In vitro effects of medium tonicity, nutrient concentration, and free chlorine content on Acanthamoeba

Abstract

The environment preferred by Acanthamoeba trophozoites and the mechanism by which the amebae enters the cornea are not yet fully understood. A better understanding of the pathogenesis of this disease may help with prevention and treatment. To define the preferred environments for Acanthamoeba survival and proliferation in vitro by examining the effect of tonicity, nutrient concentration, and free chlorine content on Acanthamoeba. Human corneal isolates of Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites were cultured at 22°C (room temperature) in PYG (peptone-yeast extract-glucose) medium. The effect of tonicity on amebae was determined by incubating trophozoites in sodium chloride solutions in concentrations ranging from 0% to 10% for 19 days. Two different sets of media were prepared-one with and the other without added nutrients. The tonicity varied from 50 to 3438 mOsm/L while the pH was maintained at 6.7-6.8. Aliquots were recovered to determine the number and morphologic type of the amebae. To test the effect of chlorine, Acanthamoeba trophozoites were incubated for 7 days in buffered solutions with free chlorine concentrations varying from 0 to 5 mg/L free chlorine at 22°C. The pH was maintained at 7.2 and the tonicity varied from 88 to 92 mOsm/L. Trophozoites were enumerated by hemocytometer. Low tonicity solutions (<300 mOsm/L) favored the trophozoite stage, but elevating tonicity encouraged encystment. Only 3.3-3.9% of the trophozoites remained in 10% NaCl, while 46-58% of the trophozoites were present in distilled water. Increasing osmolality yielded a smaller number of Acanthamoeba with a greater proportion of cysts. Nutrients improved the replication rate at lower concentrations, increased the number of trophozoites and reduced the percentage of cysts. Chlorine completely inhibited both species of Acanthamoeba at free chlorine levels of 5mg/L, while lesser concentrations were less inhibitory. Acanthamoeba prefer hypotonic environments. Nutrients merely slowed the conversion of trophozoites to cysts at higher tonicity levels. Chlorine concentrations less than 5 mg/L, ocular irritation level, did not effectively convert trophozoites into cysts. We conclude that contact lens patients should avoid hypotonic ocular exposures, especially tap water and stagnant media such as lake water, and water from poorly maintained swimming pools and hot-tubs.