Encystation in Acanthamoeba castellanii: Development of Biocide Resistance¹

Abstract

Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp. superb experimental systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides 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 Escherichia coli. Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC4(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment. For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process. After two hours, resistance to HCl was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamine isethionate, and H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetate resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated.

Resistance of Acanthamoeba Cysts to Disinfection Treatments Used in Health Care Settings

“…The second stage develops not only under unfavorable environmental conditions, such as nutrient starvation, heat, cold, and desiccation, but also in the presence of elevated concentration of various chemical compounds, with the trophozoites differentiating into cysts (13,40). During the encystment phase, amoebae become round and form two distinct layers: the endocyst, containing cellulose, and the ectocyst, containing various polysaccharides and proteins (27,34). Cysts are metabolically inactive and can remain viable for more than 20 years under dry conditions (51) and 24 years at 4°C in water (38).…”

Cysteine Protease Inhibitor (AcStefin) Is Required for Complete Cyst Formation of Acanthamoeba

“…Under challenging conditions, such as those associated with starvation, low temperatures, and biocide treatment, the trophozoite converts to the resilient cyst form (1)(2)(3)(4)(5). This differentiation, which is termed encystation, protects Acanthamoeba against host immune responses and allows it to evade the effect of chemical treatments, and thus, encystation decreases treatment efficacies (3,6). Therefore, the inhibition of encystation during medical treatment could lead to outcomes that are more favorable in cases of amoebic infection.…”

Autoimmunity in the Mediation of Granulomatous Amoebic Encephalitis: Implications for Therapy

“…Bacteria grown in Acanthamoeba show resistance to bactericides and biocides; their survival and virulence are enhanced; and they mechanically transport disease-producing agents to various target organs, thus increasing the risk of multiple infections in the affected patients (King, et al, 1988, Barker, et al, 1995, Turner, et al, 2000, Lloyd, et al, 2001, Marciano-Cabral & Cabral, 2003. The amoebae shed waste through vesicles of 2.1 to 6.4 µm diameter, and they can potentially contain pathogenic microbes.…”