bacterialinteractionswithcontactlenses;eﬀectsoflensmaterial ......spectroscopy(xps).after wear,...

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 duringasymptomatic contact lens wear lenses are colonized by low levels of bacteria with gram-positive bacteria, such as coagulase negativestaphylococci, 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 demonstratedconsiderable differences. In particular, Pseudomonas aeruginosa strains Paer1 and 6294 and Aeromonas hydrophilia strain Ahyd003adhered in larger numbers to the highly oxygen permeable contact lenses Balafilcon A compared to hydrogel lenses manufacturedfrom 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% to1393%. However, wearing Etafilcon A lenses usually resulted in a decrease in adhesion (22–48%). Bacteria were able to grow afteradhesion 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 greatestnumbers if grown under low temperature (251C compared to 371C). The changes in growth temperature was shown, using 2D gelelectrophoresis, 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 theincreased 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 visioncorrection and are worn by approximately 85 millionpeople worldwide. Two major types of contact lenses arecommonly worn. These two types are rigid gas perme-able (RGP) lenses and soft hydrogel lenses. The RGPcontact lenses are commonly composed of monomerscontaining silicone, fluorine and methylmethacrylate.Soft hydrogel lenses are commonly composed of2-hydroxyethyl methacrylate polymer alone (e.g. Poly-macon, Bausch and Lomb, Rochester, NY, USA; FDAgroup I) or containing methacrylic acid (e.g. EtafilconA, Vistakon, a division of Johnson and JohnsonVision Products Inc, Jacksonville, FL, USA; FDAgroup 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 havebeen incorporated into the soft hydrogel lens materials,including silicone polymers for increased oxygenpermeability (e.g. Lotrafilcon A, CIBA Vision, orBalafilcon A, Bausch and Lomb) and phosphoryl-choline to increase biocompatability (e.g. OmafilconA, Biocompatables Ltd., UK). Contact lenses can beworn on several wear schedules including daily wear(the wearer removes the lens each night, cleans anddisinfects the lens overnight and returns the same lensto the eye in the morning [these lenses are commonlyreplaced with fresh lenses every month]), dailydisposable wear (the wearer removes and discards thelens at the end of the day and inserts a new lens intothe eye the next morning), extended wear (the wearerwears the same lens continuously for, commonly,6 nights, then removes the lens and inserts a newlens 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 protected] (M.D.P. Willcox).

0142-9612/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 1 6 1 - 2

then discards the lens and inserts a new lens on the thirtyfirst day).

Occasionally adverse responses to contact lens wearoccur. These adverse responses are frequently caused bybacterial contamination of the contact lens surface.Contact lens induced corneal adverse responses haverecently been classified into serious sight threateningresponses (microbial keratitis [MK; incidence 0.3%]),significant adverse responses (contact lens induced acutered eye [CLARE; incidence 1.4–6.2%], contact lensinduced peripheral ulcers [CLPU; incidence 0.6–8.7%]and infiltrative keratitis [IK; incidence 1.7–5.2%]) andnon-significant adverse responses (asymptomatic infil-trative keratitis [AIK; incidence 1.5–3.9%] and asymp-tomatic infiltrates [AI; incidence 5.2%]) [1]. Of theseadverse responses, bacterial colonization of contactlenses is one of the initiating factors in MK [2], CLARE[3,4], CLPU [5] and certain IK and AIK events [6].

There have been several investigations into the effectsof contact lenses on the normal ocular microbiota. Thenormal ocular microbiota in the absence of contact lenswear is composed almost exclusively of three bacterialtypes, coagulase negative staphylococci, Corynebacter-ium sp. and Propionibacterium sp. [7,8]. The lids usuallyharbor a microbiota similar to the normal skinmicrobiota and harbor more bacteria of more speciesthan the conjunctiva [9]. During sleep the number ofbacteria colonizing the conjunctiva and lid increases[10]. An increase in the number of bacteria isolated fromthe conjunctiva and lids during daily lens wear has beenreported [9,11], although the types of micro-organismswere not found to differ from non-lens wearing eyes. Analteration in the types of micro-organisms was seen withextended lens wear (more Gram-negative bacteria beingisolated) along with an increase in the frequency ofcultures growing no micro-organisms [9,12]. The abovefinding is significant as Gram-negative organisms arecommon ocular pathogens [13]. Other studies, however,have reported no differences between wearers and non-lens wearers although an increase in positive ocularcultures was found in former lens users and inassociation with certain modes of lens wear and typesof disinfection systems [8].

Table 1 details the types of bacteria that have beenisolated from contact lenses at the time of an MK,CLARE, CLPU, IK or AIK. Pseudomonas aeruginosa isthe most common cause of MK during contact lens wear[13–16]. Gram-positive bacteria are more commonlyassociated with CLPU [5,6,17], whereas gram-negativebacteria are more commonly associated with CLARE[3,4,18]. For CLARE, Haemophilus influenzae is themost commonly isolated bacterium [4].

One of the initial steps in the development of thebacterially driven adverse responses is the binding ofbacteria to a contact lens. Several studies have examinedthe ability of bacteria to adhere to contact lenses.

Staphylococcus epidermidis or P. aeruginosa strainsadhere in larger numbers to lenses made from hydro-xyethyl methacrylate (HEMA) alone compared to lensesmade from HEMA plus methacrylic acid [19–21] andthis may be a function of differing water contents [22,23]or charges of these lens types. A contact lens wheninserted into the eye rapidly accumulates proteins,glycoproteins and lipids (known as deposits) from thetear film to its surface. Therefore, it is likely that, otherthan contamination upon insertion (which is usually bybacteria that are part of the normal microbiota),bacteria adhere to these adsorbed components ratherthan the contact lens material itself. That is not to saythe contact lens material will not still affect adhesion; thetypes of deposits are likely to be affected by thechemistry of the contact lens. Subsequent to adhesion,it is likely that bacteria further colonize the lens surfaceby growing on that lens surface. Hume and Willcox [24]demonstrated that Serratia marcescens was able to growon a contact lens after adhesion to contact lenses coatedin 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

aMK, microbial keratitis; CLARE, contact lens induced acute red

eye; CLPU, contact lens induced peripheral ulcer; IK, infiltrative

keratitis; AIK, asymptomatic infiltrative keratitis [1].bThese 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.

M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–32473236

Another important factor in the ability of a bacteriumto adhere to contact lenses is the physiology of thatbacterium. Recently Cowell et al. [25] demonstrated thatgrowing Paer1 under nitrogen limitation increased theadhesion of this strain to Etafilcon A contact lenses. Theaims of this investigation were to demonstrate the typesof bacteria that adhere to contact lenses during wear andto determine the factors, both material and microbio-logical, that can affect the adhesion of bacteria tocontact lenses.

2. Materials and methods

2.1. Comparison of bacterial colonization on soft contactlenses worn on different wear schedules

Contact lens wearing subjects wore either bilateralEtafilcon A lenses or contra-lateral Etafilcon A in oneeye and Polymacon in the other eye, or bilateralLotrafilcon A lenses over a 7 year period. The EtafilconA and Polymacon lenses were worn on a 6 nightsextended wear schedule or daily wear schedule withmonthly replacement. The Lotrafilcon A lenses wereworn on a 30 nights continuous wear schedule. We havepreviously demonstrated that there are no differences inthe bacterial colonization between Etafilcon A andPolymacon [9,26] or Etafilcon A and Lotrafilcon Alenses [27]. Seventy three subjects wearing extended/continuous wear lenses and 39 subjects wearing lenseson a daily wear schedule were involved in the study andall were free of ocular diseases, had no ocular surgeryand required visual correction for low refractive errorsonly. Informed consent was obtained from the subjectsand all procedures were approved by the University ofNew South Wales Human Ethics Committee. Eachsubject was sampled on average 3 times.

Contact lenses were removed aseptically and trans-ported to the laboratory in sterile phosphate bufferedsaline (PBS) [9]. Bacteria adherent to the contact lenseswere grown using an agar sandwich technique [9].Briefly, lenses were placed concave side up on achocolate agar plate and the plate was flooded withmolten (561C) agar and placed in a CO2-enrichedatmosphere (5%) at 351C for 48 h. Aliquots (0.4ml) ofthe remaining transport PBS were spread onto threechocolate and one Sabouraud’s agar plate. The Sabour-aud’s agar plate and one chocolate agar plate wereincubated aerobically at 351C for 48 h. The Sabouraud’sagar plate was subsequently incubated for six days atambient temperature. The two remaining chocolateplates were incubated at 351C either in a CO2-enrichedatmosphere for 48 h or anaerobically (95% N2, 5%CO2) for four days. Microbiological characterization ofthe contact lenses was conducted as described previously[9,26,28]. The incidence rates of various groups of

micro-organisms were compared using chi-square testwith Yates correction and the level of significance wasset at P ¼ o0:05:

2.2. Adhesion of bacteria to contact lens materials in vitro

P. aeruginosa 6294, P. aeruginosa Paer1, Streptococ-cus pneumoniae 001, S. pneumoniae 008, Haemophilusinfluenzae 001, H. influenzae 009, Aeromonas hydrophilia003, Stenotrophomonas maltophilia 010 were isolatedfrom cases of CLARE, with the exception of P.aeruginosa 6294 which was isolated from a case ofMK, at the time of presentation. Bacteria were grown tostationary phase in Trypicase soy broth (Oxoid, Sydney,Australia) at 351C, then cells were washed three timeswith PBS and resuspended to an OD of 1.0 (approxi-mately 1� 108 bacteria/ml) [29]. Previous studies haddemonstrated that, for all lens types used, this opticaldensity gave maximum adhesion and that opticaldensities above this did not show increased adhesion(date not shown). Bacteria (1ml) were added to lensesand adhesion was allowed to occur for 10min. Non-adherent cells were then removed by washing in PBSthree times and cells stained with crystal violet prior toenumeration by light microscopy [25]. Lenses used in theexperiments were Etafilcon A, Balafilcon A, Polymacon,Omafilcon A. All results were expressed compared to theadhesion of the bacterial strains to Etafilcon A contactlenses as these are the current market leaders in hydrogellenses. All experiments were repeated on three separateoccasions. Adhesion data were not normally distributedand therefore were analysed for differences between lenstypes non-parametrically with the Mann-WhitneyV-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 eyesfor 6 h during the day on different days. Lenses werethen removed aseptically, washed three times in PBS toremove loosely adsorbed tear film components andbacterial adhesion was measured as described above(Section 2.2). Results were expressed as a percentagedifference compared to control unworn lenses and allexperiments were repeated on at least two separate days.Adhesion data were not normally distributed andtherefore were analysed for differences between wornand unworn lenses non-parametrically with the Mann-Whitney V-Wilcoxon Rank Sum test.

2.4. Analysis of types of deposits on worn contact lenses

The types of deposits formed on Polymacon orEtafilcon A lenses that had been worn for 6 h duringthe 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 inMilliQ water, dried and sectioned. XPS analysis wasperformed as described previously [30] using a KratosAxis H1s instrument, an A1 monochromated sourcewith a spot size of 1mm. Elemental identification wasperformed from scans acquired at 160 eV.

2.5. The ability of strains to grow on contact lenses

Scanning confocal laser microscopy (SCLM) is atechnique used for the observation of bacteria attachedto surfaces. The strengths of this technique lie in itsability to observe and analyze sections of three-dimen-sional bacterial biofilms [31,32].

Multi-channel flow cells were designed by DarrylWilkie and Jason Marshall (Department of AppliedMicrobiology and Food Science, University ofSaskatchewan) and constructed using polycarbonateplastic (Fig. 1). Irrigation channels, 40mm wide, weredrilled into the plastic and a glass coverslip placed overthe channels and sealed with silicone glue (SiliconeRubber Adhesive Sealant, GE Translucent RTV118).Flow cells were connected to silicone tubing ateither end. A Watson Marlow peristaltic pump wasused to pump the media or washing solutions throughthe flow cells.

Contact lenses (Etafilcon A) were cut in half using asterile scalpel. Each half was attached to a plastic blockusing silicone glue applied around the edges of thesemicircular lens sample. The block with the affixed lenssample was placed in the flow cell and sealed with a glasscoverslip. The gap between the exposed surface of thecontact lens sample and the underside of the coverslipwas approximately 4mm.

A protein mixture comprised five proteins: lactoferrin(bovine colostrum, 1mg/ml), lysozyme (chicken egg-white, 1mg/ml), g-globulins (bovine, 1mg/ml), albumin(bovine serum, 0.1mg/ml), mucin (bovine submaxillarygland, 0.1mg/ml) was constructed in PBS. All proteinswere purchased from Sigma (St. Louis, MO, USA).Although this protein mixture represented a simplifiedversion of tear proteins, the exact composition of thesolution was less critical than the fact that it containedpotentially antibacterial proteins (lactoferrin, lysozyme)and a high concentration of glycoproteins (lactoferrin, g-globulins, 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 wereimmersed in PBS. Lens samples were left in the flowcells for 18 h at 351C. Loosely bound protein waswashed off using 10ml of PBS pumped through the flowcell. Bacteria were grown and washed as describedabove (Section 2.2). Bacterial suspension (Paer1, OD660

¼ 0:1 in PBS) was then introduced until the flowcell was filled. Initially, bacteria were allowed toadhere to the lens, then a solution (PBS, protein

mixture, or 10% TSB) was pumped through the flowcell at a very slow rate of flow. The flow cell was left at351C for 10min. Loosely attached bacteria were washedoff by the same method used for removing looselybound protein.

The growth medium (protein mixture or 10% TSB orPBS) was introduced into the flow cell at a rate of2.6ml/h for 3 days at 251C. RH795 (0.1% in PBS;Molecular Probes, Eugene, Oregon, USA), whichresponds to cell membrane potential and was used tostain the bacterial cells, was introduced (3ml) into theflow cell by injecting it into the silicone tubingimmediately prior to the flow cell and was left for 1 hat 251C. The flow of solution was then stopped and theglass coverslip was removed. Microscope work wascarried out using the Bio-Rad MRC 600 SCLMequipped with an argon laser and standard filter sets.The laser was mounted on a Nikon Microphot-FXAmicroscope. The microscope was equipped with a 20 Xwater immersion lens.

Four separate conditions were examined for theireffect on Paer1 growth on lens samples, and set up inparallel using the flow cells: (1) PBS was passed over aclean lens sample; (2) PBS passed over a lens samplecoated with the protein mixture; (3) protein mixturepassed over a lens sample coated with the proteinmixture; (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.


not colonized by bacteria were also observed usingSCLM. All experiments were repeated on two separateoccasions.

2.6. Effect of bacterial physiology on adhesion

In an effort to further elucidate the mechanisms ofbacterial adhesion, P. aeruginosa Paer1 was grown in achemostat under different environmental conditions.Paer1 was grown as described previously [25]. Briefly,the bacteria were grown in a defined medium [25] eitherat 371C or 251C and at a growth rate of either 0.3 or0.05 h�1. After growth, the bacteria were washed threetimes in PBS and their adhesion to Etafilcon A contactlenses was measured as described above (Section 2.2)and repeated three times. The cells’ adhesion tosubstituted Sepharose 6-B was also investigated [25].Sepharose 6-B, octyl-, phenyl-, CM- and DEAE-Sepharose were purchased from Pharmacia LKBBiotechnology (Uppsala, Sweden). Columns were con-structed by loading 1ml (B5 cm) of each Sepharose gelinto Pasteur pipettes plugged with glass wool. The voidvolume determined using methylene blue was found tobe 0.5ml. Columns were equilibrated with 5ml PBS.The optical density of the original suspension wasmeasured at 600 nm. Bacterial suspensions (0.5ml,OD600 ¼ 1:0) were added to the columns and the firsteluant (0.5ml) discarded. A second aliquot of bacteria(0.5ml) was added to the column and the eluantcollected with the first wash of 0.5ml PBS. Threeadditional washes (1ml) were collected and the absor-bance at 600 nm of each wash determined. Percent cellsretained on each gel, and percent cells subsequentlydesorbed in the next three washes were determined, andfrom this data the net retention (percent of originalinoculum) of bacteria on each Sepharose type wascalculated. Retention on Sepharose assays were per-formed twice for each incubation condition.

2.7. The role of bacterial cell surface proteinsand lipopolysaccharide in adhesion

Experiments were then conducted on the expressionof cell-surface proteins and lipopolysaccharide (LPS) byP. aeruginosa Paer1 grown at 371C or 251C. Cell-surfaceproteins were extracted [25,33] using 100ml of 50mm

sodium citrate buffer (sodium citrate/citric acid, pH 4.5)containing 0.1% Zwittergent (Calbiochem, La Jolla,CA), 1mm PMSF (phenylmethyl-sulfonyl fluoride,Boehringer Mannheim, Mannheim, Germany) and10mm EDTA (ethylenediaminetetraacetic acid, Sigma,St. Louis, MO). The reaction mixture was incubated for25min at 451C with occasional mixing. Bacteria werethen pelleted by centrifugation at 3200g for 2 h at 41C.The supernatant containing the extracted proteins wasdialysed overnight against distilled water containing

0.02% (w/v) sodium azide to remove the detergent.Proteins were concentrated using Centriprep-10 concen-trators (10,000 MW cut-off, Amicon, Beverly, MA).Protein samples were reduced with DTT (1,4-dithio-threitol, Boehringer Mannheim, Mannheim, Germany)prior to SDS-PAGE (sodium dodecyl sulphate-poly-acrylamide gel) analysis on 10% acrylamide gelsaccording to the method of Laemmli [34]. Gels weresilver-stained to visualise protein bands following themethod of Bjellqvist et al. [35]. Proteins preparationswere extracted from two samples and run on gels todetermine consistency. The LPS was extracted using thewater/phenol method of Westphal and Jann [36] and theamount of LPS was analysed using a Coatesttendotoxin kit (Chromogenix AB, M .ooindal, Sweden)followed by sodium dodecylsulfate gel electrophoresis(10% acrylamide) and visualized by silver diaminestaining.

3. Results

3.1. Comparison of bacterial colonization on soft contactlenses worn on different wear schedules

Table 2 shows the types of bacteria that were isolatedfrom contact lens wearers on an extended or daily wearschedule. The most common bacteria isolated weregram-positive bacteria including coagulase negativestaphylococci, Propionibacterium sp. and Corynebacter-ium sp. Of the gram-negative bacteria isolated, Pseudo-monas sp. and Stenotrophomonas sp. were isolated mostfrequently during extended wear, whereas Pseudomonassp. and Acinetobacter sp. were isolated most frequentlyduring daily wear. However, there were no differences inthe numbers, types or frequency of colonization ofcontact lenses worn on either an extended/continuouswear schedule or daily wear schedule.

3.2. Bacterial adhesion to soft contact lenses in vitro

As can be seen (Fig. 2), there was considerablevariation in adhesion between bacterial strains andcontact lenses. Three strains of bacteria, P. aeruginosaPaer1 and 6294 and Aeromonas hydrophilia 003, adheredin increased numbers to the High DK silicone hydrogellenses (Balafilcon A) compared to Etafilcon A. Theincrease may have been due to the more hydrophobicnature of the underlying contact lens material in thehigh DK lenses. The strains of Streptococcus pneumoniaewere chosen for testing against Omafilcon A lenses asthese bacteria are known to possess receptors for cholineon their surface [37]. Fig. 3 demonstrates that for onlyone of these strains, Spne 004, there was an increase inadhesion to Omafilcon A lenses that contain phosphor-ylcholine.


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

aEW, 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.bFrequency 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).


3.3. Effect of lens wear on bacterial adhesion in vitro

Using contact lenses that had been worn for 6 hduring the day, bacterial adhesion was examined. Ingeneral, the gram-negative bacteria used in the adhesionassays adhered in lower numbers to worn compared tounworn Etafilcon A lenses (Table 3). On the other hand,bacteria adhered in greater numbers to worn rather thanunworn Balafilcon A (High DK) lenses (Table 3).Statistical analysis demonstrated a significant(Po0:05) increase in adhesion of strains Paer1, Hinf001and Xmal010 to worn Balafilcon A lenses, Paer1 andHinf001 to worn Polymacon lenses and Ahyd003 toworn 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 lensadsorbed more nitrogen containing material than thePolymacon lens (approximately six times as much),indicating more protein was adsorbed to the surface.The worn Polymacon lens adsorbed very little proteinand only on its front surface. Interestingly, the smalldecrease in carbon (and increase in oxygen) on the

surface of the worn Polymacon lenses may indicate thatmucin 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 observedunder SCLM, without any further treatment. Fig. 4bshows the response of bacteria attached to a clean lenssample after exposure to PBS under laminar flowconditions. The diffuse nature of the surface mayrepresent a very loose aggregation of Paer1 thatobscures the surface of the lens sample. Fig. 4c showsa protein-coated lens sample used in place of a clean lenssample. For the next set of experimental conditions,bacteria were attached to a lens sample pre-coated withthe protein mixture. The same protein mixture was thenpassed over these bacteria under laminar flow condi-tions. Fig. 4d clearly shows the presence of discreteclumps of bacteria, presumably micro-colonies, over thesurface. These cannot represent aggregates of adsorbedprotein, as these were not visible under this magnifica-tion. The response of bacteria attached to a clean lenssample when exposed to a 10% solution of TSB wasquite distinct. Although putative bacterial aggregationswere 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 43b723 14347323c 453797c

6294 48715 NDd

ND

Ahyd003 402752c 100736 2437141

Hinf001 ND 367796c 13937253c

Xmal010 2275e 65726 303729c

aAdhesion 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.bMean7SD.cSignificant increase in adhesion over unworn lenses (Po0:05).dND, not determined.eSignificant 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

aC, carbon; O, oxygen; N, nitrogen; Si, silicon; F, fluorine.


from the micro-colonies shown in Fig. 4c and d, andprobably represented flocculation. Within 48 h thesolution inside the flow cell was observed to be veryturbid and this was not observed for any other of thesets of conditions. These enriched conditions obviouslygenerated very strong growth of bacteria in suspension.If any biofilm was present, it was very diffuse andloosely attached to the surface, and can probably be bestdescribed as flocculation. Under these extremely nu-trient-rich conditions, there was apparently no impera-tive for bacteria to attach to a solid surface.

3.6. Effect of bacterial physiology on adhesion

Table 5 demonstrates the changes that occurred in thesurface charge and hydrophobicity of Paer1 under thedifferent conditions. Low growth temperature increasedadhesion to the control Sepharose 6-B by 55% and toOctyl-Sepharose by 19% but did not appreciably alter

the adhesion to Phenyl, DEAE or CM-Sepharose. Aslow growth rate increased the adhesion to controlSepharose 6-B by 65%, to Octyl-Sepharose by 21% andto 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, g-globulins, 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 10–15mm 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

aAll cells were grown in a chemostat in defined media [25] 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, 37oC

and 0.05 h�1 dilution rate.


Phenyl-Sepharose by 56%. Fig. 5 demonstrates theadhesion of bacterial cells grown under the differentconditions to Etafilcon A lenses. Adhesion was highestwhen cells were grown at low temperature and relativelyhigh growth rates, followed by growth at high tempera-ture and slow growth rate and adhesion was least whencells were grown at high growth rates and hightemperature. Whilst the hydrophobicity (adhesion tooctyl or phenyl Sepharose) and charge (adhesion toeither DEAE or CM-Sepharose) also changed with growconditions, there was no direct correlation betweenadhesion to the lenses and adhesion to the substitutedSepharose polymers probably demonstrating the abilityof bacterial cells to utilize several mechanisms to adhere.

3.7. The role of bacterial cell surface proteins andlipopolysaccharide in adhesion

Fig. 6 demonstrates that growth at 251C altered theexpression of a number of cell-surface proteins (markedby either a red arrow head or enclosed in a red circle)compared to growth at 371C. Similarly, growth at 251Caltered the expression of LPS, yielding larger LPSmolecules that did not migrate as far into the gel matrix(Fig. 7).

4. Discussion

The current study has confirmed that there was nodifference in the colonization of contact lenses worn onan extended or daily wear schedule during asympto-matic lens wear. Lens contamination during asympto-matic lens wear appears to involve small numbers ofmicro-organisms [38]. The most common bacteriaisolated from contact lenses are coagulase-negativestaphylococci [8,12,26,39]. Contact lens contaminationcommonly occurs through lens handling [40] but itappears that during uncomplicated lens wear these

micro-organisms are readily cleared from the lenssurface by the ocular defense mechanisms. Other sourcesof contamination of lenses by the normal microbiotainclude the eyelids of wearers [41] or from environ-mental sources [41]. Contamination of lenses duringwear is sporadic. Subjects sampled on successive days ofextended lens wear, from 1 night to 13 nights, were aslikely to have contaminated lenses on Day 1 as on Day13 [42]. In other words, wearing lenses for increasinglengths of time did not result in increasing microbialcontamination.

However, some adverse responses that occur duringlens wear are known to be associated with bacterialadhesion to lenses [2–6]. These bacteria do not make uppart of the normal ocular microbiota. In order forbacteria to initiate an adverse response, they must beable to adhere to the lens surface. The current studydemonstrated that there were differences in the ability ofthe bacteria isolated from adverse responses to adhere tolens materials and for most material/strain combina-tions there were increases in adhesion to worn lenses orno differences between adhesion to worn or unwornlenses. It was demonstrated that worn lenses did adsorbtear film components, probably proteins/glycoproteins,and that bacteria were able to grow on those proteinsthat were adsorbed to a lens surface. The increases inadhesion seen for certain strains to worn lenses mayindicate that the tear molecules, most likely proteins,that bound to the contact lenses were conducive tobacterial adhesion. To date there are no publications onthe types of proteins or other molecules that bind to thehigh DK lenses which demonstrated the most noticeableincreases in adhesion to worn lenses, although oneabstract at the British Contact Lens Association annualmeeting in May 2000 reported more deposition on thesurface of Balafilcon A lenses compared to Etafilcon Alenses (however, no biochemical analysis of the depositswas reported) [43]. Protein adsorbs more readily to lesshydrophilic surfaces compared to the surface carryingan anionic charge such as that of an Etafilcon A lens[44]. The exception to this is the adsorption of lysozymeto anionic lenses [45,46]. Also, the anionic lenses tend toadsorb less lipid [47], although N-vinyl pyrrolidonecontaining anionic lenses do bind lipid [48–50]. Theworn Polymacon lenses may have bound mucin to theresurface. It is known that P. aeruginosa can bind toocular mucin [18,51]. Total protein does not correlatewith adhesion of P. aeruginosa to lenses [40]. However,deposits on lenses did increase adhesion in one study[52], although this may be due to increased surfaceroughness. No relation between the ability of P.aeruginosa to bind to worn Etafilcon A contact lensesand the presence of lysozyme or lactoferrin has beenfound, although worn lenses did usually increase theadhesion of strains [53]. Albumin coated onto thesurface 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).


increased the adhesion of P. aeruginosa [21]. Similarly,some strains of Serratia marcescens adhered better toEtafilcon A lenses coated in an artificial tear fluid [24].Lysozyme adsorbed to a contact lens increases theadhesion of Staphylococcus aureus to Etafilcon Acontact lenses [54].

Factors in addition to adhesion are likely tocontribute to the production of adverse reponses. Onesuch pathogenic trait would be the ability of the adheredbacteria to grow on the tear film components that haveadsorbed to the lens surface. Using SCLM observation,Paer1 grew under conditions in which soluble proteinwas passed over bacteria attached to the lens surface.

Micro-colony production is the prelude to biofilmformation [55]. After initial adhesion, adherent bacteriamay proliferate on the substratum within the polysac-charide-rich glycocalyx, forming micro-colonies [55]. Asthese micro-colonies grow and recruit planktonicbacteria, they coalesce with neighboring micro-coloniesto form fully-developed biofilms [55].

The affect of changing the environmental conditionsthat the bacterium P. aeruginosa Paer1 was grown underwere also measured and shown to change adhesionproperties. Growth under conditions that the bacteriaare likely to grown under in environmental conditions[41], 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.


significantly altered the surface properties of thebacterium. However, no change in the surface propertiesof the bacterium was directly correlated with thechanges in adhesion to contact lenses demonstratingthe ability of bacterial cells to utilize several mechanismsto adhere. Indeed, it has been demonstrated that P.aeruginosa can use several cell surface structures toadhere to epithelial cells [56–64].

The LPS has been demonstrated to be involved in theadhesion of P. aeruginosa to corneal epithelial cells[57,58,64]. The present study demonstrated that alteringthe LPS of P. aeruginosa Paer1 increased its adhesion toEtafilcon A lenses. Similarly, there were changes in theouter membrane proteins that were expressed on Paer1that may have affected its adhesion to contact lenses.Cowell et al. [25] demonstrated that growth of P.aeruginosa Paer1 under conditions of nitrogen or carbonlimitation also altered the ability of this strain to adhereto Etafilcon A lenses and altered the 2D protein profile.Interestingly the changes that occurred in the proteinprofile under nitrogen limited conditions, which resultedin increased adhesion to contact lenses, were not thesame as those changes that occurred during growth at251C indicating the flexibility that there is in themechanisms of adhesion of P. aeruginosa to contactlenses.

5. Conclusions

Bacterial adhesion to contact lenses is clearly involvedin the production of several adverse responses that occurduring contact lens wear. There is usually little or nochange in the ocular microbiota during asymptomatichydrogel contact lens wear and there is no majordifferences in the types of bacterial that colonize lensesduring either extended or daily wear. Contact lensesrepresent a new surface for colonization in the eye, butthe colonization is sporadic and the numbers of bacteriathat initially colonize are probably low such that growthis required to cause many of the inflammatory reactions.The adhesion to contact lenses in vitro varied with thetype of lens polymer, bacterial genus (with P. aeruginosausually adhering to lenses in greater numbers than othergenera/species [data not shown]), or species, or strain orindeed the environmental conditions individual strainswere grown under. P. aeruginosa, once adhered to acontact lens, could utilize the adsorbed tear filmcomponents (proteins, lipids, mucin) for growth.

In order to reduce or prevent the bacterially drivenadverse responses associated with contact lens wear, webelieve novel lenses that contain active substances suchas those that prevent growth (one example would beantibiotics although problems with bacterial resistancemight arise), or affect cell metabolism by interfering withglobal regulators of gene expression (such as the argsystem in S. aureus [65] or s factors in P. aeruginosa[66,67]) should be investigated.

Acknowledgements

Dr Heather St. John, CSIRO Division of MolecularSciences, Clayton, Vic, Australia for analyzing the wornlenses using XPS. Dr R. Schneider, School of Micro-biology and Immunology, University of New SouthWales, NSW, Australia, for help with bacterial growthin a chemostat. Dr Gideon Wolfaardt, Applied Micro-biology and Food Science, College of Agriculture,Saskatoon, Canada for help with the scanning confocalmicroscopy. Dr Ben Herbert, Australian ProteomeAnalysis Facility, University of Macquarie, NSW,Australia, for help with the 2D gel electrophoresis ofbacterial proteins.

References

[1] Holden BA, Sankaridurg P, Jalbert I. Adverse responses. Which

ones and how many. In: Sweeney DF, editor. Silicone hydrogels.

The rebirth of continuous wear contact lenses. Oxford: Butter-

worth Heinmann, 2000. p. 150–213.

[2] Solomon OD, Loff H, Perla B, Kellis A, Belkin J, Roth AS,

Zucker J. Testing hypotheses for risk factors for contact lens-

associated infectious keratitis in an animal model. CLAO

J 1994;20:109–13.

Fig. 7. Differential expression of lipopolysaccharide on the surface of

P. aeruginosa grown at 251C or 371C. 371C is the Paer1 grown at 371C

and a dilution rate of 0.3 h�1, 251C the Paer1 grown at 251C and a

dilution rate of 0.3 h�1.


[3] Holden BA, La Hood D, Grant T, Newton-Howes J, Baleriola-

Lucas C, Willcox MD, Sweeney DF. Gram-negative bacteria can

induce contact lens related acute red eye (CLARE) responses.

CLAO J 1996;22:47–52.

[4] Sankaridurg PR, Willcox MD, Sharma S, Gopinathan U,

Janakiraman D, Hickson S, Vuppala N, Sweeney DF, Rao GN,

et al. Haemophilus influenzae adherent to contact lenses associated

with production of acute ocular inflammation. J Clin Microbiol

1996;34:2426–31.

[5] Jalbert I, Willcox MD, Sweeney DF. Isolation of Staphylococcus

aureus from a contact lens at the time of a contact lens-induced

peripheral ulcer: case report. Cornea 2000;19:116–20.

[6] Sankaridurg PR, Sharma S, Willcox M, Sweeney DF, Naduvilath

TJ, Holden BA, Rao GN. Colonization of hydrogel lenses with

Streptococcus pneumoniae: risk of development of corneal

infiltrates. Cornea 1999;18:289–95.

[7] Perkins RE, Kundsin RB, Pratt MV, Abrahamsen I, Leibowitz

HM. Bacteriology of normal and infected conjunctiva. J Clin

Microbiol 1975;1:147–9.

[8] Fleiszig SM, Efron N. Microbial flora in eyes of current and

former contact lens wearers. J Clin Microbiol 1992;30:1156–61.

[9] Stapleton F, Willcox MD, Fleming CM, Hickson S, Sweeney DF,

Holden BA. Changes to the ocular biota with time in extended-

and daily-wear disposable contact lens use. Infect Immun

1995;63:4501–5.

[10] Ramachandran L, Sharma S, Sankaridurg PR, Vajdic CM,

Chuck JA, Holden BA, Sweeney DF, Rao GN. Examination of

the conjunctival microbiota after 8 hours of eye closure. CLAO

J 1995;21:195–9.

[11] Larkin DF, Leeming JP. Quantitative alterations of the commen-

sal eye bacteria in contact lens wear. Eye 1991;5:70–4.

[12] Hovding G. The conjunctival and contact lens bacterial flora

during lens wear. Acta Ophthalmol 1981;59:387–401.

[13] Schein OD, Ormerod LD, Barraquer E, Alfonso E, Egan KM,

Paton BG, Kenyon KR. Microbiology of contact lens-related

keratitis. Cornea 1989;8:281–5.

[14] Tabbara KF, El-Sheikh HF, Aabed B. Extended wear

contact lens related bacterial keratitis. Br J Ophthalmol

2000;84:327–8.

[15] Patrinely JR, Wilhelmus KR, Rubin JM, Key JEd. Bacterial

keratitis associated with extended wear soft contact lenses. CLAO

J 1985;11:234–6.

[16] Galentine PG, Cohen EJ, Laibson PR, Adams CP, Michaud R,

Arentsen JJ. Corneal ulcers associated with contact lens wear.

Arch Ophthalmol 1984;102:891–4.

[17] Willcox M, Sweeney D, Sharma S, Gopinathan U, Sankaridurg P,

Ramachandran L, Holden B. Culture negative peripheral ulcers

are associated with increased bacterial contamination of contact

lenses. In: The Association for Research in Vision and Ophthal-

mology, Fort Lauderdale, 1995.

[18] Willcox MDP, Sankaridurg P, Lan J, Pearce D, Thakur A, Zhu

H, Keay L, Stapleton F. Inflammation and infection and effects of

the closed eye. In: Sweeney DF, editor. Silicone hydrogels. The

rebirth of continuous wear contact lenses. Oxford: Butterworth

Heinemann, 2000. p. 45–75.

[19] Fleiszig SM, Evans DJ, Mowrey-McKee MF, Payor R, Zaidi TS,

Vallas V, Muller E, Pier GB. Factors affecting Staphylococcus

epidermidis adhesion to contact lenses. Opto Vision Sci

1996;73:590–4.

[20] Cowell BA, Willcox MD, Schneider RP. A relatively small change

in sodium chloride concentration has a strong effect on adhesion

of ocular bacteria to contact lenses. J Appl Microbiol

1998;84:950–8.

[21] Taylor RL, Willcox MD, Williams TJ, Verran J. Modulation of

bacterial adhesion to hydrogel contact lenses by albumin. Opto

Vision Sci 1998;75:23–9.

[22] Cook AD, Sagers RD, Pitt WG. Bacterial adhesion to protein-

coated hydrogels. J Biomater Appl 1993;8:72–89.

[23] Cook AD, Sagers RD, Pitt WG. Bacterial adhesion to

poly(HEMA)-based hydrogels. J Biomed Mater Res

1993;27:119–26.

[24] Hume EB, Willcox MD. Adhesion and growth of Serratia

marcescens on artificial closed eye tears soaked hydrogel contact

lenses. Aust NZ J Ophthalmol 1997;25:S39–41.

[25] Cowell BA, Willcox MD, Herbert B, Schneider RP. Effect of

nutrient limitation on adhesion characteristics of Pseudomonas

aeruginosa. J Appl Microbiol 1999;86:944–54.

[26] Gopinathan U, Stapleton F, Sharma S, Willcox MD, Sweeney

DF, Rao GN, Holden BA. Microbial contamination of hydrogel

contact lenses. J Appl Microbiol 1997;82:653–8.

[27] Keay L, Willcox M, Sweeney D, Morris C, Harmis N, Corrigan

K, Holden BA. Bacterial populations on thirty night extended

wear silicone hydrogel lenses. CLAO J 2001, in press.

[28] Leitch EC, Harmis NY, Corrigan KM, Willcox MD. Identifica-

tion and enumeration of staphylococci from the eye during soft

contact lens wear. Opto Vision Sci 1998;75:258–65.

[29] Williams TJ, Willcox MD, Schneider RP. Interactions of bacteria

with contact lenses: the effect of soluble protein and carbohydrate

on bacterial adhesion to contact lenses. Opto Vision Sci

1998;75:266–71.

[30] Garrett Q, Chatelier RC, Griesser HJ, Milthorpe BK. Effect of

charged groups on the adsorption and penetration of proteins

onto and into carboxymethylated poly(HEMA) hydrogels.

Biomaterials 1998;19:2175–86.

[31] Gorman SP, Mawhinney WM, Adair CG, Issouckis M. Confocal

laser scanning microscopy of peritoneal catheter surfaces. J Med

Microbiol 1993;38:411–7.

[32] Lawrence JR, Neu TR. Confocal laser scanning microscopy for

analysis of microbial biofilms. Methods Enzymol 1999;310:

131–44.

[33] Carnoy C, Scharfman A, Van Brussel E, Lamblin G, Ramphal R,

Roussel P. Pseudomonas aeruginosa outer membrane adhesins for

human respiratory mucus glycoproteins. Infect Immun

1994;62:1896–900.

[34] Laemmli UK. Cleavage of structural proteins during the assembly

of the head of bacteriophage T4. Nature 1970;227:680–5.

[35] Bjellqvist B, Pasquali C, Ravier F, Sanchez JC, Hochstrasser D. A

nonlinear wide-range immobilized pH gradient for two-dimen-

sional electrophoresis and its definition in a relevant pH scale.

Electrophoresis 1993;14:1357–65.

[36] Westphal O, Jann K. Bacterial lipopolysaccharides. Extraction

with phenol-water and further applications of the procedure. In:

Whistler RL, Wolfron ML, editors. Methods in carbohydrate

chemistry, vol. 5. New York: Academic Press, 1965.

[37] Rosenow C, Ryan P, Weiser JN, Johnson S, Fontan P, Ortqvist

A, Masure HR. Contribution of novel choline-binding proteins to

adherence, colonization and immunogenicity of Streptococcus

pneumoniae. Mol Microbiol 1997;25:819–29.

[38] Hart DE, Reindel W, Proskin HM, Mowrey-McKee MF.

Microbial contamination of hydrophilic contact lenses: quantita-

tion and identification of microorganisms associated with contact

lenses while on the eye. Opto Vision Sci 1993;70:185–91.

[39] Fleiszig SM, Efron N. Conjunctival flora in extended wear of rigid

gas permeable contact lenses. Opto Vision Sci 1992;69:354–7.

[40] Mowrey-McKee MF, Monnat K, Sampson HJ, Smith CM,

Davies GA, Mandt L, Proskin HM. Microbial contamination of

hydrophilic contact lenses. Part I: quantitation of microbes on

patient worn-and-handled lenses. CLAO J 1992;18:87–91.

[41] Willcox MD, Power KN, Stapleton F, Leitch C, Harmis N,

Sweeney DF. Potential sources of bacteria that are isolated from

contact lenses during wear. Opto Vision Sci 1997;74:1030–8.


[42] Sweeney DF, Stapleton F, Leitch C, Willcox MDP, Taylor J,

Holden BA. The microbial colonisation of soft contact lenses over

time. Opto Vision Sci 2001, in press.

[43] Franklin V. A study of the spoilation profiles of high DK

fluorosilicone hydrogel lenses. In: British Contact Lens Associa-

tion 24th Annual Clinical Conference and Exhibition. Birming-

ham, UK, 2000.

[44] Bohnert JL, Horbett TA, Ratner BD, Royce FH. Adsorption of

proteins from artificial tear solutions to contact lens materials.

Invest Ophthalmol Visual Sci 1988;29:362–73.

[45] Deng XM, Castillo EJ, Anderson JM. Surface modification of

soft contact lenses: silanization, wettability and lysozyme adsorp-

tion studies. Biomaterials 1986;7:247–51.

[46] Sack RA, Sathe S, Hackworth LA, Willcox MD, Holden BA,

Morris CA. The effect of eye closure on protein and comple-

ment deposition on Group IV hydrogel contact lenses:

relationship to tear flow dynamics. Curr Eye Res 1996;15:

1092–100.

[47] Jones L, Evans K, Sariri R, Franklin V, Tighe B. Lipid and

protein deposition of N-vinyl pyrrolidone-containing group II and

group IV frequent replacement contact lenses. CLAO J 1997;

23:122–6.

[48] Keith D, Hong B, Christensen M. A novel procedure for the

extraction of protein deposits from soft hydrophilic contact lenses

for analysis. Curr Eye Res 1997;16:503–10.

[49] Tighe BJ, Jones L, Evans K, Franklin V. Patient-dependent and

material-dependent factors in contact lens deposition processes.

Adv Exp Med Biol 1998;438:745–51.

[50] Maissa C, Franklin V, Guillon M, Tighe B. Influence of contact

lens material surface characteristics and replacement frequency

on protein and lipid deposition. Opto Vision Sci 1998;75:

697–705.

[51] Fleiszig SM, Zaidi TS, Pier GB. Mucus and Pseudomonas

aeruginosa adherence to the cornea. Adv Exp Med Biol 1994;

350:359–62.

[52] Butrus SI, Klotz SA. Contact lens surface deposits increase the

adhesion of Pseudomonas aeruginosa. Curr Eye Res 1990;9:

717–24.

[53] Williams TJ, Willcox MD, Schneider RP. Role of tear fluid in the

growth of gram-negative bacteria on contact lenses. Aust NZ

J Ophthalmol 1997;25:S30–2.

[54] Thakur A, Chauhan A, Willcox MD. Effect of lysozyme on

adhesion and toxin release by Staphylococcus aureus. Aust NZ

J Ophthalmol 1999;27:224–7.

[55] Hoyle BD, Costerton JW. Bacterial resistance to antibiotics: the

role of biofilms. Prog Drug Res 1991;37:91–105.

[56] Gupta SK, Berk RS, Masinick S, Hazlett LD. Pili and

lipopolysaccharide of Pseudomonas aeruginosa bind to the

glycolipid asialo GM1. Infect Immun 1994;62:4572–9.

[57] Gupta SK, Masinick S, Garrett M, Hazlett LD. Pseudomonas

aeruginosa lipopolysaccharide binds galectin-3 and other human

corneal epithelial proteins. Infect Immun 1997;65:2747–53.

[58] Hazlett LD. Bacterial lipopolysaccharide binding to host recep-

tors [letter; comment]. Trends Microbiol 1996;4:418.

[59] Hazlett L, Rudner X, Masinick S, Ireland M, Gupta S. In the

immature mouse, Pseudomonas aeruginosa pili bind a 57-kd

(alpha 2–6) sialylated corneal epithelial cell surface protein: a first

step in infection. Invest Ophthalmol Visual Sci 1995;36:634–43.

[60] Hazlett LD, Rudner XL. Investigations on the role of flagella in

adhesion of Pseudomonas aeruginosa to mouse and human corneal

epithelial proteins. Ophthalmic Res 1994;26:375–9.

[61] Hazlett LD, Moon MM, Strejc M, Berk RS. Evidence for N-

acetylmannosamine as an ocular receptor for P. aeruginosa

adherence to scarified cornea. Invest Ophthalmol Visual Sci

1987;28:1978–85.

[62] Panjwani N, Zhao Z, Raizman MB, Jungalwala F. Pathogenesis

of corneal infection: binding of Pseudomonas aeruginosa to

specific phospholipids. Infect Immun 1996;64:1819–25.

[63] Wu X, Gupta SK, Hazlett LD. Characterization of P. aeruginosa

pili binding human corneal epithelial proteins. Curr Eye Res

1995;14:969–77.

[64] Zaidi TS, Fleiszig SM, Preston MJ, Goldberg JB, Pier GB.

Lipopolysaccharide outer core is a ligand for corneal cell binding

and ingestion of Pseudomonas aeruginosa. Invest Ophthalmol

Visual Sci 1996;37:976–86.

[65] Pohlmann-Dietze P, Ulrich M, Kiser KB, Doring G, Lee JC,

Fournier JM, Botzenhart K, Wolz C. Adherence of Staphylo-

coccus aureus to endothelial cells: influence of capsular poly-

saccharide, global regulator agr, and bacterial growth phase.

Infect Immun 2000;68:4865–71.

[66] Boucher JC, Schurr MJ, Deretic V. Dual regulation of mucoidy in

Pseudomonas aeruginosa and sigma factor antagonism. Mol

Microbiol 2000;36:341–51.

[67] Latifi A, Foglino M, Tanaka K, Williams P, Lazdunski A.

A hierarchical quorum-sensing cascade in Pseudomonas aerugi-

nosa links the transcriptional activators LasR and RhIR (VsmR)

to expression of the stationary-phase sigma factor RpoS. Mol

Microbiol 1996;21:1137–46.


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