Encystation in Acanthamoeba castellanii: Development of biocide resistance

Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp, superb experiment al systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis hav e become widely recognized, it has become urgent to understand the paramete rs that determine differentiation, as cysts are much more resistant to bioc ides than are the trophozoites. Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff ), is conveniently measured by its ability to form plaques on a lawn of Esc herichia coli. Use of confocal laser scanning microscopy with Calcofluor wh ite, Congo Red or the anionic oxonol dye, DiBAC(4)(3) or how cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid a ssessment. For cysts, the plaque method is still the best, because dye excl usion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resis tance during the differentiation process. After two hours, resistance to HC l was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propami dine isethionate, pentamidine isethionate, dibromopropamine isethionate, an d H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetat e resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now bein g investigated.