Resistance of Acanthamoeba cysts to disinfection treatments used in health care settings

Abstract

Free-living amoebae that belong to the genus Acanthamoeba are widespread in the environment, including water. They are responsible for human infections and can host pathogenic microorganisms. Under unfavorable conditions, they form cysts with high levels of resistance to disinfection methods, thus potentially representing a threat to public health. In the present study we evaluated the efficacies of various biocides against trophozoites and cysts of several Acanthamoeba strains. We demonstrated that disinfectant efficacy varied depending on the strains tested, with environmental strains demonstrating greater resistance than collection strains. Trophozoites were inactivated by all treatments except those using glutaraldehyde as an active compound: for these treatments, we observed resistance even after 30 min exposure. Cysts resisted many treatments, including certain conditions with glutaraldehyde and other biocides. Moist heat at 55 degrees C was not efficient against cysts, whereas exposure at 65 degrees C was. Several chemical formulations containing peracetic acid, hydrogen peroxide, or ortho-phthalaldehyde presented greater efficacy than glutaraldehyde, as did ethanol and sodium hypochlorite; however, some of these treatments required relatively long incubation times to achieve cyst inactivation. Amoebal cysts can be highly resistant to some high-level disinfectants, which has implications for clinical practice. These results highlight the need to consider the effective disinfection of protozoa in their vegetative and resistant forms due to their intrinsic resistance. This is important not only to prevent the transmission of protozoa themselves but also due to the risks associated with a range of microbial pathogens that are found to be associated intracellularly with these microorganisms.

FIG. 1.

Phylogenetic tree showing the relationship of FLA isolates used in disinfection tests. DF3 fragment sequences were aligned using Muscle software (19), and the tree was constructed using the neighbor-joining method. Bootstrap values resulting from 2,000 replications are presented at each node when the value was >50%.

FIG. 2.

Self-aggregation of cysts and treatment-induced aggregation of cysts. Cysts were observed after 7 days of incubation in Neff's medium, after treatment with 0.5% SDS for 5 min, and after treatment with various biocides for various contact times (only treatments that induced visible aggregation are reported). 1, 7.5% hydrogen peroxide for 30 min; 2, hydrogen peroxide-based product Resert XL-HLD for 10 min; 3, hydrogen peroxide/peracetic acid-based product Sporklenz RTU for 10 min; 4, 2% glutaraldehyde-based product for 30 min; 5, 70% ethanol for 10 min; 6, 0.2% peracetic acid for 10 min; 7, 0.25% sodium hypochlorite for 10 min; 8, 2.5% sodium hypochlorite for 10 min; †, no growth observed after treatment. Magnification, ×100.

FIG. 3.

Cysts obtained from strains 1 (A), 3 (B), and 4 (C) and A. polyphaga strain CCAP 1501/18 (D). Arrows indicate ectocyst walls. Bar, 2 μm.