Molecular characterization, biofilm analysis and experimental biofouling study of Fusarium isolates from recent cases of fungal keratitis in New York State

Abstract

Background: To characterize Fusarium isolates from recent cases of fungal keratitis in contact lens wearers, and to investigate fungal association with MoistureLoc solution.

Methods: We studied six fungal isolates from recent cases of keratitis in New York State. The isolates were characterized by nucleotide sequencing and phylogenetic analyses of multiple genes, and then typed using minisatellite and microsatellite probes. Experimental fungal biofilm formation was tested by standard methods. MoistureLoc solutions were tested in biofouling studies for their efficacy in elimination of Fusarium contamination.

Results: Fusarium solani--corneal ulcers (2 isolates), lens case (1 isolate), and F. oxysporum--corneal ulcer (1 isolate), eye (1 isolate), were recovered from five patients. An opened bottle of MoistureLoc solution provided by a patient also yielded F. solani. Two distinct genotypes of F. solani as well as of F. oxysporum were present in the isolated strains. Remarkably, F. solani strains from the lens case and lens solution in one instance were similar, based on phylogenetic analyses and molecular typing. The solution isolate of F. solani formed biofilm on contact lenses in control conditions, but not when co-incubated with MoistureLoc solution. Both freshly opened and 3-month old MoistureLoc solutions effectively killed F. solani and F. oxysporum, when fungal contamination was simulated under recommended lens treatment regimen (4-hr). However, simulation of inappropriate use (15-60 min) led to the recovery of less than 1% of original inoculum of F. solani or F. oxysporum.

Conclusion: Temporary survival of F. solani and F. oxysporum in MoistureLoc suggested that improper lens cleaning regimen could be a possible contributing factor in recent infections.

Figure 1

Recovery of Fusarium from lens solution. (a) Fusarium solani colonies recovered from 100 μl of MoistureLoc cleaning solution provided by a patient (b) F. solani colonies on Sabouraud's dextrose agar slants inoculated with supernatant (left); in contrast no colonies were recovered from the pellet (right) from the same MoistureLoc solution.

Figure 2

Unrooted parsimony phylogram of Fusarium EF-1α gene sequences. Nucleotide sequences of EF-1α gene were compared by ClustalW multiple alignment using MacVector 7.1.1 software (Accelrys, San Diego, CA). Phylogenetic analyses were done with the PAUP v4 program using a bootstrap method with a Neighbor-Joining or Maximum Parsimony search [21]. The numbers on the branches indicate percent bootstrap values, based on 1000 replicates. Three distinct clades were seen in both the F. oxysporum and the F. solani strains. The six strains from our investigation came from five different patients: 23-06 (corneal ulcer), 24-06 (contact lens), 158-06 (right eye), 159-06 (corneal ulcer left eye), 237-06 and 238-06 (contact lens case swab and contact lens cleaning solution from the same patient). Three outlier controls were F. oxysporum (163-05) and F. solani (1064-05), previously isolated from unrelated keratitis cases, and F. oxysporum (974-05) isolated from finger nail.

Figure 3

Molecular typing of Fusarium isolates. Molecular typing with (a) minisatellite probe {M13}, and (b) repeat DNA probe {GACA}₄ showed distinct genotypes among Fusarium isolates. F. solani strain 24-06 genotype was easily separated from the other three strains (23-06, 237-06, and 238-06). F. solani from contact lens case (237-06) and from MoistureLoc solution (238-06) showed similar genotypes, by the two probes. In contrast, F. oxysporum strains 158-06 and 159-06 yielded two different genotypes. The control F. oxysporum (163-05, 974-05) and F. solani (1064-05) genotypes were distinct. The lane assignments for 163-05, 974-05, and 24-06 are different in panels (a) and (b).

Figure 4

Scanning electron micrographs of biofilm formation. (a) F. solani (238-06) incubated with fresh ACUVUE lens and balanced salt solution for 48 h at 25°C on a rotator (180 rpm). (b) Close-up of the same isolate showing hyphal attachment to the lens surface (c) Disappearance of biofilm when preparation from (a) was treated with MoistureLoc for 4 h.

Figure 5

Experimental biofouling studies with F. solani (238-06) and F. oxysporum (158-06). Fungal cells (1 × 10⁵) were inoculated into 1.0 ml of MoistureLoc solution, and 100-μl aliquots were plated on yeast extract-peptone-dextrose agar at intervals (a-h). Fungal colonies were recovered from MoistureLoc^® solutions sampled at 15, 30 and 60 min intervals (b-d) F. solani; (f-h) F. oxysporum. All solutions were sterile after the manufacturer recommended length of treatment, 4 hr (data not shown). The total numbers of colonies recovered are ten-fold of the dilution shown in these illustrations; the data is summarized in figure 6. Similar results were seen when 3-months old and freshly opened MoistureLoc solutions were compared. The experiments were repeated at least three times; a representative experiment is shown.

Figure 6

Summary of experimental biofouling studies. The experimental biofouling studies were carried out by introducing 1 × 10⁵ fungal cells (colony forming units; CFU) of F. solani (238-06) or F. oxysporum (158-06) into 1.0 ml of MoistureLoc solution. The recovery of CFU after 30 and 60 minutes is shown from three representative experiments (mean ± SD). The cultures were sterile after recommended 4.0 hrs of incubation (data not shown).