Cellulose is the main component of the cyst wall [9]. Cellulose synthesis inhibitor 2, 6-dichlorobenzonitrile (DCB) blocked the encystment of Acanthamoeba and improved the anti-amoebic effects [7,10]. In this study, we tested whether DCB can enhance the amoebicidal effects of the MPDS against Acanthamoeba, especially the cysts.

Acanthamoeba castellanii Castellani was obtained from the American Type Culture Collection (ATCC 30011), and axenically cultured in PYG (Protose peptone-Yeast extract-Glucose) medium. Encystation of Acanthamoeba was performed in encystment media [11]. DCB was purchased from Sigma Aldrich. Eight types of MPDS (Table 1) were used to determine amoebicidal activities against Acanthamoeba cyst by the most probable number (MPN) technique [12].

Almost all MPDS showed insufficient amoebicidal activity against the 72 hr-induced mature cysts (Fig. 1A). Even though 100 μM DCB was added to the 8 MPDS, no significant differences were found between MPDS alone and MPDS combined with DCB against the mature cysts (Fig. 1B). The amoebicidal activities of the MPDS with or without DCB were insufficient against the 48 hr-induced cyst (Supplementary Fig. S1). Three of the 8 MPDS (solution e, f, and h) showed sufficient amoebicidal activities against 24 hr-induced immature cysts (Fig. 2A). Combining 100 μM DCB to the 8 commercial MPDS, 4 (solution d, e, f, and h) out of 8 MPDS showed sufficient amoebicidal activities against the immature cysts (Fig. 2B). The amoebicidal activity of MPDS-d was increased against immature cysts upon addition of DCB. MPDS-a combined with DCB displayed slightly increased amoebicidal activity against the immature cyst (Fig. 2B). Encystation ratio revealed 0% mature cysts at 0 hr induction, 27% mature cysts at 24 hr induction, 55% mature cysts at 48 hr induction, and 84% mature cysts at 72 hr induction when incubated in encystation media [13]. We supposed that mature cyst, immature cyst (encysting cyst) and trophozoite were mixed in 24 hr-induced group (Fig. 2), and the DCB acted on immature cyst by blocking its encystation, thereby enhancing the amoebicidal effect on the immature cyst.

To determine the cytotoxicity of DCB, human corneal epithelial (HCE) cells were treated with 100 μM DCB and 8 kinds of MPDS combined with DCB. Cytotoxicity was assessed visually after Giemsa staining and optical density measurement at 590 nm after 0.1 ml of cells were solubilized in 5% sodium dodecyl sulfate. Percent cytotoxicity was calculated according to the following formula: % cytotoxicity=100−[(OD of the experimental well−OD of HCE cell alone)/OD of control cells]× 100. Compared to the control solution, 100 μM DCB did not cause cytotoxicity to HCE cells after 30 min of incubation (Fig. 3). Most MPDS combined with DCB showed no significant cytotoxicity towards HCE cells (Fig. 3). Minor levels of cytotoxicity was detected in HCE cells treated with MPDS-a and MPDS-f combined with DCB.

As detailed composition of the MPDS are confidential, it is difficult to explain the mechanism that triggered the enhanced cytotoxicity upon addition of DCB. Despite the presence of cytotoxicity, it can be assumed that DCB will be diluted with tears within minutes, thereby reducing its concentration and ultimately its cytotoxicity. Furthermore, it is unlikely that cellulose biosynthesis inhibitor such as DCB will have significant effect on human cells, which lack cellulose synthesis mechanism. Our results suggested the possibility of DCB as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba immature cyst. This may be helpful in the prevention of Acanthamoeba infection associated with contact lens usage.