (unsoaked). The bacterial growth in various cholesterol concentrations was compared using one-way analysis of variance (Post hoc multiple comparisons were adjusted using Dunnett correction when compared with one control category.). Pilot experiments indicated that a minimum of three repeats were needed in each arm to show a difference of 0.5 log with a 95% confidence and 80% power; therefore, we have performed three repeats. SPSS program 15 was used for all analysis, and p value of <0.05 was considered significant.
Ex Vivo Cholesterol Determination
Cholesterol deposition on each group of lens is listed in Table 1. Overall, there were greater levels of cholesterol deposited on PureVision than on Acuvue Oasys lenses irrespective of lens care solutions used and wear schedules. When combined with Opti-Free Replenish solution, PureVision accumulated more cholesterol (3.4 ± 0.2 μg/lens) than Acuvue Oasys (1.0 ± 0.1 μg/lens) lenses. Cholesterol deposited more on extended-wear PureVision (5.1 ± 0.5 μg/lens) lenses compared with extended-wear Acuvue Oasys (2.2 ± 0.1 μg/lens). Lenses that had been used with Opti-Free Replenish solution had less cholesterol extracted from Acuvue Oasys compared with those when Menicare Soft solution was used. Using AQuify gave lowest levels of cholesterol extracted from Acuvue Oasys lenses, but a relatively high level of cholesterol extracted from PureVision lenses29 (Table 1).
In Vitro Cholesterol Adsorption
An example of TLC analysis of lens extracts from cholesterol-soaked PureVision, Acuvue Oasys, and cholesterol standards are shown in Fig. 1. Each lens extract showed a major band (band A in Fig. 1) located in the same position to cholesterol standard. On the basis of the standard curve, the levels of cholesterol adsorbed to the PureVision and Acuvue Oasys lenses in vitro were quantified and shown in Table 2. The in vitro adsorptions of cholesterol to both PureVision and Acuvue Oasys (Table 2) were dose and cycle number dependent. For a given cycle, the increases of cholesterol adsorption were seen in both lens types when soaked in increased concentrations of cholesterol solutions. Increasing the cycling times (i.e., from 1 to 2 cycles) of test lenses in a same concentration of cholesterol solution (i.e., 20 μg/mL) increased adsorption of cholesterol to PureVision lens from 5.6 to 10.8 μg/lens, which was not significantly different (p = 0.73; Table 2). When cycled in the same conditions (i.e., one cycle at concentrations of 20 or 10 μg/mL), PureVision adsorbed a similar amount of cholesterol (5.6 or 3.2 μg/lens, respectively) to that on Acuvue Oasys (5.9 or 3.3 μg/lens, respectively, p > 0.05, Table 2).
At cholesterol concentrations ranging from 10 to 20 μg/mL for PureVision and from 1.25 to 20 μg/mL for Acuvue Oasys lenses, the amount of cholesterol adsorption to PureVision (3.2, 5.6, and 10.8 μg/lens) and to Acuvue Oasys (0.5, 1.2, 3.3, and 5.9 μg/lens) was similar to that on worn lenses (Table 2).29
Effect of Cholesterol Adsorption on Bacterial Adhesion
Fig. 2 shows the adhesion of P. aeruginosa 6294 to unsoaked, cholesterol-soaked, and worn PureVision and Acuvue Oasys contact lenses. There was no difference between the total and viable counts for unsoaked lenses or for each group of cholesterol-soaked lenses (p > 0.05). The count of total or viable P. aeruginosa 6294 on cholesterol-soaked PureVision or Acuvue Oasys was not different to that on control lenses without cholesterol treatment (p > 0.05). There was no differences between total count of P. aeruginosa 6294 to worn PureVision (6.5 ± 0.3 log units) and to unworn PureVision lenses (6.4 ± 0.1 log units, p = 1.00). However, the numbers of viable cells of P. aeruginosa 6294 adhered to worn PureVision (5.8 ± 0.4 log units) was significantly less than to unworn PureVision lenses (6.4 ± 0.2 log units, p = 0.001). There were no significant differences in total or viable adhesion of any test strains to worn Acuvue Oasys lenses compare to unworn Acuvue Oasys (p > 0.05).
Fig. 3 shows the adhesion of S. aureus 031 to unsoaked, cholesterol-soaked, and worn PureVision and Acuvue Oasys contact lenses. There was no difference between the total and viable counts for unsoaked lenses or for cholesterol-soaked lenses (p > 0.05). The count of total or viable S. aureus 031 on cholesterol-soaked PureVision or Acuvue Oasys was not different to that on control lenses without cholesterol treatment (p > 0.05). There was no differences between total count of S. aureus 031 to worn PureVision (5.5 ± 0.3 log units) and to unworn PureVision lenses (5.4 ± 0.3 log units, p = 1.00), whereas viable count of S. aureus 031 for worn PureVision (3.05 ± 0.8 log units) decreased significantly compared with unworn PureVision (4.6 ± 0.3 log units, p = 0.0001). However, there was no significant difference in total or viable adhesion to worn Acuvue Oasys lenses compared with unworn Acuvue Oasys.
The adhesion of the remaining strains of P. aeruginosa, 6206 and GSU-3, and of S. aureus, 038 and ATCC6538, to unsoaked and cholesterol-soaked (10 μg/lens) PureVision contact lenses is shown in Fig. 4. The count of total or viable P. aeruginosa 6206 and GSU-3 on cholesterol-soaked PureVision was not different to that on control lenses without cholesterol treatment (p > 0.05). There was significant difference between the total and viable counts of S. aureus 038 for unsoaked lenses (4.3 ± 0.1 vs. 5.6 ± 0.1 log units; p = 0.03) and for cholesterol-soaked lenses (4.04 ± 0.3 vs. 5.7 ± 0.1 log units; p = 0.02). There was no difference between the total and viable counts of S. aureus ATCC6538 for unsoaked lenses (4.9 ± 0.2 vs. 5.4 ± 0.5 log units; p = 0.3) and for cholesterol-soaked lenses (4.6 ± 0.4 vs. 5.4 ± 0.2; p = 0.2). The count of total or viable S. aureus 038 and ATCC6538 on cholesterol-soaked PureVision was not different to that on control lenses without cholesterol treatment (p > 0.05).
Effect of Cholesterol on Bacterial Growth
The free cholesterol in minimal medium showed no effect on the growth of any test strains (p > 0.05). Growth of P. aeruginosa 6294 was 7.5 ± 0.03 log CFU/mL in non-cholesterol containing media and was 7.4 ± 0.2 log CFU/mL in cholesterol containing media. For S. aureus 031, growth was 5.3 ± 0.3 log CFU/mL in non-cholesterol containing media and was 5.2 ± 0.3 log CFU/mL in cholesterol containing media. For S. aureus 038, growth was 6.8 ± 0.2 log CFU/mL in non-cholesterol containing media and was 6.8 ± 0.05 log CFU/mL in cholesterol containing media. For S. aureus ATCC6538, growth was 5.9 ± 0.3 log CFU/mL in non-cholesterol containing media and was 5.9 ± 0.4 log CFU/mL in cholesterol containing media.
This study reports ex vivo biochemical analyses of lipid deposits extracted from two types of silicone hydrogel contact lenses when used in combination with two types of lens care solutions, and the effect of in vitro cholesterol adsorption to PureVision and Acuvue Oasys contact lenses on the adhesion and growth of P. aeruginosa and S. aureus. Cholesterol was selected for this study because it is recognized as a frequently detected non-polar lipid on silicone hydrogel contact lenses.10,29,31,32 This is the first report examining the effect of cholesterol adsorption on adhesion of bacteria to silicone hydrogel contact lenses.
Of the silicone hydrogel lens types examined in this study, more cholesterol could be extracted from PureVision lenses than from Acuvue Oasys lenses when combined with Menicare Soft or Opti-Free Replenish solutions. This might be related to surface treatment of PureVision lenses or might be due to the fact that PureVision lenses in this study have been worn for 1 month and Acuvue Oasys lenses for 2 wk. Zhao et al.29 found that significantly higher levels of cholesterol could be extracted from PureVision lenses than Acuvue Oasys lenses after wear, which is in the agreement with the current in vivo finding. Jones et al.9 suggested that PureVision, which contains N-vinyl pyrrolidone, had high affinity to adsorb lipids that can slowly penetrate the lens matrix. This report is supported by Maissa et al.30 The reason for high propensity of vinylpyrrolidone-based materials to adsorb lipids relates to the lipid solubility of pyrrolidone derivatives,50 which are found in both contact lenses tested in this study. There was a similar amount of cholesterol extracted from extended-wear Acuvue Oasys and Acuvue Oasys on DW modality in conjunction with Menicare Soft, which suggests that cholesterol may not be removed easily by Menicare Soft.
This ex vivo study demonstrated that there was a decrease in the viable adhesion of test strains to worn PureVision lenses. This may be due to the fact that specific tear components inhibit bacterial adhesion or kill the attached bacteria. However, this in vitro study showed cholesterol was unlikely to be the major tear component that reduced the adhesion of viable P. aeruginosa to worn lens surfaces, because we did not find any differences between the number of bacteria adhered to cholesterol-soaked or unsoaked lenses. It was noticed that the overall viable adhesion of S. aureus strains was lower than total adhesion. This might be due to S. aureus failing to survive in PBS over the adhesion assay (18 h), or because of the fact that, for Staphylococci in general, the tendency for them to clump would mean that viable cell counts, arising from more than one cell (4 to 6 cells generally), will always be less than total cell counts (where incorporation of 3H-uridine into cells is likely for all cells regardless of clumping). This study demonstrated that, unlike the findings for S. pneumoniae in vitro,49 cholesterol did not reduce the numbers of viable P. aeruginosa or S. aureus.
The current findings contrast with previous published data that has shown significant effects of tear film proteins on bacterial adhesion to lenses. When lactoferrin was coated on to hydrogel contact lenses, number of total P. aeruginosa adhesion increased; however, the number of viable bacterial cells reduced.24 Lactoferrin is known as an antibacterial protein,51 which presents in high concentration in the tear fluid.51–53 This iron-binding protein is able to damage the outer membrane of the Gram negative bacteria.54 Butrus et al.26 reported that albumin, lysozyme, and lactoferrin coated on the contact lenses increased adherence of P. aeruginosa to unworn contact lenses. Stern and co-workers55 indicated that the adhesion of P. aeruginosa increased by adsorption of mucin to the lens surface. The adhesion of S. aureus can be either increased or remained unchanged by adsorption of lysozyme to the lens surface.23,27 Moreover, a direct comparison between our study and most other adhesion studies is difficult as others have used different contact lenses and bacteria for a short-term adhesion. However, Borazjani et al.45 using the initial inoculum of 108 cells/mL found that for P. aeruginosa GSU 3, 6206, 6294, and S. aureus ATCC 6538, 2 × 105, 1 × 105, 2 × 104, and 7 × 103 cells/mm2 adhered to unworn PureVision lenses after 2 h incubation, respectively, and in this study, we found that at the initial inoculum of 107, the same strains adhered to the same lens at the level of 5.2 × 103, 2.6 × 103, 8.5 × 103, and 1.2 × 103 cells/mm2, respectively. This indicates that there is probably no biofilm formation when cells are incubated for 18 h compared with 2 h, and cholesterol may not be used by bacteria as a nutrient to promote biofilm formation. This may be due to the lack of nutrients in the suspending fluid (PBS) used in the current set of experiments.
This study indicates that cholesterol adsorbs/absorbs to silicone hydrogel contact lenses. Without the potential interactive effects of other tear proteins, cholesterol adsorb/absorption alone on unworn lenses does not affect bacterial adhesion or growth. This suggests that cholesterol deposition on silicone hydrogel materials during wear may not increase the risk of microbial keratitis associated with strains tested in this study. However, the influence of the interaction between cholesterol and other tear lipids or proteins on bacterial adhesion is unclear. Further study is required to identify the specific deposition that affects adhesion of bacteria to silicone hydrogel lens during wear.
We thank Dr. Thomas John for his assistance with the statistical analysis.
Negar Babaei Omali
Brien Holden Vision Institute
Level 5, North Wing, RMB, Gate 14, Barker Street
University of New South Wales
Sydney 2052, NSW
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