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

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.


Background
Fungal keratitis due to filamentous fungi is quite common in tropical parts of the world and the southeastern United States [1][2][3]. Farmers and workers in agro-industries are most at risk for these infections, due to occupation-related corneal abrasions and subsequent fungal infections. In temperate regions, fungal keratitis is most commonly caused by the yeast Candida albicans, although mold keratitis due to Fusarium is being increasingly recognized [4][5][6]. Contact lens wearers have an elevated risk for fungal keratitis, but the incidence of Fusarium keratitis is quite rare among this group of patients [7][8][9]. There has been no report until the beginning of 2006, on the association of multipurpose contact lens solutions with fungal keratitis.
An upsurge in Fusarium keratitis among contact lens wearers was first noticed in 2005 by public health authorities in Singapore. Subsequently, 66 patients were identified from March 2005-February 2006; the characteristics in common among these patients were the use of ReNu with MoistureLoc solution and poor lens hygiene [10]. Two recent reports described a noticeable increase in Fusarium keratitis in contact lens wearers in the US, in the San Francisco Bay Area and in southern Florida [11,12]. A more comprehensive report has documented 176 cases of Fusarium keratitis in 164 patients from various regions in the US [13]. The investigators found significant use of Mois-tureLoc solution among 45 case patients in their series. Interestingly, they did not recover any Fusarium isolate through extensive sampling of lens solutions from the patients, or from environmental sampling of the plant where the solution was manufactured. Thus, the underlying causes of this perplexing outbreak remain elusive. We investigated recent fungal keratitis cases from New York State including the first case in which Fusarium was isolated from MoistureLoc solution being used by a patient. We also carried out experimental studies with Fusarium isolates in attempts to understand why these infections were associated with the use of MoistureLoc solution.

Mycology
Five Fusarium isolates, recovered from patients' corneal ulcers, contact lenses, or contact lens cases by hospital laboratories in New York and New Jersey, were forwarded to the Mycology Laboratory at the Wadsworth Center (Table  1). These isolates were studied in detail for their colony and microscopic characteristics [14,15]. An opened container of MoistureLoc solution being used by a patient for approximately three-months was also submitted for evaluation. An aliquot of this solution was directly plated on Sabouraud's dextrose agar supplemented with antibiotics (chloramphenicol 25 μg/ml, gentamicin 40 μg/ml) for fungal recovery, and the solution was centrifuged at 5000 rpm for 15 min to recover fungi from the pellet and the supernatant, by plating on culture medium. Additional unopened bottles of MoistureLoc solution were procured from local pharmacies in Albany and New York City before the global recall of this product by the manufacturer. These solutions were processed for fungal testing at the Wadsworth Center and the New York Eye and Ear Infirmary.

Molecular characterization
Specific identifications and typing of Fusarium isolates were done by PCR and nucleotide sequencing. Fungal DNA extraction involved grinding mycelia in a pestle and mortar under liquid nitrogen, suspending the slurry in lysis buffer (200 mM Tris-HCl, pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5%SDS), extraction with phenol: chloroform: isoamyl alcohol, and ethanol precipitation [16]. Fungal ribosomal DNA internal transcribed spacers 1 and 2 (ITS1, ITS2) and nuclear 28S rRNA [17], and Fusariumspecific partial β-tubulin and elongation factor (EF-1α) genes were amplified with published primers [18]. Nucleotide sequencing of the PCR amplicons was done on both strands according to the standard methods [19]. Nucleotide sequences were manually edited and compared against the NCBI database and Fusarium database at the Pennsylvania State University [20]. These sequences have been deposited in the GenBank under Accession Numbers DQ852626 -DQ852630 and DQ813505 -DQ813508. Percentage of nucleotide identity among various genes was compared by ClustalW (v1.4) multiple alignment, using MacVector 7.1.1 software (Accelrys, San Diego, CA). Phylogenetic analyses of nucleotide sequences were done with the PAUP v4 program using a bootstrap method with a Neighbor-Joining or Maximum Parsimony search [21].  [24].

Biofilm analysis
Microbial biofilms are an important potential source of infectious propagules in keratitis [25,26]. Therefore, the ability of the Fusarium isolates to form biofilms in the laboratory was tested with etafilcon A contact lenses (ACU-VUE ® , Johnson & Johnson Vision Care, Inc.) using a published procedure [25]. Briefly, F. solani 238-06 was grown on Sabouraud's dextrose agar slants for 5 days, and the conidia harvested in sterile distilled water. Fungal inoculum comprised of conidial suspension adjusted to 1 × 10 4 cells/ml with a hemacytometer. A balanced salt solution (0.49 g NaCl, 0.075 g KCl, 0.048 g CaCl 2 , 0.03 g MgCl 2 , 0.39 g sodium acetate {CH 3 COONa}, 0.17 g sodium citrate {HOC(COONa)(CH2COONa)2}, dissolved in 100 ml deionized water, and filter sterilized) was used to promote biofilm formation [25]. Five ml balanced salt solution aliquots were transferred to 25-ml flasks. Half of the flasks received sterile, fresh, ACUVUE contact lens and 10 μl of the fungal inoculum. The other flasks received contact lens, fungal inoculum and 1.0 ml MoistureLoc. The negative control was balanced salt solution with contact lens but no fungal inoculum. The flasks were incubated for 48 h at 25°C on a rotator (180 rpm). Some lenses from flasks with balanced salt solution and fungal inoculum but no MoistureLoc were removed at the end of incubation, treated with MoistureLoc for 4 h and processed for scanning electron microscopy (SEM). The contact lenses were fixed in 2% glutaraldehyde for 1 h, and dehydrated in 25-100% ethanol. Dehydrated lenses were critical point dried, coated with gold particles and examined in SEM as described previously [19].

Experimental biofouling
A previous report indicated that the time elapsed since opening and the storage conditions significantly affected the efficacy of the multipurpose contact lens solutions [27]. We designed a biofouling study based on earlier publications to test for efficacy of MoistureLoc solutions against Fusarium isolates [28,29]. MoistureLoc solutions, purchased from local pharmacies before the global recall of this product, were inoculated with freshly grown Fusarium to test for survival and growth. Both sterile glass test tubes (20 × 150 mm) and commercial lens cases (Sight Savers ® , Bausch & Lomb, Inc.) were used in these experiments. F. solani 238-06 and F. oxysporum 158-06, from 4-5 day old slants of Sabouraud's dextrose agar, were suspended in sterile distilled water. The suspension comprised mostly of conidia with rare hyphal fragments. The conidia were counted using a hemacytometer, and adjusted to a final density of 1 × 10 4 -10 7 cells/ml. Ten microliters of these suspensions were used to inoculate 1.0 ml of MoistureLoc. The mix was incubated unstirred at 23° -25°C for various intervals, and 100 μl aliquots were spread on Sabouraud's dextrose agar plates after 15, 30, 60 and 240 min. The positive control included fungal inoculum suspended in sterile water without MoistureLoc. The inoculated plates were incubated at 25°C for 48 -72 h and fungal colonies counted and photographed. [28,29].
The biofouling experiments were repeated 5 times.

Mycology
MoistureLoc solution, provided by a patient, was positive for Fusarium species, and quantification of fungal load revealed approximately 50 colonies/ml (figure 1a). Furthermore, the supernatant, and not the pellet from the contaminated solution yielded the fungus (figure 1b). The finding was consistent with moderate recovery of fungal colonies from this solution. Characterization of the morphology and spores of the MoistureLoc isolate (238-06) showed it to most closely resemble F. solani. Mycological investigations of five patient isolates also showed them to be either F. solani or F. oxysporum (Table 1).

Molecular characterization
Total of five genes including ITS1, ITS2, 28S rRNA, β-Tubulin, and EF-1α were used for confirmation of Fusarium species (GenBank Accession Numbers DQ852626 -DQ852630 and DQ813505 -DQ813508). Of these genes, sequences from ITS1, ITS2, and EF-1α possessed enough polymorphism, and therefore, were excellent marker with 99-100% accuracy for the identification of Fusarium species to be either F. solani or F. oxysporum species complex in the NCBI and Fusarium databases (hereafter referred as F. solani and F. oxysporum). On the contrary, sequences from 28S and β-tubulin genes were highly conserved and therefore, these were only of limited use in fur-

Discussion
Among the significant findings of our study was the recovery of similar F. solani strains from the MoistureLoc solution and from the contact lens case of a patient. To our knowledge, this is the first instance of matched isolations of any Fusarium strain connected with recent cases of fungal keratitis in contact lens wearers [10,13]. However, only one additional isolate of F. solani, among the four that we studied, shared this genotype. Also, two F. oxysporum strains in this study showed two distinct genotypes. These observations, although limited suggested that it is unlikely that common or clonal strains were the cause of the infections. This conclusion agrees with the findings of the report about the presence of multiple genotypes of F. solani and F. oxysporum in keratitis cases in contact lens wearers in the US [13]. The population make-up of a few F. solani isolates from Singapore and Hong Kong also showed mixed genotypes, albeit with one population predominating; the latter was common in both locales. More 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.

Molecular typing of Fusarium isolates
Scanning electron micrographs of biofilm formation comprehensive studies are indicated to rule out the presence of 'hypervirulent clones' as the cause of recent keratitis cases. It could be pertinent that a number of other investigators have pointed out that clinical strains of Fusarium are generally a mix of genotypes of plant and environmental origin [13,20,30,31].
It was reasonable to suspect that devices such as contact lenses or lens cases are important in the etiology of these infections. In fact, the storage cases are reported to pose an independent risk factor for biofilm formation in mycotic keratitis [26]. We did not receive any relevant specimens for mycology and/or microscopy that would have enabled us to further investigate this aspect. Our limited experimental analyses indicated that both F. solani and F. oxysporum strains were capable of forming biofilms on these devices under permissive conditions, but not in presence of or after recommended treatment with MoistureLoc solution. Based on this evidence, we suggest that the possible sources of Fusarium in keratitis patients are unlikely to include contact lenses or lens cases when MoistureLoc solution is properly used. However, further studies are indicated to examine if contaminated lenses or lens cases could still be important sources of infections independent of lens cleaning regimen.
Our recovery of F. solani from MoistureLoc solution provided by a patient, taken with the previous report of a strong association between MoistureLoc solution and keratitis cases, led us to evaluate the scenarios in which the efficacy of this multipurpose solution would be compromised. We found that MoistureLoc solution completely sterilized F. solani and F. oxysporum contaminations as long as recommended 4-hr treatment regimen was followed. These observations were consistent with the conclusions of many other reports about the Summary of experimental biofouling studies overall efficacy of various multipurpose solutions against fungi [28,29]. However, it has been documented that the efficacy of cleaning solutions depends on the storage temperature and the time elapsed after opening of the container [27]. Both of these variables were examined to a limited extent in our study; our experiments were carried out with freshly opened containers and with containers opened roughly 3-months earlier with storage at room temperature. Again, no appreciable differences in efficacy were noticed between fresh and aged solutions, experimentally inoculated with F. solani and F. oxysporum. The simulation of inappropriate usage of MoistureLoc solution yielded positive growth up to 60 min with F. oxysporum being 30-fold more resistant than F. solani. These results suggested that at least some occurrences of keratitis could result from temporary contamination and/or inadequate cleansing with MoistureLoc solution.

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