In Vitro Amoebicidal Activity of a Ceragenin,Cationic Steroid Antibiotic-13,Against Acanthamoeba castellanii and Its Cytotoxic Potential

Abstract

Acanthamoeba is a free-living amoeba causing a potentially blinding infection of the cornea. Acanthamoeba keratitis is difficult to treat, without total efficacy in some patients because of cysts that are less susceptible than trophozoites to the usual treatments. Contact lens wearers are most at risk and account for some 95% of cases. Cationic steroid antibiotic (CSA)-13 is a small molecule aminosterol that has been shown to mimic the activity of endogenous antimicrobial peptides and has bactericidal activity based on membrane disruption. We investigated here the in vitro effectiveness of CSA-13 with a concentration of 100, 75, 50, and 25 mg/mL on proliferation of Acanthamoeba castellanii trophozoites and cysts and cytotoxic potential. CSA-13 was evaluated for its amoebicidal activity using an inverted light microscope at 1, 2, 4, 8, 12, and 24 h. For the determination of cytotoxicity of the CSA-13 on L929 cells, agar diffusion tests were performed. CSA-13 inhibited trophozoite growth in dose- and time-dependent ways. At 1 h, no viable trophozoites were observed in the presence of CSA-13 solution in a concentration 100 mg/mL in phosphate-buffered saline. Results of cytotoxicity experiments demonstrated that CSA-13 solution had mild toxicity at 100 mg/mL concentration on cells, whereas it had no toxicity at 75 mg/mL concentration. The findings of this experiment as in vitro ameboebicidal activity for Acanthamoeba suggest that CSA-13 has a potential to be used as a new agent in lens solutions to prevent Acanthamoeba growth and infections.

Introduction

Acanthamoeba keratitis is a rare but painful and devastating infection of the cornea that is difficult to diagnose and treat.^1–5 An epidemiologic study of Acanthamoeba keratitis in the United States showed that the number of reported cases gradually increased between 1981 and 1985, with a dramatic increase beginning in 1985.⁶ The predisposing conditions most commonly associated with the development of Acanthamoeba keratitis include the use of contact lenses,⁷ the use of homemade saline,^7,8 chemical disinfection,⁹ exposure to contaminated water,¹⁰ and minor corneal trauma.¹¹ Contact lens wearers are most at risk from Acanthamoeba keratitis and account for ∼95% of reported cases.^12,13 Bacterial and fungal contamination within contact lens systems may be an important element for the survival and growth of Acanthamoeba.⁶

Safe contact lens use requires regular cleaning and disinfection of the lenses and storage in a sterile solution. Ineffective disinfectant solutions or inadequate disinfection times can enable patient-contaminant organism survival that, if the lens cases are not cleaned and air-dried properly, could subsequently proliferate during contact lens storage.^14,15 To simplify contact lens hygiene and promote better patient compliance, multipurpose solutions have been developed. These represent a single solution for the cleaning, disinfection, and storage of contact lenses and hence offer continued antimicrobial protection during lens storage.¹⁶

Membrane-active cationic steroid antibiotics (CSAs) called ceragenins were developed as nonpeptide mimics of endogenous cationic antimicrobial peptides. They have antibacterial activity against Gram-positive and Gram-negative bacteria.¹⁷ Similar to positively charged antibacterial peptides,^18–21 CSA molecules display cationic facial amphiphilicity with charges arranged on 1 side and hydrophobic residues on the other. Because CSA molecules target membranes based in part on electrostatic interactions, they effectively kill different types of bacteria¹⁷ and are unlikely to induce resistance.²²

The aim of the present study was to evaluate the in vitro effectiveness of CSA-13 on the growth and adherence of Acanthamoeba castellanii trophozoites and cysts and its cytotoxic potential.

Methods

Antimicrobial agent

CSA-13 were synthesized and provided by one of the authors (Paul B. Savage). CSA-13 was synthesized from a cholic acid scaffolding technique as previously described^23–25 (Fig. 1).

FIG. 1.

Chemical structure of CSA-13 (molecular weight, 822.94). CSA, cationic steroid antibiotic.

In vitro amoebicidal activity of CSA-13

Parasites and culture

Trophozoites. A. castellanii 1BU strain²⁶ vegetative forms were from axenic cultures in 25-cm² Corning flasks with 10 mL of PPYG (protease peptone, yeast extract, glucose) medium²⁷ and were kept at 37°C. Trophozoites in exponential growth (72–96 h) were concentrated by centrifugation at 500 g for 10 min. The amoebas, which were washed twice in sterile Neff's saline (1.2 g NaCl, 0.4 g MgSO₄·H₂O, 0.4 g CaCl₂·2H₂O, 1.42 g Na₂HPO₄, and 1.36 g KH PO₄ in 100 mL distilled water), counted in a hemacytometer, adjusted to a final concentration in Neff's saline at a density of 2 × 10³ amebae/mL (95% trophozoites), and used immediately for testing.

Cysts

Cyst inocula were prepared from cultures incubated in flasks for up to 6 weeks and were harvested as described above. Inocula with at least 95% cysts were used in the comparative experiments. The cysts were harvested, washed in sterile Neff's saline, and adjusted to a final concentration of 2 × 10³ cysts/mL.

Experimental design

Experiments were performed in 15 mL centrifugation tubes. One hundred microliters of the calibrated trophozoite and cyst suspensions was added to each tube, separately. Four milliliters of the CSA-13 solution in concentrations 100, 75, 50, and 25 mg/mL in phosphate-buffered saline (PBS) was added to each tube, and then the tubes were sealed and incubated at 37°C in a 5% CO₂ atmosphere for 1, 2, 4, 8, 12, and 24 h. Tests were repeated 6 times and controls containing only the parasites in PBS were submitted to the same procedure used for the experimental cultures (this work was perfomed at CUTFAM [Research Center of School of Medicine, Cumhuriyet University]).

Effects of the CSA-13 on trophozoite stage

After incubation in CSA-13 solutions at 37°C for indicated times, a sterile cell scraper was used to remove gently the trophozoites that adhered to the base of the tubes. Then, we gently agitated it by pipeting up and down for 1 min. For counting from each test and control wells, separately, 0.1 mL of 0.3% basic methylene blue was used. Unstained (viable) and stained (nonviable) trophozoites (Fig. 2A) were enumerated in the hemocytometer 10 min later after stain addition. More than 100 A. castellanii trophozoites were examined in each of samples.

FIG. 2.

Representative pictures of unviable Acanthamoeba castellanii trophozoites (A) and cysts (B). Arrows mark viable trophozoite and cyst. Color images available online at www.liebertonline.com/jop.

Effects of the CSA-13 on cyst stage

After each incubation period, for counting of each test and control well, separately, 0.1 mL was transferred into 0.1 mL of 0.3% basic methylene blue. Unstained (viable) and stained (nonviable) cysts (Fig. 2B) were enumerated in the hemocytometer 2 min after stain addition. More than 100 A. castellanii cysts were examined in each of samples. For cultures with no viable cyst, we performed an additional test to confirm the results. After each incubation period, cysts were inoculated onto non-nutrient agar plates covered with a lawn of Escherichia coli to evaluate their viability. The culture plates were incubated at 30°C for 14 days. Amoebic growth was observed daily by phase-contrast microscopy (Eclipse TS 100; Nikon).

Cytotoxicity of CSA-13

Cells

A mouse connective tissue fibroblast cell line, L929 (ATCC cell line, NCTC clone 929), was cultured in Dulbecco's minimum Eagle's medium (Gibco) supplemented with 10% fetal calf serum (Gibco) and 2 mM/mL L-glutamine. No antibiotics were added to the cell culture medium. The cultures were cultivated in an incubator at 37°C and 5% CO₂, until cell monolayers attained confluence, which occurred after 7 days. Assays were always performed in the exponential growth phase of the cells.

Agar diffusion method

The agar diffusion tests were performed according to international standard.²⁸ Briefly, the cultures were harvested using 0.25% trypsin solution (Gibco). Stock cultures were seeded in 35 mm diameter cell culture dishes (Nunc) at a density of 1 × l0⁶ cells/dish and subcultured once a week. After a confluent cell layer had formed, the medium was removed and replaced with complete medium containing 1.5% agarose (FMC BioProducts). After the agarose had solidified, the cells were stained with a vital dye (neutral red; Sigma). During the following procedures the cells were protected from light to prevent cell damage elicited by photoactivation of the stain. Experimental solutions were applied by using sterile round Whatman papers in diameter 6 mm. Twenty milliliters of the CSA-13 solution in concentrations 100, 75, 50, and 25 mg/mL in PBS was added to each steril rounds Whatman papers. CSA-13 solutions, 4 replicate dishes, and 4 additional dishes with positive and negative control materials were prepared. As negative control, PBS was used; as positive control, absolute phenol was used. After an exposition period of 24 h at 37°C, the cell response was evaluated by inverted microscope observation. In this study, cell lysis was scored as follows: 0 = no cell lysis detectable; 1 = <20% cell lysis; 2 = 20%–40% cell lysis; 3 = >40% to <60% cell lysis; 4 = 60%–80% cell lysis; 5 = >80% cell lysis. For each specimen, 1 score was given and the median score value for all parallels from each specimen was calculated for the lysis zone. Cytotoxicity was then classified as follows: 0–0.5 = noncytotoxic; 0.6–1.9 = mildly cytotoxic; 2.0–3.9 = moderately cytotoxic; 4.0–5.0 = markedly cytotoxic.

Statistical analysis

Data were presented as mean ± SD and analyzed by repeated measures of ANOVA followed by the Tukey test for post hoc pairwise comparisons. The difference was considered significant when the P value was <0.05.

Results

In vitro amoebicidal activity of CSA-13

CSA-13 solution was evaluated for its amoebicidal activity against A. castellanii using an inverted light microscope. According to the results obtained from the tests, CSA-13 solution showed remarkable amoebicidal effect on A. castellanii (Fig. 3). The effect of CSA-13 solution in concentrations 100, 75, 50, and 25 mg/mL on the proliferation of A. castellanii trophozoites is shown in Fig. 3A. At 1 h, no viable trophozoites were observed in the presence of CSA-13 solution in a concentration 100 mg/mL in PBS. Overall, in a concentration-dependent manner, viable trophozoite numbers decreased with the increased concentrations of the CSA-13 (P < 0.05). Viable trophozoites within the control media did not show any changes during the test (P > 0.05).

FIG. 3.

Effect of cationic steroid antibiotic-13 on the proliferation of A. castellanii trophozoite (A) and cysts (B).

The effect of CSA-13 on the proliferation of A. castellanii cysts is shown in Fig. 3B. The number of Acanthamoeba cyst at 2 h was significantly decreased that at 1 h (P < 0.05). After 8 h, no viable cysts were observed in the presence of CSA-13 solution in a concentration 100 mg/mL in PBS. Viable cysts within the control media did not show any significant changes during the test.

Cytotoxicity of CSA-13

Results of cytotoxicity experiments demonstrated that the lysis index score of CSA-13 solution was 0.9 (mild toxicity) at 100 mg/mL concentration on L929 cell lines, whereas at 75 mg/mL concentration its lysis index score was 0 (noncytotoxic). There was no decolorization zone around the samples. Although the cells are directly in contact with the extract in the culture media, they did not showed any signs of injury and kept their morphological characteristics and wholeness like those in the controls. On the overall, lysis index score was 5 (markedly cytotoxic) in the positive control group and 0 (noncytotoxic) in the negative control group.

Discussion

Acanthamoeba keratitis is a rare but severe corneal infection that, despite improvements in diagnosis and treatment, still culminates in prolonged morbidity and significant loss of visual acuity for up to 15% of patients.^13,29 Pathogenic Acanthamoeba strains have been found in biocidal solutions for lens care.^30,31 The cysts that are implicated have been known to resist desiccation for months to years; they can tolerate 2.0% HCl, peroxides, and chlorine.³ Manufacturers should state the killing times required by contact lens solution for all pathogenic Acanthamoeba isolates.³²

Disinfection is a fundamental part of the contact lens hygiene regime and serves to prevent the growth of potentially pathogenic organisms that may lead to ocular infections.³³ Noncompliant or incomplete disinfection may allow survival of bacteria and fungi that can provide a food source inside the contact lens storage case for the growth and replication of Acanthamoeba.^14,15,34 From here, the organism can adhere to the contact lens for inoculation on to the cornea to cause infection.

Membrane-active cationic antimicrobial peptides and some cationic steroids, or ceragenins, offer alternatives as antimicrobial agents that can be used to combat bacterial drug resistance often associated with chronic infection. Although some forms of ceragenins are effective against both Gram-negative and Gram-positive bacteria, they are generally more potent against Gram-positive bacteria. Surprisingly, it is not the cell wall but the high content of phosphatidylethanolamine in most Gram-negative bacteria that endows them with resistance. Ceragenins have the unusual property of forming complexes with phospholipids. Factors contributing to the mechanism of action of these agents are discussed.³⁵

Ceragenins have broad-spectrum antibacterial activities as shown by many studies. The covalent attachment of CSA-13 to a polymer backbone to generate thin films inhibited bacterial growth and may be helpful in controlling bacterial infections associated with medical devices.³⁶ Minimal inhibitory concentration (MIC) testing with common Gram-negative aerobic and facultative bacilli, strict anaerobes such as Propionibacterium acnes and Clostridium perfringens, as well as multidrug-resistant Staphylococcus aureus demonstrated that CSA-13 possess a broad spectrum of activity.³⁷ CSA-13 proved to be effective in inhibiting the growth of gatifloxacin- and moxifloxacin-resistant species of Staphylococcus. Staphylococcus is a Gram-positive organism often involved in conjunctival infections.³⁸

The main risk factor for corneal infection in contact lens wearers is the use of contact lens disinfecting systems ineffective in killing the Acanthamoeba cysts and trophozoites. Commercial solutions are not effective in eliminating Acanthamoeba cysts within a reasonable time frame.²⁴ Improvement or development of new contact lens disinfecting systems by the manufacturers is needed to prevent Acanthamoeba keratitis.

In conclusion, the findings of this experiment of in vitro ameboebicidal activity for Acanthamoeba suggest that CSA-13 decreases trophozoite numbers effectively but cyst number moderately, and that it has potential to be used as a new agent in lens solutions to prevent Acanthamoeba growth and infections.

Footnotes

Acknowledgments

We thank Julia Walochnik of the Department of Medical Parasitology, Clinical Institute of Hygiene, University of Vienna, Vienna, Austria, for providing the A. castellanii strain 1BU.

Author Disclosure Statement

The authors have no competing financial interests.

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