In vitro evaluation of commercial foam Belcils® on Acanthamoeba spp

Abstract

Interest in periocular (eyelid and eyelashes margins) hygiene has attracted attention recently and a growing number of commercials eye cleanser and shampoos have been marketed. In the present study, a particular eye cleanser foam, Belcils® has been tested against trophozoites and cysts on the facultative pathogen Acanthamoeba. Viability was tested by the alamarBlue™ method and the foam was tested for the induction of programmed cell death in order to explore its mode of action. We found that a 1% solution of the foam eliminated both trophozoite and cyst stage of Acanthamoeba spp. After 90 min of incubation, Belcils® induced, DNA condensation, collapse in the mitochondrial membrane potential and reduction of the ATP level production in Acanthamoeba. We conclude that the foam destroys the cells by the induction of an apoptosis-like process. The current eye cleanser could be used as part of AK therapy protocol and as prevention from AK infections for contact lens users and post-ocular trauma patients.

Keywords: Acanthamoeba spp.; Amoebicidal activity; Belcils® foam; PCD.

Graphical abstract

Fig. 1

Standard curve analysis for IC₅₀ and IC₉₀ calculation using increasing doses of Belcils® determined by the alamarBlue™ assay. A: trophocidal activity, B: Cysticidal activity.

Fig. 2

Effect of the Belcils® foam at 10% on Acanthamoeba spp. (A) Acanthamoeba castellanii Neff (A); Acanthamoeba polyphaga (B); Acanthamoeba quina (C); Acanthamoeba griffini (D). Black arrowheads indicate shrunken and unviable cells. Images (40X) are representative of the cell population observed in the performed experiments. Images were obtained using an EVOS FL Cell Imaging System AMF4300, Life Technologies, USA.

Fig. 3

The effect of Belcils® foam at IC₉₀ on Acanthamoeba castellanii Neff on the ATP content, using CellTiter-Glo® luminescent cell viability assay during incubation time. Results are representing in percentage relative to the negative control. Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3); NS non-significant; **p < 0.01; ***p < 0.001 significance differences when comparing the different mean values.

Fig. 4

Acanthamoeba castellanii Neff trophozoites incubated with IC₉₀ (B, D, F, H) of the tested eye cleanser and the evolution of chromatin condensation observed for 30 min (B), 1.5h (D), 4h (F), 24h (H). Hoechst stain is different in control cells (A, C, E, G), where uniformly faint-blue nuclei are observed, and in treated cells, where the nuclei are bright blue. Red fluorescence corresponds to the propidium iodide stain. Images (20X) are showing chromatin condensation (blue) in Acanthamoeba treated cells. Images (20X) are representative of the cell population observed in the performed experiments. Images were obtained using an EVOS FL Cell Imaging System AMF4300, Life Technologies, USA. The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) **p < 0.01; ***p < 0.001; ****p < 0.0001 significance differences when comparing treated cells to negative control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Images (20X) obtained from EVOS FL Cell Imaging System showing the effect of IC₉₀ of Belcils® (B, E, F) on the mitochondrial potential of Acanthamoeba castellanii Neff compared to the control (A, C, D) using JC1 kits after 1.5h (A, B); 4h (C, E) and 24h (D, F) of incubation. The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) **p < 0.01; ***p < 0.001; ****p < 0.0001 significance differences when comparing treated cells to negative control.

Fig. 6

Images (20X) obtained from EVOS FL Cell Imaging System showing the effect of IC₉₀ of Belcils® (B, D, F) on the membrane permeability of Acanthamoeba castellanii Neff compared to the control (A, C, E) using Sytox Green dye after 1.5h (A, B); 4h (C, D) and 24h of incubation (E, F). The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) **p < 0.01; ***p < 0.001; ****p < 0.0001 significance differences when comparing treated cells to negative control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Images (20X) obtained from EVOS FL Cell Imaging System showing the effect of IC₉₀ of Belcils® (B, D, F) on the ROS production of Acanthamoeba castellanii Neff compared to the control (A, C, E) after 1.5h (A, B); 4h (C, D) and 24h of incubation (E, F). The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) **p < 0.01; ***p < 0.001; ****p < 0.0001 significance differences when comparing treated cells to negative control.