Killing of diverse eye pathogens (Acanthamoeba spp., Fusarium solani, and Chlamydia trachomatis) with alcohols

Abstract

Background: Blindness is caused by eye pathogens that include a free-living protist (Acanthamoeba castellanii, A. byersi, and/or other Acanthamoeba spp.), a fungus (Fusarium solani), and a bacterium (Chlamydia trachomatis). Hand-eye contact is likely a contributor to the spread of these pathogens, and so hand washing with soap and water or alcohol-based hand sanitizers (when water is not available) might reduce their transmission. Recently we showed that ethanol and isopropanol in concentrations present in hand sanitizers kill walled cysts of Giardia and Entamoeba, causes of diarrhea and dysentery, respectively. The goal here was to determine whether these alcohols might kill infectious forms of representative eye pathogens (trophozoites and cysts of Acanthamoeba, conidia of F. solani, or elementary bodies of C. trachomatis).

Methodology/principal findings: We found that treatment with 63% ethanol or 63% isopropanol kills >99% of Acanthamoeba trophozoites after 30 sec exposure, as shown by labeling with propidium iodide (PI) and failure to grow in culture. In contrast, Acanthamoeba cysts, which contain cellulose fibers in their wall, are relatively more resistant to these alcohols, particularly isopropanol. Depending upon the strain tested, 80 to 99% of Acanthamoeba cysts were killed by 63% ethanol after 2 min and 95 to 99% were killed by 80% ethanol after 30 sec, as shown by PI labeling and reduced rates of excystation in vitro. Both ethanol and isopropanol (63% for 30 sec) kill >99% of F. solani conidia, which have a wall of chitin and glucan fibrils, as demonstrated by PI labeling and colony counts on nutrient agar plates. Both ethanol and isopropanol (63% for 60 sec) inactivate 96 to 99% of elementary bodies of C. trachomatis, which have a wall of lipopolysaccharide but lack peptidoglycan, as measured by quantitative cultures to calculate inclusion forming units.

Conclusions/significance: In summary, alcohols kill infectious forms of Acanthamoeba, F. solani, and C. trachomatis, although longer times and higher ethanol concentrations are necessary for Acanthamoeba cysts. These results suggest the possibility that expanded use of alcohol-based hand sanitizers in places where water is not easily available might reduce transmission of these important causes of blindness.

Fig 1. A. castellanii trophozoites and cysts are rapidly permeabilized and killed by ethanol and isopropanol.

(A) Differential interference contrast (DIC) micrograph shows that untreated trophozoites have prominent vacuoles and acanthopods. In contrast, trophozoites treated with 63% ethanol (B) or 63% isopropanol (C) for 30 sec are rounded and have lost acanthopods. Not captured in still photographs is mobility of untreated trophozoites versus absence of mobility of alcohol-treated trophozoites. (D) Fluorescence micrograph of untreated trophozoites shows that Syto 9 (green), a vital stain, readily penetrates the plasma membrane, while PI (red stain in live-dead kits) does not. In contrast, both Syto 9 and PI readily label trophozoites killed by incubation in ethanol (E) and isopropanol (F). Wheat germ agglutinin (WGA in green) labels glycoproteins in the walls of both untreated (G) and alcohol-treated cysts for 120 sec (H and I). PI (red) does not label untreated cysts but readily penetrates walls and plasma membrane of alcohol-treated cysts. All images are of A. castellanii MEI 0184 strain and were shot through the 100X objective.

Fig 2. Treatment with alcohols blocks all growth of A. castellanii trophozoites in culture.

Log plots of growth in culture shows untreated trophozoites (blue), which were undiluted or diluted 10-fold or 100-fold, increase their number by 23 to 47 times in culture for three days. In contrast, ethanol-treated and isopropanol-treated trophozoites (red) show no growth in culture, and all of the remaining trophozoites are dead (as judged by either lack of mobility and lack of acanthopods by DIC or PI labeling by fluorescence microscopy) (previous figure). Plots shown are for A. castellanii MEI 0184 strain, but the same results were obtained with the other Acanthamoeba spp. strains and with A. byersi. Arrows indicate number of trophozoites at start of incubation.

Fig 3. Treatment of Acanthamoeba cysts with alcohols reduces excystation and trophozoite growth.

Log plots of excystation and growth of A. castellanii MEI 0184 strain (A), Acanthamoeba spp. Shi (B) and Esbc4 (C) strains, and A. byersi (D). Compared to killing of trophozoites (Fig 2), killing of cysts was much slower with 63% ethanol and much less effective with isopropanol. However, all four sets of Acanthamoeba cysts were rapidly killed by 80% ethanol.

Fig 4. F. solani conidia are rapidly permeabilized and killed by ethanol and isopropanol.

WGA (green) labels glycoproteins in the walls of both untreated and alcohol-treated conidia (A to C). In contrast, PI (red) fails to penetrate walls of untreated conidia (A), while PI readily penetrate walls and plasma membrane and labels nuclei of alcohol-treated conidia for 30 sec (B and C). All images shot through the 100X objective.

Fig 5. Treatment of F. solani conidia with ethanol and isopropanol markedly inhibits colony formation on nutrient agar plates.

Ten (A) or 100 (B) untreated F. solani conidia form discrete colonies on nutrient agar plates that may be counted. When 10⁶ conidia treated for 30 sec with ethanol (C) or isopropanol (D), diluted, pelleted, washed, and then plated, nearly the same number of colonies appear as when 10 untreated conidia are plated.

Fig 6. Elementary bodies of C. trachomatis are killed by ethanol and isopropanol.

(A) Fluorescence micrograph of a monolayer of L929 fibroblasts infected at an MOI of 1:10 with mock-treated C. trachomatis ocular serovar A2497 EBs. C. trachomatis inclusions stain green (arrows), while L929 cells are counterstained red. In contrast, after exposure for 30 sec to 63% ethanol (B) or 63% isopropanol (C), the development of inclusions is markedly reduced. After 60 sec treatment, few to no inclusions were visible in the wells. Images shot through the 10X objective. (D) Quantitative cultures of the alcohol treated EBs were compared to the mock treated EBs to calculate the percent killing. Shown above are the mean +/- standard deviation of triplicate wells. Data is representative of three independent experiments.