Toxic effects of selected proprietary dry eye drops on Acanthamoeba

Amoebae of the genus Acanthamoeba are ubiquitous protists that have been isolated from many sources such as soils, water and the air. They are responsible for infections including fatal encephalitis and a severe keratitis in humans. To date, there is no satisfactorily effective therapeutic agent against this pathogen and the infections it causes are exacerbated by the existence of a resistant cyst stage produced by this amoeba. As dry eye syndrome is a risk factor for Acanthamoeba keratitis, we aimed to evaluate the anti-Acanthamoeba activity of a variety of proprietary eye drops intended to treat dry eye syndrome. From the nine eye drop formulations tested, “Systane Ultra” was determined to be the most active against all tested Acanthamoeba strains. During our investigations into the mode of action of Systane Ultra, we discovered that it decreases mitochondrial membrane potential and ATP levels, induces chromatin condensation, and increases the permeability of the plasma-membrane.

Acanthamoeba keratitis (AK) is increasingly being recognized as a serious infection of the cornea that can lead to a permanent visual impairment or even blindness 1 . In the developed world, AK is most often found in contact lens users particularly where poor hygiene has been practiced. AK is difficult to diagnose partly because clinicians rarely encounter this infection but also because the symptoms mimic those of other types of keratitis diseases (viral, bacterial and fungal). Patients with AK may experience eye pain and redness, blurred vision, photophobia and excessive tear production 1 . Dry eyes disease (DED) is a more common ocular surface disease that has a severe impact both on quality of live and on cost but it is also a predisposing risk factor for the development of AK 2 . DED results from either a systemic immunologic disorder known as Sjögren's syndrome 3 in which there is insufficient production of moisture in the salivary and tear-producing glands, or from the low production or high evaporation of tears caused by other means 4 . Its severity may range from mild/episodic to severe/chronic and the disease is characterized by several symptoms including visual disturbance (blurred and fluctuating vision), foreign-body sensation and eye discomfort, irritation, ocular surface inflammation, redness, excess tearing, and photophobia 5 . DED is treated with a range of proprietary eye drops which contain a variety of active ingredients. We could find no previous studies describing the potential anti-Acanthamoeba activity of eye dry drops, and so the aim of the present study was to assess the potential anti-amoebic activity of several eye dry drops solutions, against a range of Acanthamoeba strains.

Material and Methods
Chemicals. Nine proprietary eye drop solutions available commercially for topical use against DED were selected for analysis. Table 1 shows the details of the composition of these solutions.
In vitro drug sensitivity assay. Strains used. The anti-Acanthamoeba activity of the selected eye drops were initially evaluated against the Acanthamoeba castellanii Neff (ATCC 30010) type strain from the American Type Culture Collection. Subsequently, eye drop solutions were tested against three clinical isolates, CLC-16 and Acanthamoeba griffini, genotype T3 and CLC-51, genotype T1 obtained in previous studies 6,7 . Those strains were grown axenically in PYG medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose) containing 40 μg gentamicin ml −1 (Biochrom AG, Cultek, Granollers, Barcelona, Spain). In vitro effect against the trophozoite stage of Acanthamoeba. The anti-Acanthamoeba activities of the eye drop solutions were determined by the Alamar Blue assay as previously described 6,8 . Briefly, Acanthamoeba strains were seeded in duplicate on a 96-well microtiter plate with 50 μl from a stock solution of 10 4 cells ml −1 . Amoebae were allowed to adhere for 15 min and 50 μl of serial dilution series of the eye drop solution was added. Finally, the Alamar Blue Assay Reagent (Bioresource, Europe, Nivelles, Belgium) was added into each well at an amount equal to 10% of the medium volume. The plates were then incubated for 120 h at 28 °C with a slight agitation. Subsequently the plates were analyzed, during an interval of time between 72 and 144 h, with an Enspire microplate reader (PerkinElmer, Massachusetts, USA) using a test wavelength of 570 nm and a reference wavelength of 630 nm. Percentages of growth inhibition, 50% and 90% inhibitory concentrations (IC 50 and IC 90 ) were calculated by linear regression analysis with 95% confidence limits. All experiments were performed three times each in duplicate, and the mean values were calculated.
In vitro effect against the cyst stage of Acanthamoeba. The       Analysis of Mitochondrial Membrane Potential. The collapse of an electrochemical gradient across the mitochondrial membrane during apoptosis was measured using a JC-1 mitochondrial membrane potential detection kit (Cell Technology) by flow cytometry as described by the manufacturer. After being treated with IC 50 and IC 90 of the test solution for 24 h, the cells were centrifuged (1000 r.p.m. × 10 min) and resuspended in JC-1 buffer. 100 µl of each treated culture was added to a black 96 well plate than 10 µl of JC-1 was added and incubated at 26 °C for 30 min. The mean green and red fluorescence intensity was measured using flow cytometry for 30 minutes.

Measurement of ATP. ATP level was measured using a CellTiter-Glo Luminescent Cell Viability Assay.
The effect of the drug on the ATP production was evaluated by incubating (10 5 ) of cells/ml with the previously calculated IC 50 and IC 90 of the active eye drop solution.

Results
In vitro drug sensitivity assay. Initially, all eye drops were screened for their activity against the trophozoite stage of Acanthamoeba castellanii Neff strain. The IC 50 and IC 90 at /96 h were chosen as the appropriate and comparable data to give as previously described 6 . The results are illustrated in Table 2. Among the nine tested eye drops, seven of them are active against trophozoites with an IC 50 ranged from 2.036 ± 0.137% (v:v) for Systane Ultra to 47.946 ± 3.770% (v:v) for Artelac Splash. Based on their amoebicidal activity on the Neff strain, three eye drop solutions, namely Systane Ultra, Colircusi Humectante and Optiben were selected to evaluate their effect on the clinical Acanthamoeba strains. The results are illustrated in Table 3.  The analysis of variance by Multifactor ANOVA, illustrated that the biological activity was strain dependent with p = 0.0001 < 0.001. In fact, the Acanthamoeba castellanii Neff was the most sensitive strain to the eye drops. Meanwhile, A. griffini was the most resistant strain to all eye drops. The toxic effect was statistically significant with p = 0.0000 < 0.001, Systane Ultra was statistically the most effective drug against all the strains with the IC 50 ranged from 2.036 ± 0.137% for the A. castellanii Neff to 10.691 ± 1.484% for A. griffini (Fig. 1).
Systane Ultra was observed to cause a dose-dependent cysticidal affect (Fig. 2). We found that 1.35% of Systane Ultra inhibited 50% excystation from the initial inoculum of cysts. Systane Ultra induced mitochondrial malfunction. As it can be observed in Fig. 5, the curve of the mitochondrial potential fluorescence ratio demonstrated that the treatment with the IC 90 , decreased the membrane potential (ΔΨm) of A. castellanii Neff comparing to the negative control. As presented in the Fig. 6, confocal microscopy confirmed the effects of the Systane Ultra on the mitochondrial potential. The mitochondrial damage has been documented by measuring the ATP level generated in 24 h. We found out, that the IC 90 produced a pronounced decrease in the total ATP level (Fig. 7). In fact, cells treated with this dose generated only the half of ATP level produced in untreated cells.

Discussion
AK is a vision-threatening ophthalmological illness that may even result in blindness if left untreated. In the early stages of infection, this disease usually manifests with nonspecific symptoms such as eye redness, epithelial defects, photophobia, edema and intense pain. AK is often mis-diagnosed as many of these symptoms are shared with the other eye problems 11,12 . An increase in the number of AK cases is blamed on contact lens use, particularly of soft contact lenses, and their improper use and maintenance 12,13 . The use of contact lenses is also associated with DED 14 , and this is also a predisposing factor in the development of AK 2 . We have described anti-Acanthamoeba activities of 9 eye drop solutions using a range of Acanthamoeba strains. Among the tested eye drops, Systane Ultra was the most active against all the tested Acanthamoeba strains. The fact that the eye drops have an anti-cyst activity has been established by viability and proliferative assay and analyzed by microplate-based fluorescence. The Systane Ultra formulation contains 0.4% Polyethylene Glycol 400 which has been reported 15 to be effective against various pathogenic bacteria, including Klebsiella pneumonia, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus through damage to bacterial membrane 15,16 . Systane Ultra also contains 0.3% Propylene Glycol, and several reports have described the antimicrobial property of this molecule and its effectiveness as a preservative 17,18 .
The effects of Systane Ultra on Acanthamoeba that we describe here are consistent with a Programmed Cell Death (PCD) mechanism. A PCD-like process has been described in Acanthamoeba occurring 6 h after infection with Salmonella typhimurium 19 . The early stage was inferred from phosphatidylserine externalization and chromatin condensation. Since this initial report we 20,21 and others 22,23 have reported a number of PCD inducing agents in Acanthamoeba. In these reports, authors have been able to distinguish between early PCD cells, late PCD cells. This process is generally characterized by distinct morphological features that occurs in different stages from the loss of mitochondrial potential, the condensation of nuclear chromatin and exposure of phosphatidylserine (PS) on the cell exterior. At the late of PCD, the membrane starts blebbing and cell dehydration causes changes in cellular shape and size. The structural integrity and most of the functions of the cell membrane remain intact at least in the initial stages of the process 20 . In the present study, Systane Ultra at the IC 90 was found to induce chromatin condensation observed through the Hoechst fluorescence as showed in the Fig. 3. Some of the brighter staining material is associated with structures within vacuoles and these are likely to be autophagosomes 24 . However, this is still a speculative hypothesis since a complete autophagy evaluation should be performed by analyzing the autophagosome formation among other assays.
To get a better knowledge of the membrane damage caused by Systane Ultra, we measured fluorimetrically the influx of SYTOX Green into the parasites, as its fluorescence is enhanced when bound to intracellular nucleic acids. After 24 hours of treatment, this eye drop solution was able to induce lesions in the plasma-membrane with a size large allowing the entrance of the dye but without cell rupture. The maintenance of cell's shape was confirmed using confocal microscopy as showed by Fig. 4. It is well known that PCD is linked to the malfunction of the mitochondria. The loss of mitochondrial membrane potential leads to mitochondrial dysfunction and this is regarded as being an important factor in PCD 25 . In the present study, the selected eye drop produced a pronounced decrease in the mitochondrial potential and therefore in the total ATP level. It's likely that Systane Ultra induces apoptosis in Acanthamoeba cells through the mitochondrial pathway.

Conclusion
Our results suggest that Systane Ultra possess an amoebicidal activity that may be useful for the prevention or even treatment of Acanthamoeba keratitis, or form the basis for an optimized solution. We suggest that the Systane Ultra eye drop solution probably induces PCD via the intrinsic pathway. Nevertheless, a limitation of this study is whether these eye drops could be used in the future since they could not be available commercially in ten-year time. Another issue is the need to perform further studies using an in vivo model, since the in vitro methodology used has its limitations such as the lack of a water related vehicle control and/washing action of tears in the eye once the eye drops are placed on the eye and washed away. Therefore, the need for further studies in the near future using these eye drops and an in vivo model. Nevertheless, the potential use of these eye drops especially Systane Ultra due to its high anti-Acanthamoeba effects is clear and presents a promising alternative for AK treatment in the current and near future infection cases.