Acanthamoeba are opportunistic free-living protozoa with more than 24 separate identified species based on morphology.1 The organism is ubiquitous and has been isolated from seawater, lakes, rivers, and streams and in water supplies. Acanthamoeba has two stages in its life cycle, a metabolically active trophozoite stage during which it multiples by binary fission and a dormant but resistant cyst stage.1 Acanthamoeba can cause vision-threatening keratitis in contact lens wearers2–4 or after ocular trauma.5 Corneal abrasion, along with the presence of trophozoites, is necessary to produce keratitis in an animal model.6 Corneal abrasion or mild trauma results in increased expression of glycoproteins7 on the corneal surface to which the trophozoites bind. Contact lens wear by itself has been shown to upregulate the expression of mannose glycoproteins on the corneal epithelium that enhances the binding of trophozoites to the cornea.8 Showering or swimming while wearing lenses and improper storage and disinfection of contact lenses (using tap water or homemade saline) are major risk factors for Acanthamoeba keratitis in contact lens wearers.9,10
Studies in the United Kingdom and Hong Kong have estimated the incidence of Acanthamoeba keratitis in contact lens wearers at around 0.18 to 0.25 and 0.33 per 10,000 lens wearers, respectively,3,11,12 whereas in the United States, the rate has been estimated at 0.01 per 10,000 wearers.2,13 Clinical features of Acanthamoeba keratitis include severe ocular pain not commensurate with clinical signs, radial keratoneuritis, and ring infiltration in the cornea.5,14 Treatment of Acanthamoeba keratitis using chlorhexidine combined with propamidine or polyhexamethylene biguanide (PHMB) has been very successful.15–19 Chlorhexidine and PHMB are believed to act by binding their positively charged nitrogen species to the mucopolysaccharide plug of the ostiole (pore) in Acanthamoeba cells.20 A recent study by Lim et al.21 has demonstrated that either of these drugs by itself is effective in the treatment of Acanthamoeba keratitis.
In 2007, a contact lens–disinfecting solution, Complete MoisturePlus (Advanced Medical Optics [now called Abbott Medical Optics], Santa Ana, Calif), was removed from sale worldwide after reports that the solution was associated with an increased risk of developing Acanthamoeba keratitis during contact lens wear.22 At the same time as the outbreak associated with Complete MoisturePlus, there was an apparent, but independent, increase in Acanthamoeba keratitis associated with changes to disinfection of the domestic water supply in Chicago, Illinois.23,24 Although testing for Acanthamoeba efficacy of contact lens multipurpose disinfecting solutions (MPDSs) is as yet not mandatory before their sale, several authors have assessed different brands of MPDSs for their ability to kill trophozoites and cysts,25–28 many of which show varying activity against Acanthamoeba. Interestingly, propylene glycol in the Complete MoisturePlus MPDS was associated with increased encystment of Acanthamoeba,29 and this was possibly a factor in the failure of this MPDS.
Protamine is a 33–amino acid cationic peptide, containing mostly arginine amino acids, found in salmon sperm nuclei. It has broad-spectrum antimicrobial activity against bacteria and fungi.30–33 Protamine disrupts microbial cell membranes, leading to an efflux of intracellular components in addition to affecting cell metabolism.34–36 The antimicrobial efficacy of protamine is affected by pH37 and the presence of divalent cations such as Ca2+ and Mg2+.36,38 Addition of a chelating agent such as EDTA restores the antimicrobial activity and has a synergistic effect.36,39 Although protamine is an effective antimicrobial agent, its efficacy against Acanthamoeba has not been investigated. In this study, the antimicrobial activity of protamine on Acanthamoeba trophozoites and cysts and its synergistic potential with PHMB and EDTA was tested. An effective antimicrobial activity against Acanthamoeba could indicate the potential for protamine to be used in formulations of new contact lens–disinfecting solutions.
Acanthamoeba strains Acanthamoeba polyphaga Ros and Acanthamoeba castellanii 044 isolated from keratitis were used. Acanthamoeba were grown and maintained axenically in peptone–yeast extract–glucose medium (PYG). Cryopreserved Acanthamoeba cysts were inoculated into 25 mL PYG and incubated at 32°C for 7 to 10 days to obtain motile trophozoites. A sterile cell scraper was used to gently detach the trophozoites adhered to the base of the flask. Aliquots of this culture were added to flasks containing fresh PYG and incubated for a further 3 to 4 days to obtain trophozoites and 28 days to obtain cysts. Trophozoites or cysts were collected by centrifugation for 12 minutes at 300g and resuspended in Page saline (2 mM NaCl, 16 μM MgSO4 7H2O, 27 μM CaCl2 2H2O, 1 mM Na2HPO4, 1 mM KH2PO4). Cells were enumerated using a Neubauer hemocytometer, and the final inoculum was adjusted using Page saline to approximately 1.0 to 1.5 × 106 cells/mL.
Chemicals and Reagents
Protamine (molecular weight, 4381 g/mol) and EDTA were obtained from Sigma (St. Louis, Mo), and PHMB was obtained from Dayang Chemicals Co. (Hangzhou City, China). A stock solution of protamine (2.3 mM) was prepared in phosphate buffered saline ([PBS] 0.137 M NaCl, 2.6 mM KCl, 1.5 mM KH2PO4, 1.66 mM Na2HPO4; pH 7.4) as was a stock solution of 0.05% EDTA. Stock solution of PHMB (0.01%) was prepared in distilled water.
Effect of Protamine on Acanthamoeba Trophozoites and Cysts
The International Organization for Standardization (ISO) guideline number 14729:2000 provides guidelines and methodology for evaluating the antimicrobial activity of soft contact lens–disinfecting systems. However, testing for efficacy against Acanthamoeba species has not been included because of lack of a standardized testing method, variability in trophozoites and cyst quantification, and the low prevalence of Acanthamoeba keratitis. The stand-alone procedure described in ISO 14729:2000 was modified to test the amoebicidal activity.
Protamine concentrations of 57, 114, and 228 μM were evaluated for efficacy against trophozoites of both the Acanthamoeba strains. The concentration with the best activity was then tested in combination with EDTA (171 μM), PHMB (0.0001%), and both EDTA and PHMB. These concentrations of EDTA and PHMB were chosen because they have been reported to be used in multipurpose disinfecting solutions.40–42 Aliquots (900 μL) of each of the test samples were prepared in triplicate in 1.5-mL reaction tubes (Greiner Bio-One, Germany) and 100 μL of the Acanthamoeba inoculum (trophozoites or cysts) added to obtain a final count of 1.5 × 105 cells/mL. Acanthamoeba in PBS were used as control samples. All samples were incubated at 25°C for 6 hours to simulate disinfection with multipurpose disinfection solutions.
After 6 hours, the samples were serially diluted 10-fold in Dey-Engley neutralizing broth (DE broth). Quadruplicates of each dilution was placed on to non-nutrient agar (NNA) plates preseeded with Escherichia coli and incubated at 32°C for up to 2 weeks. The plates were inverted and examined under a microscope for tracks produced by trophozoites indicating viability. Survivor numbers were determined using Reed and Muench computation.43 Each sample was tested in triplicate, and each experiment was repeated twice.
Encystment of Acanthamoeba
Encystment of trophozoites of both the Acanthamoeba strains was evaluated for the three concentrations of protamine and for the protamine combinations with EDTA and PHMB. Trophozoites were grown in individual wells of 12-well cell culture plates to confluence. The number of trophozoites in the wells was approximately 5 × 105 cells/mL. Wells were washed once with Page saline, then 1 mL of the test sample was added to each well and incubated at 25°C for 6 or 24 hours. Phosphate buffered saline was used as the control. Each sample was tested in triplicate for each time point, and the experiment was repeated twice.
After incubation, the surfactant sodium lauroyl sarcosinate (2.5 mg/mL) was added to each well and mixed well by pipetting to lyse trophozoites but not the cysts.29 Numbers of cysts in the test and control wells were calculated using a hemocytometer.
Log transformation of the data was performed before data analysis. Data for the trophozoites and cysts of each of the Acanthamoeba strains was analyzed separately using analysis of variance. If the overall effect was significant, post hoc multiple comparisons were performed using Dunnett correction. Level of significance was set at 5% for each analysis.
Effect of Protamine and PHMB on Acanthamoeba Trophozoites
A dose-dependent efficacy of protamine against Acanthamoeba trophozoites was observed. Compared with PBS (Fig. 1), log reductions of 0.7 or more (57 μM; p < 0.01), 0.8 or more (114 μM; p < 0.01), and 2.0 or more (228 μM; p < 0.001) were observed for trophozoites of either Acanthamoeba strain. Polyhexamethylene biguanide (0.0001%) alone did not demonstrate any activity against trophozoites of either Acanthamoeba strain (Fig. 1). The addition of PHMB (0.0001%) to 228 μM protamine improved the antimicrobial efficacy (0.8 log reduction; p = 0.002 vs. protamine alone) for A. castellanii 44, but the effect (0.4 log reduction vs. protamine alone) was not significant for A. polyphaga Ros (Fig. 1). Addition of 171 μM EDTA to 228 μM protamine or PHMB (0.0001%) or to the protamine/PHMB combination did not have any significant additional impact over their individual efficacies against the trophozoites (Fig. 1).
Effect of Protamine and PHMB on Acanthamoeba Cysts
Protamine efficacy against Acanthamoeba cysts was dose dependant, although the activity was much lower than that against the trophozoites. Compared with PBS control (Fig. 2), there were small but insignificant log reductions of 0.2 to 0.3 for 52 μM protamine (p ≥ 0.095), but the log reductions were significantly different for 114 μM (0.4 to 0.5; p < 0.05) and 228 μM (0.6 to 0.9; p = 0.000) protamine for both Acanthamoeba strains. Polyhexamethylene biguanide (1 μg/mL) showed minimal activity against A. polyphaga Ros cysts (0.5 log reduction; p = 0.032 vs. control PBS) and no activity against A. castellanii 0.44 cysts. The combination of protamine (228 μM)/PHMB (0.0001%) showed improved activity against A. castellanii 044 cysts (0.6 log reduction; p = 0.014) compared with protamine alone, whereas no improvement was observed for A. polyphaga Ros cysts. Addition of EDTA to protamine (228 μM) or PHMB (1 μg/mL) did not have a significant impact on their activities against cysts of either Acanthamoeba strains, indeed, the addition of EDTA to either protamine or PHMB had a small protective effect (although this was not significant) on A. castellanii 044 cysts. The addition of EDTA to the protamine/PHMB combination did not result in improvement of activity against cysts of either strain compared with protamine (228 μM) alone.
Encystment of Acanthamoeba trophozoites
Six- or 24-hour exposure to protamine (57 to 228 μM) did not have any significant impact on encystment for either Acanthamoeba strain (Table 1). However, 24-hour exposure to the positive control PBS produced a large number of cysts for both strains. Addition of PHMB (0.0001%) or EDTA (171 μM) to 228 μM protamine did not have a significant effect on the degree of encystment for either of the Acanthamoeba strains after 6- and 24-hour exposure.
This study has for the first time evaluated the antimicrobial efficacy of the cationic peptide protamine against Acanthamoeba spp. trophozoites and cysts. Protamine was demonstrated to be effective against Acanthamoeba spp. trophozoites using an assay similar to the stand-alone assay specified by the ISO for testing the effectiveness of contact lens multipurpose disinfecting solutions against bacteria and fungi (ISO standard 14729:2000). The efficacy was dependent on the concentration of protamine. There was some difference in the efficacy of protamine against trophozoites of the two strains tested; trophozoites of A. polyphaga Ros were slightly more susceptible compared with those of A. castellanii 044. Addition of EDTA did not have a significant impact on protamine efficacy; however, addition of PHMB significantly enhanced the activity of protamine against trophozoites of A. castellanii 044. The results suggest synergy between protamine and PHMB because the combined activity was higher than the sum of the individual effects.
Protamine also showed some cysticidal activity against both Acanthamoeba test strains, albeit at a much reduced level compared with its effects on trophozoites. These results are consistent with other studies that report resistance of Acanthamoeba cysts to disinfection by a wide range of biocides and contact lens solutions.44,45 The limited efficacy against Acanthamoeba cysts in contrast to trophozoites could be caused by the fact that protamine requires metabolically active cells to penetrate the cell membrane.46 Also, the walls of the cysts may be resistant to the action of protamine.
Addition of PHMB had a significant impact on the efficacy of protamine on the cyst form only of A. castellanii 044. These results correlate well with studies that indicate that significantly higher concentrations of PHMB are required for the compound to have adequate efficacy against cysts.47–49 However, at higher concentrations, toxicity to mammalian cells may become an issue. Addition of EDTA has been shown to have a negative impact on the efficacy of PHMB against A. castellanii cysts.47 The present study demonstrates that addition of EDTA did not improve the cysticidal activity of protamine or PHMB. Furthermore, cysts often self-aggregate, which may further reduce the effectiveness of disinfectants.44 Surfactants can disassociate these aggregates44; therefore, further formulations of protamine/PHMB (with or without EDTA) solutions to contain surfactants that are used in MPDS (such as tectronic 1304, tectronic 904, poloxamine, poloxamer 23741,42,50,51) might improve the cysticidal activity.
Recent studies have shown that encystment of Acanthamoeba after exposure to propylene glycol present in some MPDS may be associated with the development of keratitis.29 Some of the solutions in the study by Kilvington et al.29 caused significant encystment of trophozoites after 4-hour exposure. In the current study, there was no significant encystment after 6- or 24-hour exposure to the different concentrations or combinations of protamine, but the control PBS induced significant encystment by 24 hours.
Acanthamoeba keratitis cases have been increasing.10,52,53 Some contact lens disinfection systems are ineffective against the cyst form of Acanthamoeba when used as per manufacturer’s recommendations.45,51,54 Therefore, development of new contact lens disinfection systems effective against both cyst and trophozoite forms while controlling encystment is ideal. The results of this study have shown that protamine has good ameobicidal action in isolation and excellent synergistic activity with PHMB against Acanthamoeba. In addition, the compound does not promote encystment of the trophozoites even after 24-hour exposure. Protamine should be considered as a potential ingredient in new formulations of contact lens disinfection systems.
Mark School of Optometry and Vision Science
University of New South Wales
Sydney, New South Wales 2052
The authors have no conflicts of interest to declare.
Received August 18, 2012; accepted October 8, 2012.
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