No abstract

Acanthamoeba castellanii is an organism that causes Acanthamoeba keratitis, an infectious disease. Acanthamoeba are protists that generally inhabiting soils, freshwater, and sea water, but can also infect the cornea due to dirty water infiltration if contaminated contact lenses are worn. In this study, we focused on four branched medium chain fatty acids-2-ethylhexanoic acid, 3,5,5-trimethylhexanoic acid along with their salts, potassium 2-ethylhexanoate, and potassium 3,5,5-trimethylhexanoate. We examined their antimicrobial effect toward A. castellanii and aimed to search for new drugs to be used in contact lens disinfecting solutions. The minimum inhibitory concentrations (MICs) of 2-ethylhexanoic acid and 3,5,5-trimethylhexanoic acid showed growth reduction of 4-log units (99.99% suppression) in A. castellanii upon 10 min incubation at 12.5 v/v%. In addition, at a diluted concentration of 6.25 v/v%, 2-ethylhexanoic acid produced a 4-log units reduction at 180 min and for 3,5,5-trimethylhexanoic acid, a 4-log units reduction at 60 min. The MICs of potassium 2-ethylhexanoate showed growth reduction of 4 log units in A. castellanii upon 10 min incubation at 2.50 w/v%. However, potassium 3,5,5-trimethylhexanoate showed growth reduction of 4-log units in A. castellanii upon 180 min incubation at 5.00 w/v%. Based on the results of MICs, a verification test of 3,5,5-trimethylhexanoic acid and potassium 2-ethylhexanoate was conducted. As a result, approximately 2.1 logarithmic growths were observed in the control consisting of 10% Tween 80 compared to the initial cell number, and the amoeba growth reduction with 3,5,5-trimethylhexanoic acid could not be confirmed up to a concentration of 0.0122 v/v%. Potassium 2-ethylhexanoate salt was not confirmed to reduce amoeba growth up to 0.625 w/v%. However, the amoeba growth reduction could not be confirmed with the tests where control pH-adjusted solution was applied, either. In the light of the above-mentioned results, it was revealed that 3,5,5-trimethylhexanoic acid exhibits the highest anti-amoeba effect among the four tested branched medium chain fatty acids. This result indicated the possibility to use it in contact lens disinfectant solutions as a novel drug against Acanthamoeba keratitis.

Acanthamoeba castellanii is an organism that causes the infection termed Acanthamoeba keratitis (Ishibashi et al., 1988) . The infection was first reported in Britain in 1974(Naginton et al., 1974) and in Japan in 1988(Ishibashi et al., 1988 , and it is estimated that more than hundreds of cases occur each year in contact lens users who are said to represent over 15 million people in Japan so far (Tokoro et al., 2008) . In Japan, the number of young people using soft contact lenses has increased recently (Shinoda et al., 2002; Study Group of National Surveillance of Infectious Keratitis in Japan, 2006) . Acanthamoeba are protozoans found in soil, freshwater, and seawater (Ishii, 1999) . However, non-sterile hands and water can contaminate contact lenses and cause corneal infections (Miyazaki et al., 2007;Yamaura et al., 1993;Zimmerman et al., 2017) .Patients with Acanthamoeba keratitis may experience pain presenting with photophobia, ring-like stromal infiltration, epithelial defects, and eyelid edema (Lorenzo-Morales et al., 2015) . If Acanthamoeba keratitis is not treated adequately, loss of vision may result. Currently, cold disinfection with hydrogen peroxide solution or a multipurpose solution (MPS) is commonly used for disinfecting contact lenses (Hiti et al., 2002;Morishige et al., 2012;Atkins, 2006) . In particular, an MPS can be used to clean, rinse, and disinfect soft contact lenses with the same liquid. In addition, it serves as a preservative, making it a commonly used solution (Hiti et al., 2002;Atkins, 2006) . However, the antiseptic effect of MPS is weaker than that of other disinfection methods, and it is suggested that hypersensitivity may occur (Morishige et al., 2012;Atkins, 2006) . Moreover, according to the 2009 National Consumer Center report in Japan, it has been shown that the anti-amoebic activity of commercially available MPSs is very low

House dust mites, Dermatophagoides pteronyssinus are present in the indoor environments, such as pillows and carpets. In this study, we investigated the mite control effect of branched chain fatty acids (2-ethylhexanoic acid (iso-C8) , 2-butyloctanoic acid (iso-C12) , isopalmitic acid (iso-C16) ) and their mechanism of action. These fatty acids showed a higher acaricidal activity than the straight chain fatty acids. Among these, iso-C12 had the highest acaricidal activity (LC50: 13mM) and more than 50% repellence rate at 0.20% (8.0mM) concentration. In the fumigant mortality bioassay, iso-C8 was 4 times more toxic than iso-C12 and isoC-16 in the gas phase. However, all branched chain fatty acids showed higher acaricidal activities on direct contact than fumigation. As the concentration of these fatty acids decreased, the number of deaths decreased and the number of escapes increased. There was no significant change in the mite epidermis due to contact with any of the fatty acids used. All branched chain fatty acids immobilized more than half of the mites within 90min of exposure. These results were consistent with the tendency of immobilizer type miticides targeting the respiratory system.