Roya Borazjani

Influence of Protein Deposition on Bacterial Adhesion to Contact Lenses

Purpose. The aim of the study is to determine the adhesion of Gram positive and Gram negative bacteria onto conventional hydrogel (CH) and silicone hydrogel (SH) contact lens materials with and without lysozyme, lactoferrin, and albumin coating. Methods. Four lens types (three SH—balafilcon A, lotrafilcon B, and senofilcon A; one CH—etafilcon A) were coated with lysozyme, lactoferrin, or albumin (uncoated lenses acted as controls) and then incubated in Staphylococcus aureus (Saur 31) or either of two strains of Pseudomonas aeruginosa (Paer 6294 and 6206) for 24 h at 37°C. The total counts of the adhered bacteria were determined using the 3H-thymidine method and viable counts by counting the number of colony-forming units on agar media. Results. All three strains adhered significantly lower to uncoated etafilcon A lenses compared with uncoated SH lenses (p < 0.05). Lysozyme coating on all four lens types increased binding (total and viable counts) of Saur 31 (p < 0.05). However, lysozyme coating did not influence P. aeruginosa adhesion (p > 0.05). Lactoferrin coating on lenses increased binding (total and viable counts) of Saur 31 (p < 0.05). Lactoferrin-coated lenses showed significantly higher total counts (p < 0.05) but significantly lower viable counts (p < 0.05) of adhered P. aeruginosa strains. There was a significant difference between the total and viable counts (p < 0.05) that were bound to lactoferrin-coated lenses. Albumin coating of lenses increased binding (total and viable counts) of all three strains (p < 0.05). Conclusions. Lysozyme deposited on contact lenses does not possess antibacterial activity against certain bacterial strains, whereas lactoferrin possess an antibacterial effect against strains of P. aeruginosa.

Bacterial Adhesion to Unworn and Worn Silicone Hydrogel Lenses

Purpose. The objective of this study was to determine the bacterial adhesion to various silicone hydrogel lens materials and to determine whether lens wear modulated adhesion. Methods. Bacterial adhesion (total and viable cells) of Staphylococcus aureus (31, 38, and ATCC 6538) and Pseudomonas aeruginosa (6294, 6206, and GSU-3) to 10 commercially available different unworn and worn silicone hydrogel lenses was measured. Results of adhesion were correlated to polymer and surface properties of contact lenses. Results. S. aureus adhesion to unworn lenses ranged from 2.8 × 104 to 4.4 × 105 colony forming units per lens. The highest adhesion was to lotrafilcon A lenses, and the lowest adhesion was to asmofilcon A lenses. P. aeruginosa adhesion to unworn lenses ranged from 8.9 × 105 to 3.2 × 106 colony forming units per lens. The highest adhesion was to comfilcon A lenses, and the lowest adhesion was to asmofilcon A and balafilcon A lenses. Lens wear altered bacterial adhesion, but the effect was specific to lens and strain type. Adhesion of bacteria, regardless of genera/species or lens wear, was generally correlated with the hydrophobicity of the lens; the less hydrophobic the lens surface, the greater the adhesion. Conclusions. P. aeruginosa adhered in higher numbers to lenses in comparison with S. aureus strains, regardless of the lens type or lens wear. The effect of lens wear was specific to strain and lens. Hydrophobicity of the silicone hydrogel lens surface influenced the adhesion of bacterial cells.

Effect of cholesterol deposition on bacterial adhesion to contact lenses.

Purpose. To examine the effect of cholesterol on the adhesion of bacteria to silicone hydrogel contact lenses. Methods. Contact lenses, collected from subjects wearing Acuvue Oasys or PureVision lenses, were extracted in chloroform:methanol (1:1, v/v) and amount of cholesterol was estimated by thin-layer chromatography. Unworn lenses were soaked in cholesterol, and the numbers of Pseudomonas aeruginosa strains or Staphylococcus aureus strains that adhered to the lenses were measured. Cholesterol was tested for effects on bacterial growth by incubating bacteria in medium containing cholesterol. Results. From ex vivo PureVision lenses, 3.4 ± 0.3 &mgr;g/lens cholesterol was recovered, and from Acuvue Oasys lenses, 2.4 ± 0.2 to 1.0 ± 0.1 &mgr;g/lens cholesterol was extracted. Cholesterol did not alter the total or viable adhesion of any strain of P. aeruginosa or S. aureus (p > 0.05). However, worn PureVision lenses reduced the numbers of viable cells of P. aeruginosa (5.8 ± 0.4 log units) compared with unworn lenses (6.4 ± 0.2 log units, p = 0.001). Similarly, there were fewer numbers of S. aureus 031 adherent to worn PureVision (3.05 ± 0.8 log units) compared with unworn PureVision (4.6 ± 0.3 log units, p = 0.0001). Worn Acuvue Oasys lenses did not affect bacterial adhesion. Cholesterol showed no effect on the growth of any test strain. Conclusions. Although cholesterol has been shown to adsorb to contact lenses during wear, this lipid does not appear to modulate bacterial adhesion to a lens surface.

Traversal of Multilayered Corneal Epithelia by Cytotoxic Pseudomonas aeruginosa Requires the Phospholipase Domain of ExoU

PURPOSE Pseudomonas aeruginosa isolates from microbial keratitis are invasive or cytotoxic toward mammalian cells, depending on their type III secreted toxins. Cytotoxic strains express ExoU, a phospholipase that contributes to corneal virulence. This study determined whether the ExoU phospholipase domain is required for P. aeruginosa traversal of the human corneal epithelium. METHODS P. aeruginosa traversal of airlifted, multilayered, human corneal epithelial cells was quantified in vitro up to 8 hours after apical inoculation with ∼10⁶ cfu of strain PA14, or an isogenic exoU mutant (PA14ΔexoU). In addition, PA14ΔexoU or its triple effector mutant PA14ΔexoUΔexoTΔexoY, were complemented with exoU (pUCPexoU), phospholipase-inactive exoU (pUCPexoUD344A), or control plasmid (pUCP18). Transepithelial resistance (TER) was measured (by epithelial volt ohmmeter), and cytotoxicity was determined by trypan blue staining. RESULTS PA14 traversed more efficiently than its exoU mutant at 4, 6, and 8 hours after inoculation (100-, 20-, and 8-fold, respectively; P < 0.05), but not at 2 hours. Cells exposed to PA14 lost TER to baseline (P < 0.05). Controls confirmed PA14 cytotoxicity toward these corneal epithelial cells that was absent with exoU mutants. Epithelial traversal, cytotoxicity, and lost TER were restored for PA14ΔexoU, or PA14ΔexoUΔexoTΔexoY, by complementation with pUCPexoU, but not by complementation with pUCPexoUD344A. CONCLUSIONS Traversal of multilayered corneal epithelia in vitro by cytotoxic P. aeruginosa requires ExoU with an active phospholipase domain. Correlative loss of TER with traversal by wild-type, or exoU-complemented, bacteria suggests involvement of epithelial cell death and/or lost tight junction integrity. However, traversal by exoU mutants without reduced TER suggests that additional mechanisms are also operative.

Effect of phospholipid deposits on adhesion of bacteria to contact lenses

Purpose. Protein and lipid deposits on contact lenses may contribute to clinical complications. This study examined the effect of phospholipids on the adhesion of bacteria to contact lenses. Methods. Worn balafilcon A (n = 11) and senofilcon A (n = 11) were collected after daily wear and phospholipids were extracted in chloroform:methanol. The amount of phospholipid was measured by electrospray ionization mass spectrometry. Unworn lenses soaked in phospholipids were exposed to Pseudomonas aeruginosa and Staphylococcus aureus. After 18 h incubation, the numbers of P. aeruginosa or S. aureus that adhered to the lenses were measured. Phospholipid was tested for possible effects on bacterial growth. Results. A broad range of sphingomyelins (SM) and phosphatidylcholines (PC) were detected from both types of worn lenses. SM (16:0) (m/z 703) and PC (34:2) (m/z 758) were the major phospholipids detected in the lens extracts. Phospholipids did not alter the adhesion of any strain of P. aeruginosa or S. aureus (p > 0.05). Phospholipids (0.1 mg/mL) showed no effect on the growth of P. aeruginosa 6294 or S. aureus 031. Conclusions. Phospholipids adsorb/absorb to contact lenses during wear, however, the major types of phospholipids adsorbed to lenses do not alter bacterial adhesion or growth.