Ethanol and Isopropanol in Concentrations Present in Hand Sanitizers Sharply Reduce Excystation of Giardia and Entamoeba and Eliminate Oral Infectivity of Giardia Cysts in Gerbils

Chatterjee, A. K.; Bandini, Giulia; Motari, Edwin; Samuelson, John

doi:10.1128/aac.01290-15

Cited by 3 publications

(5 citation statements)

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“…Whether a compound identified in the encystation screen exerts an effect on trophozoite growth is among the most important questions that should be addressed to further characterize the compound. For this assessment, we attempted to standardize the flow cytometry analysis using propidium iodide (PI), a membrane-impermeable dye that specifically stains dying or dead cells (Chatterjee et al, 2015 ). In the established assay system, the inhibitory effect of different compounds on trophozoite proliferation is evaluated by the live cell rate, which is calculated using the live trophozoite cell number [PI negative (PI − ) population] and the total cell number in each sample relative to those in solvent-treated controls (set as 100%) (Figure 8 ).…”

Section: Resultsmentioning

confidence: 99%

A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation

Mi-ichi

Miyake

Tam

et al. 2018

Front. Cell. Infect. Microbiol.

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Amoebiasis is caused by Entamoeba histolytica infection, a protozoan parasite belonging to the phylum Amoebozoa. This parasite undergoes a fundamental cell differentiation process from proliferative trophozoite to dormant cyst, termed “encystation.” The cysts formed by encystation are solely responsible for the transmission of amoebiasis; therefore, Entamoeba encystation is an important subject from both biological and medical perspectives. Here, we have established a flow cytometry strategy for not only determining the percentage of formed cysts but also for monitoring changes in cell populations during encystation. This strategy together with fluorescence microscopy enables visualization of the cell differentiation process of Entamoeba encystation. We also standardized another flow cytometry protocol for counting live trophozoites. These two different flow cytometry techniques could be integrated into 96-well plate-based bioassays for monitoring the processes of cyst formation and trophozoite proliferation, which are crucial to maintain the Entamoeba life cycle. The combined two systems enabled us to screen a chemical library, the Pathogen Box of the Medicine for Malaria Venture, to obtain compounds that inhibit either the formation of cysts or the proliferation of trophozoites, or both. This is a prerequisite for the development of new drugs against amoebiasis, a global public health problem. Collectively, the two different 96-well plate-based Entamoeba bioassay and flow cytometry analysis systems (cyst formation and trophozoite proliferation) provide a methodology that can not only overcome the limitations of standard microscopic counting but also is effective in applied as well as basic Entamoeba biology.

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Section: Resultsmentioning

confidence: 99%

A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation

Mi-ichi

Miyake

Tam

et al. 2018

Front. Cell. Infect. Microbiol.

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“…Labeling with PI shows that the walls and membranes of Acanthamoeba and F . solani are penetrated by the alcohols (Figs 1 and 4 ), as has been shown from cysts of Giardia and Entamoeba [ 60 ]. Although the small size makes it difficult to study the cell wall architecture, it is likely that the bacterial membranes of the chlamydial EBs are also penetrated by alcohols.…”

Section: Discussionmentioning

confidence: 68%

“…Differential interference microscopy (DIC) was used to discriminate live organisms (motile with acanthopod spikes) from dead organisms (immotile and rounded). Alternatively, untreated and alcohol-treated trophozoites were incubated with Syto 9 (Thermo Fisher Scientific, Cambridge, MA), a vital stain that penetrates the plasma membrane, and PI, a nuclear stain that does not penetrate the plasma membrane [ 60 , 62 , 63 ]. Using a Zeiss inverted fluorescence microscope, we determined how many trophozoites were live (label green with Syto 9 but not red with PI) or dead (label with both Syto 9 and PI).…”

Section: Methodsmentioning

confidence: 99%

“…In particular, might alcohol-based hand sanitizers, which are widely used to prevent spread of bacterial and fungal infections in hospitals in developing and developed countries, kill these important eye pathogens [ 50 – 59 ]? As a model for these studies, we recently showed that ethanol and isopropanol in concentrations present in hand sanitizers kill cysts of Giardia and Entamoeba , important causes of diarrhea and dysentery, respectively [ 60 ]. In each case, the alcohols penetrate the cyst walls and permeabilize the plasma membrane, so that nuclei stain with propidium iodide (PI), and trophozoites fail to excyst in culture or in animals ( Giardia ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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BackgroundBlindness 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 findingsWe 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/SignificanceIn 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.

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“…The term 'pseudocyst' has been historically used to describe rounded forms of the parasite with internalized flagella with a regular cellular membrane, in contrast to true cysts, such as those from Entamoeba spp. and Giardia spp., which possess a true cyst wall (Chatterjee et al 2015). Pioneering investigators considered pseudocysts as irreversible and degenerative forms of the parasite (Wenrich, 1939;Samuels, 1957).…”

Section: Discussionmentioning

confidence: 99%

Iron-modulated pseudocyst formation inTritrichomonas foetus

et al. 2016

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Iron is an essential element for the survival of trichomonads during host-parasite interaction. The availability of this metal modulates several metabolic pathways of the parasites and regulates the expression of virulence factors such as adhesins and proteolytic enzymes. In this study, we investigated the effect of iron depletion on the morphology and life cycle of Tritrichomonas foetus. Scanning and transmission electron microscopy analyses revealed that depletion of iron from the culture medium (named TYM-DIP inducer medium) induces morphological transformation of typical pear-shaped trophozoites into spherical and non-motile pseudocysts. Remarkably, inoculation of pseudocysts into an iron-rich medium (standard TYM medium), or addition of FeSO4 to a TYM-DIP inducer medium reverted the morphological transformation process and typical trophozoites were recovered. These results show that pseudocysts are viable forms of the parasite and highlight the role of iron as a modulator of the parasite phenotype. Although iron is required for the survival of T. foetus, iron depletion does not cause a cellular collapse of pseudocysts, but instead induces phenotypic alterations, probably in order to allow the parasite to survive conditions of nutritional stress. Together, these findings support previous studies that suggest pseudocysts are a resistance form in the life cycle of T. foetus and enable new approaches to understanding the multifactorial role of iron in the cell biology of this protozoan parasite.

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Ethanol and Isopropanol in Concentrations Present in Hand Sanitizers Sharply Reduce Excystation of Giardia and Entamoeba and Eliminate Oral Infectivity of Giardia Cysts in Gerbils

Cited by 3 publications

References 48 publications

A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation

A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation

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

Iron-modulated pseudocyst formation inTritrichomonas foetus

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