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Applied and Environmental Microbiology, May 2003, p. 2563-2567, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2563-2567.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enhanced Killing of Acanthamoeba Cysts with a Plant Peroxidase-Hydrogen Peroxide-Halide Antimicrobial System

Reanne Hughes, Peter W. Andrew, and Simon Kilvington^*

Department of Microbiology and Immunology, University of Leicester, Leicester LE1 9HN, United Kingdom

Received 30 August 2002/ Accepted 31 January 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
The activity of H₂O₂ against the resistant cyst stage of the pathogenic free-living amoeba Acanthamoeba was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase. This resulted in an increase in the cysticidal activity of 3% (wt/vol) H₂O₂, and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (P < 0.05). With 2% H₂O₂, enhancement was observed at all time points (P < 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. The activity of sublethal 1% H₂O₂ was enhanced to give 3-log killing after 8 h of exposure (P < 0.05). No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures. The H₂O₂ was not neutralized in the enhanced system during the experiments. However, in the presence of a platinum disk used to neutralize H₂O₂ in contact lens care systems, the enhanced 2% H₂O₂ system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H₂O₂ occurred by 4 h. In contrast, 2% H₂O₂ alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide. Our observations could result in significant improvement in the efficacy of H₂O₂ contact lens disinfection systems against Acanthamoeba cysts and prevention of acanthamoeba keratitis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hydrogen peroxide (H₂O₂) is an effective microbial disinfectant, destroying pathogens by oxidation that results in protein denaturation (5). As such, it is widely used for disinfection processes in the food, water treatment, health care, and contact lens industries (5). In vivo, stimulated phagocytes produce H₂O₂ and then use it in the destruction of phagocytosed microorganisms (8). In neutrophils, at least, the process is further enhanced by the action of a myeloperoxidase that forms hypochlorous acid (HOCl) from chloride ions and H₂O₂ (17). The combination of H₂O₂ and HOCl is a potent antimicrobial combination that destroys cells by halogenation and oxidation of cell surface components (14, 17, 23, 28).

This innate myeloperoxidase-mediated antimicrobial system was first described by Klebanoff in 1967 (12). Subsequently, it was demonstrated that this mechanism can be reproduced in vitro to obtain enhanced hydrogen peroxide killing of a variety of pathogenic microbes, including bacteria (10, 13, 15, 24, 25), fungi (28), human immunodeficiency virus type 1 (16), and the parasitic helminth Schistosoma mansoni (11). Peroxidases other than myeloperoxidase have been used, including eosinophil peroxidase (27), lactoperoxidase, horseradish peroxidase (HRP), and catalase (2, 3, 9, 24). In addition, bromide and iodide have been shown to be capable of replacing chloride in the reaction (9, 11, 15). Until now, the effects of this potent antimicrobial system on protozoans have not been reported.

Acanthamoeba is a genus of free-living amoebae that occur in virtually all soil and aquatic habitats (20). These organisms are characterized by a motile feeding and replicating trophozoite that can form a highly resistant cyst stage in response to adverse conditions (20). Acanthamoeba is an opportunistic pathogen of humans, causing fatal encephalitis in immunocompromised hosts and, more frequently, a potentially blinding infection of the cornea, termed acanthamoeba keratitis, in previously healthy persons (18). Contact lens wearers are most at risk from infection and account for 90% of reported cases (21). Hydrogen peroxide (3%) is a commonly used contact lens disinfectant and has been shown to be effective against Acanthamoeba cysts, giving a 3-log reduction in viability, provided that an exposure time of at least 4 to 6 h is used prior to neutralization (7). Hydrogen peroxide is toxic to the cornea and must be neutralized before lens wear to avoid pronounced stinging, lacrimation, hyperemia, and possible corneal damage (6, 26). One-step hydrogen peroxide systems which do not require a separate neutralization step are available. In these systems, neutralization is achieved in a storage case during disinfection by using a platinum-coated disk or soluble catalase tablet which catalyzes the decomposition of hydrogen peroxide to water and oxygen. However, the rapid neutralization in such systems results in little or no cysticidal efficacy (7).

In the present study we investigated the potential for enhancing the cysticidal activity of H₂O₂ through a plant peroxidase-hydrogen peroxide-halide antimicrobial system. The finding that use of this system results in significant enhancement of H₂O₂ killing of Acanthamoeba cysts, either alone or in the presence of a platinum disk used in one-step peroxide disinfection of contact lenses, prompted this report.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Acanthamoeba strains and culture.
Acanthamoeba polyphaga strain Ac-Ros isolated from a keratitis case in the United Kingdom in 1991 was used in all developmental experiments. The pathogenic species Acanthamoeba quina (strain L1a) and Acanthamoeba culbertsoni (strain ATCC 30171) and four Acanthamoeba strains from recent keratitis cases (Ak-1 to Ak-4) were also studied. Although the latter strains were not identified to the species level, they were cyst morphological type II strains, which account for the majority of Acanthamoeba strains causing keratitis (20). Two nonpathogenic species, Acanthamoeba castellanii (strain Neff [= CCAP 1501/1a]) and Acanthamoeba palestinensis (strain CCAP 1547/1), were also tested. Trophozoites were maintained in a semidefined axenic broth medium as previously described (7). Cysts were prepared from the trophozoite cultures by using Neff's chemically defined encystment medium or in 0.25x Ringer's solution containing 20 mM taurine and 15 mM MgCl₂ in the case of A. culbertsoni (7, 19). Mature cysts were stored at 4°C for use in testing within 14 days.

Test solutions.
All chemicals and enzymes used in this study were obtained from Sigma Chemical Company (Poole, United Kingdom) or BDH (Poole, United Kingdom) unless otherwise stated. Catalase (EC 1.11.1.6), HRP (EC 1.11.1.7), soybean peroxidase (SBP) (EC 1.11.1.7), and bovine lactoperoxidase (LPO) (EC 1.11.1.7) were dissolved in nanopure water, filter sterilized, and stored at -20°C. The halide stock solutions were stored at room temperature in the dark. The H₂O₂ stock solution was stored in the dark at 4°C, and the concentration was measured before use by using a molar extinction coefficient of 81 M^-1 • cm^-1 at 230 nm.

Cysticidal assay.
The assay method used to determine cyst killing was the method described previously (7). Briefly, 2 x 10⁵ cysts were inoculated into 10 ml of solution in 50-ml polypropylene tubes (Becton Dickinson, Oxford, United Kingdom). At zero time and at 1, 2, 4, and 6 h the solution was vortexed, and quadruplicate 20-µl samples were removed, added to 200 µl of 0.02% (wt/vol) bovine liver catalase in a 96-well flat-bottom microtiter plate (Triple Red Laboratory Technology, Thane, United Kingdom), and left for 5 min to neutralize. Serial dilutions of 20 µl in 200 µl of 0.25x Ringer's solution (Oxoid, Basingstoke, United Kingdom) were then made across the rows of a microtiter plate in quadruplicate. Twenty-five microliters of Escherichia coli JM101 (optical density at 600 nm, 0.4) was then added to each well containing a cyst dilution, and the plate was sealed and incubated at 32°C for up to 7 days. Viable cysts hatched (excysted) in the presence of the live E. coli, and the subsequent growth and replication of the trophozoite were observed microscopically. The plates were inspected daily for 7 days for the presence of excystment and trophozoite replication in the wells. This allowed us to assess the number of cysts killed by a most-probable-number approach (see below) (22).

The test reaction mixtures were kept in the dark between measurements. The concentration of H₂O₂ at each time point was determined by using Peroxid test strips (BDH, Darmstadt, Germany) that had a detection sensitivity ranging from 25 to 0.00005% (0.5 ppm). The pH values of the solutions were determined at the beginning and end of the experiments with a pH meter (Jenway 3310: Jenway Ltd., Essex, England).

Enhanced peroxide system.
A checkerboard experimental design was used to determine the cysticidal effects of various concentrations of HRP (50 to 300 U ml^-1) combined with various concentrations of KI (20 to 200 µM) and various concentrations of H₂O₂ (0.125 to 3% [vol/vol]) in 0.25x Ringer's solution. In control experiments we used H₂O₂ alone in the assays. The combination of HRP, H₂O₂, and KI showing the greatest enhanced cysticidal efficacy was then tested on three separate occasions. Controls in which 0.25x Ringer's solution, hydrogen peroxide, KI, HRP, and KI-HRP alone or in combination were used were included.

Assays were conducted in either 0.25x Ringer's solution (pH 4.31 to 5.62) or phosphate-buffered saline (PBS) (pH 6.55 to 7.13) to determine the effect of pH on the activity of the system. Assays were also performed in which KI was replaced with KCl (50 to 200 µM) and HRP was replaced with SBP (150 to 200 U ml^-1), LPO (150 U ml^-1), or catalase (100 to 200 U ml^-1). In addition, H₂O₂ was replaced with the peroxide-generating chemical sodium perborate (1 to 3% [wt/vol]), and tests were performed by using HRP concentrations ranging from 5 to 50 U ml^-1 and KI concentrations ranging from 20 to 50 µM.

The HRP-H₂O₂-KI combination that gave the greatest enhancement of killing of A. polyphaga Ac-Ros cysts was then tested with the additional Acanthamoeba species and strains.

Peroxidase-hydrogen peroxide-halide system with platinum neutralization.
Five milliliters of 2% (wt/vol) H₂O₂ in 0.25x Ringer's solution, 50 µM KI, and 150 U of HRP per ml were combined in a 50-ml polypropylene tube. A platinum-coated disk (Aodisc; Ciba Vision, Atlanta, Ga.) which is used for neutralization in hydrogen peroxide-based contact lens disinfectant systems was added. The system was immediately challenged with A. polyphaga Ac-Ros cysts, and viability assays were performed over a 6-h period. Experiments in which 0.25x Ringer's solution, H₂O₂ alone, and H₂O₂ in the presence of a platinum-coated disk were used were also conducted. Hydrogen peroxide levels in the reaction mixtures were recorded at each sampling point as described above.

Data analysis.
The most probable number of surviving cysts in a microtiter plate at each time point was determined by using Reed-Muench computations as previously described for Acanthamoeba cyst viability (4, 22). The reduction in the number of viable cysts was plotted as the change in the log viability for each time point compared to the zero-time viability. A statistical analysis was performed by using one-way analysis of variance with means and standard errors of the means of triplicate experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hydrogen peroxide killing.
The cysticidal activity of H₂O₂ alone in 0.25x Ringer's solution (pH 4.31 to 5.62) against A. polyphaga Ac-Ros is shown in Fig. 1. Hydrogen peroxide at a concentration of <1% showed little cysticidal activity, giving <1-log killing in 6 h (results not shown). After 4 h of exposure, H₂O₂ at concentrations of 1, 2, and 3% gave values of 1.10 ± 0.31, 2.35 ± 0.49, and 2.75 ± 0.00 logs for killing of cysts, respectively. By 6 h the values were 1.67 ± 0.38, 3.46 ± 0.08, and 4.33 ± 0.08 logs, respectively (Fig. 1). Similar results were obtained with H₂O₂ tested in PBS (pH 6.55 to 7.13), and there was no statistical difference between this cysticidal activity and that in 0.25x Ringer's solution (P > 0.05) (results not shown).

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FIG. 1. Enhanced killing of A. polyphaga cysts with the peroxidase-hydrogen peroxide-halide system. Symbols: , 1% H2O2; , 1% H2O2, 50 U of HRP ml-1, and 50 µM KI; , 2% H2O2; , 2% H2O2, 150 U of HRP ml-1, and 50 µM KI; , 3% H2O2; •, 3% H2O2, 200 U of HRP ml-1, and 50 µM KI. The error bars indicate standard errors of the means from triplicate experiments. In control experiments with only 0.25x Ringer's solution, a <0.5-log reduction in cyst viability occurred (data not shown).

Enhanced hydrogen peroxide killing.
Addition of KI and HRP enhanced the cysticidal activity of H₂O₂ (Fig. 1). The checkerboard experiments showed that maximum activity occurred with 50 µM KI and 50 U of HRP ml^-1 for 1% H₂O₂, with 150 U of HRP ml^-1 for 2% H₂O₂, and with 200 U of HRP ml^-1 for 3% H₂O₂ (Fig. 1). Omission of either the KI or HRP from the reaction mixture resulted in no enhancement of cysticidal activity (results not shown). KI, HRP, or KI-HRP alone showed no cysticidal activity (results not shown).

With 1% H₂O₂, enhancement of cysticidal activity was observed for all time points, and by 6 h the level of killing was 2.76 ± 0.27 logs, compared with 1.7 ± 0.30 logs for H₂O₂ alone (P < 0.05). With 2% H₂O₂, significant enhancement was also observed at all times (P < 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. Significant cysticidal activity was also observed with 3% H₂O₂ at all times (P < 0.05), and this activity resulted in total killing at 4 h, compared with 6 h for the peroxide solution alone.

Modifications to the enhanced H₂O₂ system.
Replacing KI with KCl (50 µM to 200 µM) resulted in no enhancement of the activity of 1 to 3% H₂O₂ with concentrations of HRP up to 200 U ml^-1 (results not shown). Replacing HRP with SBP resulted in similar statistically significant enhanced cysticidal activity with 50 µM KI and 2 or 3% H₂O₂ (P < 0.05). No difference in efficacy was observed between the two plant-derived peroxidases. For example, 2% H₂O₂ and 50 µM KI used with 150 U of HRP ml^-1 gave levels of killing of 3.50 ± 0.25 logs after 4 h, compared to 3.89 ± 0.45 logs when 150 U of SBP ml^-1 was used. These values were compared with the value obtained with 2% H₂O₂ alone at this time, 2.33 ± 0.49 logs. With 2% H₂O₂, 50 µM KI, and 150 U of LPO ml^-1, levels of killing of 1.98 ± 0.27 logs were observed after 2 h and levels of killing of 3.56 ± 0.44 logs were observed after 4 h. These values were compared with the levels of killing obtained with 2% H₂O₂ alone at these times, 1.28 ± 0.13 and 2.42 ± 0.10 logs, respectively. Although there was some suggestion that the activity was enhanced, the findings for LPO were not statistically significant (P > 0.05).

In contrast, substituting catalase (50 to 200 U ml^-1) for HRP resulted in decreased cysticidal activity. For example, 2% H₂O₂ with 100 U of catalase ml^-1 and 50 µM KI gave levels of killing of 0.20 ± 0.36 log at 4 h, compared with 2.33 ± 0.49 logs with 2% H₂O₂ alone at that time. With 3% H₂O₂, 100 U of catalase ml^-1, and 50 µM KI, levels of killing of 0.51 ± 0.27 log occurred after 4 h, compared with 2.75 ± 0.00 logs with the peroxide alone. Replacing KI with KCl (50 to 200 µM) did not alter this effect with catalase (results not shown).

Substituting PBS (pH 6.55 to 7.13) for 0.25x Ringer's solution (pH 4.31 to 5.62) in the optimized HRP-H₂O₂-KI reactions did not affect the efficacy of the enhanced cysticidal activity observed with 1 to 2% peroxide (results not shown) (P > 0.05). However, with 3% peroxide in PBS, the HRP-H₂O₂-KI optimized system gave levels of cyst killing of 2.84 ± 0.10 logs at 2 h, compared with 4.42 ± 0.08 logs in 0.25x Ringer's solution (P < 0.05); the differences at the other times were not significant, and killing values similar to those obtained with 0.25x Ringer's solution were obtained (results not shown) (P > 0.05).

Sodium perborate (1 to 3% [wt/vol] in 0.25x Ringer's solution, yielding 0.17 to 0.5% H₂O₂) was not cysticidal even after 8 h of contact time (<1-log killing), and addition of HRP (5 to 20 and 150 U ml^-1) and KI or KCl (50 µM) did not result in increased cysticidal activity (results not shown).

The peroxide levels (1 to 2% [wt/vol]) in the HRP-H₂O₂-KI and SBP-H₂O₂-KI systems remained constant throughout the experiments. However, rapid H₂O₂ neutralization occurred when catalase was used in place of peroxidase, and the levels fell to 0.2% after the first hour and then remained at this level thereafter (results not shown).

Enhanced activity against other Acanthamoeba species and strains.
When tested in 0.25x Ringer's solution, all additional Acanthamoeba species and strains showed enhanced killing with 2% H₂O₂, 150 U of HRP ml^-1, and 50 µM KI (Fig. 2). The differences were statistically significant at 4 h compared to the values for H₂O₂ alone (P < 0.05) for strains Ak-3, Ak-Ros, and Ak-4, as well as A. castellanii Neff and A. palestinensis CCAP 1547/1. By 6 h the enhanced system had achieved total killing (>3 logs) for all species and strains studied, compared with values of <3 logs for 2% H₂O₂ alone (results not shown).

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FIG. 2. Enhanced killing of cysts of Acanthamoeba species and strains with peroxidase-hydrogen peroxide-halide after 4 h of exposure. Open bars, 2% H2O2; solid bars, 2% H2O2, 150 U of HRP ml-1, and 50 µM KI. In control experiments with only 0.25x Ringer's solution, a <0.6-log reduction in cyst viability occurred (data not shown).

Peroxidase-hydrogen peroxide-halide system with platinum neutralization.
The efficacy of 2% H₂O₂ with 150 U of HRP ml^-1 and 50 µM KI in the presence of a platinum neutralizing disk is shown in Fig. 3. Little cysticidal activity was found with 2% H₂O₂ alone in the presence of the platinum disk, and <0.8-log killing of cysts occurred after 6 h of exposure. In contrast, the HRP-H₂O₂-KI system with the platinum-coated disk gave 1.4-log killing after 1 h, 2.4-log killing after 4 h, and 2.8-log killing after 6 h. Total cyst killing (>3 logs) was achieved by 8 h (results not shown). Less than 1-log killing occurred with the platinum disk alone in 0.25x Ringer's solution, KI, HRP, or KI-HRP (results not shown).

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FIG. 3. Enhanced killing of A. polyphaga cysts with the peroxidase-hydrogen peroxide-halide system in the presence of a platinum neutralizing disk. The log killing is indicated on the left y axis (, 2% H2O2; , 2% H2O2, 150 U of HRP ml-1, and 50 µM KI), and the percentage of hydrogen peroxide is indicated on the right y axis (, H2O2 levels; •, H2O2 levels in the presence of peroxidase-halide). In control experiments with only 0.25x Ringer's solution, a <0.5-log reduction in cyst viability occurred (data not shown).

The hydrogen peroxide levels in the mixtures fell to <0.5 ppm within 1 h with H₂O₂ and the platinum-coated disk. However, with HRP-H₂O₂-KI and the platinum-coated disk, the H₂O₂ levels were 0.5% after 1 and 2 h and <0.5 ppm at 4 h (Fig. 3).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hydrogen peroxide (3%) is an effective disinfectant against Acanthamoeba cysts, giving at least a 3-log reduction in viability provided that an exposure time of at least 4 to 6 h is used (7). In this study, we demonstrated that the activity of H₂O₂ against Acanthamoeba cysts can be enhanced by addition of the halide I^- and a plant-derived peroxidase. This resulted in an increased rate of cysticidal activity and total killing of the cyst inocula by 2 h with 3% H₂O₂ and by 4 h with 2% H₂O₂. The system also enabled a sublethal H₂O₂ concentration (1%) to produce 3-log killing within 6 h. Furthermore, this finding indicates that the previously observed in vitro activity of the peroxidase-peroxide-halide system against pathogenic viruses, bacteria, fungi, and the helminth S. mansoni can be extended to include the pathogenic protozoan Acanthamoeba (10, 11, 13, 15, 16, 24, 28).

It is known that in vivo myeloperoxidase plays a crucial role in killing phagocytosed bacteria in neutrophils by reacting with H₂O₂ to form an enzyme substrate complex that oxidizes halide to produce even more toxic components (13, 14). The primary agent involved is hypohalous acid, which destroys cells by halogenation and oxidation of cell surface components (14, 17, 23, 28). It has been suggested that the peroxidase-peroxide-halide system acts preferentially against pathogenic rather than nonpathogenic organisms through binding to the cell surface; the target-bound peroxidase then catalyzes halide oxidation and facilitates the disproportionation of peroxide to singlet molecular oxygen at the surface of the target microbe (1, 2, 3). As the lifetime of singlet molecular oxygen is short and its diffusion potential is proportionally limited, the target organism is killed with minimal damage to any nonpathogenic microbes or host cells (1).

The precise mode of action of the peroxidase-hydrogen peroxide-halide system described here against Acanthamoeba cysts is unclear, but it appears to be equally effective against both pathogenic and nonpathogenic species (A. castellanii Neff and A. palestinensis CCAP 1547/1). However, it seems probable that under the reaction conditions described here, this results in the formation of HOI, which enhances the cysticidal activity of the H₂O₂. Whether this reaction is localized through primary binding of the peroxidase on the cyst wall surface or occurs within the cyst has yet to be elucidated.

Replacing KI with KCl in the system did not result in enhanced cysticidal activity. This is in accordance with other studies, which have shown that I^- is the most effective halide, followed by Br^- and then Cl^- (11, 13, 15). We also found that the enzyme SBP could replace HRP in the system with comparable cysticidal enhancing activity. Enhanced activity was also observed with LPO, although this enzyme was not as efficacious as the plant peroxidases. Replacing these peroxidases with bovine liver catalase resulted in no cysticidal activity, as the H₂O₂ was rapidly neutralized within 1 h. Using lower levels of catalase and replacing KI with KCl did not result in enhanced H₂O₂ activity. This is in contrast to the findings for SBP or HRP when the H₂O₂ levels remained constant throughout the experiments. Replacing H₂O₂ with sodium perborate (3% [wt/vol], yielding 0.5% H₂O₂) in the system resulted in no cysticidal activity, presumably due to the low peroxide-generating capacity of the latter chemical (7).

Hydrogen peroxide is commonly used for disinfection of contact lenses, although it must be neutralized before the lenses are worn to avoid corneal damage (6, 26). One-step hydrogen peroxide systems which do not require a separate neutralization step are available; neutralization is achieved during disinfection in the storage case by using a platinum-coated disk or a soluble catalase tablet which catalyzes the decomposition of hydrogen peroxide. However, in one-step systems this neutralization process occurs too rapidly for cysticidal activity to occur (7). In the peroxidase-hydrogen peroxide-halide system developed here, which exhibits enhanced cysticidal activity, neutralization of the H₂O₂ does not occur during the reaction. However, when the reaction was performed in the presence of a platinum disk, not only was the enhanced killing maintained, but the peroxide was neutralized so that the concentration was <0.5 ppm by 4 h. Levels of H₂O₂ of 2 to 5 ppm are not likely to cause irritation to the eye; 30 ppm has been reported to induce cytotoxicity in the eye, and 100 ppm produces noticeable discomfort (26).

Acanthamoeba keratitis is a potentially sight-threatening infection that is most commonly seen among contact lens wearers (21). Although one-step hydrogen peroxide disinfection systems are convenient for contact lens wearers, they are not effective against the highly resistant cyst form of Acanthamoeba (7). The findings of this study demonstrate that the efficacy of H₂O₂ against Acanthamoeba cysts can be enhanced by addition of KI and a plant-derived peroxidase. When the reaction is performed in the presence of a platinum disk, the cysticidal activity is retained and complete neutralization of the H₂O₂ is obtained. This may result in a significant improvement in the efficacy of one-step H₂O₂ systems against Acanthamoeba cysts and, possibly, other contact lens-related ocular pathogens.

 
This study was supported by research grant GA324 from the British Society for Antimicrobial Chemotherapy.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Leicester, Medical Sciences Building, P.O. Box 138, University Rd., Leicester LE1 9HN, United Kingdom. Phone: 44 (0) 116 252 2950. Fax: 44 (0) 116 252 5030. E-mail: sk46{at}le.ac.uk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, May 2003, p. 2563-2567, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2563-2567.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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