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Immunology and Microbiology  |   September 2012
The Role of Type III Secretion System and Lens Material on Adhesion of Pseudomonas aeruginosa to Contact Lenses
Author Affiliations & Notes
  • Elizabeth P. Shen
    From the Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan;
    Department of Ophthalmology, Buddhist Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan;
    Department of Ophthalmology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan;
  • Ruey-Yug Tsay
    Institute of Biomedical Engineering, National Yang Ming University, Taipei, Taiwan;
  • Jean-San Chia
    Graduate Institute of Microbiology, National Taiwan University, Taipei, Taiwan;
  • Semon Wu
    Department of Life Science, Chinese Culture University, Taipei, Taiwan; and
    Department of Medical Research, Buddhist Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan.
  • Jing-Wen Lee
    Department of Ophthalmology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan;
  • Fung-Rong Hu
    Department of Ophthalmology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan;
  • Corresponding author: Fung-Rong Hu, Department of Ophthalmology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan; fungronghu@ntu.edu.tw
Investigative Ophthalmology & Visual Science September 2012, Vol.53, 6416-6426. doi:10.1167/iovs.11-8184
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      Elizabeth P. Shen, Ruey-Yug Tsay, Jean-San Chia, Semon Wu, Jing-Wen Lee, Fung-Rong Hu; The Role of Type III Secretion System and Lens Material on Adhesion of Pseudomonas aeruginosa to Contact Lenses. Invest. Ophthalmol. Vis. Sci. 2012;53(10):6416-6426. doi: 10.1167/iovs.11-8184.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To determine the distribution of invasive and cytotoxic genotypes among ocular isolates of P. aeruginosa and investigate the influence of the type III secretion system (T3SS) on adhesion to conventional, cosmetic, and silicone hydrogel contact lenses (CL).

Methods.: Clinical isolates from 2001 to 2010 were analyzed by multiplex PCR for exoS, exoU, and exoT genes. Bacterial adhesion to etafilcon, nelfilcon (gray colored), balafilcon, and galyfilcon CL with or without artificial tear fluid (ATF) incubation were compared. Surface characteristics were determined with scanning electron microscopy (SEM).

Results.: Among 87 total isolates, 64 strains were from microbial keratitis cases. CL-related microbial keratitis (CLMK) isolates were mostly of the cytotoxic genotype (expressing exoU) (P = 0.002). No significant differences were found in bacterial adhesion to all types of CL between the genotypes under T3SS-inducing conditions. A trend for least bacterial adhesion of galyfilcon compared to the other CL was noted for both genotypes. Needle complex pscC mutants adhered less to all materials than the wild type (P < 0.05), indicating a role of the T3SS in contact lens adhesion. ATF-incubated CL had significantly more bacterial adhesion (P < 0.05). SEM showed most of the bacteria adhering on CL surfaces.

Conclusions.: CLMK isolates were mostly of cytotoxic genotype. Different genotypes did not significantly differ in its adhesion to various CL. T3SS and other adhesins are involved in bacteria–contact lens adhesion through complex interactions. Contact lens materials may also play an important role in the adherence of both genotypes of P. aeruginosa .

Introduction
P s eudomonas aeruginosa is the most commonly isolated Gram-negative bacteria causing microbial keratitis (MK) in contact lens wearers. 16 The incidence of contact lens (CL)-related MK (CLMK) is reported to be approximately 3.5 to 20.9 per 10,000 wearers, depending on the contact lens material and wearing schedules. 2,6,7 This incidence is expected to rise with the increasing popularity of cosmetic color-tinted soft contact lenses for emmetropic individuals. 8,9 Because CLMK often occurs in the younger population, 10,11 there have been extensive efforts to prevent this sight-threatening complication and find the perfect contact lens. 5,12 Research has shown that corneal hypoxia due to contact lens wear induces lipid raft formation which increases susceptibility to P. aeruginosa infection. 13,14 Silicone hydrogel materials, which are highly permeable to oxygen, were developed to prevent possible corneal hypoxia induced by conventional hydrogel materials and thus hopefully reduce the incidence of CLMK. However, recent reports indicate that the annual incidence of CLMK for daily wear silicone hydrogel CL wearers is approximately 6 times greater than for daily wear or for daily disposable conventional soft CL wearers. 15 Even after other risk factors are adjusted for, daily wearers of silicone hydrogels still showed a higher risk of infection than conventional hydrogel wearers, albeit not reaching a statistically significant difference (odds ratio, 2.6; 95% confidence interval [CI], 1.0–7.1). 15 Clearly, increasing the oxygen permeability of contact lenses did not significantly lower the occurrence of CLMK. Other factors related to the contact lens material or bacterial and ocular surface interactions must also be considered. 
Development of CLMK starts with bacterial contamination and subsequent bacterial adhesion to contact lenses, causing prolonged exposure of the cornea to microbial pathogens. 14,16 The material of each type of contact lens displays various surface properties such as hydrophobicity, wettability, and roughness. These surface properties may influence the propensity of bacteria to adhere to contact lens. 1719 The relatively hydrophobic silicone hydrogel contact lens balafilcon (Purevision; Bausch & Lomb, Taipei, Taiwan) was previously shown to have greater adherence of P. aeruginosa bacteria. 2022 However, other investigators reported contradictory results. 23 These disparate results may be due to differences between bacterial serotypes and its growth conditions as previous reports had indicated (i.e., nutrient limitation, growth temperature, and surface hydrophobicity). 21,2426  
Two phenotypes of P. aeruginosa , based on the secretion of certain type III secretion system (T3SS) toxins, were previously discovered. 1 The T3SS is a specialized protein export system that forms a needle-like complex between bacterial and host cells for the transport and secretion of four exotoxins, ExoS, ExoU, ExoT, and ExoY. 27 The PscC protein is an essential structural component of the needle complex located on the outer membrane of P. aeruginosa cells. 27,28 Mutation in the PscC protein results in loss of cytotoxicity and the T3SS exotoxin secretion. 2830 As most strains carry the exoT and exoY genes, P. aeruginosa strains that have the exoS gene encoding the protein ExoS but not the exoU gene can invade corneal epithelial cells and are, thus, known as invasive strains. 1,3134 Cytotoxic strains, on the other hand, carrying the exoU-positive (exoU +) and exoS-negative (exoS ) genotypes cause acute host cell lysis by the production of ExoU, a phospholipase. 32,35 Invasive and cytotoxic phenotypes with their respective exoS and exoU genotypes were found to be mutually exclusive in nearly all strains. 1 Previously, reports with a sufficient number of clinical P. aeruginosa isolates found a significantly higher prevalence of cytotoxic strains among contact lens wearers. 36,37 Cytotoxic strains were also found to be highly correlated with antibiotic resistance. 38,39 Interestingly, environmental and clinical isolates from lung, urinary tract, or burn wound infections were mostly invasive. 37,40,41 Thus, the higher prevalence of the cytotoxic genotype specifically to contact lens wearers poses the possibility of T3SS involvement either directly or indirectly with adhesion to contact lens materials. 
Expression of the T3SS usually requires close cell contact in vivo or low calcium growth conditions in vitro. 4244 Contamination of contact lenses with adhering bacteria may provide a chance for relatively close contact with corneal epithelium. However, calcium normally present in tears 4547 may repress activation of the T3SS. Thus, interaction of contact lenses with tear fluid should also be considered in experiments involving P. aeruginosa adhesion to contact lens materials to better mimic physiologic conditions. 
In this study, we first determined the distribution of invasive and cytotoxic genotypes among clinical ocular isolates and confirmed the higher distribution of the cytotoxic genotype specifically among CLMK isolates. We then compared the adhesion of different genotypes of P. aeruginosa to conventional hydrogel, cosmetic hydrogel lenses, and silicone hydrogel contact lenses and found less bacterial adhesion to non–surface-treated silicone hydrogel lenses. Strains with the needle complex PscC protein mutation with a different T3SS genotype background were shown to adhere significantly less to various contact lens materials than wild-type strains. A general increase in bacterial adhesion to all contact lenses incubated with artificial tear fluid (ATF) was also shown. Scanning electron microscopy (SEM) of CL surfaces demonstrated an association between CL surface morphology and bacterial adhesion. 
Materials and Methods
Contact Lenses
Four types of contact lenses used in this study were provided by their corresponding manufacturers. Their characteristics are listed in Table 1. 19,48 Etafilcon A (Acuvue 2, Vistakon; Johnson & Johnson, Taipei, Taiwan) is a conventional hydrogel contact lens material. Nelfilcon A (FreshLook; Ciba Vision, Taipei, Taiwan) is a color-tinted cosmetic hydrogel lens. All nelficon A lenses used in this study were tinted gray. Balafilcon A (Purevision; Baush & Lomb) and galyfilcon A (Acuvue advance, Vistakon; Johnson & Johnson) are silicone hydrogel contact lenses. 
Table 1. 
 
Characteristics of Soft Contact Lenses Used in this Study
Table 1. 
 
Characteristics of Soft Contact Lenses Used in this Study
Characteristic US Adopted Name
Etafilcon A Nelfilcon A Balafilcon A Galyfilcon A
Commercial name Acuvue 2 Freshlook Purevision Acuvue Advance
Manufacturer Vistakon Ciba Vision GmbH Bausch & Lomb Vistakon
FDA group IV II III I
Material Hydrogel Color-tinted hydrogel (gray) Silicone hydrogel Silicone hydrogel
Water content (%) 58 69 36 47
Oxygen permeability (Barrers) 28 26 100 60
Surface treatment None None Plasma oxidation treatment No surface treatment; internal wetting agent (PVP)
Artificial Tears
Artificial tears were constructed with phosphate buffered saline (PBS; One-Star Biotechnology, Taipei, Taiwan), 0.3 mM CaCl2, and the following proteins: lactoferrin (bovine colostrum, 1 mg/mL), lysozyme (chicken egg white, 1mg/mL), γ-globulin (bovine, 1mg/mL), mucin (bovine submaxillary gland, 0.1 mg/mL), and bovine serum albumin (0.1mg/mL). 21 All proteins were purchased from Sigma-Aldrich (St. Louis, MO). 
Bacterial Strains and Culture Conditions
Clinical isolates of P. aeruginosa were collected from cases of ocular infection seen at the Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan, from 2001 to 2010. All strains were confirmed as P. aeruginosa by green pigment production and positive cytochrome oxidase test (BD oxidase reagent droppers; BD Biosciences, San Jose, CA). Isolates were kept as frozen stocks and were thawed when needed for experimentation. Bacteria were grown to stationary phase in 15 mL of trypticase soy broth (TSB), considered as the noninducing condition (BD Biosciences), or grown in TSB supplemented with 1% glycerol, 100 mM monosodium glutamate, and 2 mM EGTA, which was considered the inducing medium, at 37°C and then harvested by centrifugation at 9600g for 10 minutes. The pellet was washed once with 5 mL of normal saline and resuspended to various concentrations as required for experimentation. 
Genotyping
Multiplex PCR was performed with all clinical P. aeruginosa strains to determine the presence of the exoU, exoS, and exoT genes. Using primer pairs reported by Ajayi et al, 49 we amplified the following gene fragments: exoU (134 basepairs [bp]), exoS (118 bp), and exoT (153 bp). Bacteria were grown overnight at 37°C in TSB, and DNA was isolated by using a DNA purification kit according to the manufacturer's protocol (Viogene, Taipei, Taiwan). The PCR mixture consisted of 1 μL of DNA template (500 ng), 1 μL of total PCR primers (MDBio, Inc., Taipei, Taiwan), a final 40 mM concentration of each primer, 12.5 μL of 2× GoTaq Green Master Mix (Promega Corp., Madison, WI), and 10.5 μL of sterile water. The negative control contained GoTaq Green Master Mix, no DNA, and 11.5 μL of sterile water. The standard reaction mixture included 1 μL each of bacterial DNA and GoTaq Green Master Mix and 10.5 μL of sterile water. PCR amplification was carried out as follows: initial denaturation at 94°C for 10 minutes; 35 cycles at 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 45 seconds; and a final extension step at 72°C for 7 minutes. The reaction was run in a 3% agarose gel (Sea Kem LE agarose; BMA, Rockland, MD) with 0.5 mg of ethidium bromide/mL (Sigma-Aldrich). 
Serotyping
Clinical isolates were serotyped using antiserum from Denka Seiken Co. Live P. aeruginosa cells were used. The O antigen serotype was determined by agglutination reaction to specific antiserum. The Lanyi 50 digital coding corresponding to the Japanese letter coding as specified by instruction manual was used. 
Detection of ExoS and ExoU mRNA by RT-PCR
Total RNA was extracted from bacterial cells grown under inducing or noninducing conditions using Aurum Total RNA mini-kit (Bio-Rad, Hercules, CA). cDNA was transcribed with reverse transcriptase from 1 μg of total RNA following the manufacturer's instructions (ImProm-II reverse transcription system; Promega). A sample of 30 ng of DNA was amplified by PCR under the following conditions: 30 cycles each of 94°C for 45 seconds, 62°C for 45 seconds, and 72°C for 45 seconds. Primers used in the reactions were 5′-GGGAATACTTTCCGGGAAGTT-3′ (exoU sense) and 5′-CGATCTCGCTGCTAATGTGTT-3′ (exoU antisense); 5′-GCGAGGTCAGCAGAGTATCG-3′ (exoS sense) and 5′-TTCGGCGTCACTGTGGATGC-3′ (exoS antisense). Results were visualized by electrophoresis in a 1.5 % agarose gel, followed by ethidium bromide staining. 
SDS-PAGE and Immunoblotting
P. aeruginosa were grown under inducing and noninducing conditions as specified above. Culture supernatant were collected and then precipitated with 55% ammonium sulfate at 4°C. Precipitated protein were collected by centrifugation (12,500g) for 15 minutes and washed with 50 mM Tris-HCl (pH 7.6) before being concentrated through a centrifugation filter device (Ultra Centricon tubes; molecular weight cutoff, 10,000; Amicon; Millipore, Billerica, MA). Protein concentrations were determined using DC protein assay kit (Bio-Rad) before equal concentrations of each sample were loaded onto a 10% SDS polyacrylamide gel. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad) and incubated with blocking solution (5% non-fat dry milk in PBS) for 2 hours. Membranes were then incubated with hen anti-ExoS antibody (1:5000 dilution; Agrisera AB, Inc., Vännäs, Sweden) or anti-ExoU antibody (1:20,000 dilution; provided by Professor DW Frank, University of Wisconsin) overnight, followed by washing with Tris-buffered saline (TBS; 0.1 M Tris, 0.1M Nacl, pH 7.5) and 0.05% Tween20. Goat anti-chicken immunoglobulin Y (IgY)-horsradish peroxidase (HRP) antibody (1:10,000 dilution; Immunology Consultants Laboratory, Inc., Portland, OR) or goat anti-mouse IgG HRP (1:10,000 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used as secondary antibody and incubated for 1 hour. After final washes with TBS-0.05% Tween20, the signal was enhanced using ECL Plus chemiluminescent system (GE Healthcare; Bio-Sciences, Taipei, Taiwan). To verify the status of the T3SS during bacterial adhesion experiments, bacterial adhesion solutions with or without EGTA for inducing or noninducing conditions, respectively, were also collected for immunoblot analysis. All experiments were repeated at least three times. 
Bacterial Adhesion to Contact Lenses
Assessment of P. aeruginosa adhesion to various contact lens materials was done by the viable cell culturing method. Characteristics of strains used in this experiment are listed in Table 2. 29,30,32 Five invasive strains (PAK, 6294, 2007AX44, 2007A01, and PAKΔpscC) and five cytotoxic strains (PA103, 6206, 2002AP68, 2007AD46, and PA103ΔpscC) were used in these experiments. Strains grown in inducing conditions were resuspended to an optical density at 660 nm (OD660) of 0.1 (∼108 bacteria/mL) in normal saline supplemented with 2% TSB and 2 mM EGTA to maintain the bacteria under inducing conditions. For maintenance under noninducing conditions, bacteria grown under noninducing conditions were also resuspended to OD660 of 0.1 (∼108 bacteria/mL) with normal saline supplemented with 2% TSB and 0.3 mM calcium. After contact lenses were removed from manufacturers' storage case, they were aseptically cut in half. The lenses were then immersed in either 1 mL of PBS or ATF for 18 hours and kept in an incubator at 37°C with a rotatory shaker at 130 rpm. Lenses were transferred to a new tissue culture plate (24-well tissue culture plate; Sarstedt, Beaumont Leys, Leicester, UK) with the concave surface facing up, and 1 mL of bacterial solution was added. The plate was incubated at 37°C for 2 hours with a rotatory shaker at 130 rpm. After incubation, each lens was picked up with aseptic fine-tip forceps and washed 3 times by careful dipping in 3 mL of PBS. Each lens was then placed in 1 mL of PBS and homogenized with a tissue homogenizer (Polytron model PT 4000; Kinematica, Inc., New York, NY) and serially diluted and plated on Muller-Hinton agar plates (BD Biosciences). The agar plates are incubated at 37°C overnight before being counted the next day. Bacterial adhesion experiments for each strain were repeated at least three times, and results were then averaged. Mean bacterial adhesion values of the four wild-type strains of each genotype were then averaged to obtain the bacterial adhesions for each genotype. 
Table 2. 
 
Characteristics of Bacterial Strains Used in Bacterial Experiments
Table 2. 
 
Characteristics of Bacterial Strains Used in Bacterial Experiments
Strain Genotype Serotype Presence of Flagella Source or Reference
PAK Invasive O6 Yes ref. 45
6294 Invasive O6 Yes ref. 28
PAO1 Invasive O2/O5 Yes Laboratory collection
2007AX44 Invasive O6 Unknown CLMK isolate
2007A01 Invasive O15 Unknown CLMK isolate
PA103 Cytotoxic O11 No ref. 46
6206 Cytotoxic O11 Yes ref. 28
2002AP68 Cytotoxic O11 Unknown CLMK isolate
2007AD46 Cytotoxic O7 Unknown CLMK isolate
PAKΔpscC Invasive O6 Yes ref. 45
PA103ΔpscC Cytotoxic O11 No TL Yahr
Scanning Electron Microscopy
Contact lenses were incubated for 2 hours with PAO1 and 6206, following the aforementioned protocol, and were fixed in half-strength Karnovsky fixative (2% paraformaldehyde and 2.5% glutaraldehyde) after the washing step. The fixative was removed, and distilled water was added for rinsing. Contact lenses without bacterial adhesion were removed from their containers and rinsed in distilled water before freeze drying. Rapid freezing was done by liquid nitrogen immersion for all lenses before lenses were placed under vacuum overnight. The concave side of the samples was mounted on aluminum stubs and sputter-coated with gold for examination under SEM (JSM model 5300; JEOL Ltd., Tachikawa, Tokyo, Japan) at 15 kV under various magnifications. 
Statistics
Data were entered into a Windows Excel spreadsheet (Microsoft, Inc., Taipei, Taiwan) and analyzed with SPSS statistical software (version 11.0; SPSS, Inc., Armonk, NY). Because bacterial adhesion data were not normally distributed, the Mann-Whitney nonparametric test was used to compare adhesions between each group. Fisher's exact test was used to compare the genotype frequencies between CLMK and noncontact lens-related MK and CLMK with other ocular isolates. A P value of less than 0.05 was considered statistically significant. 
Results
From August 2001 to December 2010, a total of 87 clinical ocular isolates of P. aeruginosa were collected. There were 64 isolates from cases with MK and 23 isolates from other ocular infectious diseases including endophthalmitis, scleritis, dacryocystitis, and conjunctivitis. Genotyping revealed that 56% of the total isolates were exoS + exoU invasive strains and that 36% were exoSexoU + cytotoxic strains. Among the total isolates, the exoS and exoU genes were nearly mutually exclusive with the exception of 3 isolates positive for both and 4 isolates negative for both. ExoT was present in all isolates. 
Among the MK isolates, 35 isolates were from non-contact lens-related cases, while 29 isolates were from CLMK cases. Isolates from cases with non-contact lens-related MK were exoS + exoU and exoSexoU + strains in 71% and 20% of the isolates, respectively (Table 3). For CLMK isolates, exoS+exoU and exoS exoU+ strains were 31% and 62%, respectively. The cytotoxic genotype was found more frequently in CLMK isolates than in isolates from non-contact lens-related cases (P = 0.002, Fisher exact test). The cytotoxic genotype was also significantly more common in CLMK cases than in isolates from cases of ocular infections other than MK (P = 0.02, Fisher exact test). 
Table 3. 
 
Distribution of Type III Secretion Genes among P. aeruginosa Ocular Isolates
Table 3. 
 
Distribution of Type III Secretion Genes among P. aeruginosa Ocular Isolates
Genotype Number of Isolates (%)
Non-Contact Lens-Related MK CLMK Ocular Infections Other than MK
exoS + exoU (invasive) 25 (71.4) 9 (31.0) 15 (68.2)
exoS exoU + (cytotoxic) 7 (20) 18 (62.1)*† 6 (27.3)
exoS + exoU + 0 (0) 2 (6.9) 1 (4.5)
exoS exoU 3 (8.6) 0 (0) 1 (4.5)
Total 35 (100) 29 (100) 23 (100)
RT-PCR was used to detect ExoU and ExoS mRNA of each strain used in contact lens bacterial adhesion under both inducing and noninducing conditions. Under inducing conditions, the presence of ExoU or ExoS mRNA that corresponded to each strain's T3SS genotype was detected for both wild-type strains and for the pscC needle complex mutants (Fig. 1A). However, pscC mutants grown under inducing conditions were unable to secrete exotoxins, indicating that the PscC needle complex protein is essential for exotoxin secretion (Fig. 1C). Under noninducing conditions, neither exotoxin gene expression nor secretion was detected (Figs. 1B, 1D). The inducing and noninducing conditions of growth were maintained after transfer to respective bacterial adhesion solutions with or without EGTA as indicated in Figures 1E and 1F. 
Figure 1. 
 
RT-PCR analysis of exoU (428 bp) and exoS (118 bp) genes of strains used in bacterial adhesion experiments under inducing conditions (A) and noninducing conditions (B). M, molecular weight markers; N, negative control (distilled water). Immunoblot analysis of ExoU (74 kDa) or ExoS (49 kDa) proteins in supernatants of all strains grown under inducing (C) and noninducing (D) conditions. Under inducing conditions, exoU and exoS gene expression were detected for all strains. However, the pscC mutants were unable to secrete exotoxins under inducing conditions. Bacteria grown under inducing or noninducing conditions were respectively transferred to bacterial adhesion solutions with or without EGTA. After 2 hours of incubation, supernatants were collected and tested for the presence of the T3SS exotoxins. T3SS exotoxins were detected in bacterial adhesion solutions with EGTA (E) but not in bacterial solutions without EGTA and containing 0.3 mM of calcium (F).
Figure 1. 
 
RT-PCR analysis of exoU (428 bp) and exoS (118 bp) genes of strains used in bacterial adhesion experiments under inducing conditions (A) and noninducing conditions (B). M, molecular weight markers; N, negative control (distilled water). Immunoblot analysis of ExoU (74 kDa) or ExoS (49 kDa) proteins in supernatants of all strains grown under inducing (C) and noninducing (D) conditions. Under inducing conditions, exoU and exoS gene expression were detected for all strains. However, the pscC mutants were unable to secrete exotoxins under inducing conditions. Bacteria grown under inducing or noninducing conditions were respectively transferred to bacterial adhesion solutions with or without EGTA. After 2 hours of incubation, supernatants were collected and tested for the presence of the T3SS exotoxins. T3SS exotoxins were detected in bacterial adhesion solutions with EGTA (E) but not in bacterial solutions without EGTA and containing 0.3 mM of calcium (F).
Table 4 compares the mean bacterial adhesion of each strain to four types of contact lens materials in bacterial adhesion solutions maintained under inducing and noninducing conditions. Under inducing conditions, significantly more bacterial adhesion was noted for all wild-type strains than for adhesion under noninducing conditions, indicating a possible role of the T3SS in bacterial adhesion. Figure 2 further compares the bacterial adhesion between the wild-type strains PAK (invasive genotype) and PA103(cytotoxic genotype) with that of the pscC isogenic mutants PAKΔpscC and PA103ΔpscC, respectively. Under inducing growth conditions, the pscC isogenic mutants of both genotypes adhered significantly less than bacteria for each type of contact lens material, suggesting that functional T3SS apparatus enhanced bacterial adhesion (P < 0.05, Mann-Whitney U test) (Figs. 2A, 2B). As a negative control, under noninducing conditions, the differences in bacterial adhesion between wild-type and pscC mutants were diminished (Figs. 3A, 3B). Under noninducing conditions, ATF incubation of contact lenses significantly increased bacterial adhesion of both genotypes for all lens materials, indicating tear fluid proteins may substantially influence bacteria−lens adhesion, even without T3SS activation (Table 4). 
Figure 2. 
 
Comparison of bacterial adhesion between wild-type and pscC isogenic mutant strains (mean CFU/mm2 ± SD) grown and maintained under inducing conditions. For both invasive (A) and cytotoxic (B) strains, there was significantly less bacterial adhesion of mutant strains to each contact lens material (*Mann-Whitney U test, P < 0.05).
Figure 2. 
 
Comparison of bacterial adhesion between wild-type and pscC isogenic mutant strains (mean CFU/mm2 ± SD) grown and maintained under inducing conditions. For both invasive (A) and cytotoxic (B) strains, there was significantly less bacterial adhesion of mutant strains to each contact lens material (*Mann-Whitney U test, P < 0.05).
Figure 3. 
 
Comparison of bacterial adhesion between wild-type and pscC isogenic mutant (mean CFU/mm2 ± SD) grown and maintained under noninducing conditions. No statistically significant differences were found between wild-type and mutant strains for both invasive (A) and cytotoxic (B) strains.
Figure 3. 
 
Comparison of bacterial adhesion between wild-type and pscC isogenic mutant (mean CFU/mm2 ± SD) grown and maintained under noninducing conditions. No statistically significant differences were found between wild-type and mutant strains for both invasive (A) and cytotoxic (B) strains.
Table 4. 
 
Bacterial Adhesion to Various Contact Lens Materials Maintained under Inducing (EGTA+) or Noninducing (EGTA−) Conditions of Growth
Table 4. 
 
Bacterial Adhesion to Various Contact Lens Materials Maintained under Inducing (EGTA+) or Noninducing (EGTA−) Conditions of Growth
Strain Mean Bacterial Adhesions × 104 ± SD
EGTA
Growth condition + + + +
Bacterial adhesion solution + + + +
Contact lens material Etafilcon Etafilcon Etafilcon + ATF Nelfilcon Nelfilcon Nelfilcon + ATF Balafilcon Balafilcon Balafilcon + ATF Galyfilcon Galyfilcon Galyfilcon + ATF
Invasive strains
 PAK 57.0 ± 8.5* 24.0 ± 3.6 168.4 ± 9.6 † 67.0 ± 9.5* 23.3 ± 2.2 163.3 ± 7.6† 125.8 ± 9.9* 32.7 ± 2.5 209.6 ± 11.6† 38.3 ± 2.4* 17.8 ± 3.1 80.8 ± 5.9†
 6294 64.5 ± 3.7* 23.0 ± 4.2 238.9 ± 7.1† 67.8 ± 11.6* 41.0 ± 4.2 214.7 ± 2.0† 170.0 ± 18.3* 66.0 ± 2.6 291.9 ± 12.0† 44.5 ± 7.5* 21.0 ± 1.4 191.8 ± 6.4†
 2007AX44 68.3 ± 8.1* 16.0 ± 2.8 210.7 ± 13.4† 81.5 ± 5.9* 17.8 ± 3.6 254.8 ± 3.5† 148.8 ± 15.5* 36.0 ± 1.4 275.0 ± 9.3† 45.8 ± 2.9* 15.2 ± 1.3 182.0 ± 7.9†
 2007A01 130.0 ± 18.3* 31.0 ± 5.7 138.0 ± 4.1† 147.5 ± 17.1* 30.5 ± 3.5 198.0 ± 8.7† 172.5 ± 17.1* 51.0 ± 5.7 192.0 ± 4.5† 58.3 ± 8.7* 26.5 ± 2.1 87.0 ± 2.1†
 PAKΔpscC 26.0 ± 2.8 24.8 ± 2.2 67.2 ± 11.4† 25.7 ± 1.5 22.8 ± 3.9 63.7 ± 10.0† 35.5 ± 2.6 33.5 ± 5.4 101.7 ± 7.9† 19.3 ± 1.7 17.3 ± 2.5 52.3 ± 7.2†
Cytotoxic strains
 PA103 41.0 ± 2.6* 15.9 ± 0.8 130.0 ± 18.9† 44.7 ± 4.5* 17.5 ± 1.3 136.4 ± 6.0† 82.0 ± 7.8* 30.8 ± 5.6 204.9 ± 25.3† 32.8 ± 5.3* 11.1 ± 1.6 76.3 ± 11.7†
 6206 55.3 ± 3.8* 15.0 ± 2.8 159.8 ± 9.0† 55.5 ± 7.6* 16.5 ± 0.7 166.7 ± 9.2† 175.0 ± 12.9* 41.5 ± 3.5 298.9 ± 15.0† 44.3 ± 1.7* 14.1 ± 1.3 145.1 ± 5.0†
 2002AP68 82.5 ± 6.8* 23.5 ± 3.3 210.5 ± 12.2† 61.0 ± 4.3* 38.5 ± 7.0 233.4 ± 2.4† 127.5 ± 13.2* 53.3 ± 1.7 241.2 ± 12.4† 55.5 ± 4.2* 16.8 ± 1.7 146.8 ± 8.9†
 2007AD46 73.8 ± 2.8* 29.0 ± 3.7 153.0 ± 7.6† 90.0 ± 2.6* 33.3 ± 0.6 155.0 ± 9.8† 143.0 ± 10.9* 36.0 ± 2.2 211.0 ± 13.0† 43.5 ± 2.6* 24.3 ± 2.2 119.0 ± 11.5†
 PA103ΔpscC 16.7 ± 2.5 15.5 ± 2.1 41.7 ± 7.8† 19.0 ± 1.8 17.6 ± 2.5 35.6 ± 6.4† 31.8 ± 2.8 32.0 ± 3.9 79.0 ± 19.1† 12.3 ± 2.1 11.7 ± 2.4 23.6 ± 13.7†
To demonstrate genotype differences and reduce interstrain variability among P. aeruginosa population, we averaged the mean bacterial adhesion of four wild-type strains for each genotype (invasive strains used were PAK, 6294, 2007AX44, and 2007A01; cytotoxic strains used were PA103, 6206, 2002AP68, and 2007AD46) (Fig. 4). Figures 4A and 4B show bacterial adhesion to various materials with strains grown under inducing and noninducing conditions, respectively. Compared to cytotoxic strains, invasive strains generally adhered in greater numbers to all types of contact lens material, although they did not reach a statistically significant difference (Fig. 4A). A general trend of the least bacterial adhesion to galyfilcon lenses compared to that of all other types of lenses was found. This trend was seen for both invasive and cytotoxic genotypes (Fig. 4). A statistically significant difference was noted while comparing bacterial adhesions between galyfilcon and balafilcon lenses for both genotypes (P < 0.05 , Mann-Whitney U test) (Fig. 4). With the lenses incubated in ATF and strains grown under noninducing conditions to mimic physiologic conditions, galyfilcon consistently showed a trend for the least bacterial adhesion (Fig. 5). Statistically significant differences in bacterial adhesions were found between ATF-incubated galyfilcon and balafilcon lenses for both genotypes (P < 0.05 , Mann-Whitney U test) (Fig. 5). 
Figure 4. 
 
Mean bacterial adhesion of four wild-type P. aeruginosa strains maintained under inducing (A) and noninducing (B) conditions to four types of soft contact lens materials (mean CFU/mm2 ± SD). For both cytotoxic and invasive strains of P. aeruginosa , galyfilcon significantly adhered less bacteria than balafilcon (*Mann-Whitney U test, P < 0.05). Under both inducing and noninducing conditions, a general trend of the least bacterial adhesion was observed for galyfilcon lenses than for the other three types of contact lenses for both genotypes.
Figure 4. 
 
Mean bacterial adhesion of four wild-type P. aeruginosa strains maintained under inducing (A) and noninducing (B) conditions to four types of soft contact lens materials (mean CFU/mm2 ± SD). For both cytotoxic and invasive strains of P. aeruginosa , galyfilcon significantly adhered less bacteria than balafilcon (*Mann-Whitney U test, P < 0.05). Under both inducing and noninducing conditions, a general trend of the least bacterial adhesion was observed for galyfilcon lenses than for the other three types of contact lenses for both genotypes.
Figure 5. 
 
Mean bacterial adhesion of four wild-type P. aeruginosa strains maintained under noninducing conditions to four types of soft contact lens materials incubated with ATF (mean CFU/mm2 ± SD). For both cytotoxic and invasive strains of P. aeruginosa , galyfilcon adhered significantly less bacteria than balafilcon (*Mann-Whitney U test, P < 0.05). For cytotoxic genotype, balafilcon lenses had significantly more bacterial adhesion than etafilcon lenses (Mann-Whitney U test, P < 0.05). A general trend of the least bacterial adhesion for galyfilcon lenses compared to the other three types of contact lenses was seen for both genotypes.
Figure 5. 
 
Mean bacterial adhesion of four wild-type P. aeruginosa strains maintained under noninducing conditions to four types of soft contact lens materials incubated with ATF (mean CFU/mm2 ± SD). For both cytotoxic and invasive strains of P. aeruginosa , galyfilcon adhered significantly less bacteria than balafilcon (*Mann-Whitney U test, P < 0.05). For cytotoxic genotype, balafilcon lenses had significantly more bacterial adhesion than etafilcon lenses (Mann-Whitney U test, P < 0.05). A general trend of the least bacterial adhesion for galyfilcon lenses compared to the other three types of contact lenses was seen for both genotypes.
Figures 6 to 9 exhibit SEM images of four types of soft contact lenses with and without bacterial adhesion. SEM of the new conventional hydrogel etafilcon, the cosmetic hydrogel nelfilcon, and the silicone hydrogel galyfilcon showed relatively homogeneous polymeric structures over the entire lens surface, although some debris or artifacts may be noted (Figs. 6A, 7A, 9A). Balafilcon exhibited areas with small pores on seemingly flat surfaces with a disarray of fine lines or cracks, more clearly seen at a higher magnification in the background of Figure 8B. This may be the glassy islands produced by plasma oxidation coating of balafilcon lenses. After incubation with P. aeruginosa PAO1 (invasive genotype) or 6206 (cytotoxic genotype) for 2 hours, bacterial attachments were seen mostly on the surfaces of etafilcon, balafilcon, and galyfilcon contact lenses and were not found to be associated with the porous structures of the lenses (Figs. 6B, 6C, 8B, 8C, 9B, 9C). However, numerous bacteria were seen clogged within the relatively large pores of nelfilcon lenses (Figs. 7B, 7C). 
Figure 6. 
 
(A) SEM of new etafilcon contact lens exhibiting relatively wavy yet homogeneous polymeric structures (original magnification ×2000). SEM of etafilcon lens incubated for 2 hours with PAO1 (B) and 6206 (C). Bacterial clumping was seen on the surfaces and not within the polymeric structure (original magnification ×2000).
Figure 6. 
 
(A) SEM of new etafilcon contact lens exhibiting relatively wavy yet homogeneous polymeric structures (original magnification ×2000). SEM of etafilcon lens incubated for 2 hours with PAO1 (B) and 6206 (C). Bacterial clumping was seen on the surfaces and not within the polymeric structure (original magnification ×2000).
Figure 7. 
 
(A) SEM of new nelfilcon contact lens exhibiting relatively homogeneous polymeric structures under low magnification (original magnification ×2000). SEM of nelfilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Numerous bacteria were seen within the relatively large porous structures (original magnification ×2000).
Figure 7. 
 
(A) SEM of new nelfilcon contact lens exhibiting relatively homogeneous polymeric structures under low magnification (original magnification ×2000). SEM of nelfilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Numerous bacteria were seen within the relatively large porous structures (original magnification ×2000).
Figure 8. 
 
(A) SEM of new balafilcon contact lens. The surface displays pores and a disarray of lines or cracks composing a mosaic-like morphology, probably indicative of “silicate islands” produced from plasma surface oxidation treatment (original magnification ×2000). SEM of balafilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Numerous bacterial clumps are seen on the surface of the lens (original magnification ×2000).
Figure 8. 
 
(A) SEM of new balafilcon contact lens. The surface displays pores and a disarray of lines or cracks composing a mosaic-like morphology, probably indicative of “silicate islands” produced from plasma surface oxidation treatment (original magnification ×2000). SEM of balafilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Numerous bacterial clumps are seen on the surface of the lens (original magnification ×2000).
Figure 9. 
 
(A) SEM of new galyfilcon contact lens. The surface exhibits a relatively smooth and homogeneous sponge-like structure (original magnification ×2000). SEM of galyfilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Most bacteria are on the surface of the lens, although a few are seen within the porous structure (original magnification ×2000).
Figure 9. 
 
(A) SEM of new galyfilcon contact lens. The surface exhibits a relatively smooth and homogeneous sponge-like structure (original magnification ×2000). SEM of galyfilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Most bacteria are on the surface of the lens, although a few are seen within the porous structure (original magnification ×2000).
Discussion
In this study, we collected clinical ocular isolates of P. aeruginosa and found that the exoS and exoU genes were mostly mutually exclusive in 92% of our total isolates. 1,51 Invasive strains were more prevalent in all ocular isolates except in isolates from CLMK cases. CLMK isolates had a significantly higher prevalence of P. aeruginosa with the cytotoxic genotype than isolates from patients who did not use CL. Although many previous studies have studied the prevalence of each genotype among P. aeruginosa isolates from clinical MK cases, only two reports compare the genotype distribution between CLMK and non-CLMK isolates. Among those studies, one with only nine isolates noted no statistically significant difference in genotype distribution between CLMK and non-CLMK isolates. 52 However, in the only other report with a larger number of isolates, the authors claimed to be the first to note a significantly higher prevalence of the cytotoxic genotype among CLMK cases. 36 Thus, the possibility of a stronger or higher adherence of cytotoxic strains to contact lenses was proposed. 36  
Even though the cytotoxic genotype was more prevalent among CLMK isolates, the degree of bacterial adhesion to the same contact lens material under inducing conditions was generally found to be similar between cytotoxic and invasive strains. To our knowledge, we are the first to compare adhesion differences between invasive and cytotoxic genotypes for conventional, color-tinted, and silicone hydrogel contact lenses. Of the two studies that compared adhesion differences between strains of different genotypes, only etafilcon lenses were used. 53,54 Bacterial adhesion to etafilcon lenses between cytotoxic (strain 6206) and invasive (Paer1, Paer8, and 6294) strains was found to be not statistically different between the two genotypes. 53,54 Thus, the higher prevalence of the cytotoxic genotype among CLMK isolates may not be related to the quantity of bacteria attached to the contact lens but more possibly associated with the virulence of the cytotoxic strains themselves. As previously reported, cell death due to infection from cytotoxic strains occurs faster than from invasive strains. 27 Cytotoxic strains cause rapid disruption of plasma membrane integrity and also loss of epithelial barrier function. 27,55,56  
Although bacterial adhesion was not significantly different between the two genotypes, mutation in the needle complex protein PscC resulted in decreased adhesion to all type of lenses, indicating a possible role of the T3SS in bacterial adhesion to lens materials. Previously, reports have indicated a possible cooperative association between the T3SS and other adhesins such as lipopolysaccharide O antigens and flagella. 57,58 Strains with lower O antigen complexity and without flagella seemed to promote expression of the T3SS. 57,58 Although our study was not designed to address the association or interaction among various adhesins with the T3SS in conjunction with bacteria–contact lens adhesion, serotype analysis found a higher prevalence of O11 (51.7%, unpublished data) serotype among our CLMK isolates. The predominance of O11 serotype among keratitis isolates was also previously noted by Stewart et al. 59 Strains used in bacterial adhesion experiments shown in Table 2 were mostly of the O6 and O11 serotypes. Although the type of material of contact lens was not specified, Thuruthyil et al. 26 reported better adherence of strains with serotypes I (O1), G (O6), and E (O11) to contact lenses. Thus, the T3SS and a multitude of other factors including possible adhesins may be involved in P. aeruginosa adhesion to contact lenses. Further studies to clarify the complex coordination of various adhesins with the T3SS and contact lens material adherence under physiologic conditions are required. 
For the same genotype, we generally found the least bacterial adherence to galyfilcon lenses than to the other types of lenses under all conditions tested (Figs. 4, 5). In conformity with other studies comparing pseudomonal adhesion between silicone hydrogel lenses, balafilcon lenses comparatively adhered greater numbers of bacteria than galyfilcon. 20,60 This may be due to the greater hydrophobicity of balafilcon lenses. 21,6163 The relatively hydrophilic hydrogel material etafilcon was also shown to have less bacterial adhesion than balafilcon lenses. 21 To render the lens surface more hydrophilic, balafilcon lenses were surface treated in plasma-reactive chambers to transform the silicone surface components on the lenses into hydrophilic silicate compounds. 48 Thus, glassy discontinuous silicate “islands” on the surfaces of balafilcon lenses were produced and seen by others 48 and us by SEM (Fig. 8A). Nevertheless, this surface treatment does not completely shield the hydrophobic silicone exposed and therefore still creates a relative hydrophobic lens surface with poor wettability. 60,64 Galyfilcon lenses, on the other hand, are non–surface-treated, and because of the incorporation of a new internal wetting agent (Hydraclear) a hydrophilic surface was created, decreasing the tendency for microbial adhesion. 20,64,65 Thus, properties related to contact lens materials may play a crucial role in bacterial adhesion. 
To mimic physiologic conditions, contact lenses were incubated with ATF prior to bacterial adhesion assay with bacteria cultivated under noninducing conditions to account for the high calcium conditions in tears. The artificial tears formula used in our study follows suggestions provided by Willcox et al. 21 This formula, although simplified, contains essential antibacterial proteins (lactoferrin, lysozyme), glycoproteins (lactoferrin, mucin, γ-globulin), and calcium found in natural tears. 21,46,47 Under noninducing conditions, we found a significant increase in pseudomonal adhesion to all types of contact lens material after protein incubation, indicating that tear proteins may more significantly affect bacterial adhesion than the T3SS. Because a mixture of proteins was used, we cannot determine which protein specifically mediated or promoted bacterial adhesion. Nevertheless, in comparison, the influence of tear fluid proteins in vivo was previously shown to affect bacterial lens adhesion. Worn hydrophobic balafilcon lenses were previously shown to attach significantly greater numbers of P. aeruginosa than worn hydrophilic galyfilcon lenses or worn conventional hydrogel lenses. 21,60 Although bacterial adhesion to worn lenses was not tested in our study, the artificial tear incubation of the lenses may provide clues to the extent and complexity of bacterial adhesion to various contact lens materials under in vivo conditions. 
SEM is commonly used to analyze the surface structures of contact lenses. 19,48 Etafilcon, nelfilcon, and galyfilcon lenses showed relatively homogeneous sponge-like polymeric structures over the entire surface, while balafilcon lenses had a combination of lines or cracks with oval pores. The striking difference in surface morphology between these lenses is most likely due to the plasma oxidation surface treatment of balafilcon A lenses that cause mosaic-like patterns, which was previously shown by others. 48 Our study found that after 2 hours of incubation with the standard strains PAO1 and 6206, most of the bacteria were clumped together and were distributed randomly on the lens surfaces. Although bacterial adhesion to nelfilcon lenses was similar to that of the other conventional hydrogel etafilcon, nelfilcon lenses had comparatively more bacteria within their relatively large porous structure. Thus, for individuals wearing contaminated contact lenses, corneal surfaces are in direct contact with the attached bacteria, increasing the probability of infection. Because the attached bacteria are mostly on the surfaces of these lenses, frequent and effective cleaning of soft contact lenses could remove surface- adhering bacteria to lower the risk of infection. The previously no-rub recommendation for contact lens cleaning should be discouraged. Daily disposable cosmetic contact lenses are recommended over planned disposables as bacteria tend to be embedded within the polymeric structure. 
There are some limitations to our study. First, the viable cell culture method used in this study to enumerate bacterial adhesion to contact lenses depends on the removal of bacteria from the attached surfaces by maceration of the lens material, shaking, and/or ultrasonography. Although other methods such as phase-contrast microscopy may be used for direct enumeration of attached bacteria, it can only provide total counts and cannot determine the viability of these organisms, which is crucial in clinical situations. Second, further experimentation with bacterial adhesion with worn contact lenses will best mimic physiologic conditions and further consider the effects of lens surface structures during in vivo conditions. 
Conclusions
In conclusion, isolates from CLMK cases were more commonly of the cytotoxic genotype than isolates from cases with non-contact lens-related MK. The invasive genotype dominated all other ocular isolates. A general and consistent trend of least bacterial adhesion to galyfilcon lenses for both genotypes and in both inducing and noninducing growth mediums was found. There were no genotype-related differences in bacterial adhesion to all contact lens materials studied. SEM showed a difference in surface morphology between surface-treated (balafilcon) and non–surface-treated lenses (etafilicon, nelfilcon, and galyfilcon), which may contribute to their differences in P. aeruginosa adhesion. The correlation between the T3SS and pseudomonal adhesions to various contact lens materials may be through complex interactions with other adhesins and tear fluid proteins. Differences in contact lens material may crucially affect the adherence of cytotoxic and invasive strains of P. aeruginosa
Acknowledgments
The authors thank Suzanne M. J. Fleiszig (School of Optometry, University of California at Berkeley) for providing P. aeruginosa strains 6294 and 6206; Timothy L. Yahr (Department of Microbiology, University of Iowa) for providing P. aeruginosa strains PA103 and PA103ΔpscC; and Stephen Lory (Department of Microbiology and Molecular Genetics, Harvard Medical School) for providing P. aeruginosa strains PAK and PAKΔpscC. We also thank Dara W. Frank (Department of Microbiology and Molecular Genetics, Medical College of Wisconsin) for generosity in providing ExoU monoclonal antibodies. 
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Footnotes
 Supported in part by National Science Council, Executive Yuan, Taiwan, Grant NSC99-2628-B-002-045-MY3 and Buddhist Tzu Chi General Hospital, Taipei Branch, Grant TCRD-TPE-101-34. The authors have no proprietary interest in the research presented herein.
Footnotes
 Disclosure: E.P. Shen, None; R.-Y. Tsay, None; J.-S. Chia, None; S. Wu, None; J.-W. Lee, None; F.-R. Hu, None
Figure 1. 
 
RT-PCR analysis of exoU (428 bp) and exoS (118 bp) genes of strains used in bacterial adhesion experiments under inducing conditions (A) and noninducing conditions (B). M, molecular weight markers; N, negative control (distilled water). Immunoblot analysis of ExoU (74 kDa) or ExoS (49 kDa) proteins in supernatants of all strains grown under inducing (C) and noninducing (D) conditions. Under inducing conditions, exoU and exoS gene expression were detected for all strains. However, the pscC mutants were unable to secrete exotoxins under inducing conditions. Bacteria grown under inducing or noninducing conditions were respectively transferred to bacterial adhesion solutions with or without EGTA. After 2 hours of incubation, supernatants were collected and tested for the presence of the T3SS exotoxins. T3SS exotoxins were detected in bacterial adhesion solutions with EGTA (E) but not in bacterial solutions without EGTA and containing 0.3 mM of calcium (F).
Figure 1. 
 
RT-PCR analysis of exoU (428 bp) and exoS (118 bp) genes of strains used in bacterial adhesion experiments under inducing conditions (A) and noninducing conditions (B). M, molecular weight markers; N, negative control (distilled water). Immunoblot analysis of ExoU (74 kDa) or ExoS (49 kDa) proteins in supernatants of all strains grown under inducing (C) and noninducing (D) conditions. Under inducing conditions, exoU and exoS gene expression were detected for all strains. However, the pscC mutants were unable to secrete exotoxins under inducing conditions. Bacteria grown under inducing or noninducing conditions were respectively transferred to bacterial adhesion solutions with or without EGTA. After 2 hours of incubation, supernatants were collected and tested for the presence of the T3SS exotoxins. T3SS exotoxins were detected in bacterial adhesion solutions with EGTA (E) but not in bacterial solutions without EGTA and containing 0.3 mM of calcium (F).
Figure 2. 
 
Comparison of bacterial adhesion between wild-type and pscC isogenic mutant strains (mean CFU/mm2 ± SD) grown and maintained under inducing conditions. For both invasive (A) and cytotoxic (B) strains, there was significantly less bacterial adhesion of mutant strains to each contact lens material (*Mann-Whitney U test, P < 0.05).
Figure 2. 
 
Comparison of bacterial adhesion between wild-type and pscC isogenic mutant strains (mean CFU/mm2 ± SD) grown and maintained under inducing conditions. For both invasive (A) and cytotoxic (B) strains, there was significantly less bacterial adhesion of mutant strains to each contact lens material (*Mann-Whitney U test, P < 0.05).
Figure 3. 
 
Comparison of bacterial adhesion between wild-type and pscC isogenic mutant (mean CFU/mm2 ± SD) grown and maintained under noninducing conditions. No statistically significant differences were found between wild-type and mutant strains for both invasive (A) and cytotoxic (B) strains.
Figure 3. 
 
Comparison of bacterial adhesion between wild-type and pscC isogenic mutant (mean CFU/mm2 ± SD) grown and maintained under noninducing conditions. No statistically significant differences were found between wild-type and mutant strains for both invasive (A) and cytotoxic (B) strains.
Figure 4. 
 
Mean bacterial adhesion of four wild-type P. aeruginosa strains maintained under inducing (A) and noninducing (B) conditions to four types of soft contact lens materials (mean CFU/mm2 ± SD). For both cytotoxic and invasive strains of P. aeruginosa , galyfilcon significantly adhered less bacteria than balafilcon (*Mann-Whitney U test, P < 0.05). Under both inducing and noninducing conditions, a general trend of the least bacterial adhesion was observed for galyfilcon lenses than for the other three types of contact lenses for both genotypes.
Figure 4. 
 
Mean bacterial adhesion of four wild-type P. aeruginosa strains maintained under inducing (A) and noninducing (B) conditions to four types of soft contact lens materials (mean CFU/mm2 ± SD). For both cytotoxic and invasive strains of P. aeruginosa , galyfilcon significantly adhered less bacteria than balafilcon (*Mann-Whitney U test, P < 0.05). Under both inducing and noninducing conditions, a general trend of the least bacterial adhesion was observed for galyfilcon lenses than for the other three types of contact lenses for both genotypes.
Figure 5. 
 
Mean bacterial adhesion of four wild-type P. aeruginosa strains maintained under noninducing conditions to four types of soft contact lens materials incubated with ATF (mean CFU/mm2 ± SD). For both cytotoxic and invasive strains of P. aeruginosa , galyfilcon adhered significantly less bacteria than balafilcon (*Mann-Whitney U test, P < 0.05). For cytotoxic genotype, balafilcon lenses had significantly more bacterial adhesion than etafilcon lenses (Mann-Whitney U test, P < 0.05). A general trend of the least bacterial adhesion for galyfilcon lenses compared to the other three types of contact lenses was seen for both genotypes.
Figure 5. 
 
Mean bacterial adhesion of four wild-type P. aeruginosa strains maintained under noninducing conditions to four types of soft contact lens materials incubated with ATF (mean CFU/mm2 ± SD). For both cytotoxic and invasive strains of P. aeruginosa , galyfilcon adhered significantly less bacteria than balafilcon (*Mann-Whitney U test, P < 0.05). For cytotoxic genotype, balafilcon lenses had significantly more bacterial adhesion than etafilcon lenses (Mann-Whitney U test, P < 0.05). A general trend of the least bacterial adhesion for galyfilcon lenses compared to the other three types of contact lenses was seen for both genotypes.
Figure 6. 
 
(A) SEM of new etafilcon contact lens exhibiting relatively wavy yet homogeneous polymeric structures (original magnification ×2000). SEM of etafilcon lens incubated for 2 hours with PAO1 (B) and 6206 (C). Bacterial clumping was seen on the surfaces and not within the polymeric structure (original magnification ×2000).
Figure 6. 
 
(A) SEM of new etafilcon contact lens exhibiting relatively wavy yet homogeneous polymeric structures (original magnification ×2000). SEM of etafilcon lens incubated for 2 hours with PAO1 (B) and 6206 (C). Bacterial clumping was seen on the surfaces and not within the polymeric structure (original magnification ×2000).
Figure 7. 
 
(A) SEM of new nelfilcon contact lens exhibiting relatively homogeneous polymeric structures under low magnification (original magnification ×2000). SEM of nelfilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Numerous bacteria were seen within the relatively large porous structures (original magnification ×2000).
Figure 7. 
 
(A) SEM of new nelfilcon contact lens exhibiting relatively homogeneous polymeric structures under low magnification (original magnification ×2000). SEM of nelfilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Numerous bacteria were seen within the relatively large porous structures (original magnification ×2000).
Figure 8. 
 
(A) SEM of new balafilcon contact lens. The surface displays pores and a disarray of lines or cracks composing a mosaic-like morphology, probably indicative of “silicate islands” produced from plasma surface oxidation treatment (original magnification ×2000). SEM of balafilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Numerous bacterial clumps are seen on the surface of the lens (original magnification ×2000).
Figure 8. 
 
(A) SEM of new balafilcon contact lens. The surface displays pores and a disarray of lines or cracks composing a mosaic-like morphology, probably indicative of “silicate islands” produced from plasma surface oxidation treatment (original magnification ×2000). SEM of balafilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Numerous bacterial clumps are seen on the surface of the lens (original magnification ×2000).
Figure 9. 
 
(A) SEM of new galyfilcon contact lens. The surface exhibits a relatively smooth and homogeneous sponge-like structure (original magnification ×2000). SEM of galyfilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Most bacteria are on the surface of the lens, although a few are seen within the porous structure (original magnification ×2000).
Figure 9. 
 
(A) SEM of new galyfilcon contact lens. The surface exhibits a relatively smooth and homogeneous sponge-like structure (original magnification ×2000). SEM of galyfilcon lens incubated for 2 hours with PAO1(B) and 6206 (C). Most bacteria are on the surface of the lens, although a few are seen within the porous structure (original magnification ×2000).
Table 1. 
 
Characteristics of Soft Contact Lenses Used in this Study
Table 1. 
 
Characteristics of Soft Contact Lenses Used in this Study
Characteristic US Adopted Name
Etafilcon A Nelfilcon A Balafilcon A Galyfilcon A
Commercial name Acuvue 2 Freshlook Purevision Acuvue Advance
Manufacturer Vistakon Ciba Vision GmbH Bausch & Lomb Vistakon
FDA group IV II III I
Material Hydrogel Color-tinted hydrogel (gray) Silicone hydrogel Silicone hydrogel
Water content (%) 58 69 36 47
Oxygen permeability (Barrers) 28 26 100 60
Surface treatment None None Plasma oxidation treatment No surface treatment; internal wetting agent (PVP)
Table 2. 
 
Characteristics of Bacterial Strains Used in Bacterial Experiments
Table 2. 
 
Characteristics of Bacterial Strains Used in Bacterial Experiments
Strain Genotype Serotype Presence of Flagella Source or Reference
PAK Invasive O6 Yes ref. 45
6294 Invasive O6 Yes ref. 28
PAO1 Invasive O2/O5 Yes Laboratory collection
2007AX44 Invasive O6 Unknown CLMK isolate
2007A01 Invasive O15 Unknown CLMK isolate
PA103 Cytotoxic O11 No ref. 46
6206 Cytotoxic O11 Yes ref. 28
2002AP68 Cytotoxic O11 Unknown CLMK isolate
2007AD46 Cytotoxic O7 Unknown CLMK isolate
PAKΔpscC Invasive O6 Yes ref. 45
PA103ΔpscC Cytotoxic O11 No TL Yahr
Table 3. 
 
Distribution of Type III Secretion Genes among P. aeruginosa Ocular Isolates
Table 3. 
 
Distribution of Type III Secretion Genes among P. aeruginosa Ocular Isolates
Genotype Number of Isolates (%)
Non-Contact Lens-Related MK CLMK Ocular Infections Other than MK
exoS + exoU (invasive) 25 (71.4) 9 (31.0) 15 (68.2)
exoS exoU + (cytotoxic) 7 (20) 18 (62.1)*† 6 (27.3)
exoS + exoU + 0 (0) 2 (6.9) 1 (4.5)
exoS exoU 3 (8.6) 0 (0) 1 (4.5)
Total 35 (100) 29 (100) 23 (100)
Table 4. 
 
Bacterial Adhesion to Various Contact Lens Materials Maintained under Inducing (EGTA+) or Noninducing (EGTA−) Conditions of Growth
Table 4. 
 
Bacterial Adhesion to Various Contact Lens Materials Maintained under Inducing (EGTA+) or Noninducing (EGTA−) Conditions of Growth
Strain Mean Bacterial Adhesions × 104 ± SD
EGTA
Growth condition + + + +
Bacterial adhesion solution + + + +
Contact lens material Etafilcon Etafilcon Etafilcon + ATF Nelfilcon Nelfilcon Nelfilcon + ATF Balafilcon Balafilcon Balafilcon + ATF Galyfilcon Galyfilcon Galyfilcon + ATF
Invasive strains
 PAK 57.0 ± 8.5* 24.0 ± 3.6 168.4 ± 9.6 † 67.0 ± 9.5* 23.3 ± 2.2 163.3 ± 7.6† 125.8 ± 9.9* 32.7 ± 2.5 209.6 ± 11.6† 38.3 ± 2.4* 17.8 ± 3.1 80.8 ± 5.9†
 6294 64.5 ± 3.7* 23.0 ± 4.2 238.9 ± 7.1† 67.8 ± 11.6* 41.0 ± 4.2 214.7 ± 2.0† 170.0 ± 18.3* 66.0 ± 2.6 291.9 ± 12.0† 44.5 ± 7.5* 21.0 ± 1.4 191.8 ± 6.4†
 2007AX44 68.3 ± 8.1* 16.0 ± 2.8 210.7 ± 13.4† 81.5 ± 5.9* 17.8 ± 3.6 254.8 ± 3.5† 148.8 ± 15.5* 36.0 ± 1.4 275.0 ± 9.3† 45.8 ± 2.9* 15.2 ± 1.3 182.0 ± 7.9†
 2007A01 130.0 ± 18.3* 31.0 ± 5.7 138.0 ± 4.1† 147.5 ± 17.1* 30.5 ± 3.5 198.0 ± 8.7† 172.5 ± 17.1* 51.0 ± 5.7 192.0 ± 4.5† 58.3 ± 8.7* 26.5 ± 2.1 87.0 ± 2.1†
 PAKΔpscC 26.0 ± 2.8 24.8 ± 2.2 67.2 ± 11.4† 25.7 ± 1.5 22.8 ± 3.9 63.7 ± 10.0† 35.5 ± 2.6 33.5 ± 5.4 101.7 ± 7.9† 19.3 ± 1.7 17.3 ± 2.5 52.3 ± 7.2†
Cytotoxic strains
 PA103 41.0 ± 2.6* 15.9 ± 0.8 130.0 ± 18.9† 44.7 ± 4.5* 17.5 ± 1.3 136.4 ± 6.0† 82.0 ± 7.8* 30.8 ± 5.6 204.9 ± 25.3† 32.8 ± 5.3* 11.1 ± 1.6 76.3 ± 11.7†
 6206 55.3 ± 3.8* 15.0 ± 2.8 159.8 ± 9.0† 55.5 ± 7.6* 16.5 ± 0.7 166.7 ± 9.2† 175.0 ± 12.9* 41.5 ± 3.5 298.9 ± 15.0† 44.3 ± 1.7* 14.1 ± 1.3 145.1 ± 5.0†
 2002AP68 82.5 ± 6.8* 23.5 ± 3.3 210.5 ± 12.2† 61.0 ± 4.3* 38.5 ± 7.0 233.4 ± 2.4† 127.5 ± 13.2* 53.3 ± 1.7 241.2 ± 12.4† 55.5 ± 4.2* 16.8 ± 1.7 146.8 ± 8.9†
 2007AD46 73.8 ± 2.8* 29.0 ± 3.7 153.0 ± 7.6† 90.0 ± 2.6* 33.3 ± 0.6 155.0 ± 9.8† 143.0 ± 10.9* 36.0 ± 2.2 211.0 ± 13.0† 43.5 ± 2.6* 24.3 ± 2.2 119.0 ± 11.5†
 PA103ΔpscC 16.7 ± 2.5 15.5 ± 2.1 41.7 ± 7.8† 19.0 ± 1.8 17.6 ± 2.5 35.6 ± 6.4† 31.8 ± 2.8 32.0 ± 3.9 79.0 ± 19.1† 12.3 ± 2.1 11.7 ± 2.4 23.6 ± 13.7†
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