Amoebicidal Effect of Chlorine Dioxide Gas against Pathogenic Naegleria fowleri and Acanthamoeba polyphaga

doi:10.21203/rs.3.rs-3969220/v1

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Amoebicidal Effect of Chlorine Dioxide Gas against Pathogenic Naegleria fowleri and Acanthamoeba polyphaga

https://doi.org/10.21203/rs.3.rs-3969220/v1

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published 01 Apr, 2024

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The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO₂) gas is a potent disinfectant that is harmless to humans. In this study, we examined the amoebicidal effects of ClO₂ gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO₂ gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO₂ gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO₂ gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO₂ gas. ClO₂ gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO₂ gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Naegleri fowleri

Acanthamoeba polyphaga

chlorine dioxide (ClO2) gas

disinfectant

amoebicidal effect

The ubiquitous free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. Naegleria fowleri can cause a sudden and severe brain infection known as primary amoebic meningoencephalitis (PAM) in humans and animals (Ma et al., 1990; Marciano-Cabral and Cabral, 2007; Visvesvara, 2013). Some Acanthamoeba spp. are the causative agents of severe medical complications including fatal granulomatous encephalitis (GAE) and Acanthamoeba keratitis (AK) (Lorenzo-Morales et al., 2015; Marciano-Cabral et al., 2000).

Humans are often infected with N. fowleri through water activities and pollution and develop PAM, leading to serious medical and social problems (Marciano-Cabral, 1988; Siddiqui et al., 2016; Taravaud et al., 2018). The infection primarily occurs when contaminated water is inhaled through the nose; the amoeba enters the nasal cavity and olfactory nerve, traverses the central nervous system (CNS), and kills its host within 5–9 days (Marciano-Cabral and Cabral, 2007; Martinez et al., 2010; Schuster and Visvesvara, 2004). As PAM is acute, and cerebrospinal fluid (CSF) is the main diagnostic specimen, early diagnosis is difficult. Anti-fungal agents (amphotericin B and/or miltefosine) have been used as treatment drugs; however, their effects are often unsatisfactory. Consequently, PAM has a high mortality rate of 95% (Kim et al., 2008; Visvesvara et al., 2007). AK is a rare but painful and severe corneal infection often observed in patients who wear contact lenses (Marciano-Cabral and Cabral, 2003). As the diagnosis of AK is difficult and delayed, a rapid and accurate diagnosis method is urgently required for AK.

Chlorine dioxide (ClO₂) is a highly effective disinfectant. It is a yellowish-green gas at room temperature that is relatively easily dissolved in water; therefore, it can be used in various forms. This compound is a free radical, strongly oxidizing disinfectant with a stronger biocidal capacity than chlorine-based disinfectants and alcohols. ClO₂ has a sterilizing effect over a broad pH range of 2–10 and is useful as a food-washing solution and disinfectant owing to its relative stability and high solubility. It eliminates pigments, oxidizes iron and manganese, and neutralizes odors (Vogt et al., 2010). ClO₂ gas is well-known for its outstanding deodorizing, sterilizing, and viral amplification inhibitory properties (Kaly-Kullai et al., 2020; O Young, 2016). It is cost-effective, safe, and has eco-friendly qualities such as rapid light-induced breakdown (Wood et al., 2010). ClO₂ gas has been extensively used to disinfect various medical equipment; without direct application to the surface of an object, the growth of many bacterial species in the immediate vicinity can be prevented.

A previous study on the microbiocidal mechanism of a ClO₂ solution reported that the primary mechanism in the treatment of Escherichia coli was the disruption of protein synthesis (Benarde et al., 1967). Bacillus subtilis is also inactivated by free chlorine and ClO₂ solutions owing to alterations in cell permeability (Young and Setlow, 2003). ClO₂ applications have recently expanded to include food disinfection and medical therapy. Moreover, ClO₂ in the gas form is an excellent disinfectant that can be used in daily life at low concentrations. However, owing to technical challenges in maintaining a consistent concentration of ClO₂ gas for evaporation, its practical application is limited. Although ClO₂ solution has antimicrobial effects, the reports documenting these effects are limited. Furthermore, our understanding of the amoebicidal mechanism of ClO₂ gas is still lacking, especially in free-living amoebae. Therefore, in this study, we investigated the amoebicidal effect of ClO₂ gas on N. fowleri and A. polyphaga, which was confirmed by observing amoebic viability by cell counting and mRNA expression of the N. fowleri and A. polyphaga actin genes nf-actin and ap-actin, respectively.

Preparation of N. fowleri and A. polyphaga

The trophozoites of N. fowleri (Carter NF69 strain, ATCC NO. 30215) and A. polyphaga were axenically cultured at 37 °C in Nelson's medium containing 10% fetal bovine serum and at 30 °C in PYG medium, respectively (Visvesvara and Balamuth, 1975; Willaert, 1971). The amoebae were washed twice with phosphate-buffered saline (PBS) and centrifuged at 1500 rpm for 3 min. The cell pellets of N. fowleri and A. polyphaga were suspended in Nelson’s medium (2 × 10⁵ cells/mL) and PYG medium (2 × 10⁵ cells/mL), respectively, and then placed in 40φ culture dishes.

Exposure of N. fowleri and A. polyphaga to ClO₂ gas

ClO₂ gas from ‘PURISTIC’, a ready-to-use product from Purgofarm Co. Ltd. (Yongin City, Korea), was activated and placed in a small chamber (210 mm high × 297 mm wide × 250 mm deep), and the concentration of ClO₂ gas was measured using a gas detector (C-16, Analytical Technology, Collegeville, PA, USA). To begin the experiment, we pre-activated PURISTIC for 5 h, and then exposed N. fowleri and A. polyphaga to ClO₂ gas at 0.36 ppmv/h for 12, 24, 36, and 48 h. A group of N. fowleri and A. polyphaga were maintained without ClO₂ treatment as a control.

Viability observation

During the experimental period, amoebic viability, which is demonstrated through morphological changes after ClO₂ gas treatment, was observed using an inverted microscope (Olympus, Tokyo, Japan). The viable ClO₂ gas-treated amoeba samples were re-cultured with fresh Nelson’s or PYG media to observe their ability to recover into trophozoites. Amoebic viability and growth rates were estimated by cell counting with a hemocytometer.

Ultrastructural characteristics of N. fowleri and A. polyphaga trophozoites exposed to ClO₂ gas

Amoebic trophozoites exposed to ClO₂ gas for 24 h in 40φ culture dishes were harvested, fixed with fixation buffer (2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2), post fixed with 1% osmium tetroxide at room temperature, dehydrated with increasing concentrations of ethanol, and embedded in epoxy resin. Subsequently, the samples were cut into thin sections and stained with uranyl acetate and lead citrate and observed using a Sigma 500 transmission electron microscope (TEM; Carl Zeiss, Oberkochen, Germany).

RT-PCR for the mRNA expression of actin gene in N. fowleri and A. polyphaga

To confirm amoebic viability, reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the nf-actin (actin gene of N. fowleri) and ap-actin (actin gene of A. polyphaga) products according to a previously described method (Sohn et al., 2019). Amoebae at each stage of incubation were used to generate cDNA (5 µg), and total RNA was prepared using a Superscript First Strand synthesis system (Invitrogen, Carlsbad, CA, USA) and RNeasy®Mini kit (QIAGEN, Germantown, MD, USA), respectively. PCR conditions were as follows: 95°C for 5 min, 30 cycles at 95°C for 1 min, 50°C for 30 s, 72°C for 30 s, and a final extension for 10 min at 72°C. The amplified products were separated by electrophoresis on an ethidium bromide-stained 1% agarose gel and detected under UV light.

Morphological changes of N. fowler and A. polyphaga exposed to ClO₂ gas

An inverted microscope analysis revealed that N. fowleri exposed to ClO₂ gas showed some immobility after 3 h and became wrinkled in shape after 12 h (Fig. 1). At 12 h, N. fowleri trophozoites were converted to a precystic form (round shape). Unlike the N. fowleri trophozoites of the control group (no ClO₂ gas treatment), those in the treatment group were round, ruptured, and died after 24 h of treatment (Fig. 1). No amoeba trophozoites were detected after 36 h. N. fowleri exposed to ClO₂ gas could not be recovered in fresh Nelson’s medium (Fig. 1). In A. polyphaga, trophozoites of A. polyphaga exposed to ClO₂ gas showed some morphological changes and wrinkled forms after 24 h (Fig. 2). At 36 h, A. polyphaga trophozoites were converted to a precystic or cystic form. The trophozoites were rounded, ruptured, and died after 36 h of treatment, compared to those in the control group (Fig. 2). After 48 h, no trophozoites were detected and no recovery was observed in fresh PYG medium (Fig. 2).

Ultrastructural characteristics of N. fowleri and A. polyphaga exposed to ClO₂ gas

When N. fowleri and A. polyphaga were exposed to ClO₂ gas for 36 h, TEM analysis of the cellular response revealed increased breakdown of the nuclear membrane layers. With increased exposure to ClO₂ gas, trophozoites developed abnormal shapes and became fragmented. Ultrastructural changes within the trophozoites exposed to ClO₂ gas were investigated using TEM (Figs. 3 and 4). A loss of extracellular material was observed, which progressed to a loss of cellular membrane integrity in N. fowleri exposed to ClO₂ gas for 36 h (Fig. 3). When A. polyphaga was exposed to ClO₂ gas for 36 h, the trophozoites showed the same morphological changes as N. fowleri (Fig. 4). Cellular membrane destruction, cytoplasmic damage, and lipid droplets were evident in both cases (Figs. 3 and 4).

Growth rates of N. fowleri and A. polyphaga exposed to ClO₂ gas

After treatment with ClO₂ gas, the growth rate of N. fowleri trophozoites decreased remarkably at 24 h, showing an approximately 80% reduction compared to that of the control group (Fig. 5A). At 24 h, the number of trophozoites of N. fowleri significantly decreased, and cysts appeared to increase slightly; however, the number of cysts also decreased at 36 and 48 h (Fig. 5A). In A. polyphaga, the growth rate of trophozoites decreased after 36 h (Fig. 5B). Similar to the results for N. fowleri, the number of cysts increased at 24 h and then decreased significantly at 36 and 48 h (Fig. 5B). These results show that ClO₂ gas had cytotoxic effects on N. fowleri and A. polyphaga in a time-dependent manner (Fig. 5).

mRNA expression of actin gene in N. fowleri and A. polyphaga exposed to ClO₂ gas

RT-PCR was performed to confirm the viability of N. fowleri and A. polyphaga exposed to ClO₂. The mRNA expression levels of nf-actin and ap-actin mRNA decreased in a time-dependent manner (Fig. 6). In particular, no nf-actin mRNA expression was observed in N. fowleri exposed to ClO₂ gas for 48 h (Fig. 6A).

Patients with PAM are mostly infected through various amoeba-contaminated freshwater sources, such as bathing in ponds and hot springs, diving and skiing in rivers, and drinking tap water (Chen et al., 2019; Izumiyama et al., 2003; Maclean et al., 2004; Marciano-Cabral, 1988). The disinfection or decontamination of freshwater contaminated with N. fowleri is still in its nascent stage. Additionally, PAM is difficult to diagnose, and no existing treatment has achieved satisfactory results (Kim et al., 2008; Visvesvara et al., 2007). In addition, the incidence of AK among patients who wear contact lenses has been increasing worldwide (Lorenzo-Morales et al., 2015). Acanthamoeba spp. infiltrate the corneal stroma and epithelial cells, which is followed by the progressive infiltration of inflammatory cells. Therefore, the removal of pathogenic N. fowleri and A. polyphaga from fresh and polluted water is important.

The US Environmental Protection Agency (US EPA) first approved the liquid form of ClO₂ for use as a disinfectant and sanitizer at various sites, including animal farms, bottling plants, and food processing, handling, and storage plants, in 1967, under the authority of the Federal Insecticide, Fungicide, and Rodenticide Act (Benarde et al., 1967). In 1988, the US EPA approved ClO₂ gas as a disinfectant for use on tools, clean rooms, environmental surfaces, manufacturing, and laboratory equipment and in 2006, they published a re-registration eligibility decision regarding ClO₂ as a pesticide (EPAGuidanceManual, 2006). ClO₂ is rapidly decomposed to the byproducts chlorite (ClO₂-) and chlorate (ClO₃-) upon contact with naturally occurring organic and inorganic substances. Evaluation of these byproducts are reported to have acceptable daily intake (ADI) values of 0.03 mg/kg body weight (bw) per day for chlorite and 0.01 mg/kg bw per day for chlorate (‎JEFCA, 2007; 2008). Clinical research has shown that ClO₂ is an effective disinfectant and cytotoxic to bacteria, fungi, and viruses (Kuo-Shan Yao, 2010; Sanekata et al., 2010; Wei et al., 2008). ClO₂ gas is several times more potent than sodium hypochlorite and has been shown to be effective in various situations.

Sifaoui et al. (Sifaoui et al., 2021) reported the effects of a commercial disinfectant (CLORICAN) on Acanthamoeba spp. and N. fowleri viability. They reported that CLORICAN, a commercial liquid form of chlorine dioxide used to disinfect swimming pools, had a stronger effect on N. fowleri than Acanthamoeba spp. (Sifaoui et al., 2021). In this study, the growth rate of N. fowleri trophozoites decreased remarkably after 24 h of exposure to ClO₂ gas, showing an approximately 80% reduction compared to the control group. In addition, the growth rate of A. polyphaga trophozoites decreased after 24 h of exposure to ClO₂. In both cases, recovery of amoebae treated with ClO₂ gas was not possible. The cell membrane is an important structure in both prokaryotes and eukaryotes, and disruption of the nuclear and cytoplasmic membrane layers and cytoplasmic organelle degradation were observed by TEM analysis in both N. fowleri and A. polyphaga exposed to ClO₂ gas. These findings reveal its efficacy as a disinfectant, similar to the effects observed in bacteria, fungi, and viruses (Kuo-Shan Yao, 2010; Sanekata et al., 2010; Wei et al., 2008). In our study, ClO₂ gas was shown to have high sensitivity on both N. fowleri and A. polyphaga. ClO₂ gas alters the amoeboid shape and induces cell shrinkage.

Additionally, we investigated actin gene expression in N. fowleri and A. polyphaga exposed to ClO₂ gas. The actin gene is a major component of the cytoskeleton of pathogenic free-living amoebae such as N. fowleri and A. polyphaga (Sohn et al., 2019). Overexpression of the actin genes in N. fowleri or A. polyphaga trophozoites increases their amoebic motility and phagocytosis (Sohn et al., 2010). Therefore, we analyzed the expression levels of actin mRNA using RT-PCR to confirm the physiological changes in N. fowleri and A. polyphaga treated with ClO₂ gas. In both cases, the levels of actin mRNA in N. fowleri and A. polyphaga exposed to ClO₂ gas decreased in a time-dependent manner. Specifically, no actin mRNA expression was observed in N. fowleri exposed to ClO₂ gas for 48 h, indicating that ClO₂ gas completely eliminated N. fowleri trophozoites. These results suggest that ClO₂ gas exerts amoebicidal effects on N. fowleri and A. polyphaga by reducing their motility and phagocytic activity. However, further studies are required to elucidate this mechanism. Therefore, ClO₂ gas has been proposed as an efficient agent for the prevention and control of pathogenic free-living amoebae such as N. fowleri and A. polyphaga.

Acknowledgements

We would like to thank to Jong-Rak Kim, CEO of Purgofarm Co. for providing the materials.

Author contribution

Conceptualization, Hae-Jin Sohn, Kyoung-Ju Song and Ho-Joon Shin; Data curation, Hae-Jin Sohn, A-Young Park, Kyu-Hwa Yun and Jong-Hyun Kim; Formal analysis, Hae-Jin Sohn, Jeong-Heon Lee and Kyu-Hwa Yun; Investigation, Hae-Jin Sohn; Methodology, Hae-Jin Sohn and A-Young Park; Resources, Kyoung-Ju Song; Software, Kyoung-Ju Song; Supervision, Ho-Joon Shin; Validation, Hae-Jin Sohn; Visualization, Hae-Jin Sohn, A-Young Park and Jeong-Heon Lee; Writing – original draft, Hae-Jin Sohn; Writing – review & editing, Jong-Hyun Kim and Ho-Joon Shin.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1C1C2009518).

Data availability

All data generated or analyzed during this study are included in this published article.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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Amoebicidal Effect of Chlorine Dioxide Gas against Pathogenic Naegleria fowleri and Acanthamoeba polyphaga

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