Bacterial interactions with contact lenses; effects of lens material, lens wear and microbial physiology
Bacterial interactions with contact lenses; effects of lens material, lens wear and microbial physiology
Biomaterials 22 (2001) 3235–3247 Bacterial interactions with contact lenses; eﬀects of lens material, lens wear and microbial physiology M.D.P. Willcox*, N. Harmis, B.A. Cowell, T. Williams, B.A. Holden Co-operative Research Centre for Eye Research and Technology, University of New South Wales, Sydney, NSW 2052, Australia Abstract Contact lens wear is a successful form of vision correction. However, adverse responses can occur during wear. Many of these adverse responses are produced as a consequence of bacterial colonization of the lens. The present study demonstrated that during asymptomatic contact lens wear lenses are colonized by low levels of bacteria with gram-positive bacteria, such as coagulase negative staphylococci, predominating.
Gram-negative bacteria are frequently the causative agents of adverse responses during contact lens wear. Measuring the adhesion of diﬀerent strains and/or species of bacteria to diﬀerent contact lens materials demonstrated considerable diﬀerences. In particular, Pseudomonas aeruginosa strains Paer1 and 6294 and Aeromonas hydrophilia strain Ahyd003 adhered in larger numbers to the highly oxygen permeable contact lenses Balaﬁlcon A compared to hydrogel lenses manufactured from either Etaﬁlcon A or HEMA. Furthermore, after Balaﬁlcon A lenses had been worn for 6 h during the day bacteria were able to adhere in greater numbers to the worn lenses compared to the unworn lenses with increases in adhesion ranging from 243% to 1393%.
However, wearing Etaﬁlcon A lenses usually resulted in a decrease in adhesion (22–48%). Bacteria were able to grow after adhesion to lenses soaked in artiﬁcial tear ﬂuid and formed bioﬁlms, visualized by scanning confocal microscopy. Chemostat grown bacterial cultures were utilized to enable control of bacterial growth conditions and bacteria were shown to adhere in the greatest numbers if grown under low temperature (251C compared to 371C). The changes in growth temperature was shown, using 2D gel electrophoresis, to change the experssion of cell-surface proteins and, using 1D gel electrophoresis, to change the expression of surface lipopolysaccharide of P.
aeruginosa Paer1. Thus, these surface changes would have been likely to have mediated the increased adhesion to Etaﬁlcon A contact lenses. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bacterial adhesion; Pseudomonas aeruginosa; Ocular microbiology; 2D gel electrophoresis; Scanning confocal microscopy 1. Introduction Contact lenses are a successful form of vision correction and are worn by approximately 85 million people worldwide. Two major types of contact lenses are commonly worn. These two types are rigid gas permeable (RGP) lenses and soft hydrogel lenses. The RGP contact lenses are commonly composed of monomers containing silicone, ﬂuorine and methylmethacrylate.
Soft hydrogel lenses are commonly composed of 2-hydroxyethyl methacrylate polymer alone (e.g. Polymacon, Bausch and Lomb, Rochester, NY, USA; FDA group I) or containing methacrylic acid (e.g. Etaﬁlcon A, Vistakon, a division of Johnson and Johnson Vision Products Inc, Jacksonville, FL, USA; FDA group IV) and/or N-vinyl pyrrolidone (e.g. Viﬁlcon A, CIBA Vision, Atlanta, GA, USA; FDA group IV). Furthermore, in recent years new co-polymers have been incorporated into the soft hydrogel lens materials, including silicone polymers for increased oxygen permeability (e.g. Lotraﬁlcon A, CIBA Vision, or Balaﬁlcon A, Bausch and Lomb) and phosphorylcholine to increase biocompatability (e.g.
Omaﬁlcon A, Biocompatables Ltd., UK). Contact lenses can be worn on several wear schedules including daily wear (the wearer removes the lens each night, cleans and disinfects the lens overnight and returns the same lens to the eye in the morning [these lenses are commonly replaced with fresh lenses every month]), daily disposable wear (the wearer removes and discards the lens at the end of the day and inserts a new lens into the eye the next morning), extended wear (the wearer wears the same lens continuously for, commonly, 6 nights, then removes the lens and inserts a new lens on the seventh day), and continuous wear (wearers wear lenses continuously for 30 nights, *Corresponding author.
Tel.: +61-2-9385-7524; fax: +61-2-9385- 7401.
E-mail address: email@example.com (M.D.P. Willcox). 0142-9612/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: 2
then discards the lens and inserts a new lens on the thirty ﬁrst day). Occasionally adverse responses to contact lens wear occur. These adverse responses are frequently caused by bacterial contamination of the contact lens surface. Contact lens induced corneal adverse responses have recently been classiﬁed into serious sight threatening responses (microbial keratitis [MK; incidence 0.3%]), signiﬁcant adverse responses (contact lens induced acute red eye [CLARE; incidence 1.4–6.2%], contact lens induced peripheral ulcers [CLPU; incidence 0.6–8.7%] and inﬁltrative keratitis [IK; incidence 1.7–5.2%]) and non-signiﬁcant adverse responses (asymptomatic inﬁltrative keratitis [AIK; incidence 1.5–3.9%] and asymptomatic inﬁltrates [AI; incidence 5.2%]) .
Of these adverse responses, bacterial colonization of contact lenses is one of the initiating factors in MK , CLARE [3,4], CLPU  and certain IK and AIK events . There have been several investigations into the eﬀects of contact lenses on the normal ocular microbiota. The normal ocular microbiota in the absence of contact lens wear is composed almost exclusively of three bacterial types, coagulase negative staphylococci, Corynebacterium sp. and Propionibacterium sp. [7,8]. The lids usually harbor a microbiota similar to the normal skin microbiota and harbor more bacteria of more species than the conjunctiva .
During sleep the number of bacteria colonizing the conjunctiva and lid increases . An increase in the number of bacteria isolated from the conjunctiva and lids during daily lens wear has been reported [9,11], although the types of micro-organisms were not found to diﬀer from non-lens wearing eyes. An alteration in the types of micro-organisms was seen with extended lens wear (more Gram-negative bacteria being isolated) along with an increase in the frequency of cultures growing no micro-organisms [9,12]. The above ﬁnding is signiﬁcant as Gram-negative organisms are common ocular pathogens .
Other studies, however, have reported no diﬀerences between wearers and nonlens wearers although an increase in positive ocular cultures was found in former lens users and in association with certain modes of lens wear and types of disinfection systems .
Table 1 details the types of bacteria that have been isolated from contact lenses at the time of an MK, CLARE, CLPU, IK or AIK. Pseudomonas aeruginosa is the most common cause of MK during contact lens wear [13–16]. Gram-positive bacteria are more commonly associated with CLPU [5,6,17], whereas gram-negative bacteria are more commonly associated with CLARE [3,4,18]. For CLARE, Haemophilus inﬂuenzae is the most commonly isolated bacterium . One of the initial steps in the development of the bacterially driven adverse responses is the binding of bacteria to a contact lens. Several studies have examined the ability of bacteria to adhere to contact lenses.
Staphylococcus epidermidis or P. aeruginosa strains adhere in larger numbers to lenses made from hydroxyethyl methacrylate (HEMA) alone compared to lenses made from HEMA plus methacrylic acid [19–21] and this may be a function of diﬀering water contents [22,23] or charges of these lens types. A contact lens when inserted into the eye rapidly accumulates proteins, glycoproteins and lipids (known as deposits) from the tear ﬁlm to its surface. Therefore, it is likely that, other than contamination upon insertion (which is usually by bacteria that are part of the normal microbiota), bacteria adhere to these adsorbed components rather than the contact lens material itself.
That is not to say the contact lens material will not still aﬀect adhesion; the types of deposits are likely to be aﬀected by the chemistry of the contact lens. Subsequent to adhesion, it is likely that bacteria further colonize the lens surface by growing on that lens surface. Hume and Willcox  demonstrated that Serratia marcescens was able to grow on a contact lens after adhesion to contact lenses coated in an artiﬁcial tear ﬁlm.
Table 1 Bacteria isolated from contact lenses at the time of an adverse response Bacteria Adverse responsea Gram positive bacteria Abiotrophia defectiva IK Bacillus sp. MK Coagulase-negative staphylococcib MK Corynebacterium sp.b MK Micrococcus sp.b MK Nocardia sp. MK Propionibacterium acnesb MK Non-hemolytic Streptococcus sp. IK Staphylococcus aureus MK, CLPU Streptococcus pneumoniae MK, CLARE, CLPU, IK, AIK Viridans streptococci MK, IK, AIK Gram-negative bacteria Acinetobacter sp. MK, CLARE, IK, AIK Aeromonas hydrophilia CLARE Alcaligenes xylosoxidans subsp. denitriﬁcans IK Enterobacter sp.
MK, AIK Escherichia coli MK, CLARE Haemophilus inﬂuenzae MK, CLARE, IK, AIK Klebsiella sp. MK, CLARE, IK, AIK Morganella morgani MK Moraxella sp. MK Neisseria sp. IK Proteus sp. MK Pseudomonas sp. MK, CLARE, CLPU, AIK Serratia sp. MK, CLARE, CLPU, IK Stenotrophomonas maltophilia MK, CLARE, AIK a MK, microbial keratitis; CLARE, contact lens induced acute red eye; CLPU, contact lens induced peripheral ulcer; IK, inﬁltrative keratitis; AIK, asymptomatic inﬁltrative keratitis . b These bacteria are part of the normal ocular microbiota and as such their signiﬁcance in the production of adverse responses must be viewed with caution as they could be present as contaminants.
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Another important factor in the ability of a bacterium to adhere to contact lenses is the physiology of that bacterium. Recently Cowell et al.  demonstrated that growing Paer1 under nitrogen limitation increased the adhesion of this strain to Etaﬁlcon A contact lenses. The aims of this investigation were to demonstrate the types of bacteria that adhere to contact lenses during wear and to determine the factors, both material and microbiological, that can aﬀect the adhesion of bacteria to contact lenses.
2. Materials and methods 2.1. Comparison of bacterial colonization on soft contact lenses worn on diﬀerent wear schedules Contact lens wearing subjects wore either bilateral Etaﬁlcon A lenses or contra-lateral Etaﬁlcon A in one eye and Polymacon in the other eye, or bilateral Lotraﬁlcon A lenses over a 7 year period.
The Etaﬁlcon A and Polymacon lenses were worn on a 6 nights extended wear schedule or daily wear schedule with monthly replacement. The Lotraﬁlcon A lenses were worn on a 30 nights continuous wear schedule. We have previously demonstrated that there are no diﬀerences in the bacterial colonization between Etaﬁlcon A and Polymacon [9,26] or Etaﬁlcon A and Lotraﬁlcon A lenses . Seventy three subjects wearing extended/ continuous wear lenses and 39 subjects wearing lenses on a daily wear schedule were involved in the study and all were free of ocular diseases, had no ocular surgery and required visual correction for low refractive errors only.
Informed consent was obtained from the subjects and all procedures were approved by the University of New South Wales Human Ethics Committee. Each subject was sampled on average 3 times. Contact lenses were removed aseptically and transported to the laboratory in sterile phosphate buﬀered saline (PBS) . Bacteria adherent to the contact lenses were grown using an agar sandwich technique . Brieﬂy, lenses were placed concave side up on a chocolate agar plate and the plate was ﬂooded with molten (561C) agar and placed in a CO2-enriched atmosphere (5%) at 351C for 48 h. Aliquots (0.4 ml) of the remaining transport PBS were spread onto three chocolate and one Sabouraud’s agar plate.
The Sabouraud’s agar plate and one chocolate agar plate were incubated aerobically at 351C for 48 h. The Sabouraud’s agar plate was subsequently incubated for six days at ambient temperature. The two remaining chocolate plates were incubated at 351C either in a CO2-enriched atmosphere for 48 h or anaerobically (95% N2, 5% CO2) for four days. Microbiological characterization of the contact lenses was conducted as described previously [9,26,28]. The incidence rates of various groups of micro-organisms were compared using chi-square test with Yates correction and the level of signiﬁcance was set at P ¼ o0:05: 2.2.
Adhesion of bacteria to contact lens materials in vitro P. aeruginosa 6294, P. aeruginosa Paer1, Streptococcus pneumoniae 001, S. pneumoniae 008, Haemophilus inﬂuenzae 001, H. inﬂuenzae 009, Aeromonas hydrophilia 003, Stenotrophomonas maltophilia 010 were isolated from cases of CLARE, with the exception of P. aeruginosa 6294 which was isolated from a case of MK, at the time of presentation. Bacteria were grown to stationary phase in Trypicase soy broth (Oxoid, Sydney, Australia) at 351C, then cells were washed three times with PBS and resuspended to an OD of 1.0 (approximately 1
108 bacteria/ml) .
Previous studies had demonstrated that, for all lens types used, this optical density gave maximum adhesion and that optical densities above this did not show increased adhesion (date not shown). Bacteria (1 ml) were added to lenses and adhesion was allowed to occur for 10 min. Nonadherent cells were then removed by washing in PBS three times and cells stained with crystal violet prior to enumeration by light microscopy . Lenses used in the experiments were Etaﬁlcon A, Balaﬁlcon A, Polymacon, Omaﬁlcon A. All results were expressed compared to the adhesion of the bacterial strains to Etaﬁlcon A contact lenses as these are the current market leaders in hydrogel lenses.
All experiments were repeated on three separate occasions. Adhesion data were not normally distributed and therefore were analysed for diﬀerences between lens types non-parametrically with the Mann-Whitney V-Wilcoxon Rank Sum test.
2.3. Eﬀect of lens wear on bacterial adhesion in vitro Five subjects were instructed to wear contact lenses (Etaﬁlcon A, Polymacon or Balaﬁlcon A) in both eyes for 6 h during the day on diﬀerent days. Lenses were then removed aseptically, washed three times in PBS to remove loosely adsorbed tear ﬁlm components and bacterial adhesion was measured as described above (Section 2.2). Results were expressed as a percentage diﬀerence compared to control unworn lenses and all experiments were repeated on at least two separate days. Adhesion data were not normally distributed and therefore were analysed for diﬀerences between worn and unworn lenses non-parametrically with the MannWhitney V-Wilcoxon Rank Sum test.
2.4. Analysis of types of deposits on worn contact lenses The types of deposits formed on Polymacon or Etaﬁlcon A lenses that had been worn for 6 h during the day were investigated using X-ray photoelectron M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247 3237
spectroscopy (XPS). After wear, lenses were washed in MilliQ water, dried and sectioned. XPS analysis was performed as described previously  using a Kratos Axis H1s instrument, an A1 monochromated source with a spot size of 1 mm. Elemental identiﬁcation was performed from scans acquired at 160 eV.
2.5. The ability of strains to grow on contact lenses Scanning confocal laser microscopy (SCLM) is a technique used for the observation of bacteria attached to surfaces. The strengths of this technique lie in its ability to observe and analyze sections of three-dimensional bacterial bioﬁlms [31,32].
Multi-channel ﬂow cells were designed by Darryl Wilkie and Jason Marshall (Department of Applied Microbiology and Food Science, University of Saskatchewan) and constructed using polycarbonate plastic (Fig. 1). Irrigation channels, 40 mm wide, were drilled into the plastic and a glass coverslip placed over the channels and sealed with silicone glue (Silicone Rubber Adhesive Sealant, GE Translucent RTV118). Flow cells were connected to silicone tubing at either end. A Watson Marlow peristaltic pump was used to pump the media or washing solutions through the ﬂow cells.
Contact lenses (Etaﬁlcon A) were cut in half using a sterile scalpel.
Each half was attached to a plastic block using silicone glue applied around the edges of the semicircular lens sample. The block with the aﬃxed lens sample was placed in the ﬂow cell and sealed with a glass coverslip. The gap between the exposed surface of the contact lens sample and the underside of the coverslip was approximately 4 mm. A protein mixture comprised ﬁve proteins: lactoferrin (bovine colostrum, 1 mg/ml), lysozyme (chicken eggwhite, 1 mg/ml), g-globulins (bovine, 1 mg/ml), albumin (bovine serum, 0.1 mg/ml), mucin (bovine submaxillary gland, 0.1 mg/ml) was constructed in PBS.
All proteins were purchased from Sigma (St. Louis, MO, USA). Although this protein mixture represented a simpliﬁed version of tear proteins, the exact composition of the solution was less critical than the fact that it contained potentially antibacterial proteins (lactoferrin, lysozyme) and a high concentration of glycoproteins (lactoferrin, gglobulins, mucin). To coat the lens samples with protein, the ﬂow cell was gently ﬁlled with the protein mixture. Lens samples that were to be left uncoated were immersed in PBS. Lens samples were left in the ﬂow cells for 18 h at 351C. Loosely bound protein was washed oﬀ using 10 ml of PBS pumped through the ﬂow cell.
Bacteria were grown and washed as described above (Section 2.2). Bacterial suspension (Paer1, OD660 ¼ 0:1 in PBS) was then introduced until the ﬂow cell was ﬁlled. Initially, bacteria were allowed to adhere to the lens, then a solution (PBS, protein mixture, or 10% TSB) was pumped through the ﬂow cell at a very slow rate of ﬂow. The ﬂow cell was left at 351C for 10 min. Loosely attached bacteria were washed oﬀ by the same method used for removing loosely bound protein.
The growth medium (protein mixture or 10% TSB or PBS) was introduced into the ﬂow cell at a rate of 2.6 ml/h for 3 days at 251C. RH795 (0.1% in PBS; Molecular Probes, Eugene, Oregon, USA), which responds to cell membrane potential and was used to stain the bacterial cells, was introduced (3 ml) into the ﬂow cell by injecting it into the silicone tubing immediately prior to the ﬂow cell and was left for 1 h at 251C. The ﬂow of solution was then stopped and the glass coverslip was removed. Microscope work was carried out using the Bio-Rad MRC 600 SCLM equipped with an argon laser and standard ﬁlter sets.
The laser was mounted on a Nikon Microphot-FXA microscope. The microscope was equipped with a 20 X water immersion lens.
Four separate conditions were examined for their eﬀect on Paer1 growth on lens samples, and set up in parallel using the ﬂow cells: (1) PBS was passed over a clean lens sample; (2) PBS passed over a lens sample coated with the protein mixture; (3) protein mixture passed over a lens sample coated with the protein mixture; (4) 10% TSB passed over a clean lens sample. In addition to these conditions, lens samples which were Fig. 1. Flow cell used for bacterial adhesion and scanning confocal microscopy. Multi-channel ﬂow cell used in the SCLM study, showing the ﬂow cell from above and as a side view in section.
Polycarbonate plastic is lightly shaded, and the irrigation channels are unshaded. The hatched line indicates the passage of the irrigation channels through the plastic. The contact lens, mounted on the plastic block, is indicated in both views. Direction of laminar ﬂow is indicated by the arrowheads.
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not colonized by bacteria were also observed using SCLM. All experiments were repeated on two separate occasions. 2.6. Eﬀect of bacterial physiology on adhesion In an eﬀort to further elucidate the mechanisms of bacterial adhesion, P. aeruginosa Paer1 was grown in a chemostat under diﬀerent environmental conditions. Paer1 was grown as described previously . Brieﬂy, the bacteria were grown in a deﬁned medium  either at 371C or 251C and at a growth rate of either 0.3 or 0.05 h
1 . After growth, the bacteria were washed three times in PBS and their adhesion to Etaﬁlcon A contact lenses was measured as described above (Section 2.2) and repeated three times.
The cells’ adhesion to substituted Sepharose 6-B was also investigated . Sepharose 6-B, octyl-, phenyl-, CMand DEAESepharose were purchased from Pharmacia LKB Biotechnology (Uppsala, Sweden). Columns were constructed by loading 1 ml (B5 cm) of each Sepharose gel into Pasteur pipettes plugged with glass wool. The void volume determined using methylene blue was found to be 0.5 ml. Columns were equilibrated with 5 ml PBS. The optical density of the original suspension was measured at 600 nm. Bacterial suspensions (0.5 ml, OD600 ¼ 1:0) were added to the columns and the ﬁrst eluant (0.5 ml) discarded.
A second aliquot of bacteria (0.5 ml) was added to the column and the eluant collected with the ﬁrst wash of 0.5 ml PBS. Three additional washes (1 ml) were collected and the absorbance at 600 nm of each wash determined. Percent cells retained on each gel, and percent cells subsequently desorbed in the next three washes were determined, and from this data the net retention (percent of original inoculum) of bacteria on each Sepharose type was calculated. Retention on Sepharose assays were performed twice for each incubation condition. 2.7. The role of bacterial cell surface proteins and lipopolysaccharide in adhesion Experiments were then conducted on the expression of cell-surface proteins and lipopolysaccharide (LPS) by P.
aeruginosa Paer1 grown at 371C or 251C. Cell-surface proteins were extracted [25,33] using 100 ml of 50 mm sodium citrate buﬀer (sodium citrate/citric acid, pH 4.5) containing 0.1% Zwittergent (Calbiochem, La Jolla, CA), 1 mm PMSF (phenylmethyl-sulfonyl ﬂuoride, Boehringer Mannheim, Mannheim, Germany) and 10 mm EDTA (ethylenediaminetetraacetic acid, Sigma, St. Louis, MO). The reaction mixture was incubated for 25 min at 451C with occasional mixing. Bacteria were then pelleted by centrifugation at 3200g for 2 h at 41C. The supernatant containing the extracted proteins was dialysed overnight against distilled water containing 0.02% (w/v) sodium azide to remove the detergent.
Proteins were concentrated using Centriprep-10 concentrators (10,000 MW cut-oﬀ, Amicon, Beverly, MA). Protein samples were reduced with DTT (1,4-dithiothreitol, Boehringer Mannheim, Mannheim, Germany) prior to SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel) analysis on 10% acrylamide gels according to the method of Laemmli . Gels were silver-stained to visualise protein bands following the method of Bjellqvist et al. . Proteins preparations were extracted from two samples and run on gels to determine consistency. The LPS was extracted using the water/phenol method of Westphal and Jann  and the amount of LPS was analysed using a Coatestt endotoxin kit (Chromogenix AB, M.
o oindal, Sweden) followed by sodium dodecylsulfate gel electrophoresis (10% acrylamide) and visualized by silver diamine staining.
3. Results 3.1. Comparison of bacterial colonization on soft contact lenses worn on diﬀerent wear schedules Table 2 shows the types of bacteria that were isolated from contact lens wearers on an extended or daily wear schedule. The most common bacteria isolated were gram-positive bacteria including coagulase negative staphylococci, Propionibacterium sp. and Corynebacterium sp. Of the gram-negative bacteria isolated, Pseudomonas sp. and Stenotrophomonas sp. were isolated most frequently during extended wear, whereas Pseudomonas sp. and Acinetobacter sp. were isolated most frequently during daily wear.
However, there were no diﬀerences in the numbers, types or frequency of colonization of contact lenses worn on either an extended/continuous wear schedule or daily wear schedule. 3.2. Bacterial adhesion to soft contact lenses in vitro As can be seen (Fig. 2), there was considerable variation in adhesion between bacterial strains and contact lenses. Three strains of bacteria, P. aeruginosa Paer1 and 6294 and Aeromonas hydrophilia 003, adhered in increased numbers to the High DK silicone hydrogel lenses (Balaﬁlcon A) compared to Etaﬁlcon A. The increase may have been due to the more hydrophobic nature of the underlying contact lens material in the high DK lenses.
The strains of Streptococcus pneumoniae were chosen for testing against Omaﬁlcon A lenses as these bacteria are known to possess receptors for choline on their surface . Fig. 3 demonstrates that for only one of these strains, Spne 004, there was an increase in adhesion to Omaﬁlcon A lenses that contain phosphorylcholine.
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Table 2 Median and frequency of microbial contamination for contact lenses worn on either an extended or daily wear schedule Bacterial type Extended wear (N ¼ 73)a Daily wear (N ¼ 39)a Median number of CFU/lens Range CFU/ml Frequency of bacterial contamination of lensesb Median number of CFU/lens Range CFU/ml Frequency of bacterial contamination of lensesb Gram-positive bacteria Coagulase negative staphylococci 6 6–>300 39.0 6 1–>300 38.5 Propionibacterium sp. 10 3–>300 25.8 10 6–>300 23.0 Corynebacterium sp.
6 1–>300 4.6 6 1–>300 6.0 Streptococcus sp. 6 1–>300 2.8 6 1–282 3.6 Bacillus sp. 6 1–65 2.2 6 1–20 2.2 Micrococcus sp. 6 1–>300 1.9 6 1–16 2.4 Staphylococcus aureus 6 1–>300 1.6 6 1–222 2.1 Stomatococcus sp. 6 1–143 1.0 6 1–10 0.9 Planococcus sp. 5 1–6 0.3 6 6 0.2 Nocardia sp. 6 1–10 0.2 6 2–6 0.3 Listeria sp. >300c 10–>300 0.1 0 F 0 Peptococcus sp. 6 6 0.1 0 F 0 Gram-negative bacteria Pseudomonas sp. 6 1–>300 2.0 8 1–>300 1.6 Stenotrophomonas maltophilia 93 1–>300 1.5 40 6–>300 1.4 Serratia sp. >300 1–>300 1.1 9 1–>300 1.4 Acinetobacter sp. 10 1–>300 1.1 6 1–>300 1.6 Enterobacter sp. 6 1–>300 0.6 9 1–>300 1.4 Moraxella sp.
10 1–>300 0.6 6 1–212 0.4 Flavobacterium sp. 45 6–>300 0.5 45 6–>300 1.0 Commonas sp. 40 1–>300 0.4 12 6–>300 0.3 Neisseria sp. 6 1–16 0.3 6 1–66 0.4 Acromobacter sp. >300 28–>300 0.3 >300 227–>300 0.4 Klebsiella sp. 161 6–>300 0.2 142 1–>300 0.6 Alcaligenes sp. 16 4–46 0.2 15 1–36 0.4 Haemophilus sp. 6 1–26 0.2 9 3–30 0.2 Escherichia coli 7 1–13 0.1 >300 7–>300 0.2 Agrobacter sp. 2 1–>300 0.1 1 1 0.1 Sphingobacterium sp. 0 F 0 3 1–270 0.4 a EW, Etaﬁlcon A or Polymacon lenses worn on a 6 night schedule or Lotraﬁlcon A lenses worn on a 30 night schedule. DW, Etaﬁlcon A or Polymacon lenses worn on a daily wear schedule with monthly replacement and daily disinfection with a multi-purpose solution.
Each subject was sampled on average 3 times/yr.
b Frequency is the number of times cultured/total number of cultures performed. c >300 CFU/lens indicates conﬂuent growth of the bacteria on the agar plate. Fig. 2. Adhesion of bacteria to various contact lens materials. *Signiﬁcantly diﬀerent to adhesion to Etaﬁlcon A (po0:05 statistical analysisFMann–Whitney U-test). Fig. 3. Adhesion of bacteria to Omaﬁlcon A in comparison to Etaﬁlcon A hydrogel contact lenses. *Signiﬁcantly diﬀerent to adhesion to Etaﬁlcon A (po0:05; statistical analysisFMann–Whitney U-test).
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Eﬀect of lens wear on bacterial adhesion in vitro Using contact lenses that had been worn for 6 h during the day, bacterial adhesion was examined. In general, the gram-negative bacteria used in the adhesion assays adhered in lower numbers to worn compared to unworn Etaﬁlcon A lenses (Table 3). On the other hand, bacteria adhered in greater numbers to worn rather than unworn Balaﬁlcon A (High DK) lenses (Table 3). Statistical analysis demonstrated a signiﬁcant (Po0:05) increase in adhesion of strains Paer1, Hinf001 and Xmal010 to worn Balaﬁlcon A lenses, Paer1 and Hinf001 to worn Polymacon lenses and Ahyd003 to worn Etaﬁlcon A lenses.
Xmal010 showed a reduced (Po0:025) adhesion to worn Etaﬁlcon A lenses. 3.4. Analysis of the type of deposit on soft contact lenses Table 4 demonstrates that the Etaﬁlcon A lens adsorbed more nitrogen containing material than the Polymacon lens (approximately six times as much), indicating more protein was adsorbed to the surface. The worn Polymacon lens adsorbed very little protein and only on its front surface. Interestingly, the small decrease in carbon (and increase in oxygen) on the surface of the worn Polymacon lenses may indicate that mucin had adsorbed to these lenses.
3.5. The ability of strains to grow on contact lenses Fig. 4a shows a clean lens sample which was observed under SCLM, without any further treatment. Fig. 4b shows the response of bacteria attached to a clean lens sample after exposure to PBS under laminar ﬂow conditions. The diﬀuse nature of the surface may represent a very loose aggregation of Paer1 that obscures the surface of the lens sample. Fig. 4c shows a protein-coated lens sample used in place of a clean lens sample. For the next set of experimental conditions, bacteria were attached to a lens sample pre-coated with the protein mixture.
The same protein mixture was then passed over these bacteria under laminar ﬂow conditions. Fig. 4d clearly shows the presence of discrete clumps of bacteria, presumably micro-colonies, over the surface. These cannot represent aggregates of adsorbed protein, as these were not visible under this magniﬁcation. The response of bacteria attached to a clean lens sample when exposed to a 10% solution of TSB was quite distinct. Although putative bacterial aggregations were evident, these were very diﬀuse and quite diﬀerent Table 3 The eﬀect of wear on the adhesion of bacteria to contact lenses Bacterial strain Etaﬁlcon A Polymacon Balaﬁlcon A % adhesiona Paer1 43b 723 14347323c 453797c 6294 48715 NDd ND Ahyd003 402752c 100736 2437141 Hinf001 ND 367796c 13937253c Xmal010 2275e 65726 303729c a Adhesion was compared to that on unworn lenses.
Adhesion >100% indicates that bacteria were able to adhere to worn lenses in greater amounts than to unworn lenses; 100% adhesion indicates no diﬀerence between adhesion to worn or unworn lenses; o100% indicates greater adhesion to unworn lenses.
b Mean7SD. c Signiﬁcant increase in adhesion over unworn lenses (Po0:05). d ND, not determined. e Signiﬁcant decrease in adhesion compared to unworn lenses (Po0:025). Table 4 XPS analysis of worn Etaﬁlcon A and Polymacon contact lenses Lens Side %Ca %Oa %Na %Sia %Fa Etaﬁlcon A (control unworn) Front 69.3 29.6 0.2 1.0 0 Back 70.8 28.5 0.4 0.3 0 Polymacon (control unworn) Front 71.1 28.4 0.5 0 0 Back 71.6 28.0 0.3 0.1 0 Etaﬁlcon A (worn) Front 68.7 25.6 5.5 0.2 0 Back 72.2 25.9 2.0 0 0 Polymacon (worn) Front 68.8 30.4 0.9 0 0 Back 69.1 30.6 0.3 0 0 a C, carbon; O, oxygen; N, nitrogen; Si, silicon; F, ﬂuorine.
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from the micro-colonies shown in Fig. 4c and d, and probably represented ﬂocculation. Within 48 h the solution inside the ﬂow cell was observed to be very turbid and this was not observed for any other of the sets of conditions. These enriched conditions obviously generated very strong growth of bacteria in suspension. If any bioﬁlm was present, it was very diﬀuse and loosely attached to the surface, and can probably be best described as ﬂocculation. Under these extremely nutrient-rich conditions, there was apparently no imperative for bacteria to attach to a solid surface. 3.6. Eﬀect of bacterial physiology on adhesion Table 5 demonstrates the changes that occurred in the surface charge and hydrophobicity of Paer1 under the diﬀerent conditions.
Low growth temperature increased adhesion to the control Sepharose 6-B by 55% and to Octyl-Sepharose by 19% but did not appreciably alter the adhesion to Phenyl, DEAE or CM-Sepharose. A slow growth rate increased the adhesion to control Sepharose 6-B by 65%, to Octyl-Sepharose by 21% and to CM-Sepharose by 55% but decreased adhesion to Fig. 4. Scanning confocal microscopy of bacterial adhesion to contact lenses. (a) A clean lens sample observed under SCLM. This surface was not coated with protein or exposed to any bacterial suspension before observation. The texture of the lens surface was smooth, although the surface of the entire lens was uneven.
The ﬂuorescence is due to the uptake of the stain RH795 by the hydrogel lens polymer. (b) SCLM image of Paer1 attached to a clean lens sample when exposed to PBS under laminar ﬂow conditions. The structures visible on the surface may represent diﬀuse aggregations of Paer1, but there is no visible micro-colonies. (c) SCLM image of Paer1 attached to a lens sample which had been pre-coated with a mixture of protein (lactoferrin, lysozyme, g-globulins, albumin, mucin). The solution passing over the lens sample was PBS. There appeared to be evidence of bacterial micro-colonies on these lenses. (d) Paer1 attached to a lens sample that had been pre-coated with a mixture of protein (lactoferrin, lysozyme, gglobulins, albumin, mucin).
This same protein mixture was then passed over these bacteria under laminar ﬂow conditions. This SCLM image shows the presence of putative micro-colonies on the surface. Each micro-colony is approximately 10–15 mm in diameter. Table 5 The eﬀect of growth conditions on retention of P. aeruginosa to Sepharose Sepharose type Growth conditionsa Control Low temperature Slow growth rate Sepharose 6-B (control) 1371 2973 3775 Octyl 5275 6474 6675 Phenyl 7274 7173 5674 DEAE 9971 10070 9970 CM 2071 1972 4475 a All cells were grown in a chemostat in deﬁned media  with the following conditions: control, 371C and 0.3 h
1 dilution rate; low temperature, 251C and 0.3 h
1 dilution rate; slow growth rate, 37o C and 0.05 h
1 dilution rate.
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Phenyl-Sepharose by 56%. Fig. 5 demonstrates the adhesion of bacterial cells grown under the diﬀerent conditions to Etaﬁlcon A lenses. Adhesion was highest when cells were grown at low temperature and relatively high growth rates, followed by growth at high temperature and slow growth rate and adhesion was least when cells were grown at high growth rates and high temperature. Whilst the hydrophobicity (adhesion to octyl or phenyl Sepharose) and charge (adhesion to either DEAE or CM-Sepharose) also changed with grow conditions, there was no direct correlation between adhesion to the lenses and adhesion to the substituted Sepharose polymers probably demonstrating the ability of bacterial cells to utilize several mechanisms to adhere.
3.7. The role of bacterial cell surface proteins and lipopolysaccharide in adhesion Fig. 6 demonstrates that growth at 251C altered the expression of a number of cell-surface proteins (marked by either a red arrow head or enclosed in a red circle) compared to growth at 371C. Similarly, growth at 251C altered the expression of LPS, yielding larger LPS molecules that did not migrate as far into the gel matrix (Fig. 7).
4. Discussion The current study has conﬁrmed that there was no diﬀerence in the colonization of contact lenses worn on an extended or daily wear schedule during asymptomatic lens wear. Lens contamination during asymptomatic lens wear appears to involve small numbers of micro-organisms . The most common bacteria isolated from contact lenses are coagulase-negative staphylococci [8,12,26,39]. Contact lens contamination commonly occurs through lens handling  but it appears that during uncomplicated lens wear these micro-organisms are readily cleared from the lens surface by the ocular defense mechanisms.
Other sources of contamination of lenses by the normal microbiota include the eyelids of wearers  or from environmental sources . Contamination of lenses during wear is sporadic. Subjects sampled on successive days of extended lens wear, from 1 night to 13 nights, were as likely to have contaminated lenses on Day 1 as on Day 13 . In other words, wearing lenses for increasing lengths of time did not result in increasing microbial contamination.
However, some adverse responses that occur during lens wear are known to be associated with bacterial adhesion to lenses [2–6]. These bacteria do not make up part of the normal ocular microbiota. In order for bacteria to initiate an adverse response, they must be able to adhere to the lens surface. The current study demonstrated that there were diﬀerences in the ability of the bacteria isolated from adverse responses to adhere to lens materials and for most material/strain combinations there were increases in adhesion to worn lenses or no diﬀerences between adhesion to worn or unworn lenses.
It was demonstrated that worn lenses did adsorb tear ﬁlm components, probably proteins/glycoproteins, and that bacteria were able to grow on those proteins that were adsorbed to a lens surface. The increases in adhesion seen for certain strains to worn lenses may indicate that the tear molecules, most likely proteins, that bound to the contact lenses were conducive to bacterial adhesion. To date there are no publications on the types of proteins or other molecules that bind to the high DK lenses which demonstrated the most noticeable increases in adhesion to worn lenses, although one abstract at the British Contact Lens Association annual meeting in May 2000 reported more deposition on the surface of Balaﬁlcon A lenses compared to Etaﬁlcon A lenses (however, no biochemical analysis of the deposits was reported) .
Protein adsorbs more readily to less hydrophilic surfaces compared to the surface carrying an anionic charge such as that of an Etaﬁlcon A lens . The exception to this is the adsorption of lysozyme to anionic lenses [45,46]. Also, the anionic lenses tend to adsorb less lipid , although N-vinyl pyrrolidone containing anionic lenses do bind lipid [48–50]. The worn Polymacon lenses may have bound mucin to there surface. It is known that P. aeruginosa can bind to ocular mucin [18,51]. Total protein does not correlate with adhesion of P. aeruginosa to lenses . However, deposits on lenses did increase adhesion in one study , although this may be due to increased surface roughness.
No relation between the ability of P. aeruginosa to bind to worn Etaﬁlcon A contact lenses and the presence of lysozyme or lactoferrin has been found, although worn lenses did usually increase the adhesion of strains . Albumin coated onto the surface of Etaﬁlcon A or Polymacon contact lenses Fig. 5. Eﬀect of growth condition on adhesion of P. aeruginosa to Etaﬁlcon A contact lenses. *Signiﬁcantly diﬀerent to adhesion to control (po0:02; statistical analysisFMann–Whitney U-test). M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247 3243
increased the adhesion of P. aeruginosa . Similarly, some strains of Serratia marcescens adhered better to Etaﬁlcon A lenses coated in an artiﬁcial tear ﬂuid . Lysozyme adsorbed to a contact lens increases the adhesion of Staphylococcus aureus to Etaﬁlcon A contact lenses . Factors in addition to adhesion are likely to contribute to the production of adverse reponses. One such pathogenic trait would be the ability of the adhered bacteria to grow on the tear ﬁlm components that have adsorbed to the lens surface. Using SCLM observation, Paer1 grew under conditions in which soluble protein was passed over bacteria attached to the lens surface.
Micro-colony production is the prelude to bioﬁlm formation . After initial adhesion, adherent bacteria may proliferate on the substratum within the polysaccharide-rich glycocalyx, forming micro-colonies . As these micro-colonies grow and recruit planktonic bacteria, they coalesce with neighboring micro-colonies to form fully-developed bioﬁlms .
The aﬀect of changing the environmental conditions that the bacterium P. aeruginosa Paer1 was grown under were also measured and shown to change adhesion properties. Growth under conditions that the bacteria are likely to grown under in environmental conditions , such as slow growing and decreased temperature, Fig. 6. 2D gel electrophoresis of cell-surface proteins extracted from P. aeruginosa Paer1 under diﬀerent growth conditions. A is the Paer1 grown at 371C and a dilution rate of 0.3 h
1 , B the Paer1 grown at 251C and a dilution rate of 0.3 h
1 . Red arrows indicate proteins that were diﬀerentially expressed.
Red circles highlight areas where multiple proteins were diﬀerentially expressed. Green spots indicate proteins that appear on both gels. Numbers one to ten indicate protein spots that were chosen as reference spots between the two gels.
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signiﬁcantly altered the surface properties of the bacterium. However, no change in the surface properties of the bacterium was directly correlated with the changes in adhesion to contact lenses demonstrating the ability of bacterial cells to utilize several mechanisms to adhere. Indeed, it has been demonstrated that P. aeruginosa can use several cell surface structures to adhere to epithelial cells [56–64]. The LPS has been demonstrated to be involved in the adhesion of P. aeruginosa to corneal epithelial cells [57,58,64].
The present study demonstrated that altering the LPS of P. aeruginosa Paer1 increased its adhesion to Etaﬁlcon A lenses. Similarly, there were changes in the outer membrane proteins that were expressed on Paer1 that may have aﬀected its adhesion to contact lenses. Cowell et al.  demonstrated that growth of P. aeruginosa Paer1 under conditions of nitrogen or carbon limitation also altered the ability of this strain to adhere to Etaﬁlcon A lenses and altered the 2D protein proﬁle. Interestingly the changes that occurred in the protein proﬁle under nitrogen limited conditions, which resulted in increased adhesion to contact lenses, were not the same as those changes that occurred during growth at 251C indicating the ﬂexibility that there is in the mechanisms of adhesion of P.
aeruginosa to contact lenses.
5. Conclusions Bacterial adhesion to contact lenses is clearly involved in the production of several adverse responses that occur during contact lens wear. There is usually little or no change in the ocular microbiota during asymptomatic hydrogel contact lens wear and there is no major diﬀerences in the types of bacterial that colonize lenses during either extended or daily wear. Contact lenses represent a new surface for colonization in the eye, but the colonization is sporadic and the numbers of bacteria that initially colonize are probably low such that growth is required to cause many of the inﬂammatory reactions.
The adhesion to contact lenses in vitro varied with the type of lens polymer, bacterial genus (with P. aeruginosa usually adhering to lenses in greater numbers than other genera/species [data not shown]), or species, or strain or indeed the environmental conditions individual strains were grown under. P. aeruginosa, once adhered to a contact lens, could utilize the adsorbed tear ﬁlm components (proteins, lipids, mucin) for growth. In order to reduce or prevent the bacterially driven adverse responses associated with contact lens wear, we believe novel lenses that contain active substances such as those that prevent growth (one example would be antibiotics although problems with bacterial resistance might arise), or aﬀect cell metabolism by interfering with global regulators of gene expression (such as the arg system in S.
aureus  or s factors in P. aeruginosa [66,67]) should be investigated.
Acknowledgements Dr Heather St. John, CSIRO Division of Molecular Sciences, Clayton, Vic, Australia for analyzing the worn lenses using XPS. Dr R. Schneider, School of Microbiology and Immunology, University of New South Wales, NSW, Australia, for help with bacterial growth in a chemostat. Dr Gideon Wolfaardt, Applied Microbiology and Food Science, College of Agriculture, Saskatoon, Canada for help with the scanning confocal microscopy. Dr Ben Herbert, Australian Proteome Analysis Facility, University of Macquarie, NSW, Australia, for help with the 2D gel electrophoresis of bacterial proteins.
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