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In vitro efficacy of a buffered chelating solution as an antimicrobial potentiator for antifungal drugs against fungal pathogens obtained from horses with mycotic keratitis

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  • 1 Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 2 Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 3 Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 4 Athens Diagnostic Laboratory, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 5 Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 6 Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 7 Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

Abstract

Objective—To determine whether a novel third-generation chelating agent (8mM disodium EDTA dehydrate and 20mM 2-amino-2-hydroxymethyl-1, 3-propanediol) would act as an antimicrobial potentiator to enhance in vitro activity of antifungal medications against fungal isolates obtained from horses with mycotic keratitis.

Sample Population—Fungal isolates (3 Aspergillus isolates, 5 Fusarium isolates, 1 Penicillium isolate, 1 Cladosporium isolate, and 1 Curvularia isolate) obtained from horses with mycotic keratitis and 2 quality-control strains obtained from the American Type Culture Collection (ATCC; Candida albicans ATCC 90028 and Paecilomyces variotii ATCC 36257).

Procedure—Minimum inhibitory concentrations (MICs) against fungal isolates for 4 antifungal drugs (miconazole, ketoconazole, itraconazole, and natamycin) were compared with MICs against fungal isolates for the combinations of each of the 4 antifungal drugs and the chelating agent. The Clinical and Laboratory Standards Institute microdilution assay method was performed by use of reference-grade antifungal powders against the fungal isolates and quality-control strains of fungi.

Results—Values for the MIC at which the antifungal drugs decreased the growth of an organism by 50% (MIC50) and 90% (MIC90) were decreased for the control strains and ophthalmic fungal isolates by 50% to 100% when the drugs were used in combination with the chelating agent at a concentration of up to 540 μg/mL.

Conclusions and Clinical Relevance—The chelating agent increased in vitro activity of antifungal drugs against common fungal pathogens isolated from eyes of horses with mycotic keratitis.

Abstract

Objective—To determine whether a novel third-generation chelating agent (8mM disodium EDTA dehydrate and 20mM 2-amino-2-hydroxymethyl-1, 3-propanediol) would act as an antimicrobial potentiator to enhance in vitro activity of antifungal medications against fungal isolates obtained from horses with mycotic keratitis.

Sample Population—Fungal isolates (3 Aspergillus isolates, 5 Fusarium isolates, 1 Penicillium isolate, 1 Cladosporium isolate, and 1 Curvularia isolate) obtained from horses with mycotic keratitis and 2 quality-control strains obtained from the American Type Culture Collection (ATCC; Candida albicans ATCC 90028 and Paecilomyces variotii ATCC 36257).

Procedure—Minimum inhibitory concentrations (MICs) against fungal isolates for 4 antifungal drugs (miconazole, ketoconazole, itraconazole, and natamycin) were compared with MICs against fungal isolates for the combinations of each of the 4 antifungal drugs and the chelating agent. The Clinical and Laboratory Standards Institute microdilution assay method was performed by use of reference-grade antifungal powders against the fungal isolates and quality-control strains of fungi.

Results—Values for the MIC at which the antifungal drugs decreased the growth of an organism by 50% (MIC50) and 90% (MIC90) were decreased for the control strains and ophthalmic fungal isolates by 50% to 100% when the drugs were used in combination with the chelating agent at a concentration of up to 540 μg/mL.

Conclusions and Clinical Relevance—The chelating agent increased in vitro activity of antifungal drugs against common fungal pathogens isolated from eyes of horses with mycotic keratitis.

Horses are more susceptible to the development of fungal keratitis, compared with the susceptibility of other domestic species.1–3 It has been suggested3 that they are useful for the study of fungal keratitis in humans.3 The reported number of horses with fungal keratitis is increasing in the United States2,3 and parallels an increase in the number of humans with fungal keratitis.4,5 In clinically normal horses, saprophytic fungi can be isolated from the conjunctiva.1,6-8 It is speculated that the environment of horses, contaminated soils, and plant materials serve as the source for these saprophytic fungi.9–11 Fungi invade the cornea when the corneal epithelium is compromised,10,11 and disease progresses rapidly when left untreated or when steroids or antimicrobials are overused during treatment.10,11

Aspergillus spp,2,12-16 Fusarium spp,2,10,12–14 Penicillium spp,2,10,12 and dematiaceous mold species2,10 are common fungal organisms associated with mycotic keratitis in horses, and Aspergillus spp2,12-17 and Fusarium spp2,3,10,12,14 are the 2 organisms detected most commonly. Fusarium spp are the fungi most frequently reported in humans with mycotic keratitis.18

Topical antifungal medications are the most practical and economic way to treat horses with fungal keratitis. Natamycin is the only commercially available and FDA-approved topical antifungal medication for the treatment of fungal keratitis,19,20 and it reportedly is effective against fungal organisms associated with mycotic keratitis in horses.2,10,14 Extralabel use of topical azole derivatives, imidazoles (miconazole and ketoconazole), and triazoles (itraconazole and fluconazole) has been reported9,10,13,14 for the treatment of horses with fungal keratitis. Azoles have a broad spectrum and good penetration of the cornea after debridement of corneal epithelium.5,21 When corneal disease is advanced, infective pathogens can become refractory to the currently available antifungal medications, which results in the need for surgical intervention.5,12 Therefore, improvements in antifungal treatments are needed to treat this challenging clinical ophthalmic problem in horses and humans.

Early generations of buffered chelators22–26 as well as a third-generation buffered chelating agent27 are effective for potentiating actions of antimicrobials. Buffered chelators can enhance the bactericidal effects of antimicrobials in dogs with refractory otitis,23–25,28 pyoderma,24 osteomyelitis,22 multiple fistulas,22,29 rhinitis,30 and cystitis24,31 and horses with metritis.32 Buffered chelators reportedly have minimum adverse effects when used in joints,22 bones,22 the uterus,32 ears,23,25,28 the bladder,24,31 and mammary glands.22 The third-generation chelator used in the study reported here can potentiate the effects of antimicrobials against bacteria and fungi27 and is reportedly effective when treating dogs with chronic otitis externa,27 birds with web dermatitis,27 fish with superficial bacterial infections,27 and cows with mastitis.33 The purpose of the study reported here was to evaluate a third-generation buffered chelating agenta as a potentiator for topical antifungal drugs used against ophthalmic fungal strains isolated from horses with mycotic keratitis.

Materials and Methods

Sample population—Filamentous fungal isolates were obtained from horses with mycotic keratitis examined at 3 veterinary medical teaching hospitals (University of Georgia, Auburn University, and University of Florida). The isolates included 3 Aspergillus isolates (1 from Georgia, 1 from Auburn, and 1 from Florida), 5 Fusarium isolates (3 from Georgia and 2 from Florida), 1 Penicillium isolate (from Auburn), 1 Cladosporium isolate (from Georgia), and 1 Curvularia isolate (from Auburn). Filamentous fungi were cultured on plates containing potato dextrose agar; plates were incubated at 35°C for 5 to 7 days. Quality-control strains of bacteria (Candida albicans ATCC 90028b, and Paecilomyces variotii ATCC 36257c) were obtained from a commercial source. Candida albicans ATCC 90028 was cultured on plates containing potato dextrose agar; plates were incubated at 35°C for 24 to 48 hours.

Determinations of MICs—The MICs were determined for 4 antifungal drugs (miconazole, ketoconazole, itraconazole, and natamycin) and the combination of a third-generation chelator (8mM disodium EDTA dehydrate and 20mM 2-amino-2-hydroxymethyl-1, 3-propanediol) with each of the antifungal drugs against ophthalmic fungal isolates (3 Aspergillus isolates, 5 Fusarium isolates, 1 Penicilliumisolate, 1 Cladosporium isolate, and 1 Curvularia isolate) and ATCC quality-control strains (C albicans ATCC 90028 and P variotii ATCC 36257). The MIC50 and MIC90 values were determined by use of a microdilution assay method established by the CLSI (formerly known as the National Committee for Clinical Laboratory Standards).34,35 The percentage decrease in MIC50 and MIC90 values for the antifungal drugs were determined after combination with the buffered chelator and were used to quantify effectiveness of the buffered chelator as a potentiator of antimicrobial effects. Concentrations of the chelating agent were considered effective as an antimicrobial potentiator when it reduced the MIC50 or MIC90 of the antifungal drug by ≥ 50%.

Preparation of antifungal drugs—Serial dilutions of antifungal drugs were prepared as described for the established CLSI microdilution method34,35 by use of referencegrade powders for miconazole,d ketoconazole,e itraconazole,f and natamycin.g Test concentrations were formulated for the azoles (0.002 to 128.0 μg/mL) and natamycin (0.15 to 19.2 μg/mL) as described for the CLSI method. Reference-grade powders of miconazole, ketoconazole, and itraconazole were dissolved in dimethyl sulfoxideh to formulate stock solutions (12,800 μg/mL). Reference-grade natamycin powder was dissolved in 0.5N NaOH,i neutralized to pH 6.0 to 7.0 by the addition of 0.5N HCl,j and dissolved in sterile water to formulate a stock solution (1,920 μg/mL). All stock solutions of antifungal drugs were sterilized by use of 0.2-μm nylon syringe filters.k For all antifungal drugs, 0.1 mL of the stock solution was placed in sterile polypropylene tubes and diluted by the addition of 4.9 mL of RPMI-1640 medial containing L-glutamate without sodium bicarbonate and with 3-(N-morpholino) propanesulfonic acid buffer to achieve drug concentrations of 256 and 38.4 μg/mL for the azoles and natamycin, respectively. An aliquot (0.1 mL of the 256 μg/mL solution for the azoles or 38.4 μg/mL solution for natamycin) was placed in wells of sterile multiple-well plates; plates were then frozen at −70°C for later use. For water-insoluble antifungal drugs, RPMI-1640 media and dimethyl sulfoxide were added to the control wells. For natamycin, RPMI 1640 medium and sterile water were added to the control wells.

Preparation of chelator solutions—A stock solution (108,000 μg/mL) of the third-generation chelator was used to formulate a range of test concentrations (0.0098 to 540 μg/mL). The chelator solutions were placed in control wells, and the antifungal solutions were combined with the chelator in the remaining wells. Plates were frozen at −70°C for later use.

Preparation of inoculum—Broth inoculum was prepared for yeast (C albicans ATCC 90028) and filamentous fungi (P variotti ATCC 36257, Aspergillus spp, Fusarium spp, Penicillium sp, Cladosporium sp, and Curvularia sp) as described for the CLSI method.34,35Candida albicans was grown on plates containing Sabouraud dextrose agar; plates were incubated at 35°C for 24 to 48 hours. Colonies were suspended in sterile 0.85% saline (NaCl) solution and adjusted by use of a spectrophotometer to 85% transmittance (0.5 McFarland standard) at a wavelength of 530 nm. The suspension was diluted 1:50 in sterile 0.85% saline solution and then further diluted 1:20 in RPMI-1640 media. To obtain a yeast concentration of 0.5 × 103 to 2.5 × 103 CFU/mL, 100 μL of the suspension was placed in each well. Filamentous organisms were grown on plates containing potato dextrose agar; plates were incubated at 35°C for 5 to 7 days. Colonies were suspended in sterile 0.85% NaCl solution and adjusted by use of a spectrophotometer to match a 0.5 McFarland standard. Suspensions were further diluted 1:50 in RPMI broth to achieve a density of 0.4 × 104 to 5 × 104 CFU/mL.

Antifungal susceptibility testing—An aliquot (0.1 mL) of fungal inoculum was combined with 0.1 mL of an antifungal drug (with or without the chelator) in each well. Multiple-well plates were incubated at 35°C for 48 hours. Wells were visually scored by use of a grading scale that corresponded to the amount of fungal growth.34,35 The MIC50 and MIC90 values were defined as the lowest drug concentration that decreased fungal growth by 50% or ≥ 90%, respectively.

Percentage decreases in MIC50 and MIC90 for the antifungal drugs were determined after combination with the buffered chelator and were used to quantify effectiveness of the chelator as a potentiator of antimicrobial effects. A concentration of the chelator was considered effective as a potentiator when it reduced the MIC50 or MIC90 of the antifungal drugs by ≥ 50%. The lowest concentration of chelator that decreased the MIC50 and MIC90 of each antifungal drug against the fungal isolates by ≥ 50% was identified. Fungal organisms were considered susceptible to the antifungal drugs on the basis of tentative breakpoints published elsewhere14,26,34-37 for miconazole and ketoconazole (susceptible, < 8 μg/mL; resistant, > 16 μg/mL), itraconazole (susceptible, < 0.5 μg/mL; resistant, > 1 μg/mL), and natamycin (susceptible, < 16 μg/mL; resistant, > 64 μg/mL).

Statistical analysis—The Fisher exact test was used to evaluate the effectiveness of the reduction in the MIC of each antifungal tested against the fungal isolates. The Spearman rank correlation coefficient was used to evaluate correlation of the buffered concentrations with the reduction in MICs of the antifungal drugs against the fungal isolates. Values of P < 0.05 were considered significant. Statistical analyses were performed by use of a commercial program.m

Results

MICs for antifungal drugs against control strains—The MIC50 and MIC90 were determined for miconazole, ketoconazole, itraconazole, and natamycin against the control strains C albicans ATCC 90028 and P variotii ATCC 36257 (Table 1). Mean MIC50 for miconazole, ketoconazole, and itraconazole against C albicans ATCC 90028 and P variotii ATCC 36257 were within CLSI reference ranges. The CLSI reference range MIC50 for natamycin and MIC90 for the antifungal drugs against C albicans ATCC 90028 and P variotii ATCC 36257 have not been determined; however, on the basis of the tentative antifungal breakpoints, the control strains were susceptible to miconazole, ketaconazole, itraconazole, and natamycin.34–39

Table 1—

The MICs of antifungal drugs alone or when combined with a chelating agent against 2 quality-control fungi and percentage decreases in MICs achieved by use of the chelating agent.

Fungal organismConcentration of chelating agent (μg/mL)MIC categoryMIC (μg/mL)Reduction in MIC (%)
MicoKetoItraNataMicoKetoItraNata
Candida albicans ATCC 900280MIC500.5000.0310.201>14.400NANANANA
MIC90>1.000>1.000>1.000>19.200NANANANA
6.25MIC50000010010010099.3
MIC900001.92010010010090.0
Paecilomyces variotti ATCC 362570MIC500.0940.0650.0594.800NANANANA
MIC900.6000.6250.3109.600NANANANA
6.25MIC500.0090.0170.0320.51890.373.644.889.2
MIC900.4770.2610.3414.36320.458.2054.5

Mico = Miconazole. Keto = Ketoconazole. Itra = Itraconazole. Nata = Natamycin. NA = Not applicable.

MICs for antifungal drugs against fungal isolates from horses with mycotic keratitis—The MIC50 and MIC90 were determined for miconazole, ketoconazole, itraconazole, and natamycin against the filamentous fungi isolated from horses with mycotic keratitis (Tables 2–4). For the ophthalmic filamentous fungi tested, the order of susceptibility to the antifungal drugs was Penicillium sp >Curvularia sp > Cladosporium sp >Aspergillus spp >Fusarium spp.

MICs for antifungal drugs combined with the buffered chelator against control strains—The third-generation chelator decreased the MIC50 and MIC90 against the control strains C albicans ATCC 90028 and P variotii ATCC 36257 for all antifungal drugs (Table 1). The chelator at a concentration of 6.25 μg/mL decreased the MIC50s and MIC90s against C albicans ATCC 90028 by 90% to 100%. With the addition of chelator at a concentration of 6.25 μg/mL, the MIC50 and MIC90 values for the antifungal drugs against P variotii ATCC 36257 were decreased by 45% to 90% and 0% to 58%, respectively.

MICs for antifungal drugs combined with the buffered chelator against fungal isolates from horses with mycotic keratitis—Buffered chelator at a concentration of 200 μg/mL reduced the MIC50s for all antifungal drugs against Aspergillusisolates by 82% to 96% and the MIC90s for miconazole, ketoconazole, and itraconazole against Aspergillus isolates by 84% to 96%. The buffered chelator at a concentration of 400 μg/mL reduced the MIC90s for natamycin by 84% to 100%. There was a significant (P = 0.003) reduction in the MIC50s against the Aspergillus isolates with the addition of the chelator at a concentration of 200 μg/mL and the MIC90s with the addition of the chelator at a concentration of 400 μg/mL (Table 2).

Table 2—

The MICs of antifungal drugs alone or when combined with a chelating agent against Aspergillis isolates from horses with mycotic keratitis and percentage decreases in MICs achieved by use of the chelating agent.

Aspergillius spConcentration of chelating agent (μg/mL)MIC categoryMIC (μg/mL)Reduction in MIC (%)
MicoKetoItraNataMicoKetoItraNata
Isolate No. 10MIC503.1102.0001.000>19.200NANANANA
MIC9012.44012.0004.000>19.200NANANANA
100MIC501.000–2.0001.000–2.0001.0001.20036.0–68.00–50.00>93.8
MIC902.0002.0004.000>19.20084.083.000
200MIC500.12500.2500.1251.20096.087.587.5>93.8
MIC902.0000.5000.250>19.20084.095.893.80
400MIC50NENENE0NENENE100
MIC90NENENE0NENENENE
Isolate No. 20MIC503.2501.6201.380>19.200NANANANA
MIC9025.14016.0005.500>19.200NANANANA
100MIC503.0001.7501.1251.1257.7018.594.1
MIC9020.0008.5006.000>19.20020.446.900
200MIC500.2340.3120.2030.75093.081.085.0>96.0
MIC902.7501.1880.750>19.20089.093.086.00
400MIC500.1880.2030.1410.17894.088.090.0>99.0
MIC901.0940.5940.4692.40096.096.091.087.5
Isolate No. 30MIC503.5002.0001.380>19.200NANANANA
MIC9034.00020.0006.500>19.200NANANANA
100MIC502.3751.2501.0620.97532.137.523.094.9
MIC9017.0007.0006.500>19.20050.065.000
200MIC500.3120.3120.2190.75091.084.084.096.0
MIC903.0001.3750.938>19.20091.093.086.00
400MIC500.1720.2030.1250.18895.090.091.0>99.0
MIC901.2500.8120.4843.00096.090.093.0>84.0

NE = Not examined.

See Table 1 for remainder of key.

Buffered chelator at a concentration of 540 μg/mL reduced the MIC50s and MIC90s for all antifungal drugs against Fusariumisolates by 99% to 100%. There was a significant (P = 0.004) reduction in the MIC50s against Fusarium isolates with the addition of the chelator at a concentration of 400 μg/mL (Table 3). Buffered chelator at a concentration of 200 μg/mL significantly reduced the MIC50s and MIC90s for all antifungal drugs against the Penicillium isolate by 100%, against the Cladosporium isolate by 50% to 98%, and against the Curvularia isolate by 74% to 100% (Table 4).

Table 3—

The MICs of antifungal drugs alone or when combined with a chelating agent against Fusarium isolates from horses with mycotic keratitis and percentage decreases in MICs achieved by use of the chelating agent.

Aspergillius spConcentration of chelating agent (μg/mL)MIC categoryMIC (μg/mL)Reduction in MIC (%)
MicoKetoItraNataMicoKetoItraNata
Isolate No. 10MIC50>128.000>128.000>128.000>19.200NANANANA
MIC90>128.000>128.000>128.000>19.200NANANANA
100MIC500.2500.250<0.250>19.200>99.8>99.8>99.8NE
MIC90>128.000>128.000>128.000>19.200000NE
200MIC50<0.250<0.250<0.250>19.200>99.8>99.8>99.8NE
MIC900.5000.25016.000>19.200>99.6>99.8>87.5NE
540MIC50<0.050<0.050<0.0500>99.9>99.9>99.9100
MIC900.0500.0500.050099.999.999.9100
Isolate No. 20MIC50>128.00011.200>128.000>19.200NANANANA
MIC90>128.00064.000>128.000>19.200NANANANA
100MIC508.0002.00032.000>19.200>93.882.0>75.00
MIC90>128.00064.000>128.000>19.2000000
200MIC504.0001.0001.00019.200>96.0>91.0>99.20
MIC90>128.00032.000>128.000>19.20005000
540MIC50<0.050<0.500<0.0500>99.9>99.5>99.9100
MIC900.050<0.050<0.0501.92099.9>99.9>99.9>90.0
Isolate No. 30MIC50>128.000>128.000>128.000>19.200NANANANA
MIC90>128.000>128.000>128.000>19.200NANANANA
100MIC50128.00016.000>128.000>19.2000>87.50NE
MIC90>128.000>128.000>128.000>19.200000NE
200MIC5032.0008.000128.000>19.20075.0>93.80NE
MIC90>128.000128.000>128.000>19.200000NE
540MIC500000100100100100
MIC900000100100100100
Isolate No. 40MIC50>128.000109.710>128.000>19.200NANANANA
MIC90>128.000>128.000>128.000>19.200NANANANA
100MIC50>128.000109.700>128.000>19.2000000
MIC90>128.000>128.000>128.000>19.2000000
200MIC5012.0004.75056.000>19.20091.096.056.00
MIC90>128.00020.000104.000>19.200084.019.00
540MIC500000100100100100
MIC900000100100100100
Isolate No. 50MIC50>128.000>128.000>128.000>19.200NANANANA
MIC90>128.000>128.000>128.000>19.200NANANANA
100MIC5096.00026.00056.000>19.20025.079.956.20
MIC90>128.00080.000>128.000>19.200037.500
200MIC5028.0005.50012.000>19.20078.096.091.00
MIC90>128.00017.000>128.000>19.200087.000
540MIC500000100100100100
MIC900000100100100100

See Tables 1 and 2 for key.

Table 4—

The MICs of antifungal drugs alone or when combined with a chelating agent against Penicillium sp, Cladosporium sp, and Curvularia sp isolates from horses with mycotic keratitis and percentage decreases in MICs achieved by use of the chelating agent.

Fungal organismConcentration of chelating agent (μg/mL)MIC categoryMIC (μg/mL)Reduction in MIC (%)
MicoKetoItraNataMicoKetoItraNata
Penicillium sp0MIC500.1810.1810.5009.600NANANANA
MIC900.3610.5000.72219.200NANANANA
100MIC50<0.062000>65.0100100100
MIC900.0620.0620083.087.5100100
200MIC500000100100100100
MIC900000100100100100
Cladosporium sp0MIC503.5000.6884.500>19.200NANANANA
MIC9015.002.12514.000>19.200NANANANA
12.5MIC500.1560.0620.281>19.20095.553.593.80
MIC904.5000.8753.750>19.2007058.873.20
200MIC500.0780.0320.2199.00098.095.095.053.0
MIC900.2810.1090.469>19.20098.095.097.00
Curvularia sp0MIC503.1100.6670.7789.600NANANANA
MIC904.0001.5602.00019.200NANANANA
200MIC500.0250.0250.025099.096.397.0100
MIC900.4000.4000.400090.074.080.0100

See Table 1 for key.

Concentration of the buffered chelator was significantly (P < 0.001) correlated with the reduction in the MIC50s for all the antifungal drugs. There also was a significant correlation between the concentration of the chelator and reduction in the MIC90s for each of the antifungal drugs (miconazole, P = 0.007; ketoconazole, P = 0.002; itraconazole, P = 0.006; and natamycin, P < 0.001).

Discussion

Fungal keratitis is a common cause of ocular disease in horses, and the incidence of mycotic keratitis is increasing in horses and humans. This condition can be challenging and expensive to treat with currently available topical medications. Vision loss is reported in 7.7% to 54.5% of affected horses, and the rate for enucleation reportedly ranges from 5.1% to 42.9% after unsuccessful treatment (medical or a combination of medical and surgical).2,3,10,12,16 Topical treatments provide the most effective means for delivery of medications to the cornea. However, natamycin is currently the only FDA-approved topical antifungal medication for the treatment of fungal keratitis. Other antifungal agents have been used in an extralabel manner with some success,9,10,13,14 but in patients with advanced disease, the infections are often refractory to medical treatment and require surgical intervention.5,12 Therefore, improved antifungal agents are needed to treat patients with this challenging ophthalmic problem.

The third-generation chelating agent evaluated in the study reported here can potentiate the effects of antimicrobials against gram-positive and gram-negative bacteria, yeast, and fungi.27 The mechanism of action for the buffered chelating agent against fungi is not known, but it is believed that the chelating agent removes divalent cations from the outer membrane of bacteria, which alters the integrity and permeability of the cell wall.27 The cell walls of fungi are composed mainly of polysaccharides (β-glucans and chitin) and protein.40 The removal of divalent cations may alter membrane proteins that are important in maintaining the construction and maintenance of polysaccharides in the cell wall of fungi. The in vitro study reported here revealed that this third-generation buffered chelator at a concentration ≤ 540 μg/mL decreased the concentration of antifungal drugs required to inhibit fungal growth.

The MICs in our study are comparable to those in other in vitro studies14,15,41-45 of Aspergillusisolates. Studies14,15 of antifungal susceptibility for ophthalmic isolates of horses revealed similar results, with lower MICs for the azoles against Aspergillusisolates, compared with the MIC for natamycin; however, in 1 of those studies,14 there was not a significant difference in susceptibility between the azoles and natamycin. All 3 of the Aspergillus isolates in the study reported here were susceptible to the azoles, and the MICs for natamycin were higher than the MICs for the azoles against the Aspergillusisolates. The buffered chelator at a concentration ≤ 400 μg/mL was effective as a potentiator of antimicrobial effects for the azoles and natamycin against the ophthalmic Aspergillus isolates.

Of the 5 Fusarium isolates tested in the study reported here, none was susceptible to the antifungal drugs, except Fusarium isolate No. 2, which was susceptible to ketoconazole. The azoles consistently had higher MICs, compared with the MICs for natamycin, against the Fusarium isolates, which is consistent with results of other in vitro studies.14,15,41,42 The Fusariumisolates required the highest concentrations (up to 540 μg/mL) of the buffered chelator to achieve effective reductions in the MICs.

In other in vitro studies, the MICs for nonophthalmic Penicillium spp, Curvularia spp, and Cladosporium spp isolates45–47 and ophthalmic Penicilliumspp isolates14,15 were consistent with those for the study reported here. Penicillium and Curvularia isolates were susceptible to the azole antifungals and natamycin. Cladosporiumisolates were susceptible to the azoles, but the MICs for natamycin were higher, compared with the MICs for the azoles. The buffered chelator at a concentration ≤ 200 μg/mL was effective as a potentiator for the antimicrobial effects of the azoles and natamycin against the ophthalmic Penicillium sp, Curvularia sp, and Cladosporium sp isolates.

The buffered chelating agent effectively potentiated (decreased the MICs by 50% to 100%) the effect of all antifungals against all ophthalmic fungal isolates tested, and the decreased MICs corresponded to the susceptibility of the fungal isolates (ie, Penicillium sp > Curvularia sp >Cladosporium sp >Aspergillus spp > Fusarium spp). Higher concentrations of the chelator (up to 540 μg/mL) were needed to reduce the MICs of the antifungal drugs against the Fusarium isolates. Chelator at a concentration of 200 μg/mL was needed to decrease the MICs for the antifungal drugs against Aspergillus spp, Penicillium sp, Cladosporium sp, and Curvularia sp. The control strains (C albicans ATCC 90028 and P variotii ATCC 36257) required the lowest concentrations of the chelator (6.25 μg/mL) to achieve effective reduction in the MIC50s for all the antifungal drugs and the MIC90s for ketoconazole and natamycin.

Analysis of results for the in vitro study reported here suggests that the third-generation chelating agent may hold promise as an adjunctive agent for use in the treatment of animals with fungal keratitis. Azole antifungal drugs inhibit ergosterol synthesis, and polyenes bind to ergosterol in the cell membrane, which increases cell permeability.32 The third-generation chelating agent evaluated in our study may interfere with fungal growth by another mechanism than that for the azoles or polyene antifungal drugs, thereby increasing their spectrum of activity. However, additional studies are needed to determine the mechanism of action against fungi for this third-generation chelating agent, evaluate its ability to penetrate the cornea, and assess its effectiveness in animals with mycotic keratitis.

ABBREVIATIONS

ATCC

American Type Culture Collection

MIC

Minimum inhibitory concentration

MIC50

MIC at which 50% of the growth of an organism is inhibited

MIC90

MIC at which 90% of the growth of an organism is inhibited

CLSI

Clinical and Laboratory Standards Institute

a.

Tricide, Molecular Therapeutic LLC, Athens, Ga.

b.

Quality-control strain Candida albicans ATCC 90028, American Type Culture Collection, Manassas, Va.

c.

Quality-control strain Paecilomyces variotii ATCC 36257, American Type Culture Collection, Manassas, Va.

d.

Reference-grade antifungal powder—miconazole, Research Diagnostics Inc, Flanders, NJ.

e.

Reference-grade antifungal powder—ketaconazole, Research Diagnostics Inc, Flanders, NJ.

f.

Reference-grade antifungal powder—itraconazole, Research Diagnostics Inc, Flanders, NJ.

g.

Reference-grade antifungal powder—natamycin, Alcon Research LTD, Fort Worth, Tex.

h.

Dimethyl sulfoxide, Sigma Chemical Co, St Louis, Mo.

i.

0.5N NaOH, JT Baker, Phillipsburg, Pa.

j.

0.5N HCL, JT Baker, Phillipsburg, Pa.

k.

Nylon syringe filters, Nalgene, Rochester, NY.

l.

RPMI 1640 medium, Sigma Chemical Co, St Louis, Mo.

m.

GB-Stat, Dynamic Microsystems Inc, Silver Spring, Md.

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Contributor Notes

Supported by the Veterinary Ophthalmology Research Fund at the University of Georgia.

Drs. Wooley and Ritchie are inventors of the antibiotic potentiator technology marketed under the name Tricide. Rights to the technology marketed under the name Tricide are licensed by the University of Georgia Research Foundation (UGARF) to Molecular Therapeutics, which pays a royalty to UGARF for those rights. A percentage of the royalties is paid to the inventors. Dr. Ritchie is a principle in Molecular Therapeutics. Drs. Weinstein, Moore, Wooley, and Ritchie and UGARF have submitted a patent application for the ophthalmic use of chelating solutions.

The authors thank Drs. Ellen Belknap and Dennis Brooks for providing ophthalmic fungal isolates, Alcon Research Incorporated for donating reference-grade natamycin, and Melinda Pethel for technical assistance.