Bacterial interactions with contact lenses; effects of lens material, lens wear and microbial physiology
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
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 CIBA Vision, Atlanta, GA, USA; FDA group IV).
Furthermore, in recent years new co-polymers have
Contact lenses are a successful form of vision been incorporated into the soft hydrogel lens materials,
correction and are worn by approximately 85 million including silicone polymers for increased oxygen
people worldwide. Two major types of contact lenses are permeability (e.g. Lotrafilcon A, CIBA Vision, or
commonly worn. These two types are rigid gas perme- Balafilcon A, Bausch and Lomb) and phosphoryl-
able (RGP) lenses and soft hydrogel lenses. The RGP choline to increase biocompatability (e.g. Omafilcon
contact lenses are commonly composed of monomers A, Biocompatables Ltd., UK). Contact lenses can be
containing silicone, fluorine and methylmethacrylate. worn on several wear schedules including daily wear
Soft hydrogel lenses are commonly composed of (the wearer removes the lens each night, cleans and
2-hydroxyethyl methacrylate polymer alone (e.g. Poly- disinfects the lens overnight and returns the same lens
macon, Bausch and Lomb, Rochester, NY, USA; FDA to the eye in the morning [these lenses are commonly
group I) or containing methacrylic acid (e.g. Etafilcon replaced with fresh lenses every month]), daily
A, Vistakon, a division of Johnson and Johnson disposable wear (the wearer removes and discards the
Vision Products Inc, Jacksonville, FL, USA; FDA lens at the end of the day and inserts a new lens into
group IV) and/or N-vinyl pyrrolidone (e.g. Vifilcon A, the eye the next morning), extended wear (the wearer
wears the same lens continuously for, commonly,
*Corresponding author. Tel.: +61-2-9385-7524; fax: +61-2-9385-
6 nights, then removes the lens and inserts a new
7401. lens on the seventh day), and continuous wear
E-mail address: m.willcox@cclru.unsw.edu.au (M.D.P. Willcox). (wearers wear lenses continuously for 30 nights,
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 - 23236 M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247
then discards the lens and inserts a new lens on the thirty Table 1
first day). Bacteria isolated from contact lenses at the time of an adverse response
Occasionally adverse responses to contact lens wear Bacteria Adverse responsea
occur. These adverse responses are frequently caused by
Gram positive bacteria
bacterial contamination of the contact lens surface. Abiotrophia defectiva IK
Contact lens induced corneal adverse responses have Bacillus sp. MK
recently been classified into serious sight threatening Coagulase-negative staphylococcib MK
responses (microbial keratitis [MK; incidence 0.3%]), Corynebacterium sp.b MK
Micrococcus sp.b MK
significant adverse responses (contact lens induced acute
Nocardia sp. MK
red eye [CLARE; incidence 1.4–6.2%], contact lens Propionibacterium acnesb MK
induced peripheral ulcers [CLPU; incidence 0.6–8.7%] Non-hemolytic Streptococcus sp. IK
and infiltrative keratitis [IK; incidence 1.7–5.2%]) and Staphylococcus aureus MK, CLPU
non-significant adverse responses (asymptomatic infil- Streptococcus pneumoniae MK, CLARE, CLPU, IK, AIK
Viridans streptococci MK, IK, AIK
trative keratitis [AIK; incidence 1.5–3.9%] and asymp-
tomatic infiltrates [AI; incidence 5.2%]) [1]. Of these Gram-negative bacteria
adverse responses, bacterial colonization of contact Acinetobacter sp. MK, CLARE, IK, AIK
lenses is one of the initiating factors in MK [2], CLARE Aeromonas hydrophilia CLARE
[3,4], CLPU [5] and certain IK and AIK events [6]. Alcaligenes xylosoxidans subsp. IK
denitrificans
There have been several investigations into the effects
Enterobacter sp. MK, AIK
of contact lenses on the normal ocular microbiota. The Escherichia coli MK, CLARE
normal ocular microbiota in the absence of contact lens Haemophilus influenzae MK, CLARE, IK, AIK
wear is composed almost exclusively of three bacterial Klebsiella sp. MK, CLARE, IK, AIK
types, coagulase negative staphylococci, Corynebacter- Morganella morgani MK
Moraxella sp. MK
ium sp. and Propionibacterium sp. [7,8]. The lids usually
Neisseria sp. IK
harbor a microbiota similar to the normal skin Proteus sp. MK
microbiota and harbor more bacteria of more species Pseudomonas sp. MK, CLARE, CLPU, AIK
than the conjunctiva [9]. During sleep the number of Serratia sp. MK, CLARE, CLPU, IK
bacteria colonizing the conjunctiva and lid increases Stenotrophomonas maltophilia MK, CLARE, AIK
[10]. An increase in the number of bacteria isolated from a
MK, microbial keratitis; CLARE, contact lens induced acute red
the conjunctiva and lids during daily lens wear has been eye; CLPU, contact lens induced peripheral ulcer; IK, infiltrative
reported [9,11], although the types of micro-organisms keratitis; AIK, asymptomatic infiltrative keratitis [1].
b
These bacteria are part of the normal ocular microbiota and as
were not found to differ from non-lens wearing eyes. An
such their significance in the production of adverse responses must be
alteration in the types of micro-organisms was seen with viewed with caution as they could be present as contaminants.
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 Staphylococcus epidermidis or P. aeruginosa strains
finding is significant as Gram-negative organisms are adhere in larger numbers to lenses made from hydro-
common ocular pathogens [13]. Other studies, however, xyethyl methacrylate (HEMA) alone compared to lenses
have reported no differences between wearers and non- made from HEMA plus methacrylic acid [19–21] and
lens wearers although an increase in positive ocular this may be a function of differing water contents [22,23]
cultures was found in former lens users and in or charges of these lens types. A contact lens when
association with certain modes of lens wear and types inserted into the eye rapidly accumulates proteins,
of disinfection systems [8]. glycoproteins and lipids (known as deposits) from the
Table 1 details the types of bacteria that have been tear film to its surface. Therefore, it is likely that, other
isolated from contact lenses at the time of an MK, than contamination upon insertion (which is usually by
CLARE, CLPU, IK or AIK. Pseudomonas aeruginosa is bacteria that are part of the normal microbiota),
the most common cause of MK during contact lens wear bacteria adhere to these adsorbed components rather
[13–16]. Gram-positive bacteria are more commonly than the contact lens material itself. That is not to say
associated with CLPU [5,6,17], whereas gram-negative the contact lens material will not still affect adhesion; the
bacteria are more commonly associated with CLARE types of deposits are likely to be affected by the
[3,4,18]. For CLARE, Haemophilus influenzae is the chemistry of the contact lens. Subsequent to adhesion,
most commonly isolated bacterium [4]. it is likely that bacteria further colonize the lens surface
One of the initial steps in the development of the by growing on that lens surface. Hume and Willcox [24]
bacterially driven adverse responses is the binding of demonstrated that Serratia marcescens was able to grow
bacteria to a contact lens. Several studies have examined on a contact lens after adhesion to contact lenses coated
the ability of bacteria to adhere to contact lenses. in an artificial tear film.M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247 3237
Another important factor in the ability of a bacterium micro-organisms were compared using chi-square test
to adhere to contact lenses is the physiology of that with Yates correction and the level of significance was
bacterium. Recently Cowell et al. [25] demonstrated that set at P ¼ o0:05:
growing Paer1 under nitrogen limitation increased the
adhesion of this strain to Etafilcon A contact lenses. The 2.2. Adhesion of bacteria to contact lens materials in vitro
aims of this investigation were to demonstrate the types
of bacteria that adhere to contact lenses during wear and P. aeruginosa 6294, P. aeruginosa Paer1, Streptococ-
to determine the factors, both material and microbio- cus pneumoniae 001, S. pneumoniae 008, Haemophilus
logical, that can affect the adhesion of bacteria to influenzae 001, H. influenzae 009, Aeromonas hydrophilia
contact lenses. 003, Stenotrophomonas maltophilia 010 were isolated
from cases of CLARE, with the exception of P.
aeruginosa 6294 which was isolated from a case of
2. Materials and methods MK, at the time of presentation. Bacteria were grown to
stationary phase in Trypicase soy broth (Oxoid, Sydney,
2.1. Comparison of bacterial colonization on soft contact Australia) at 351C, then cells were washed three times
lenses worn on different wear schedules with PBS and resuspended to an OD of 1.0 (approxi-
mately 1 108 bacteria/ml) [29]. Previous studies had
Contact lens wearing subjects wore either bilateral demonstrated that, for all lens types used, this optical
Etafilcon A lenses or contra-lateral Etafilcon A in one density gave maximum adhesion and that optical
eye and Polymacon in the other eye, or bilateral densities above this did not show increased adhesion
Lotrafilcon A lenses over a 7 year period. The Etafilcon (date not shown). Bacteria (1 ml) were added to lenses
A and Polymacon lenses were worn on a 6 nights and adhesion was allowed to occur for 10 min. Non-
extended wear schedule or daily wear schedule with adherent cells were then removed by washing in PBS
monthly replacement. The Lotrafilcon A lenses were three times and cells stained with crystal violet prior to
worn on a 30 nights continuous wear schedule. We have enumeration by light microscopy [25]. Lenses used in the
previously demonstrated that there are no differences in experiments were Etafilcon A, Balafilcon A, Polymacon,
the bacterial colonization between Etafilcon A and Omafilcon A. All results were expressed compared to the
Polymacon [9,26] or Etafilcon A and Lotrafilcon A adhesion of the bacterial strains to Etafilcon A contact
lenses [27]. Seventy three subjects wearing extended/ lenses as these are the current market leaders in hydrogel
continuous wear lenses and 39 subjects wearing lenses lenses. All experiments were repeated on three separate
on a daily wear schedule were involved in the study and occasions. Adhesion data were not normally distributed
all were free of ocular diseases, had no ocular surgery and therefore were analysed for differences between lens
and required visual correction for low refractive errors types non-parametrically with the Mann-Whitney
only. Informed consent was obtained from the subjects V-Wilcoxon Rank Sum test.
and all procedures were approved by the University of
New South Wales Human Ethics Committee. Each 2.3. Effect of lens wear on bacterial adhesion in vitro
subject was sampled on average 3 times.
Contact lenses were removed aseptically and trans- Five subjects were instructed to wear contact lenses
ported to the laboratory in sterile phosphate buffered (Etafilcon A, Polymacon or Balafilcon A) in both eyes
saline (PBS) [9]. Bacteria adherent to the contact lenses for 6 h during the day on different days. Lenses were
were grown using an agar sandwich technique [9]. then removed aseptically, washed three times in PBS to
Briefly, lenses were placed concave side up on a remove loosely adsorbed tear film components and
chocolate agar plate and the plate was flooded with bacterial adhesion was measured as described above
molten (561C) agar and placed in a CO2-enriched (Section 2.2). Results were expressed as a percentage
atmosphere (5%) at 351C for 48 h. Aliquots (0.4 ml) of difference compared to control unworn lenses and all
the remaining transport PBS were spread onto three experiments were repeated on at least two separate days.
chocolate and one Sabouraud’s agar plate. The Sabour- Adhesion data were not normally distributed and
aud’s agar plate and one chocolate agar plate were therefore were analysed for differences between worn
incubated aerobically at 351C for 48 h. The Sabouraud’s and unworn lenses non-parametrically with the Mann-
agar plate was subsequently incubated for six days at Whitney V-Wilcoxon Rank Sum test.
ambient temperature. The two remaining chocolate
plates were incubated at 351C either in a CO2-enriched 2.4. Analysis of types of deposits on worn contact lenses
atmosphere for 48 h or anaerobically (95% N2, 5%
CO2) for four days. Microbiological characterization of The types of deposits formed on Polymacon or
the contact lenses was conducted as described previously Etafilcon A lenses that had been worn for 6 h during
[9,26,28]. The incidence rates of various groups of the day were investigated using X-ray photoelectron3238 M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247
spectroscopy (XPS). After wear, lenses were washed in
MilliQ water, dried and sectioned. XPS analysis was
performed as described previously [30] 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-dimen-
sional 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 Fig. 1. Flow cell used for bacterial adhesion and scanning confocal
the channels and sealed with silicone glue (Silicone microscopy. Multi-channel flow cell used in the SCLM study, showing
Rubber Adhesive Sealant, GE Translucent RTV118). the flow cell from above and as a side view in section. Polycarbonate
Flow cells were connected to silicone tubing at plastic is lightly shaded, and the irrigation channels are unshaded. The
hatched line indicates the passage of the irrigation channels through
either end. A Watson Marlow peristaltic pump was the plastic. The contact lens, mounted on the plastic block, is indicated
used to pump the media or washing solutions through in both views. Direction of laminar flow is indicated by the
the flow cells. arrowheads.
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 mixture, or 10% TSB) was pumped through the flow
semicircular lens sample. The block with the affixed lens cell at a very slow rate of flow. The flow cell was left at
sample was placed in the flow cell and sealed with a glass 351C for 10 min. Loosely attached bacteria were washed
coverslip. The gap between the exposed surface of the off by the same method used for removing loosely
contact lens sample and the underside of the coverslip bound protein.
was approximately 4 mm. The growth medium (protein mixture or 10% TSB or
A protein mixture comprised five proteins: lactoferrin PBS) was introduced into the flow cell at a rate of
(bovine colostrum, 1 mg/ml), lysozyme (chicken egg- 2.6 ml/h for 3 days at 251C. RH795 (0.1% in PBS;
white, 1 mg/ml), g-globulins (bovine, 1 mg/ml), albumin Molecular Probes, Eugene, Oregon, USA), which
(bovine serum, 0.1 mg/ml), mucin (bovine submaxillary responds to cell membrane potential and was used to
gland, 0.1 mg/ml) was constructed in PBS. All proteins stain the bacterial cells, was introduced (3 ml) into the
were purchased from Sigma (St. Louis, MO, USA). flow cell by injecting it into the silicone tubing
Although this protein mixture represented a simplified immediately prior to the flow cell and was left for 1 h
version of tear proteins, the exact composition of the at 251C. The flow of solution was then stopped and the
solution was less critical than the fact that it contained glass coverslip was removed. Microscope work was
potentially antibacterial proteins (lactoferrin, lysozyme) carried out using the Bio-Rad MRC 600 SCLM
and a high concentration of glycoproteins (lactoferrin, g- equipped with an argon laser and standard filter sets.
globulins, mucin). To coat the lens samples with protein, The laser was mounted on a Nikon Microphot-FXA
the flow cell was gently filled with the protein mixture. microscope. The microscope was equipped with a 20 X
Lens samples that were to be left uncoated were water immersion lens.
immersed in PBS. Lens samples were left in the flow Four separate conditions were examined for their
cells for 18 h at 351C. Loosely bound protein was effect on Paer1 growth on lens samples, and set up in
washed off using 10 ml of PBS pumped through the flow parallel using the flow cells: (1) PBS was passed over a
cell. Bacteria were grown and washed as described clean lens sample; (2) PBS passed over a lens sample
above (Section 2.2). Bacterial suspension (Paer1, OD660 coated with the protein mixture; (3) protein mixture
¼ 0:1 in PBS) was then introduced until the flow passed over a lens sample coated with the protein
cell was filled. Initially, bacteria were allowed to mixture; (4) 10% TSB passed over a clean lens sample.
adhere to the lens, then a solution (PBS, protein In addition to these conditions, lens samples which wereM.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247 3239
not colonized by bacteria were also observed using 0.02% (w/v) sodium azide to remove the detergent.
SCLM. All experiments were repeated on two separate Proteins were concentrated using Centriprep-10 concen-
occasions. trators (10,000 MW cut-off, Amicon, Beverly, MA).
Protein samples were reduced with DTT (1,4-dithio-
2.6. Effect of bacterial physiology on adhesion threitol, Boehringer Mannheim, Mannheim, Germany)
prior to SDS-PAGE (sodium dodecyl sulphate-poly-
In an effort to further elucidate the mechanisms of acrylamide gel) analysis on 10% acrylamide gels
bacterial adhesion, P. aeruginosa Paer1 was grown in a according to the method of Laemmli [34]. Gels were
chemostat under different environmental conditions. silver-stained to visualise protein bands following the
Paer1 was grown as described previously [25]. Briefly, method of Bjellqvist et al. [35]. Proteins preparations
the bacteria were grown in a defined medium [25] either were extracted from two samples and run on gels to
at 371C or 251C and at a growth rate of either 0.3 or determine consistency. The LPS was extracted using the
0.05 h1. After growth, the bacteria were washed three water/phenol method of Westphal and Jann [36] and the
times in PBS and their adhesion to Etafilcon A contact amount of LPS was analysed using a Coatestt
lenses was measured as described above (Section 2.2) endotoxin kit (Chromogenix AB, Mo. indal, Sweden)
and repeated three times. The cells’ adhesion to followed by sodium dodecylsulfate gel electrophoresis
substituted Sepharose 6-B was also investigated [25]. (10% acrylamide) and visualized by silver diamine
Sepharose 6-B, octyl-, phenyl-, CM- and DEAE- staining.
Sepharose were purchased from Pharmacia LKB
Biotechnology (Uppsala, Sweden). Columns were con-
structed by loading 1 ml (B5 cm) of each Sepharose gel 3. Results
into Pasteur pipettes plugged with glass wool. The void
volume determined using methylene blue was found to 3.1. Comparison of bacterial colonization on soft contact
be 0.5 ml. Columns were equilibrated with 5 ml PBS. lenses worn on different wear schedules
The optical density of the original suspension was
measured at 600 nm. Bacterial suspensions (0.5 ml, Table 2 shows the types of bacteria that were isolated
OD600 ¼ 1:0) were added to the columns and the first from contact lens wearers on an extended or daily wear
eluant (0.5 ml) discarded. A second aliquot of bacteria schedule. The most common bacteria isolated were
(0.5 ml) was added to the column and the eluant gram-positive bacteria including coagulase negative
collected with the first wash of 0.5 ml PBS. Three staphylococci, Propionibacterium sp. and Corynebacter-
additional washes (1 ml) were collected and the absor- ium sp. Of the gram-negative bacteria isolated, Pseudo-
bance at 600 nm of each wash determined. Percent cells monas sp. and Stenotrophomonas sp. were isolated most
retained on each gel, and percent cells subsequently frequently during extended wear, whereas Pseudomonas
desorbed in the next three washes were determined, and sp. and Acinetobacter sp. were isolated most frequently
from this data the net retention (percent of original during daily wear. However, there were no differences in
inoculum) of bacteria on each Sepharose type was the numbers, types or frequency of colonization of
calculated. Retention on Sepharose assays were per- contact lenses worn on either an extended/continuous
formed twice for each incubation condition. wear schedule or daily wear schedule.
2.7. The role of bacterial cell surface proteins 3.2. Bacterial adhesion to soft contact lenses in vitro
and lipopolysaccharide in adhesion
As can be seen (Fig. 2), there was considerable
Experiments were then conducted on the expression variation in adhesion between bacterial strains and
of cell-surface proteins and lipopolysaccharide (LPS) by contact lenses. Three strains of bacteria, P. aeruginosa
P. aeruginosa Paer1 grown at 371C or 251C. Cell-surface Paer1 and 6294 and Aeromonas hydrophilia 003, adhered
proteins were extracted [25,33] using 100 ml of 50 mm in increased numbers to the High DK silicone hydrogel
sodium citrate buffer (sodium citrate/citric acid, pH 4.5) lenses (Balafilcon A) compared to Etafilcon A. The
containing 0.1% Zwittergent (Calbiochem, La Jolla, increase may have been due to the more hydrophobic
CA), 1 mm PMSF (phenylmethyl-sulfonyl fluoride, nature of the underlying contact lens material in the
Boehringer Mannheim, Mannheim, Germany) and high DK lenses. The strains of Streptococcus pneumoniae
10 mm EDTA (ethylenediaminetetraacetic acid, Sigma, were chosen for testing against Omafilcon A lenses as
St. Louis, MO). The reaction mixture was incubated for these bacteria are known to possess receptors for choline
25 min at 451C with occasional mixing. Bacteria were on their surface [37]. Fig. 3 demonstrates that for only
then pelleted by centrifugation at 3200g for 2 h at 41C. one of these strains, Spne 004, there was an increase in
The supernatant containing the extracted proteins was adhesion to Omafilcon A lenses that contain phosphor-
dialysed overnight against distilled water containing ylcholine.3240 M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247
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 Range CFU/ml Frequency of Median number Range Frequency of bacterial
of CFU/lens bacterial contamination of CFU/lens CFU/ml contamination of lensesb
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. 3. Adhesion of bacteria to Omafilcon A in comparison to
Fig. 2. Adhesion of bacteria to various contact lens materials. Etafilcon A hydrogel contact lenses. *Significantly different to
*Significantly different to adhesion to Etafilcon A (po0:05 statistical adhesion to Etafilcon A (po0:05; statistical analysisFMann–Whitney
analysisFMann–Whitney U-test). U-test).M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247 3241
3.3. Effect of lens wear on bacterial adhesion in vitro surface of the worn Polymacon lenses may indicate that
mucin had adsorbed to these lenses.
Using contact lenses that had been worn for 6 h
during the day, bacterial adhesion was examined. In 3.5. The ability of strains to grow on contact lenses
general, the gram-negative bacteria used in the adhesion
assays adhered in lower numbers to worn compared to Fig. 4a shows a clean lens sample which was observed
unworn Etafilcon A lenses (Table 3). On the other hand, under SCLM, without any further treatment. Fig. 4b
bacteria adhered in greater numbers to worn rather than shows the response of bacteria attached to a clean lens
unworn Balafilcon A (High DK) lenses (Table 3). sample after exposure to PBS under laminar flow
Statistical analysis demonstrated a significant conditions. The diffuse nature of the surface may
(Po0:05) increase in adhesion of strains Paer1, Hinf001 represent a very loose aggregation of Paer1 that
and Xmal010 to worn Balafilcon A lenses, Paer1 and obscures the surface of the lens sample. Fig. 4c shows
Hinf001 to worn Polymacon lenses and Ahyd003 to a protein-coated lens sample used in place of a clean lens
worn Etafilcon A lenses. Xmal010 showed a reduced sample. For the next set of experimental conditions,
(Po0:025) adhesion to worn Etafilcon A lenses. bacteria were attached to a lens sample pre-coated with
the protein mixture. The same protein mixture was then
3.4. Analysis of the type of deposit on soft contact lenses passed over these bacteria under laminar flow condi-
tions. Fig. 4d clearly shows the presence of discrete
Table 4 demonstrates that the Etafilcon A lens clumps of bacteria, presumably micro-colonies, over the
adsorbed more nitrogen containing material than the surface. These cannot represent aggregates of adsorbed
Polymacon lens (approximately six times as much), protein, as these were not visible under this magnifica-
indicating more protein was adsorbed to the surface. tion. The response of bacteria attached to a clean lens
The worn Polymacon lens adsorbed very little protein sample when exposed to a 10% solution of TSB was
and only on its front surface. Interestingly, the small quite distinct. Although putative bacterial aggregations
decrease in carbon (and increase in oxygen) on the 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 43b723 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.3242 M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247
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 10–15 mm in diameter.
from the micro-colonies shown in Fig. 4c and d, and Table 5
probably represented flocculation. Within 48 h the The effect of growth conditions on retention of P. aeruginosa to
Sepharose
solution inside the flow cell was observed to be very
turbid and this was not observed for any other of the Sepharose type Growth conditionsa
sets of conditions. These enriched conditions obviously Control Low temperature Slow growth rate
generated very strong growth of bacteria in suspension.
Sepharose 6-B (control) 1371 2973 3775
If any biofilm was present, it was very diffuse and
Octyl 5275 6474 6675
loosely attached to the surface, and can probably be best Phenyl 7274 7173 5674
described as flocculation. Under these extremely nu- DEAE 9971 10070 9970
trient-rich conditions, there was apparently no impera- CM 2071 1972 4475
tive for bacteria to attach to a solid surface. a
All cells were grown in a chemostat in defined media [25] with the
following conditions: control, 371C and 0.3 h1 dilution rate; low
3.6. Effect of bacterial physiology on adhesion temperature, 251C and 0.3 h1 dilution rate; slow growth rate, 37oC
and 0.05 h1 dilution rate.
Table 5 demonstrates the changes that occurred in the
surface charge and hydrophobicity of Paer1 under the the adhesion to Phenyl, DEAE or CM-Sepharose. A
different conditions. Low growth temperature increased slow growth rate increased the adhesion to control
adhesion to the control Sepharose 6-B by 55% and to Sepharose 6-B by 65%, to Octyl-Sepharose by 21% and
Octyl-Sepharose by 19% but did not appreciably alter to CM-Sepharose by 55% but decreased adhesion toM.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247 3243
Phenyl-Sepharose by 56%. Fig. 5 demonstrates the micro-organisms are readily cleared from the lens
adhesion of bacterial cells grown under the different surface by the ocular defense mechanisms. Other sources
conditions to Etafilcon A lenses. Adhesion was highest of contamination of lenses by the normal microbiota
when cells were grown at low temperature and relatively include the eyelids of wearers [41] or from environ-
high growth rates, followed by growth at high tempera- mental sources [41]. Contamination of lenses during
ture and slow growth rate and adhesion was least when wear is sporadic. Subjects sampled on successive days of
cells were grown at high growth rates and high extended lens wear, from 1 night to 13 nights, were as
temperature. Whilst the hydrophobicity (adhesion to likely to have contaminated lenses on Day 1 as on Day
octyl or phenyl Sepharose) and charge (adhesion to 13 [42]. In other words, wearing lenses for increasing
either DEAE or CM-Sepharose) also changed with grow lengths of time did not result in increasing microbial
conditions, there was no direct correlation between contamination.
adhesion to the lenses and adhesion to the substituted However, some adverse responses that occur during
Sepharose polymers probably demonstrating the ability lens wear are known to be associated with bacterial
of bacterial cells to utilize several mechanisms to adhere. adhesion to lenses [2–6]. These bacteria do not make up
part of the normal ocular microbiota. In order for
3.7. The role of bacterial cell surface proteins and bacteria to initiate an adverse response, they must be
lipopolysaccharide in adhesion able to adhere to the lens surface. The current study
demonstrated that there were differences in the ability of
Fig. 6 demonstrates that growth at 251C altered the the bacteria isolated from adverse responses to adhere to
expression of a number of cell-surface proteins (marked lens materials and for most material/strain combina-
by either a red arrow head or enclosed in a red circle) tions there were increases in adhesion to worn lenses or
compared to growth at 371C. Similarly, growth at 251C no differences between adhesion to worn or unworn
altered the expression of LPS, yielding larger LPS lenses. It was demonstrated that worn lenses did adsorb
molecules that did not migrate as far into the gel matrix tear film components, probably proteins/glycoproteins,
(Fig. 7). 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,
4. Discussion that bound to the contact lenses were conducive to
bacterial adhesion. To date there are no publications on
The current study has confirmed that there was no the types of proteins or other molecules that bind to the
difference in the colonization of contact lenses worn on high DK lenses which demonstrated the most noticeable
an extended or daily wear schedule during asympto- increases in adhesion to worn lenses, although one
matic lens wear. Lens contamination during asympto- abstract at the British Contact Lens Association annual
matic lens wear appears to involve small numbers of meeting in May 2000 reported more deposition on the
micro-organisms [38]. The most common bacteria surface of Balafilcon A lenses compared to Etafilcon A
isolated from contact lenses are coagulase-negative lenses (however, no biochemical analysis of the deposits
staphylococci [8,12,26,39]. Contact lens contamination was reported) [43]. Protein adsorbs more readily to less
commonly occurs through lens handling [40] but it hydrophilic surfaces compared to the surface carrying
appears that during uncomplicated lens wear these an anionic charge such as that of an Etafilcon A lens
[44]. The exception to this is the adsorption of lysozyme
to anionic lenses [45,46]. Also, the anionic lenses tend to
adsorb less lipid [47], 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 [40]. However,
deposits on lenses did increase adhesion in one study
[52], 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
Fig. 5. Effect of growth condition on adhesion of P. aeruginosa to found, although worn lenses did usually increase the
Etafilcon A contact lenses. *Significantly different to adhesion to adhesion of strains [53]. Albumin coated onto the
control (po0:02; statistical analysisFMann–Whitney U-test). surface of Etafilcon A or Polymacon contact lenses3244 M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247 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 h1, B the Paer1 grown at 251C and a dilution rate of 0.3 h1. 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. increased the adhesion of P. aeruginosa [21]. Similarly, Micro-colony production is the prelude to biofilm some strains of Serratia marcescens adhered better to formation [55]. After initial adhesion, adherent bacteria Etafilcon A lenses coated in an artificial tear fluid [24]. may proliferate on the substratum within the polysac- Lysozyme adsorbed to a contact lens increases the charide-rich glycocalyx, forming micro-colonies [55]. As adhesion of Staphylococcus aureus to Etafilcon A these micro-colonies grow and recruit planktonic contact lenses [54]. bacteria, they coalesce with neighboring micro-colonies Factors in addition to adhesion are likely to to form fully-developed biofilms [55]. contribute to the production of adverse reponses. One The affect of changing the environmental conditions such pathogenic trait would be the ability of the adhered that the bacterium P. aeruginosa Paer1 was grown under bacteria to grow on the tear film components that have were also measured and shown to change adhesion adsorbed to the lens surface. Using SCLM observation, properties. Growth under conditions that the bacteria Paer1 grew under conditions in which soluble protein are likely to grown under in environmental conditions was passed over bacteria attached to the lens surface. [41], such as slow growing and decreased temperature,
M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247 3245
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
Fig. 7. Differential expression of lipopolysaccharide on the surface of antibiotics although problems with bacterial resistance
P. aeruginosa grown at 251C or 371C. 371C is the Paer1 grown at 371C might arise), or affect cell metabolism by interfering with
and a dilution rate of 0.3 h1, 251C the Paer1 grown at 251C and a global regulators of gene expression (such as the arg
dilution rate of 0.3 h1.
system in S. aureus [65] or s factors in P. aeruginosa
[66,67]) should be investigated.
significantly altered the surface properties of the
bacterium. However, no change in the surface properties
of the bacterium was directly correlated with the Acknowledgements
changes in adhesion to contact lenses demonstrating
the ability of bacterial cells to utilize several mechanisms Dr Heather St. John, CSIRO Division of Molecular
to adhere. Indeed, it has been demonstrated that P. Sciences, Clayton, Vic, Australia for analyzing the worn
aeruginosa can use several cell surface structures to lenses using XPS. Dr R. Schneider, School of Micro-
adhere to epithelial cells [56–64]. biology and Immunology, University of New South
The LPS has been demonstrated to be involved in the Wales, NSW, Australia, for help with bacterial growth
adhesion of P. aeruginosa to corneal epithelial cells in a chemostat. Dr Gideon Wolfaardt, Applied Micro-
[57,58,64]. The present study demonstrated that altering biology and Food Science, College of Agriculture,
the LPS of P. aeruginosa Paer1 increased its adhesion to Saskatoon, Canada for help with the scanning confocal
Etafilcon A lenses. Similarly, there were changes in the microscopy. Dr Ben Herbert, Australian Proteome
outer membrane proteins that were expressed on Paer1 Analysis Facility, University of Macquarie, NSW,
that may have affected its adhesion to contact lenses. Australia, for help with the 2D gel electrophoresis of
Cowell et al. [25] demonstrated that growth of P. bacterial proteins.
aeruginosa Paer1 under conditions of nitrogen or carbon
limitation also altered the ability of this strain to adhere
References
to Etafilcon A lenses and altered the 2D protein profile.
Interestingly the changes that occurred in the protein [1] Holden BA, Sankaridurg P, Jalbert I. Adverse responses. Which
profile under nitrogen limited conditions, which resulted ones and how many. In: Sweeney DF, editor. Silicone hydrogels.
in increased adhesion to contact lenses, were not the The rebirth of continuous wear contact lenses. Oxford: Butter-
same as those changes that occurred during growth at worth Heinmann, 2000. p. 150–213.
[2] Solomon OD, Loff H, Perla B, Kellis A, Belkin J, Roth AS,
251C indicating the flexibility that there is in the Zucker J. Testing hypotheses for risk factors for contact lens-
mechanisms of adhesion of P. aeruginosa to contact associated infectious keratitis in an animal model. CLAO
lenses. J 1994;20:109–13.3246 M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247
[3] Holden BA, La Hood D, Grant T, Newton-Howes J, Baleriola- [22] Cook AD, Sagers RD, Pitt WG. Bacterial adhesion to protein-
Lucas C, Willcox MD, Sweeney DF. Gram-negative bacteria can coated hydrogels. J Biomater Appl 1993;8:72–89.
induce contact lens related acute red eye (CLARE) responses. [23] Cook AD, Sagers RD, Pitt WG. Bacterial adhesion to
CLAO J 1996;22:47–52. poly(HEMA)-based hydrogels. J Biomed Mater Res
[4] Sankaridurg PR, Willcox MD, Sharma S, Gopinathan U, 1993;27:119–26.
Janakiraman D, Hickson S, Vuppala N, Sweeney DF, Rao GN, [24] Hume EB, Willcox MD. Adhesion and growth of Serratia
et al. Haemophilus influenzae adherent to contact lenses associated marcescens on artificial closed eye tears soaked hydrogel contact
with production of acute ocular inflammation. J Clin Microbiol lenses. Aust NZ J Ophthalmol 1997;25:S39–41.
1996;34:2426–31. [25] Cowell BA, Willcox MD, Herbert B, Schneider RP. Effect of
[5] Jalbert I, Willcox MD, Sweeney DF. Isolation of Staphylococcus nutrient limitation on adhesion characteristics of Pseudomonas
aureus from a contact lens at the time of a contact lens-induced aeruginosa. J Appl Microbiol 1999;86:944–54.
peripheral ulcer: case report. Cornea 2000;19:116–20. [26] Gopinathan U, Stapleton F, Sharma S, Willcox MD, Sweeney
[6] Sankaridurg PR, Sharma S, Willcox M, Sweeney DF, Naduvilath DF, Rao GN, Holden BA. Microbial contamination of hydrogel
TJ, Holden BA, Rao GN. Colonization of hydrogel lenses with contact lenses. J Appl Microbiol 1997;82:653–8.
Streptococcus pneumoniae: risk of development of corneal [27] Keay L, Willcox M, Sweeney D, Morris C, Harmis N, Corrigan
infiltrates. Cornea 1999;18:289–95. K, Holden BA. Bacterial populations on thirty night extended
[7] Perkins RE, Kundsin RB, Pratt MV, Abrahamsen I, Leibowitz wear silicone hydrogel lenses. CLAO J 2001, in press.
HM. Bacteriology of normal and infected conjunctiva. J Clin [28] Leitch EC, Harmis NY, Corrigan KM, Willcox MD. Identifica-
Microbiol 1975;1:147–9. tion and enumeration of staphylococci from the eye during soft
[8] Fleiszig SM, Efron N. Microbial flora in eyes of current and contact lens wear. Opto Vision Sci 1998;75:258–65.
former contact lens wearers. J Clin Microbiol 1992;30:1156–61. [29] Williams TJ, Willcox MD, Schneider RP. Interactions of bacteria
[9] Stapleton F, Willcox MD, Fleming CM, Hickson S, Sweeney DF, with contact lenses: the effect of soluble protein and carbohydrate
Holden BA. Changes to the ocular biota with time in extended- on bacterial adhesion to contact lenses. Opto Vision Sci
and daily-wear disposable contact lens use. Infect Immun
1998;75:266–71.
1995;63:4501–5. [30] Garrett Q, Chatelier RC, Griesser HJ, Milthorpe BK. Effect of
[10] Ramachandran L, Sharma S, Sankaridurg PR, Vajdic CM,
charged groups on the adsorption and penetration of proteins
Chuck JA, Holden BA, Sweeney DF, Rao GN. Examination of
onto and into carboxymethylated poly(HEMA) hydrogels.
the conjunctival microbiota after 8 hours of eye closure. CLAO
Biomaterials 1998;19:2175–86.
J 1995;21:195–9.
[31] Gorman SP, Mawhinney WM, Adair CG, Issouckis M. Confocal
[11] Larkin DF, Leeming JP. Quantitative alterations of the commen-
laser scanning microscopy of peritoneal catheter surfaces. J Med
sal eye bacteria in contact lens wear. Eye 1991;5:70–4.
Microbiol 1993;38:411–7.
[12] Hovding G. The conjunctival and contact lens bacterial flora
[32] Lawrence JR, Neu TR. Confocal laser scanning microscopy for
during lens wear. Acta Ophthalmol 1981;59:387–401.
analysis of microbial biofilms. Methods Enzymol 1999;310:
[13] Schein OD, Ormerod LD, Barraquer E, Alfonso E, Egan KM,
131–44.
Paton BG, Kenyon KR. Microbiology of contact lens-related
[33] Carnoy C, Scharfman A, Van Brussel E, Lamblin G, Ramphal R,
keratitis. Cornea 1989;8:281–5.
Roussel P. Pseudomonas aeruginosa outer membrane adhesins for
[14] Tabbara KF, El-Sheikh HF, Aabed B. Extended wear
contact lens related bacterial keratitis. Br J Ophthalmol human respiratory mucus glycoproteins. Infect Immun
2000;84:327–8. 1994;62:1896–900.
[15] Patrinely JR, Wilhelmus KR, Rubin JM, Key JEd. Bacterial [34] Laemmli UK. Cleavage of structural proteins during the assembly
keratitis associated with extended wear soft contact lenses. CLAO of the head of bacteriophage T4. Nature 1970;227:680–5.
J 1985;11:234–6. [35] Bjellqvist B, Pasquali C, Ravier F, Sanchez JC, Hochstrasser D. A
[16] Galentine PG, Cohen EJ, Laibson PR, Adams CP, Michaud R, nonlinear wide-range immobilized pH gradient for two-dimen-
Arentsen JJ. Corneal ulcers associated with contact lens wear. sional electrophoresis and its definition in a relevant pH scale.
Arch Ophthalmol 1984;102:891–4. Electrophoresis 1993;14:1357–65.
[17] Willcox M, Sweeney D, Sharma S, Gopinathan U, Sankaridurg P, [36] Westphal O, Jann K. Bacterial lipopolysaccharides. Extraction
Ramachandran L, Holden B. Culture negative peripheral ulcers with phenol-water and further applications of the procedure. In:
are associated with increased bacterial contamination of contact Whistler RL, Wolfron ML, editors. Methods in carbohydrate
lenses. In: The Association for Research in Vision and Ophthal- chemistry, vol. 5. New York: Academic Press, 1965.
mology, Fort Lauderdale, 1995. [37] Rosenow C, Ryan P, Weiser JN, Johnson S, Fontan P, Ortqvist
[18] Willcox MDP, Sankaridurg P, Lan J, Pearce D, Thakur A, Zhu A, Masure HR. Contribution of novel choline-binding proteins to
H, Keay L, Stapleton F. Inflammation and infection and effects of adherence, colonization and immunogenicity of Streptococcus
the closed eye. In: Sweeney DF, editor. Silicone hydrogels. The pneumoniae. Mol Microbiol 1997;25:819–29.
rebirth of continuous wear contact lenses. Oxford: Butterworth [38] Hart DE, Reindel W, Proskin HM, Mowrey-McKee MF.
Heinemann, 2000. p. 45–75. Microbial contamination of hydrophilic contact lenses: quantita-
[19] Fleiszig SM, Evans DJ, Mowrey-McKee MF, Payor R, Zaidi TS, tion and identification of microorganisms associated with contact
Vallas V, Muller E, Pier GB. Factors affecting Staphylococcus lenses while on the eye. Opto Vision Sci 1993;70:185–91.
epidermidis adhesion to contact lenses. Opto Vision Sci [39] Fleiszig SM, Efron N. Conjunctival flora in extended wear of rigid
1996;73:590–4. gas permeable contact lenses. Opto Vision Sci 1992;69:354–7.
[20] Cowell BA, Willcox MD, Schneider RP. A relatively small change [40] Mowrey-McKee MF, Monnat K, Sampson HJ, Smith CM,
in sodium chloride concentration has a strong effect on adhesion Davies GA, Mandt L, Proskin HM. Microbial contamination of
of ocular bacteria to contact lenses. J Appl Microbiol hydrophilic contact lenses. Part I: quantitation of microbes on
1998;84:950–8. patient worn-and-handled lenses. CLAO J 1992;18:87–91.
[21] Taylor RL, Willcox MD, Williams TJ, Verran J. Modulation of [41] Willcox MD, Power KN, Stapleton F, Leitch C, Harmis N,
bacterial adhesion to hydrogel contact lenses by albumin. Opto Sweeney DF. Potential sources of bacteria that are isolated from
Vision Sci 1998;75:23–9. contact lenses during wear. Opto Vision Sci 1997;74:1030–8.M.D.P. Willcox et al. / Biomaterials 22 (2001) 3235–3247 3247
[42] Sweeney DF, Stapleton F, Leitch C, Willcox MDP, Taylor J, [55] Hoyle BD, Costerton JW. Bacterial resistance to antibiotics: the
Holden BA. The microbial colonisation of soft contact lenses over role of biofilms. Prog Drug Res 1991;37:91–105.
time. Opto Vision Sci 2001, in press. [56] Gupta SK, Berk RS, Masinick S, Hazlett LD. Pili and
[43] Franklin V. A study of the spoilation profiles of high DK lipopolysaccharide of Pseudomonas aeruginosa bind to the
fluorosilicone hydrogel lenses. In: British Contact Lens Associa- glycolipid asialo GM1. Infect Immun 1994;62:4572–9.
tion 24th Annual Clinical Conference and Exhibition. Birming- [57] Gupta SK, Masinick S, Garrett M, Hazlett LD. Pseudomonas
ham, UK, 2000. aeruginosa lipopolysaccharide binds galectin-3 and other human
[44] Bohnert JL, Horbett TA, Ratner BD, Royce FH. Adsorption of corneal epithelial proteins. Infect Immun 1997;65:2747–53.
proteins from artificial tear solutions to contact lens materials. [58] Hazlett LD. Bacterial lipopolysaccharide binding to host recep-
Invest Ophthalmol Visual Sci 1988;29:362–73. tors [letter; comment]. Trends Microbiol 1996;4:418.
[45] Deng XM, Castillo EJ, Anderson JM. Surface modification of [59] Hazlett L, Rudner X, Masinick S, Ireland M, Gupta S. In the
soft contact lenses: silanization, wettability and lysozyme adsorp- immature mouse, Pseudomonas aeruginosa pili bind a 57-kd
tion studies. Biomaterials 1986;7:247–51. (alpha 2–6) sialylated corneal epithelial cell surface protein: a first
[46] Sack RA, Sathe S, Hackworth LA, Willcox MD, Holden BA, step in infection. Invest Ophthalmol Visual Sci 1995;36:634–43.
Morris CA. The effect of eye closure on protein and comple- [60] Hazlett LD, Rudner XL. Investigations on the role of flagella in
ment deposition on Group IV hydrogel contact lenses: adhesion of Pseudomonas aeruginosa to mouse and human corneal
relationship to tear flow dynamics. Curr Eye Res 1996;15: epithelial proteins. Ophthalmic Res 1994;26:375–9.
1092–100. [61] Hazlett LD, Moon MM, Strejc M, Berk RS. Evidence for N-
[47] Jones L, Evans K, Sariri R, Franklin V, Tighe B. Lipid and acetylmannosamine as an ocular receptor for P. aeruginosa
protein deposition of N-vinyl pyrrolidone-containing group II and adherence to scarified cornea. Invest Ophthalmol Visual Sci
group IV frequent replacement contact lenses. CLAO J 1997; 1987;28:1978–85.
23:122–6. [62] Panjwani N, Zhao Z, Raizman MB, Jungalwala F. Pathogenesis
[48] Keith D, Hong B, Christensen M. A novel procedure for the of corneal infection: binding of Pseudomonas aeruginosa to
extraction of protein deposits from soft hydrophilic contact lenses specific phospholipids. Infect Immun 1996;64:1819–25.
for analysis. Curr Eye Res 1997;16:503–10. [63] Wu X, Gupta SK, Hazlett LD. Characterization of P. aeruginosa
[49] Tighe BJ, Jones L, Evans K, Franklin V. Patient-dependent and pili binding human corneal epithelial proteins. Curr Eye Res
material-dependent factors in contact lens deposition processes. 1995;14:969–77.
Adv Exp Med Biol 1998;438:745–51. [64] Zaidi TS, Fleiszig SM, Preston MJ, Goldberg JB, Pier GB.
[50] Maissa C, Franklin V, Guillon M, Tighe B. Influence of contact Lipopolysaccharide outer core is a ligand for corneal cell binding
lens material surface characteristics and replacement frequency and ingestion of Pseudomonas aeruginosa. Invest Ophthalmol
on protein and lipid deposition. Opto Vision Sci 1998;75: Visual Sci 1996;37:976–86.
697–705. [65] Pohlmann-Dietze P, Ulrich M, Kiser KB, Doring G, Lee JC,
[51] Fleiszig SM, Zaidi TS, Pier GB. Mucus and Pseudomonas Fournier JM, Botzenhart K, Wolz C. Adherence of Staphylo-
aeruginosa adherence to the cornea. Adv Exp Med Biol 1994; coccus aureus to endothelial cells: influence of capsular poly-
350:359–62. saccharide, global regulator agr, and bacterial growth phase.
[52] Butrus SI, Klotz SA. Contact lens surface deposits increase the Infect Immun 2000;68:4865–71.
adhesion of Pseudomonas aeruginosa. Curr Eye Res 1990;9: [66] Boucher JC, Schurr MJ, Deretic V. Dual regulation of mucoidy in
717–24. Pseudomonas aeruginosa and sigma factor antagonism. Mol
[53] Williams TJ, Willcox MD, Schneider RP. Role of tear fluid in the Microbiol 2000;36:341–51.
growth of gram-negative bacteria on contact lenses. Aust NZ [67] Latifi A, Foglino M, Tanaka K, Williams P, Lazdunski A.
J Ophthalmol 1997;25:S30–2. A hierarchical quorum-sensing cascade in Pseudomonas aerugi-
[54] Thakur A, Chauhan A, Willcox MD. Effect of lysozyme on nosa links the transcriptional activators LasR and RhIR (VsmR)
adhesion and toxin release by Staphylococcus aureus. Aust NZ to expression of the stationary-phase sigma factor RpoS. Mol
J Ophthalmol 1999;27:224–7. Microbiol 1996;21:1137–46.You can also read
