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 × 10 to 4.4 × 10 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 × 10 to 3.2 × 10 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.

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.

A new method for evaluation of compatibility of contact lenses and lens cases with contact lens disinfecting solutions.

PURPOSE: The purpose of this article is to describe new methodology, antimicrobial efficacy endpoint methodology to determine compatibility of contact lens solutions, lens cases and hydrogel lenses for disinfection (AEEMC), to evaluate the effect of a contact lens and a lens case on disinfection efficacy, and to present the ring test used to justify the use of the method in multiple laboratories. MATERIALS AND METHODS: A prototype solution containing chlorhexidine as the disinfecting agent and four representative lens types (group I and IV hydrogels and two silicone hydrogels) were used in these ring tests. Five laboratories participated in the chemical and microbiologic analyses. The residual chlorhexidine in lens cases containing the contact lenses was determined using high-performance liquid chromatography; uptake by the lenses was then determined by extrapolation. For the microbiologic part of the study, a contact lens was placed in the well of the lens case, inoculated at 10 to 10 cfu (colony forming units) per lens with microorganisms in 10% organic soil. The microorganisms, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Candida albicans, and Fusarium solani, were prepared as in International Organization for Standardization (ISO) 14729. After a 3- to 10-min exposure time, the prototype solution was dispensed into each well. Aliquots of the inoculated solutions were removed at 4 and 24 hrs and 7 and 30 days and cultured in neutralizing media for determination of survivors; lenses were also cultured for survivors. RESULTS: Chemical uptake data confirmed the differences observed in kill of the challenge organisms according to lens type. It was observed that the culturing of the solution provided adequate data to show the effect of a lens on disinfection efficacy of a lens care product. The findings of the ring test indicated that the separate culturing of the contact lenses is not necessary for routine assessment. CONCLUSIONS: The methodology in the November 12, 2008, draft standard (AEEMC), meets the stated objective of demonstrating the effect of a contact lens on the disinfection efficacy of a simulated lens care product. This method, used in combination with the methodology in ISO 14729, should provide for a more robust evaluation of applicable contact lens care disinfecting products.

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.

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 H-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.

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 µg/lens cholesterol was recovered, and from Acuvue Oasys lenses, 2.4 ± 0.2 to 1.0 ± 0.1 µg/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.

In vitro deposition of lysozyme on etafilcon A and balafilcon A hydrogel contact lenses: effects on adhesion and survival of Pseudomonas aeruginosa and Staphylococcus aureus.

PURPOSE: To compare lysozyme adsorption and absorption and bacterial adhesion interactions on conventional (etafilcon A) and silicone (balafilcon A) hydrogel contact lenses. METHOD: Lysozyme concentrations and activities associated with the lenses were determined after solvent extraction (trifluoroacetic acid/acetonitrile) and directly on the lenses without extraction with micrococcal- and micro-bicinchoninic acid (BCA) assays. Cells of bacteria with radiolabeled leucine and a cell recovery procedure were used in determinations of bacterial adhesion to lenses. RESULTS: Lysozyme was adsorbed and absorbed to the conventional etafilcon A lens at about a 10-fold greater concentration than to the balafilcon A silicone hydrogel lens. Enzyme activities on the surfaces of both lenses were similar but replenished after saline extraction only with the etafilcon A lens. Lysozyme on the lens surface showed significant lysis of Micrococcus luteus but had a negligible effect on the adhesion and survival of Staphylococcus aureus. Lysozyme did not appear to affect the survival of Pseudomonas aeruginosa on lenses. CONCLUSION: In vitro experiments show that concentrations of active lysozyme on the surface of the etafilcon A lens, unlike the balafilcon A lens which showed negligible absorption, may be sustained from the lens matrix. Lysozyme deposited on hydrogel lenses had marked activity against M. luteus but relatively minor effects on the primary adhesion of P. aeruginosa and S. aureus.

Effect of a multipurpose contact lens solution on the survival and binding of Acanthamoeba species on contact lenses examined with a no-rub regimen.

PURPOSE: To determine the effect of a multipurpose contact lens solution (ReNu MultiPlus Multi-Purpose Solution [RMP]) on the relative survival and binding of trophozoites and cysts of Acanthamoeba on hydrogel lenses with a no-rub regimen. METHODS: A stand-alone test procedure with RMP was conducted with and without the presence of organic soil (1 x 10(7) colony-forming units/mL heat-killed cells of Saccharomyces cerevisiae in heat-inactivated fetal bovine serum). Survival of amoebae on hydrogel contact lenses exposed to RMP was determined with a no-rub care regimen. RESULTS: ReNu MultiPlus Multi-Purpose Solution reduced the number of recoverable amoebae by more than 95% within 4 hours of inocula of 10(5) trophozoites and cysts, regardless of the presence or absence of an organic soil. Amoebae, particularly cysts, were readily rinsed from contact lenses, including silicone hydrogels, without rubbing after exposure to RMP. CONCLUSIONS: The efficacy of RMP for Acanthamoeba was not appreciably altered in the presence of organic soil in a no-rub protocol. The antimicrobial activity, in part, appeared to be a combination of reducing the capacity for binding of representative Acanthamoeba to the lens by alteration of morphology, often followed by lysis of the amoebae.

Relative primary adhesion of Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus to HEMA-type contact lenses and an extended wear silicone hydrogel contact lens of high oxygen permeability.

PURPOSE: To compare multiple strains of Pseudomonas aeruginosa and representative isolates of Staphylococcus aureus and Serratia marcescens for their relative primary adhesion to a high Dk silicone hydrogel lens (36% H2O) with that of a HEMA-type lens (58% H2O). METHODS: A radiolabeled cell procedure with a 2-h cell exposure was employed for enumerating bacteria on unworn and worn silicone hydrogel (balafilcon A) and HEMA-type (etafilcon A) hydrogel lenses. RESULTS: The degree of primary adhesion of P. aeruginosa to 7-day worn balafilcon A lens was similar to the degree of adhesion to unworn balafilcon A lenses. The degree of primary adhesion by clinical strains to unworn balafilcon A lenses and etafilcon lenses was strain variable, but did not differ markedly for the two lenses with a given strain of P. aeruginosa. Relative to most of the pseudomonad strains, adhesion to the hydrogel lenses by S. aureus and S. marcescens was non-significant. CONCLUSION: Adhesion of strains of P. aeruginosa to a hydrogel contact lens does not appear to differ appreciably between the HEMA-type etafilcon A and the high Dk silicone hydrogel balafilcon A lens.

Validation of the Scan RDI for routine microbiological analysis of process water.

In this report, we review the validation methods and criteria specified in the PDA Technical Report No. 33 "Evaluation, Validation, and Implementation of New Microbiological Testing Methods" against data generated on the Chemunex Scan RDI. For each parameter, we have either reported data obtained in-house or reviewed information and documentation available from the manufacturer of the system. For all specified requirements, the Scan RDI produced data that was within the specifications suggested in the PDA Technical Report or suitable information was obtained from the manufacturer. The results of the study indicate that the Scan RDI can be validated for routine use in a pharmaceutical environment and provide results equivalent to current methods, but in a much shorter time frame.