The tear film lubricates the entire ocular surface and is proposed to be a three layered structure with an outer lipid layer, an aqueous layer, and a mucin layer.1,2 Meibomian glands secrete lipids to produce the lipid layer. It is likely that the lipid layer consists of two phases, a thin polar phase and a thick non-polar phase.3,4 The polar phase consists of phospholipids such as phosphatidylethanolamine, phosphatidylcholine (PC), sphingomyelin (SM), ceramides, and cerebrosides,5–7 and the non-polar phase consists of cholesterol and wax esters.5 The polar phase is likely to be the interface between the aqueous tears and the non-polar lipids.8
Similar to tear proteins, lipids can form deposits on contact lenses immediately after lens insertion into the eye.9–12 Tear film deposition on contact lenses is affected by lens material,13,14 duration of lens wear,15,16 and the types of lens care regime.13,17 Silicone hydrogel contact lenses have been shown to accumulate more lipids than conventional soft lens materials because of their relatively increased hydrophobicity.11,13
A number of studies have reported lipid deposits on contact lenses.9–13,18–21 Lipid deposits on worn contact lenses consist of SM or PC,18 oleic acid, oleic acid methyl ester, and cholesterol.11 Tear film deposits on contact lenses may contribute to clinical complications such as reduced vision,22 dryness, and discomfort.23–25 Moreover, these deposits may increase the risk of bacterial colonization of the contact lens surface.26–30 The first step in the development of microbial keratitis (MK) and certain inflammatory responses is thought to be microbial contamination of contact lens.31 Phosphatidylserine and phosphatidylinositol may affect the initial binding of Pseudomonas aeruginosa to the cornea.32 Some studies have shown that protein or mucin deposits affected bacterial adhesion to contact lenses.26,30,33,34 To date, there have been no reports on the possible effect of phospholipid adsorption on bacterial adhesion to contact lenses. Therefore, this study examined the types of phospholipids deposited onto silicone hydrogel lenses during daily wear in conjunction with two polyhexanide containing lens disinfecting solutions, and examined the effect that phospholipids had on the adhesion of P. aeruginosa and Staphylococcus aureus to the contact lenses.
MATERIALS AND METHODS
Materials, Contact Lenses, and Lens Care Solutions
Phospholipid and SM standards, 1,2-dinonadecanoyl-sn-glycerol-3-PC (19:0/19:0), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PC 16:0/18:2), N-palmityl-D-erythro-sphingosylphosphorylcholine (SM 16:0) and N-lauroyl-D-erythro-sphingosylphosphorylcholine (SM 12:0) were purchased from Avanti Polar Lipids (Alabaster, AL). High-performance liquid chromatography grade chloroform, methanol, and analytical grade butylated hydroxytoluene (BHT) were supplied by Sigma Aldrich (Castle Hill, NSW, Australia). Phosphate free borosilicate glass tubes with lids were purchased from Crown Scientific (Minto BC, NSW, Australia). 03-FISV 0.3 mL screw top insert vials with lids were purchased from Phenomenex (Lane Cove, NSW, Australia).
The commercially available silicone hydrogel lens materials examined in this study were balafilcon A (PureVision, Bausch and Lomb, Rochester, NY), FDA group III ionic lens containing N-vinyl pyrrolidone polymer, and senofilcon A (Acuvue, Oasys, Johnson and Johnson Vision Care, Jacksonville, FL), FDA group I non-ionic lens containing polyvinyl pyrrolidone.
The commercially available multipurpose disinfecting solutions used in this study for lens disinfection were AQuify (containing polyhexanide as a disinfectant and poloxamer 407 as surfactant; CIBA Vision, Atlanta, GA) and ReNu MultiPlus (containing polyhexanide as disinfectant and poloxamine as surfactant; Bausch & Lomb).
Collection of Daily Wear Contact Lenses
Contact lenses were collected from 11 subjects who wore balafilcon A (n = 11) or senofilcon A (n = 11) in a daily wear schedule in conjunction with use of AQuify or ReNu MultiPlus solution. Senofilcon A lenses were replaced every 2 weeks and balafilcon A lenses were replaced monthly. Worn contact lenses were removed from patients' eyes by an optometrist wearing gloves and kept temporarily at 4°C, then transferred to −80°C before deposits examination. This study was conducted in compliance with the Declaration of Helsinki, and the protocols were approved by the Institutional Human Ethics Review Committee. Informed consent was obtained before enrolling participants into the study, and the study was registered on a clinical trials web site (Australian New Zealand Clinical Trials Registry, ANZCTR, No. 12608000329369).
In Vitro Adsorption of SM or PC to Contact Lenses
One lens of each type was soaked in various concentrations (0.05, 0.075, 0.1, 0.25, and 0.5 μg/mL) of two phospholipids (SM 16:0 or PC 16:0/18:2). Lenses (n = 3 for each lens type) were removed from their blister packs and washed three times with phosphate-buffered saline (PBS) (1 mL), then soaked in SM or PC solutions for 24 h at 37°C to allow adsorption of the phospholipids to the lenses. After soaking, lenses were removed, rinsed three times in Milli-Q water (1 mL for each wash) and then extracted immediately. Extraction was repeated three times for each type of lens soaked in each concentration of phospholipid.
Determination of Types and Amounts of Phospholipids Extracted from Worn and Phospholipid-Soaked Contact Lenses
Worn lenses or lenses soaked in SM or PC solutions were extracted individually according to the method reported by Saville et al.18 In brief, contact lenses spiked with 100 pmol internal standards (IS) (PC 19:0/19:0 and SM 12:0) were extracted in methanol:chloroform (1:2 v/v) containing 0.01% BHT for 15 min. Organic phase of the lens extracts was dried down under nitrogen gas and reconstituted in 50 μL methanol:chloform (2:1 v/v, containing 0.01% BHT). Unsoaked lenses were included as control.
Mass Spectroscopy of Polar Lipids
The type and concentration of phospholipids was analyzed according to Lay et al.35 In brief, nanoelectrospray ionization quadrupole time of flight tandem mass spectrometry with positive ion PC/SM precursor ion scanning (m/z 184.07) and product ion scanning (accumulation time 2 s, collision energy 37 eV) were performed on a Q-STAR Pulsar i (MDS Sciex, Concord, Canada). The Lipid Profiler software36 in conjunction with 100 pmol IS (PC 19:0/19:0 and SM 12:0) was used for quantitative analysis. Quantitative analysis was performed by comparing the peaks of individual phospholipids to IS after isotope correction.18,36,37
Phospholipid species at ion counts above the limit of detection (LOD) were reported in this study. LOD and limit of quantification of the instrument was determined according to the method described previously.18,38 A control experiment on the extraction directly from contact lenses was performed to exclude the possibility that the detected peaks were obtained from contact lenses themselves, and no other peaks attributed to the peaks of interest that were above the LOD could be detected.
Bacterial Adhesion to Phospholipid-Soaked Contact Lenses
Bacterial adhesion assay was performed according to the report by Omali et al.39 In brief, P. aeruginosa strains 6294 (MK isolate), 6206 (MK isolate), GSU-3 (MK isolate), S. aureus strains 031 (Contact Lens Peripheral Ulcer isolate), 038 (MK isolate), and ATCC 6538 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 L distilled H2O, pH 7.2]40 containing 3H-uridine (0.2%) overnight at 37°C. Bacterial cells were harvested by centrifugation (10 min), washed and diluted in PBS to obtain a final concentration of 107 CFU/mL. Unworn lenses (total number of eight for each group) with or without a phospholipid soaked were washed three times in PBS and incubated with 1 mL of bacterial suspension at 37°C for 18 h. Number of viable cells were counted after incubation of the agar plates at 37°C for 18 h. Total number of bacteria adhered on lens surfaces were estimated by a modification of a previously reported technique.34,40–44 Pringle and Fletcher41 measured the number of radiolabeled bacteria attached to a hydrogel substrata using a calibration curve, which was prepared from known numbers of radiolabeled cells. In this study, total number of bacteria adhered on lens surfaces were estimated by measuring the β scintillation (Wallac 1400 DSA, PerkinElmer, Gaithersburg, MD) of the 3H-labeled cells, and then converted to log of colony forming units/lens using a standard curve. To prepare a calibration curve, the original inoculum was serially diluted in PBS and spread plated onto nutrient agar plates. Counts per minute measurements of the dilutions were taken, and a calibration curve relating counts per minute to viable cell counts (after incubation on agar plates for 24 h at 37°C) was generated.44 Lenses without phospholipid treatment were included as controls. Experiments were repeated twice and four lens of each type were included in each adhesion experiment.
Effect of PC or SM on Bacterial Growth
The effect of free SM or PC (0.1 mg/mL) in minimal medium on the growth of S. aureus 031 or P. aeruginosa 6294 was determined using a previously published method.45 In brief, P. aeruginosa 6294 and S. aureus 031 were grown in minimal medium containing SM or PC (0.1 mg/mL) overnight. Absorbance at 660 nm was determined and bacteria were then enumerated by viable count.45 Experiments were repeated three times.
Pilot experiment indicated that a minimum of 10 lenses were needed to show an ex vivo difference of 20 ± 15.01 pmol/lens with a 95% confidence and 80% power, and we used 11 worn lenses. Linear Mixed Model (general linear model with random factors) was used to analyze the differences between phospholipid species within each phospholipid type, lens, and solution types ex vivo. Sample ID was factored as random intercepts in the linear mixed model to account for the within sample correlation. Within the totals of each phospholipid type, differences between lens types were similarly investigated. Pilot experiment indicated that a minimum of three lenses were needed to show an in vitro difference of 20 ± 7.2 pmol/lens with a 95% confidence and 80% power, therefore, we used three lenses. One-way analysis of variance with post hoc multiple comparisons adjusted using Benferroni was used to compare the amounts of phospholipid absorbed to lenses in vitro. Phospholipid detection rate was compared between solutions of the same lens or lenses of the same solution using Fisher exact test.
As previously reported,39 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 ± 0.3 with a 95% confidence and 80% power, and we used total number of eight lenses for each group of phospholipid-soaked and unsoaked lenses. Bacterial adhesion in CFU/lens was log transformed before data analysis. Bacterial adhesion was compared between various concentrations of SM or PC soaked and unsoaked lenses using Linear Mixed Model for each strain and lens type. Post hoc multiple comparisons were adjusted using Bonferroni correction when compared with one control category (unsoaked). p value of <0.05 was considered statistically significant (SPSS program 15 was used for analysis).
Types of Phospholipids That Deposit on Lenses during Wear
A representative mass spectrum of an extract from worn balafilcon A in combination with ReNu MultiPlus care solution is shown in Fig. 1A. Mass spectrometry spectra of the major phospholipids detected from a contact lens extract are shown in Fig. 1B, C. A broad range of SMs and PCs were detected from both types of worn lenses. The individual PC or SM detected at ion counts above the calculated detection limit are reported in Table 1. Eighteen phospholipid species were detected from balafilcon A lenses whereas nine phospholipid species were detected from senofilcon A lenses when ReNu MultiPlus was used as the disinfecting solution. Assuming that the maximum number of phospholipids are 18, this difference is significant at (p = 0.001) (Table 1). Moreover, 11 phospholipid species were detected from both lens types when AQuify was used as the disinfecting solution (Table 1). There is a significant (p = 0.007) difference between phospholipid profiles of different solutions used in this study extracted from the balafilcon A lenses, with 18 and 11 phospholipid species detected when ReNu MultiPlus or AQuify were used, respectively. SM (16:0) (m/z 703) and PC (34:2) (m/z 758) were the major phospholipids detected in the lens extracts (Fig. 2). Within each lens type, the amount of SM (16:0) (m/z 703) or PC (34:2) (m/z 758) was significantly higher than other species of SM or PC (p < 0.05). Furthermore, for the balafilcon A lens, using ReNu Multiplus significantly increased the level of PC34:1 and PC 36:2 (p < 0.05) extracted from lenses compared with other species of PC. The total amount of SM (51.6 ± 18.4 vs. 32.8 ± 7.02 or 31.3 ± 13.2 vs. 35.5 ± 10.1 pmol/lens) or PC (67.1 ± 47.5 vs. 45.4 ± 28.8 or 39.4 ± 27.5 vs. 53.5 ± 9.1 pmol/lens) accumulated on balaficon A was not different to that on senofilcon A lenses when ReNu MultiPlus or AQuify were used, respectively (p > 0.05). Amount of phospholipids (ng/lens) extracted from worn balafilcon A and senofilcon A is also shown in Table 2[the conversion, in terms of (ng/lens) was obtained by multiplying the number of pmol/lens with the molecular mass of each phospholipid species and then divided by 1000]. However, senofilcon A lenses bound more PC than SM when AQuify was used (Fig. 2B, p = 0.001).
In Vitro Phospholipid Adsorption
A representative mass spectrum of an extract from SM-soaked senofilcon A (0.5 μg/mL for 24 h at 37°C) is shown in Fig. 3. In vitro adsorption of SM or PC to both balafilcon A and senofilcon A (Fig. 4) was dose dependent. When soaked in the same amounts of PC or SM, balafilcon A adsorbed similar amounts of each compared with senofilcon A lenses (Fig. 4) (p > 0.05). Increasing the concentration of SM from 0.05 to 0.5 μg/mL increased its adsorption to balafilcon A from 20.45 ± 6.06 to 118 ± 20.1 pmol/lens (14.3 ± 4.2 to 82.9 ± 14.4 ng/lens) (p = 0.0001) or to senofilcon A lenses from 13.3 ± 1.1 to 151.2 ± 11.1 pmol/lens (9.7 ± 0.8 to 106.2 ± 7.8 ng/lens) (p = 0.0001). An increase in the concentration of PC from 0.05 to 0.5 μg/mL gave an increase in adsorption from 10.4 ± 3.9 to 120.2 ± 47.8 pmol/lens (7.9 ± 2.9 to 91.1 ± 36.8 ng/lens) for balafilcon A (p = 0.002) or 14.4 ± 2.5 to 143.2 ± 10.2 pmol/lens (10.9 ± 1.9 to 108.5 ± 7.7 ng/lens) for senofilcon A (p = 0.0001) lenses (Fig. 4).
At SM or PC concentrations ranging from 0.075 to 0.1 μg/mL for balafilcon A and of 0.1 μg/mL for senofilcon A lenses, the amount of SM or PC adsorption to balafilcon A and to senofilcon A was similar to that on worn lenses (Fig. 2).
Effect of Phospholipid Adsorption to Contact Lenses on Bacterial Adhesion
The adhesion of P. aeruginosa 6294 to unsoaked and phospholipid-soaked balafilcon A and senofilcon A contact lenses is shown in Fig. 5A. Coating lenses with either phospholipid did not alter the number (total or viable) of P. aeruginosa 6294 adhered to balafilcon A or senofilcon A compared with unsoaked lenses (p > 0.05). Similarly for S. aureus (Fig. 5B), coating lenses with either phospholipid did not alter the adhesion (total or viable) to balafilcon A or senofilcon A compared with unsoaked lenses (p > 0.05). However, for S. aureus, there was a significant difference in the total numbers of bacterial cells adhered to lenses compared with the numbers of viable cells (p < 0.001).
The adhesion of the other strains of P. aeruginosa (6206 and GSU-3) and of S. aureus (038 and ATCC6538) to unsoaked and SM or PC-soaked balafilcon A contact lenses is shown in Fig. 6. The number (total or viable) of P. aeruginosa 6206 or GSU-3, or S. aureus 38 or ATCC6538 on phospholipid-soaked balafilcon A was not different to that on unsoaked lenses (p > 0.05). Again, for these two strains of S. aureus, the total counts were significantly higher than the viable counts (p = 0.0001).
Effect of PC or SM on Bacterial Growth
PC or SM (0.1 mg/mL) in minimal medium had no effect on the growth of P. aeruginosa 6294 or S. aureus 031 (p > 0.05). Growth of P. aeruginosa 6294 was 7.54 ± 0.1 log CFU/mL in non-phospholipid containing media and was 7.57 ± 0.07 or 7.59 ± 0.08 log CFU/mL in SM or PC containing media, respectively. For S. aureus 031, growth was 7.01 ± 0.07 log CFU/mL in non-phospholipid containing media and was 6.96 ± 0.02 or 6.98 ± 0.05 log CFU/mL in SM or PC containing media, respectively.
This is the first study identifying the effect of phospholipid adsorption on adhesion of Gram negative and Gram positive bacteria to silicone hydrogel contact lenses. We selected phospholipids for this study because they have been detected in extracts from contact lenses.18 This study showed that SM (16:0) (m/z 703) and PC (34:2) (m/z 758) were the major phospholipids species that could be extracted from balafilcon A and senofilcon A materials. This finding is consistent with the report by Saville et al.18
Our preliminary experiments demonstrate that phospholipid extraction efficiency is >90% for both types of contact lenses used in this study (data not shown), which is in agreement with the report by Lorentz et al.46 Lorentz et al.46 reported that a solution containing 2:1 chloroform:methanol was capable of removing >90% of deposited lipid from Pure Vision (balafilcon A) contact lenses.
Overall, the phospholipid profile of contact lens extracts in this study shows greater number of phospholipid species detected from balafilcon A contact lenses than senofilcon A, which might reflect the fact that balafilcon A and senofilcon A lenses in this study have been worn for 1 month and for 2 weeks, respectively. However, in vitro study by Carney et al.14 showed that both lens types (balafilcon A and senofilcon A) were saturated after 14 h exposure to polar lipids, which may support our current finding showing that total amount of phospholipid absorbed to both lens type is not different. Moreover, some of the phospholipids reported by Saville et al.18 were below the LOD in our study, which may be due to using different care solutions.
The use of different polyhexanide-containing care solutions had no effect on the amount of phospholipids extracted from silicone hydrogel contact lenses during wear. The total amount of PC extracted from senofilcon A or balafilcon A in conjunction with ReNu MultiPlus or AQuify care solutions was not significantly different to total amount of SM extracted. Saville et al.18 found that total amount of PC on senofilcon A was significantly higher than that on balafilcon A lens material. Saville et al.18 analyzed that worn lenses after use of the multipurpose disinfecting solution OPTI-FREE EXPRESS contains polyquaternium-1 and myristamindopropyl dimethylamine as disinfectants and poloxamine as a surfactant. In this study, ReNu MultiPlus was used, which contains polyaminopropyl biguanide as disinfectants and poloxamine as its surfactant. These differences between studies may be due to other excipients in the different multipurpose disinfecting solutions having an effect on lipid deposition and ease of extraction.
The results of this study demonstrate that PC and SM deposited onto the contact lenses do not affect the adhesion of P. aeruginosa and S. aureus. This finding is in agreement with the report by Omali et al.,39 showing cholesterol absorption in vitro has no effect on the adhesion of P. aeruginosa or S. aureus to silicone hydrogel contact lenses. However, this is in contrast to the effects of protein deposition on lens surfaces. Lysozyme,29 albumin,26,29 lactoferrin,29 and mucin47 absorption to contact lenses increased the adhesion of P. aeruginosa, with lactoferrin also killing the adhered bacterial cells.30 Adsorption of lysozyme to the lens surface may either increase or show no effect on the adhesion of S. aureus.33,34 Moreover, our results indicate that PC and SM deposited onto the lenses do not affect the initial adhesion (2 h incubation) of P. aeruginosa or S. aureus (data not presented).
Similar to our previous report,39 this study shows that the overall viable adhesion of S. aureus strains was lower than total adhesion. This might be due to the fact that PBS does not contain any nutrient for bacteria to survive over the period of the adhesion assay (18 h), or that Staphylococci have the tendency to clump resulting in viable cell counts, arising from more than one cell (4 to 6 cells generally) being always less than total cell counts (where incorporation of 3H-uridine into cells is likely for all cells regardless of clumping). The total concentration of PC and SM in tears was reported as 11 ± 2 ng/mg.48 However, we used 0.1 and 0.5 μg/mL phospholipid concentrations in vitro as at 0.1 μg/mL, the amount of SM or PC adsorption to balafilcon A and to senofilcon A was similar to that on worn lenses. Our preliminary experiments indicated that 0.1 and 0.5 μg/mL phospholipid had no effect on bacterial growth (data not shown). To confirm this, we tested 0.1 mg/mL phospholipid according to the report by Panjwani et al.32 This study demonstrated that PC or SM at 0.1 mg/mL was not used as a nutrient for growth of P. aeruginosa or S. aureus nor were the phospholipids growth inhibitory substances. This finding is in agreement with the report by Panjwani et al.32
This study suggests that phospholipids accumulate on contact lenses during wear; however, that phospholipids adsorbed onto lenses, in the absence of other tear components, do not affect bacterial adhesion or growth. Further studies are required to investigate whether there are interactions between phospholipids and other tear components including other lipids and proteins on bacterial adhesion and growth.
We thank Dr Thomas John Nadviluth 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 2033, New South Wales
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Keywords:© 2012 American Academy of Optometry
phospholipid; Pseudomonas aeruginosa; Staphylococcus aureus; silicone hydrogel contact lenses; tear film