Fungal strains.

The three keratitis-associated fusaria included in this study were characterized using DNA sequence data from three loci as previously described (16). Results of multilocus DNA sequence typing indicate that the fusaria represent three phylogenetically distinct species. Two of these are members of the species-richFusarium solani species complex (FSSC)] (51), while the third is a member of theFusarium oxysporum species complex (FOSC) 3-a widespread clonal lineage (40). FSSC multilocus haplotype 1-b strain MRL8609 (= NRRL 47513) was isolated from a patient with fungal keratitis not associated with contact lens use at University Hospitals Case Medical Center, Cleveland, OH, while the FOSC 3-a strain MRL8996 (= NRRL 47514) was isolated from a patient with contact lens-associated fungal keratitis at Cleveland Clinic Foundation. ATCC 36031 (= NRRL 47512) FSSC multilocus haplotype 2-c, which was originally isolated from a human corneal ulcer in Nigeria in the mid-1970s, was acquired from the American Type Culture Collection (Manassas, VA) and used as a reference isolate in ourFusarium-related experiments. The ATCC 36031 strain is the recommended reference isolate in the International Organization for Standardization (ISO 14729) guidelines for testing of the antimicrobial activity of lens care solutions in vitro.C. albicans strain SC5314, a clinical isolate obtained from a candidiasis patient, was a generous gift from William Fonzi (Georgetown University, Washington, DC). All of the fusaria are available upon request from the Agricultural Research Service Culture Collection (NRRL), National Center for Agricultural Utilization Research, Peoria, IL.

Fungal growth conditions.

Fusarium isolates were grown at 37°C for 40 h in Sabouraud dextrose broth (SDB) (Difco Laboratories, Detroit, MI). To collect conidia following incubation, conidia of theFusarium species were harvested and hyphae were removed by filtration through sterile gauze. Conidia were washed with phosphate-buffered saline (PBS) and standardized to 1 × 10³ conidia/ml for growth rate studies and 1 × 10⁶ conidia/ml for biofilm formation experiments.C. albicans was grown overnight at 37°C in yeast nitrogen base medium (YNB) (Difco Laboratories) supplemented with 50 mM glucose.Candida cells were harvested, washed with PBS, and standardized to 1 × 10⁷ blastospores/ml for biofilm formation experiments. Growth ofFusarium in SDB, YNB, yeast potato dextrose (YPD) broth (Difco Laboratories), and RPMI 1640 (Mediatech, Inc., Herndon, VA) was monitored to identify the optimal medium to be used for biofilm formation by fusaria.

Soft contact lenses and lens care solutions.

Soft contact lenses used in the present study included the following: etafilcon A (Vistakon; Johnson & Johnson, Jacksonville, FL), galyfilcon A (Vistakon), lotrafilcon A (CIBA Vision, Duluth, GA), balafilcon A (Bausch & Lomb, Rochester, NY), alphafilcon A (Bausch & Lomb), and polymacon (Bausch & Lomb). Properties (water content and surface charge) of these contact lenses are shown in Table1. All the lenses used had a power of +1.50 diopters. The lens care solutions tested were MoistureLoc (lot number GG5033; Bausch & Lomb) and MultiPlus (lot number GF6002; Bausch & Lomb). Lens care solutions were stored at room temperature as per the manufacturer's suggestions.

Biofilm formation.

To evaluate biofilm formation byFusarium andCandida isolates, soft contact lenses were washed with PBS, placed in 12-well tissue culture plates with 4 ml standardized cell suspension (1 × 10⁷ cells/ml forC. albicans and 1 × 10⁶ conidia/ml for the fusaria), and incubated for 90 min at 37°C (adherence phase). Nonadherent cells were removed from soft contact lenses by gentle washing with 4 ml PBS. Next, soft contact lenses were immersed in medium (SDB for the fusaria, and YNB forC. albicans) and incubated for 48 h at 37°C on a rocker. Biofilms were quantified using a tetrazolium XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay as described previously (13). Planktonic cells were grown in 12-well plates in the absence of a contact lens.

Gross morphology of biofilms formed on contact lenses.

To characterize the gross morphology of biofilms formed on contact lenses, lenses were seeded with fungi and allowed to adhere and then form biofilms for 48 h. Next, a digital camera was used to capture images of the biofilms to compare their gross morphologies and appearances.

CSLM.

The architecture of biofilms formed on soft contact lenses was analyzed using confocal scanning laser microscopy (CSLM), following our previously described method (13). Briefly, soft contact lenses containing biofilms were transferred to 12-well plates and incubated for 45 min at 37°C in 4 ml of PBS containing the fluorescent stains FUN-1 (10 mM) and concanavalin A-Alexa Fluor 488 conjugate (ConA) (25 mg/ml). FUN-1 (long-pass filter; excitation wavelength, 543 nm; emission, 560 nm) is converted to orange-red cylindrical intravacuolar structures by metabolically active cells, while ConA (long-pass filter; excitation wavelength, 488 nm; emission, 505 nm) binds to glucose and mannose residues of fungal cell wall polysaccharides and emits green fluorescence. After incubation with the dyes, the lenses were flipped and placed on a 35-mm-diameter glass-bottom petri dish (MatTek Corp., Ashland, MA). Stained biofilms were observed by using a Zeiss LSM510 confocal scanning laser microscope equipped with argon and HeNe lasers and mounted on a Zeiss Axiovert100 M microscope (Carl Zeiss, Inc.). All observations were conducted with a water immersion C-apochromat objective (403; numerical aperture, 1.2).

Evaluation of antifungal activities of contact lens care solutions.

The antifungal activities of MoistureLoc and MultiPlus lens care solutions against planktonic forms ofFusarium andCandida were evaluated using the ISO 14729 standalone contact lens disinfectant test (24) and the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based metabolic activity assay. Activities of the lens care solutions against fungal biofilms were determined by measuring their metabolic activities using the XTT-based assay.

(i) Evaluation of activities of lens care solutions against planktonic cells using ISO 14729 methodology.

ISO 14729 guidelines are the standard used by the industry to demonstrate activities of contact lens care solutions against planktonically grown microorganisms (24). These guidelines specify that an active contact lens disinfectant must reduce the viability of fungal species by 1 log (90%) within the time recommended by the product's manufacturer. For evaluation of the antifungal activities of MoistureLoc and MultiPlus solutions againstFusarium strains ATCC 36031 (FSSC 2-c), MRL8609 (FSSC 1-b), and MRL8996 (FOSC 3-a), isolates were grown on potato dextrose agar plates for 10 days at 25°C. Conidia were harvested using a procedure based on the ISO standard and washed three times with Dulbecco's PBS plus 0.05% Tween 80. Conidial suspensions were adjusted to 5.0 × 10⁷ conidia/ml using a hemacytometer. Subsequently, a 0.1-ml suspension of eachFusarium strain was mixed with 10 ml of each lens care solution and incubated at 25°C for 1, 2, 3, 4, and 20 h. At each time point, 1 ml of the resultant mixture was taken, diluted with Dey-Engley neutralizing broth (DEB) (Difco Laboratories), and spread on Sabouraud dextrose agar plates. After the plates were incubated for 2 days at 25°C, viable CFU were counted. Each strain was tested three independent times. A similar ISO 14729-based approach was used to assess the inhibitory activities of these lens care solutions againstC. albicans SC5314.

(ii) Determination of metabolic activity of fungi using the XTT assay.

The XTT-based assay has been used previously to monitor fungal damage (38), to quantifyCandida andCryptococcus biofilms, and also to determine antibiofilm activities of antifungal agents (11,13,14,23,27,28,31,35,42). In this study, we used this method to evaluate the abilities of contact lens care solutions to inhibit planktonic and biofilm forms ofFusarium andCandida. Briefly, planktonic cells grown in the absence or presence of lens care solutions were incubated with XTT (1 mg/ml) and menadione (1 mM; Sigma Chemical Co., St. Louis, MO) at 37°C for 5 h. Mitochondrial dehydrogenase-mediated conversion of XTT tetrazolium salt to a formazan product in live cells resulted in a colorimetric change, which was measured using a microtiter plate reader (Bio-Rad Laboratories, Hercules, CA) at 492 nm.

To evaluate the ability of MoistureLoc or MultiPlus to inhibit contact lens-associated fungal biofilms,Fusarium orCandida biofilms formed on contact lenses were incubated with 4 ml of either solution in a 12-well plate for 4 h or 20 h, followed by incubation with 4 ml of DEB for 10 min to stop the action of these solutions (as per ISO recommendations). Cells treated with only DEB served as controls. Next, lens-associated biofilms were washed with PBS and their metabolic activity was determined as described above.

Statistical analyses.

All experiments were performed in triplicate. Statistical analysis was performed using analysis of variance followed by the Bonferroni/Dunn post hoc test for comparisons of biofilms on different contact lenses and an unpairedt test for evaluating the activity of lens care solutions. AP value of <0.05 was considered statistically significant.

Nucleotide sequence accession numbers.

The DNA sequence data generated in this study have been deposited in GenBank under accession numbersEU251484 to EU251492.

Optimization of growth conditions forFusarium biofilm formation.

To understand the pathogenesis and mechanisms involved inFusarium biofilm formation on contact lenses, it is necessary to establish a robust in vitro model of such biofilms. As the first step in developing such a model, we monitored growth of MRL8609 FSSC 1-b in four different media (SDB, YNB, YPD, and RPMI) by determining the number of microconidia and percent hyphal elements formed in each medium. No significant difference was observed in the numbers of conidia/growth rates of MRL8609 FSSC 1-b grown in SDB, YPD, or RPMI (growth rate = 6.61 ± 0.095, 7.11 ± 0.17, or 6.15 ± 0.37 conidia/h, respectively;P > 0.05) (Fig.1A and B). However, the growth rate of MRL8609 was significantly lower in YNB medium than in the other three media tested (growth rate = 4.77 ± 0.018 conidia/h;P < 0.05 for each comparison). Although no difference in the growth rates was observed between MRL8609 grown in SDB, YPD, or RPMI, the percentage of hyphae harvested from MRL8609 grown in SDB or YNB was higher than that when cultured in YPD or RPMI (Fig.1C). It is important to note that MRL8609 growth was not influenced by enriched medium, since SDB (an enriched medium) induced abundant hyphal formation while YPD (also an enriched medium) induced only minimal hyphal formation. Moreover, YNB (a minimal medium) induced significantly more hyphal formation by MRL8609 than YPD. Since clinical presentation ofFusarium keratitis is commonly associated with the presence of a large number of hyphal elements in affected tissues and hyphal elements are known to assist fungal invasion of tissue (12), and since SDB supported an optimal growth rate and maximum hyphal formation, we selected it as the optimal growth medium and incubation for 48 h as the optimal time needed to form a fully matureFusarium biofilm.

Gross morphologies of biofilms formed on soft contact lenses.

Analysis of the gross morphologies of biofilms formed on contact lenses showed that although MRL8609 FSSC 1-b was able to form biofilms on all lenses examined (Fig.1D to H), there was a difference in the abilities of the biofilms to adhere to the contact lenses tested. In this regard, biofilms formed by strain MRL8609 on etafilcon A and polymacon lenses were loosely attached to the surfaces of soft contact lenses and very easily dislodged when manipulated. In contrast, biofilms formed by strain MRL8609 on lotrafilcon A, balafilcon A, and alphafilcon A lenses adhered firmly to the surfaces of soft contact lenses. In general,C. albicans biofilms adhered firmly to the lenses, were compact, and were not easily detached, and no differences were observed in gross morphology and adhesion of biofilms formed by this pathogen on different lens types (data not shown).

Abilities ofFusaria andC. albicans to form biofilms on contact lenses vary with lens type.

Since the lenses tested in this study differ in their surface properties (surface charge and water content; Table1), we hypothesized that the ability ofFusarium to form biofilm will vary with different contact lenses. To test this hypothesis, we quantified biofilms formed on six commonly used lenses using the XTT-based method. Our analyses revealed that as measured by metabolic activity, strain MRL8609 FSSC 1-b formed significantly more biofilms on lotrafilcon A than on balafilcon A (P < 0.0001), galyfilcon A (P = 0.0029), etafilcon A (P = 0.0030), or alphafilcon A (P < 0.0001) lenses (Fig.2). Additionally, significantly more biofilms were formed on polymacon lenses than on balafilcon A (P = 0.0003) or alphafilcon A (P = 0.0021) lenses. We also found that strain MRL8609 formed significantly more biofilms on both etafilcon A and galyfilcon A lenses than were formed on balafilcon A lenses (P = 0.001 for both comparisons). By way of contrast, the ISO 14729-recommended reference isolate ATCC 36031 FSSC 2-c failed to form biofilm on the lenses tested (data not shown). These studies revealed that among silicone hydrogel lenses, maximum biofilms were formed by strain MRL8609 FSSC 1-b on lotrafilcon A lenses while minimum biofilms were formed on balafilcon A lenses.

SinceCandida is the second most common fungus causing keratitis, we evaluated its ability to form biofilms on different lenses. Metabolic activity assays revealed that in similarity toFusarium isolates, theCandida isolate tested also formed significantly more biofilms on lotrafilcon A than on galyfilcon A (P < 0.0001), balafilcon A (P < 0.0001), etafilcon A (P = 0.0012), or alphafilcon A (P < 0.0001) lenses (Fig.2). No difference was observed inCandida biofilms formed on lotrafilcon A and polymacon A lenses (P > 0.05).

Taken together, these results demonstrate thatFusarium andCandida can form biofilms on contact lenses and that this ability is influenced by the lens type.

Lens-associated FSSC biofilms are hypha rich and have homogeneous architecture.

Since the architecture ofC. albicans biofilm is influenced by the substrate used (e.g., dentures or catheters) (15) and since contact lenses differ in their surface properties (Table1), we hypothesized that the architecture ofFusarium biofilms formed on different contact lenses may also be influenced by the type of lenses used in this study. As shown in Fig.3, CSLM analyses showed that biofilms formed by strain MRL8609 FSSC 1-b on soft contact lenses were composed of profuse hyphae. Biofilms formed on lotrafilcon A and balafilcon A were characterized by numerous filaments with yellow staining within the hyphal elements, which resulted from dual staining with carbohydrate (green; ConA) and metabolically active (red; FUN-1) dyes (Fig.3C and D). Biofilms formed on etafilcon A, galyfilcon A, and alphafilcon A lenses contained abundant extracellular matrix (Fig.3A, B, and E, arrows). Moreover, biofilms formed on the polymacon lens had a diffuse, granular appearance, with ConA staining visible throughout the biofilm (Fig.3F). Additionally, biofilms formed by strain MRL8609 exhibited similar architecture and thicknesses at the center and periphery of the soft contact lenses (data not shown).

Analyses of biofilm thickness revealed that biofilms formed by strain MRL8609 FSSC 1-b on lenses with low water content were thinner than those formed on lenses with high water content. This pattern was observed for both nonionic and ionic lenses. Thus, for nonionic lenses, biofilms formed on the low-water-content lotrafilcon A and polymacon lenses (water content = 24% and 28%, respectively) were thinner than those formed on galyfilcon A and alphafilcon A, which have high water content (47% and 64%, respectively) (Fig.4). Similarly, among ionic lens-associated biofilms, those formed on balafilcon A (with 36% water content) were thinner than biofilms formed on the etafilcon A lens (with 58% water content). These studies suggested a water content-dependent effect of the lens type on the ability ofFusarium to form biofilms on lenses. However, more-detailed studies are needed to determine the relevance of such an effect.

Taken together, our results clearly demonstrate that contact lens-associatedFusarium biofilms are hypha rich and have homogenous architecture, with some surface-dependent differences in biofilm architecture.

Architecture ofC. albicans biofilms formed on contact lenses is heterogeneous and dependent on lens surface properties.

Next, we used CSLM to determine whether the architecture of lens-associated biofilms formed byC. albicans is also dependent on lens surface properties and whether this architecture is similar to that ofFusarium biofilms. As shown in Fig.3G to L,C. albicans biofilm architecture was distinctive for each lens type. For example,C. albicans biofilm formed on etafilcon A lenses was composed primarily of yeast cells with an abundant extracellular matrix (Fig.3G), whereas biofilms formed on lotrafilcon A lenses consisted of sparse yeast cells interspersed within a diffuse matrix (Fig.3I). Moreover,C. albicans biofilms formed on balafilcon A were rich in hyphae with few yeast cells and a distinct granular extracellular matrix (Fig.3J). In contrast,C. albicans biofilms formed on galyfilcon A, alphafilcon A, and polymacon lenses were a mix of yeast and hyphae, with minimal detectable extracellular matrix (Fig.3H, K, and L). Our analyses showed that the architecture ofC. albicans biofilms formed at the periphery of lotrafilcon A and polymacon contact lenses differed from those formed at the center. Specifically, among biofilms formed on lotrafilcon A, the central area contained matrix-rich, dense, “mushroom-like” structures (data not shown), similar to those seen in bacterial biofilms (9,17,43,44). InCandida biofilms formed on a polymacon lens, the peripheral region contained sparse yeast cells, while biofilms formed in the centers of the lenses contained abundant yeast and hyphal elements. Analyses of biofilm thickness formed on lenses revealed thatC. albicans biofilms formed on balafilcon A lenses were significantly thicker than those formed on the other lenses (P < 0.0001 for all comparisons) (Fig.4). There was no significant difference in thickness between biofilms formed byC. albicans on the remaining five lenses (P > 0.05), suggesting a lack of correlation between lens water content and biofilm thickness. These results suggested that lens surface properties modulate the morphology and architecture ofCandida biofilms but not their thickness.

Clinical isolates ofFusarium form biofilms on contact lenses.

Having established the in vitro biofilm model, we used it to examine the abilities of three human keratitis-associatedFusarium isolates to form biofilms on the lotrafilcon A lens using the XTT assay. This contact lens was selected because it allowed abundant biofilm formation. As per ISO 14729 recommendations for testing activities of lens care solutions, the reaction of these disinfectants was stopped after the manufacturer-recommended contact time by adding DEB. Therefore, in our assays, we used DEB-treated cells as controls. To determine whether DEB by itself interfered with the ability of fungi to form biofilms on contact lenses, we compared the abilities of the threeFusarium species FSSC 1-b (MRL8609), FSSC 2-c (ATCC 36031), and FOSC 3-a (MRL8996) andC. albicans SC5314 to form a biofilm on lotrafilcon A lens in the absence or presence of DEB. The XTT assay revealed that exposure of cells to DEB reduced the XTT activity of the biofilm by almost 50% compared to that of the untreated control (Fig.5). Although DEB reduced the level of biofilms formed by the isolates tested, the biofilms formed were significant and did not interfere with our ability to study the activity of contact lens care solutions against fungal biofilms. In contrast to the clinical isolates MRL8609 FSSC 1-b and MRL8996 FOSC 3-a, the ATCC 36031 reference isolate FSSC 2-c lacked the ability to form a biofilm in the presence or absence of DEB (Fig.5).

Planktonically grownFusaria andCandida albicans are susceptible to MoistureLoc and MultiPlus solutions.

Determination of CFU after 4 h of incubation (ISO 14729 guidelines) is the standard method for evaluating activities of lens care solutions against planktonically grown microbes. Moreover, since biofilm growth is assessed by using the XTT assay and not by CFU determination and to allow comparison of the activities of lens care solutions against planktonic and biofilm-associated cells, in the current study we evaluated the effects of MoistureLoc and MultiPlus against planktonically grown fusaria andC. albicans using both the CFU and XTT assays. As shown in Fig.6, planktonically grown fusaria (MRL8609 [FSSC 1-b], ATCC 36031 [FSSC 2-c], and MRL8996 [FOSC 3-a]) andC. albicans isolate SC5314 were susceptible to both MoistureLoc and MultiPlus solutions. Incubation of planktonically grown cells in the lens care solutions for 4 h, as suggested by the manufacturer and the ISO document, resulted in a >1 log reduction of CFU for the three fusaria isolates (Fig.6A to C) and theC. albicans isolate (Fig.6G) tested. Results from the XTT assay also revealed similar patterns (Fig.6B, D, F, and H), with complete inhibition of metabolic activity observed after 4 h of incubation. These results demonstrated that the ISO- and XTT-based methods generate similar results regarding the activities of lens case solutions, showing that both MoistureLoc and MultiPlus are active against planktonically grownFusarium andC. albicans.

Contact lens-associated fungal biofilms exhibit reduced susceptibility to lens care solutions.

Next, we used the in vitro model we developed to test whether MoistureLoc and MultiPlus could inhibit biofilm formation by MRL8609 (FSSC 1-b), MRL8996 (FOSC 3-a), orC. albicans SC5314 on the lotrafilcon A lens. In contrast to the activities of lens care solutions against planktonically grown fungi (percent reductions of 53 ± 1% and 51 ± 4% compared to that for the untreated control [mean ± standard deviation [SD]) (Table2), these solutions only partially inhibited biofilm formation following a 4-h exposure. Extending the treatment period for both solutions to 20 h resulted in slightly more inhibition of biofilms formed by MRL8906. In contrast, biofilms formed by MRL8996 were not significantly reduced by MultiPlus or MoistureLoc solutions (Table2). However, in both cases, the lens care solutions failed to completely inhibit biofilm formation by the two phylogenetically divergent species ofFusarium tested. In contrast to the partial inhibitory effect of MoistureLoc and MultiPlus solutions againstFusarium biofilms, these agents had no inhibitory effect on biofilms formed on contact lenses at 4 h or 20 h byC. albicans isolate SC5314 (Table2).

Taken together, our studies showed that biofilms formed byFusarium isolates exhibited reduced susceptibility to MoistureLoc and MultiPlus lens care solutions, whileC. albicans biofilm was completely resistant to these solutions.

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