Effect of a Commercial Disinfectant CLORICAN® on Acanthamoeba Spp

Article Effect of a Commercial Disinfectant CLORICAN® on Acanthamoeba spp. and Naegleria fowleri Viability

Ines Sifaoui 1,2,3,*,†, Aitor Rizo-Liendo 1,2,3,†, María Reyes-Batlle 1,2,3, Iñigo Arberas-Jiménez 1,2,3, Rubén L. Rodríguez-Expósito 1,2,3 , José E. Piñero 1,2,3,* and Jacob Lorenzo-Morales 1,2,3,*

1 Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna (ULL), 38206 Tenerife, Spain; [email protected] (A.R.-L.); [email protected] (M.R.-B.); [email protected] (I.A.-J.); [email protected] (R.L.R.-E.) 2 Departamento de Obstetricia, Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Universidad De La Laguna, La Laguna, Tenerife, 38203 Islas Canarias, Spain 3 Red de Investigación Cooperativa en Enfermedades Tropicales (RICET), Universidad de Salamanca, 37008 Salamanca, Spain * Correspondence: [email protected] (I.S.); [email protected] (J.E.P.); [email protected] (J.L.-M.) † Contributed equally to this work.

Abstract: Swimming pool water treatment by chemicals is an essential step to avoid microbial proliferation and infections namely caused by free living amoeba such as, for example, primary amebic meningoencephalitis and Acanthamoeba keratitis. In the present study, a commercial reactive, CLORICAN, based on chlorine dioxide, was evaluated against Acanthamoeba spp. and Naegleria fowleri. We observed that CLORICAN could eliminate in a short period of incubation time both amoebae. Citation: Sifaoui, I.; Rizo-Liendo, A.; Reyes-Batlle, M.; Arberas-Jiménez, I.; Furthermore, Naegleria fowleri’s trophozoites were more sensitive than those of Acanthamoeba spp. By Rodríguez-Expósito, R.L.; Piñero, J.E.; means of inverted microscopy, the chlorine dioxide was found to greatly affect morphology shape by Lorenzo-Morales, J. Effect of a increasing the cell size shrinkage. Commercial Disinfectant CLORICAN® on Acanthamoeba spp. Keywords: CLORICAN; Acanthamoeba spp.; Naegleria fowleri; amoebicidal effect and Naegleria fowleri Viability. Parasitologia 2021, 1, 119–129. https://doi.org/10.3390/ parasitologia1030013 1. Introduction Free-living amoebae (FLA) are ubiquitous protozoa found in natural habitats, namely Academic Editor: Geoff Hide soil and water, and artificial habitats such as swimming pools. FLA act as pathogens, as parasites, as carries, or as vehicles of pathogenic microorganisms such as as bacteria or Received: 8 April 2021 viruses [1]. At present, multiple species have been reported as being pathogens and oppor- Accepted: 8 July 2021 Published: 13 July 2021 tunistic, namely Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, Vahlkampfia, Vermamoeba and Sappinia. Based on the literature, Acanthamoeba spp., Naegleria fowleri and Balamuthia mandrillaris Publisher’s Note: MDPI stays neutral are the most common FLA, with severe pathogenesis. WHO have with regard to jurisdictional claims in defined Acanthamoeba spp. and Naegleria fowleri as quality indicator microorganisms in published maps and institutional affil- recreational water [2]. iations. Some Acanthamoeba species or genotypes are the causative agents of severe medical complications including pneumonitis, fatal granulomatous encephalitis, skin infections and Acanthamoeba keratitis (AK) [3–5]. Based on the small ribosomal subunit gene (18S rDNA), 22 different genotypes were identified as T1 to T22 [6]. Even though genotype T4 is the most abundant species in both clinical and environmental samples [7], other Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. genotypes such as T2, T3, T5, T6, T11, T13, and T15 were isolated from AK and amoebic This article is an open access article encephalitis cases [8]. Acanthamoeba are widely presented in freshwater and marine habitats distributed under the terms and such as rivers, tap water, swimming pools, and sea water, among others [9]. Overall, 25% conditions of the Creative Commons of Acanthamoeba sp. isolated from the different habitats host and protect microorganisms Attribution (CC BY) license (https:// from adverse environmental conditions and chemical factors [10,11]. Free living amoebae creativecommons.org/licenses/by/ can be a reservoir of pathogenic bacteria such as Vibrio cholerae, Mycobacterium tuberculosis, 4.0/). Listeria monocytogenes, Helicobacter pylori, Legionella pneumophila, and Klebsiella pneumoniae,

Parasitologia 2021, 1, 119–129. https://doi.org/10.3390/parasitologia1030013 https://www.mdpi.com/journal/parasitologia Parasitologia 2021, 1 120

among others. Virus such herpes virus, enteroviruses, pandoraviruses and mimivirus have also been isolated from FLA [12–14]. Nevertheless, the amoebae from the Naegleria genus were isolated particularly from fresh warm water such as hot springs, swimming pools, and lagoons, among others. Even though more than 40 species of Naegleria have been identified, only Naegleria fowleri, “the brain-eating amoeba”, has been reported to induce a rare and fatal disease, primary amoebic meningoencephalitis (PAM) [15,16]. In order to ensure the quality of recreational water such as swimming pools and so on, several water treatments are conducted, namely filtration, coagulation and disinfection [2]. Disinfection of recreational water is fundamental to eliminate any microbial hazards [17]. During this treatment, chemical agents (e.g., chlorination) and physical methods (e.g., UV-C radiation) are used in order to deactivate the present germs [2]. The CDC recommend a free chlorine concentration of at least 1 ppm in pools and at least 3 ppm in hot tubs/spas and a pH of 7.2–7.8 [18]. Chlorine dioxide has been reported as a strong disinfectant with numerous applications in wastewater treatment, environment and food disinfection, and medicine [19]. For instance, Dupuy et al. (2014) reported the efficacy of chlorine dioxide in inhibiting three different FLA strains [20]. In the present study, the efficacy of a new formulation based on chlorine dioxide was tested against four FLA strains—three Acanthamoeba strains and Naegleria fowleri. The protozoa were analyzed during the time using inverted microscopy.

2. Results Figures1–3 illustrates that a concentration above 0.063% of chlorine dioxide turned out to be lethal for Acanthamoeba castellanii Neff. In fact, at this concentration, the trophozoites seemed to lose any motility and appeared as fixed cells. After 24 h of incubation with chlorine dioxide at a concentration of 0.063% (Figure2C), the trophozoites do not show their pseudopodia and become rounded cells. At the same concentration and after 96 h of incubation, we noticed that the rounded trophozoites were not able to encyst in order to remain as pseudo-cysts (Figure3B). The same effect was observed for Acan- thamoeba polyphaga and Acanthamoeba griffini, although both strains were more resistant to CLORICAN. As shown by Figure4 for Acanthamoeba griffini and Figure5 for Acanthamoeba polyphaga, a chlorine dioxide concentration of 0.126% was effective to prevent the encystation process. A concentration of 0.063% was able to eliminate 83% and 30%, respectively, Parasitologia 2021, 1, FOR PEER REVIEW 3 of Acanthamoeba castellanii Neff and Acanthamoeba griffini. This dose was ineffective in Acanthamoeba polyphaga.

Figure 1. Cont.

Figure 1. Effect of CLORICAN on Acanthamoeba castellanii Neff trophozoite after 3 h of incubation at 0.252% (A), 0.126% (B), 0.063% (C) of chlorine dioxide and negative control (D). At these concentrations, trophozoites seems to lose any motility and appeared as fixed cells (A and B). All images (×40) are representative of the population of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. Scale bar = 50 µm.

Figure 2. Effect of CLORICAN on Acanthamoeba castellanii Neff trophozoite after 24 h of incubation at concentrations of 0.252% (A), 0.126% (B), and 0.063% (C) chlorine dioxide, and the negative control (D). The characteristic pseudopodia of Acanthamoeba castellanii Neff cannot be observed in treated cells. (A–C) All images (×40) are representative of the population of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. Scale bar = 50 µm. Parasitologia 2021, 1, FOR PEER REVIEW 3

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FigureFigure 1. Effect 1. Effect of CLORICAN of CLORICAN on on AcanthamoebaAcanthamoeba castellanii Neff Neff trophozoite trophozoite after after 3 hof 3 incubationh of incubation at 0.252% at 0.252% (A), 0.126% (A), (B 0.126%), (B), 0.063%0.063% (C (C)) of of chlorine chlorine dioxidedioxide and and negative negative control control (D). (D At). these At these concentrations, concentrations, trophozoites trophozoites seems to seems lose any to lose motility any motility andand appearedappeared as as ﬁxed fixed cells cells (A ,(AB). and All images B). All ( ×images40) are (×40) representative are representative of the population of the ofpopulation treated amoebae of treated and areamoebae based and are basedon the on live the cell live imaging cell imaging microscope microscope EVOS FL EV cellOS imaging FL cell system. imaging Scale syst barem. = 50Scaleµm. bar = 50 µm.

FigureFigure 2. Effect 2. Effect of CLORICAN of CLORICAN on onAcanthamoebaAcanthamoeba castellanii castellanii NeffNeff trophozoite after after 24 24 h ofh of incubation incubation at concentrations at concentrations of of 0.252%0.252% (A), 0.126% (A), 0.126% (B), (andB), and 0.063% 0.063% (C ()C chlorine) chlorine dioxide, dioxide, and and the negativenegative control control (D ().D The). The characteristic characteristic pseudopodia pseudopodia of of AcanthamoebaAcanthamoeba castellanii castellanii NeffNeff cannot cannot be be observed observed inintreated treated cells. cells. (A (–AC–)C All) All images images (×40) (×40) are representative are representative of the population of the population ofof treated treated amoebae and and are are based based on theon the live live cell imagingcell imaging microscope microscope EVOS FL EVOS cell imaging FL cell system.imaging Scale system bar =. Scale 50 µm. bar = 50 µm. ParasitologiaParasitologia 2021,, 12021,, FORFOR, 1 PEERPEER REVIEWREVIEW 122 4

FigureFigure 3. Growth 3. Growth inhibition inhibition of Acanthamoeba of Acanthamoeba castellanii castellanii NeffNeffNeff afterafter 969696h hhof ofof incubation. incubation.incubation.Acanthamoeba Acanthamoebaspp. sppspp cells cellscells were werewere incubated incubatedincubated with awith concentration a concentration of 0.126% of 0.126% chlorine chlorine dioxide dioxide (A (A).). AcanthamoebaAcanthamoeba cellscellscellswere werewere incubated incubatedincubated with withwith a concentration aa concentrationconcentration of 0.063% ofof 0.063%0.063% chlorinechlorine dioxide dioxide (B). (NegativeB). Negative control control (C ().C ).Histograms Histograms of of cells’ cells’ unviabilityunviability percentages percentages for for each each tested tested concentration concentration of of chlorinechlorine dioxide dioxide (D). ( DAll). Allimages images (×40) (×40) are are representative representative ofof the the population population of treatedof treated amoebae amoebae and are and based are on based the live oncell the live cell imagingimaging microscope microscope EVOSEVOS FL FL cell cell imaging imaging system. system. ** p <** 0.01; p << ***0.01;0.01;p < ****** 0.001; p << **** 0.001;0.001;p < 0.0001******** p signiﬁcance << 0.00010.0001 significancesignificance differences amongdifferencesdifferences amongdifferent different doses doses of chlorine of chlorine dioxide. dioxide. Scale Scale bar = 50barµ m.= 50 µm.

Figure 4. Cont.

Figure 4. Growth inhibition of Acanthamoeba griffini afterafter 9696 hh ofof incubation.incubation. Acanthamoeba sppspp cellscells werewere incubatedincubated withwith aa concentration of 0.126% chlorine dioxide (A). Acanthamoeba cellscells werewere incubatedincubated withwith aa concentrationconcentration ofof 0.063%0.063% chlorinechlorine dioxide (B). Negative control (C). Histograms of cells’ unviability percentages for each tested concentration of chlorine Parasitologia 2021, 1, FOR PEER REVIEW 4

Figure 3. Growth inhibition of Acanthamoeba castellanii Neff after 96 h of incubation. Acanthamoeba spp cells were incubated with a concentration of 0.126% chlorine dioxide (A). Acanthamoeba cells were incubated with a concentration of 0.063% chlorine dioxide (B). Negative control (C). Histograms of cells’ unviability percentages for each tested concentration of chlorine dioxide (D). All images (×40) are representative of the population of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. ** p < 0.01; *** p < 0.001; **** p < 0.0001 significance differences among different doses of chlorine dioxide. Scale bar = 50 µm.

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Parasitologia Figure2021, 1,4. FORGrowth PEER inhibition REVIEW of Acanthamoeba grifﬁni after 96 h of incubation. Acanthamoeba spp. cells were incubated with a 5 Figure 4. Growth inhibition of Acanthamoeba griffini after 96 h of incubation. Acanthamoeba spp cells were incubated with a concentration of 0.126% chlorine dioxide (A). Acanthamoeba cells were incubated with a concentration of 0.063% chlorine concentration of 0.126% chlorine dioxide (A). Acanthamoeba cells were incubated with a concentration of 0.063% chlorine dioxide (B). Negative control (C). Histograms of cells’ unviability percentages for each tested concentration of chlorine dioxide (B). Negative control (C). Histograms of cells’ unviability percentages for each tested concentration of chlorine dioxidedioxide (D). All (D). images All images (×40) (× are40) representative are representative of of the the population population of treated amoebae amoebae and and are are based based on theon livethe live cell imagingcell imaging microscopemicroscope EVOS EVOS FL cell FL cellimaging imaging system. system. ** p *** < p0.01;< 0.001; *** p **** < 0.001;p < 0.0001 **** p signiﬁcance < 0.0001 significance differences amongdifferences different among doses different of doses chlorineof chlorine dioxide. dioxide. Scale Scale bar = bar 50 µ =m. 50µm.

FigureFigure 5. Growth 5. Growth inhibition inhibition of Acanthamoeba of Acanthamoeba polyphaga polyphaga;; after after 9696 hh of of incubation. incubation.Acanthamoeba Acanthamoebaspp. spp cells cells were were incubated incubated with awith concentration a concentration of 0.137% of 0.137% chlorine chlorine dioxide dioxide (A (A).). AcanthamoebaAcanthamoeba cellscells were were incubated incubated with with a concentration a concentration of 0.063% of 0.063% chlorinechlorine dioxide dioxide (B). (NegativeB). Negative control control (C ().C ).Histograms Histograms of of cells’ cells’ unviabilityunviability percentages percentages for for each each tested tested concentration concentration of of chlorinechlorine dioxide dioxide (D). ( DAll). Allimages images (×40) (×40) are are representative representative ofof the the population population oftreated of treated amoebae amoebae and are and based are on based the live on cell the live cell imagingimaging microscope microscope EVOSEVOS FL FL cell cell imaging imaging system. system. Data Data are presentedare presented as means as means± SD (N± SD = 3) (N **** = 3)p < ** 0.0001 p < 0.01; signiﬁcance *** p < 0.001; **** p differences< 0.0001 significance among different differences doses of among chlorine different dioxide. doses Scale bar of =chlorine 50 µm. dioxide. Scale bar = 50 µm. Parasitologia 2021, 1 124

On the other hand, a chlorine dioxide concentration of up to 0.055 % on the Naegleria fowleri trophozoites, and a strong change in morphology was detected after 15 min of incubation; in fact, most of the amoebae lost their amoeboid shape, leading them to be rounded (Figure6). This effect persists for at least 24 h (see Figures7–9). At a lower concentration of Parasitologia 2021, 1, FOR PEER REVIEW chlorine dioxide, precisely 0.0275%, the trophozoite showed slight morphological changes 6 as well as in the number of intracellular vesicles after 1 h of incubation (Figure7). After 24 h of incubation with a 0.055% concentration of chlorine dioxide, we noticed the presence of Parasitologia 2021, 1, FOR PEER REVIEW 6 pseudo-cyst cells; nevertheless, these cells are not able to complete the encystation process, as shown in Figure9B.

FigureFigure 6. Effect 6. Effect of CLORICAN of CLORICANCLORICAN on on onNaegleriaNaegleria Naegleria fowleri fowleri fowleri trophozoitestrophozoites trophozoites after afterafter 15 15 min15 min min of of incubation of incuba incubation attion chlorineat chlorineat chlorine dioxide dioxide dioxide concentrations concentra- concentra- tions oftions 0.11%0.11% of 0.11% ( A(A),), 0.055% (0.055%A), 0.055% (B ),(B and), ( Band 0.0275%), and 0.0275% 0.0275% (C) and (C (C the) )and and negative the the negativenegative control ( controlDcontrol). The Naegleria( D(D). ).The The Naegleria fowleri Naegleriatrophozoites fowleri fowleri trophozoites losttrophozoites their amoeboidlost their lost their amoeboidamoeboidmorphology. morphology. morphology. All images All Allimages (×40) images are (×40) representative (×40) ar are representativee representative of the population ofof the population ofpopulation treated amoebae of of treated treated and amoebae areamoebae based and on and are the basedare live based cell on imagingthe on live the live cell imagingcellmicroscope imaging microscope EVOSmicroscope FL EVOS cell EVOS imaging FL FLcell system.cell imaging imaging Scale system. system. bar =50 Scale Scaleµm. bar == 5050 µm. µm.

Figure 7. Cont.

Figure 7. Effect of CLORICAN on Naegleria fowleri trophozoites after 1 h of incubation at chlorine dioxide concentrations of 0.11% (A), 0.055% (B), and 0.0275% (C) and the negative control (D). All images (×40) are representative of the population of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. Scale bar = 50 Figureµm. 7. Effect of CLORICAN on Naegleria fowleri trophozoites after 1 h of incubation at chlorine dioxide concentrations of 0.11% (A), 0.055% (B), and 0.0275% (C) and the negative control (D). All images (×40) are representative of the population of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. Scale bar = 50 µm. Parasitologia 2021, 1, FOR PEER REVIEW 6

Figure 6. Effect of CLORICAN on Naegleria fowleri trophozoites after 15 min of incubation at chlorine dioxide concentrations of 0.11% (A), 0.055% (B), and 0.0275% (C) and the negative control (D). The Naegleria fowleri trophozoites lost their amoeboid morphology. All images (×40) are representative of the population of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. Scale bar = 50 µm.

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Figure 7. Effect of CLORICAN on Naegleria fowleri trophozoites after 1 h of incubation at chlorine dioxide concentrations of ParasitologiaFigure 2021 7. Effect, 1, FOR of PEER CLORICAN REVIEW on Naegleria fowleri trophozoites after 1 h of incubation at chlorine dioxide concentrations 7 0.11% (A), 0.055% (B), and 0.0275% (C) and the negative control (D). All images (×40) are representative of the population of 0.11% (A), 0.055% (B), and 0.0275% (C) and the negative control (D). All images (×40) are representative of the popula- of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. Scale bar = 50 µm. tion of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. Scale bar = 50 µm.

FigureFigure 8. Effect 8. Effect of CLORICAN of CLORICAN on onNaegleriaNaegleria fowleri fowleri trophozoitetrophozoite after after 24 24 h ofh of incubation incubation at chlorine at chlorine dioxide dioxide concentrations concentrations of × of 0.11%0.11% (A), (A 0.055%), 0.055% (B (B),), and and 0.0275% 0.0275% ((CC)) andand the the negative negative control control (D). (D All). imagesAll images ( 40) (×40) are representative are representative of the population of the popula- µ tion ofof treated treated amoebae amoebae andand areare based based on on the the live live cell cell imaging imaging microscope microscope EVOS EVOS FL cell FL imaging cell imaging system. system. Scale bar Scale = 50 barm. = 50µm.

Figure 9. Effect of CLORICAN on Naegleria fowleri trophozoite after 48 h of incubation at chlorine dioxide concentrations of 0.11% (A), 0.055% (B), and 0.0275% (C) and the negative control (D). All images (×40) are representative of the population of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. (E) Data are presented as means ± SD (N = 3). ** p < 0.01; *** p < 0.001; **** p < 0.0001 significance differences among different doses of chlorine dioxide. Scale bar = 50 µm.

3. Discussion Several free-living amoebae including Naegleria fowleri and some species from the Acanthamoeba genus represent a potential threat to human health due to their intrinsic pathogenicity and their role as protective and disseminating hosts for other pathogenic Parasitologia 2021, 1, FOR PEER REVIEW 7

ParasitologiaFigure2021 8., 1 Effect of CLORICAN on Naegleria fowleri trophozoite after 24 h of incubation at chlorine dioxide concentrations 126 of 0.11% (A), 0.055% (B), and 0.0275% (C) and the negative control (D). All images (×40) are representative of the population of treated amoebae and are based on the live cell imaging microscope EVOS FL cell imaging system. Scale bar = 50µm.

FigureFigure 9. 9.Effect Effect of of CLORICAN CLORICAN on onNaegleria Naegleria fowleri fowleritrophozoite trophozoite after after 48 h48 of h incubation of incubation at chlorine at chlorine dioxide dioxide concentrations concentrations of 0.11%of 0.11% (A), 0.055%(A), 0.055% (B), and(B), 0.0275%and 0.0275% (C) and (C) theand negative the negative control control (D). All(D). images All images (×40) (×40) are representative are representative of the of population the popula- oftion treated of treated amoebae amoebae and are and based are on based the live on cellthe imaginglive cell microscopeimaging microscope EVOS FL cellEVOS imaging FL cell system. imaging (E) system. Data are (E presented) Data are aspresented means ± SDas means (N = 3). ± ***SDp (N< 0.001;= 3). ** **** p

3.3. Discussion Discussion SeveralSeveral free-living free-living amoebae amoebae including includingNaegleria Naegleria fowleri fowleriand and some some species species from from the the AcanthamoebaAcanthamoebagenus genus represent represent a a potential potential threat threat to to human human health health due due to to their their intrinsic intrinsic pathogenicitypathogenicity and and their their role role as as protective protective and and disseminating disseminating hosts hosts for for other other pathogenic pathogenic microorganisms [21]. Due to their higher resistance to diverse water treatments (physical and chemical), FLA may act as a Trojan horse transporting pathogenic bacteria such as Legionella spp., Mycobacterium avium and Listeria monocytogenes, among others [22]. Despite the fact that FLA have recently gained considerable attention due to their potential to develop illnesses, there is still a lack of information in methods to eliminate them [21]. Even though the disinfection treatment recommendations for recreational water are detailed by the WHO, CDC, and Commission for the Environment, Climate Change and Energy, among others, none of them include a specific protocol for free living amoeba eradication. The chemical disinfectants that are mostly used to eliminate pathogens are chlorine, chlorine dioxide and bromochlorodimethylhydantoin (BCDMH). In the WHO guideline, it is announced that the residual free chlorine level should not exceed 3 mg/L in public and semi-public pools and should not exceed 5 mg/l in public and semi-public hot tubs [2]. Chlorine dioxide, a potent oxidant and antimicrobial, is a disinfectant widely used in paper bleaching, surface disinfection, biofilm removal in water distribution systems, and, increasingly, in water treatment to produce potable water [18]. Several authors have reported the higher efficacy of chlorine dioxide than chlorine to eliminate free living amoeba cells [19]. Mogoa et al. (2010) reported that a concentration of free chlorine lower than 3 mg/L was not able to inhibit Acanthamoeba castellanii Neff, whereas in this study, a concentration of 3 mg/L was able to inhibit the growth of 60% of the cell culture [23]. On the other hand, the CDC recommends a concentration of 1–3 mg/L [18] of free chlorine to avoid the growth of Naegleria fowleri in swimming pools. Moreover, according to the Spanish recreational swimming pool regulation (RD 742/2013 27th of September), the recommended parametric value of free chlorine remains between 0.5 and 2 mg/L [24], while the CLORICAN product’s indications establish a concentration of 0.4 mg/L for biocidal activity. Parasitologia 2021, 1 127

The amoebicidal capacity of CLORICAN on the trophozoite stage of Acanthamoeba spp. and Naegleria fowleri was studied over 48 h using an inverted light-microscope. In Acanthamoeba castellanii (ATCC 30234), Mogoa et al. (2010) reported that chlorine treatment led to the loss of pseudopodia and shrinking in the cell size and membrane permeabilization [23]. The same authors proved in a second paper that chlorine dioxide was as effective as chlorine in eliminating Acanthamoeba castellanii Neff by reducing the cell size more more and inducing membrane permeabilization, as shown by propidium iodide staining [21]. In our study, CLORICAN was shown to have a stronger effect on Naegleria fowleri than Acanthamoeba spp. Several authors have mentioned the higher sensitivity of Naegleria fowleri to chlorine and chlorine dioxide compared to non-pathogenic Naegleria, namely N. gruberi and Acanthamoeba spp. [25]. The chlorine dioxide did affect Naegleria fowleri cells by altering the amoeboid shape and inducing cell shrinking.

4. Materials and Methods 4.1. Chemical CLORICAN®, a commercial liquid form of chlorine dioxide, is used to disinfect swimming pools. The reactive is produced by Vadeguas company, Canary Islands, Spain. The tested solution has a pH of 4.5–6 and a chlorine dioxide concentration of 0.75% (w/w).

4.2. Cell Culture The strains used in this study were: Acanthamoeba castellanii Neff (ATCC 30010), Acanthamoeba polyphaga, genotype T4 (ATCC 30461) and Acanthamoeba grifﬁni, genotype T3 obtained in previous studies [17]. Those strains were axenically grown in PYG medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose) containing 40 µg/mL of gentamicin (Biochrom AG, Cultek, Granollers, Barcelona, Spain). The Naegleria strain used was Naegleria fowleri (ATCC® 30808™). This strain was kept in the biological security facilities level 3 of our institution following Spanish biosafety guidelines for this pathogen. It was axenically cultured at 37 ◦C in 2% (w/v) bactocasitone medium (Thermo Fisher Scientiﬁc, Madrid, Spain) supplemented with 10% (v/v) fetal bovine serum (FBS), containing 0.5 mg/mL of streptomycin sulfate (Sigma-Aldrich, Madrid, Spain) and 0.3 µg/mL of penicillin G sodium salt (Sigma-Aldrich, Madrid, Spain).

4.3. In Vitro Activity Against Trophozoite Stage The amoebicidal activity experiments were carried out using an inverted light-microscope Leica DM IL (Wetzlar, Germany). Initially, the trophozoites were counted using a Countess II FL automatic cell counter (Thermo Fisher Scientific, Madrid, Spain) to prepare a working concentration of 5.104 trophozoites/well and 105 cells/well, respectively, for Acanthamoeba spp. and Naegleria fowleri strains), and 50 µL per well was seeded in triplicate in a 96-well plate (Thermo Fisher Scientific, Madrid, Spain). After that, a serial dilution of CLORICAN in the same culture medium was added to the plate (50 µL per well). As a negative control, the trophozoites were incubated with the medium alone. Plates were incubated with slight agitation for 96 h at 26 ◦C for Acanthamoeba spp. and 48 h at 37 ◦C for Naegleria. Subsequently, the plates were observed during the incubation time with an inverted light-microscope (15 min, 1 h, 24 h, 48 h and 96 h). The numbers of viable and nonviable trophozoite were counted using a Countess II FL automatic cell counter (Thermo Fisher Scientific, Madrid, Spain) at 96 h for Acanthamoeba spp. and 48 h for Naegleria fowleri using an inverted microscope and confirmed with trypan blue (0.4%) staining using the Countess II FL (Thermo Fisher Scientific, Madrid, Spain)

4.4. Statistical Analysis All data are expressed as the mean ± standard deviation of at least three independent experiments. To highlight the chlorine dioxide’s effect on Acanthamoeba spp., a statistical comparison was conducted using the t-test and one-way analysis of variance (ANOVA). Parasitologia 2021, 1 128

All the analysis and the graphics were done by GraphPad Prism version 8.0. Statistical signiﬁcance was set at p < 0.05.

5. Conclusions The CLORICAN reactive was highly effective at eliminating both amoeba strains tested in vitro. Moreover, Naegleria fowleri’s trophozoites were more sensitive than those of Acanthamoeba spp. The chlorine dioxide greatly affected the morphology shape and increased the cell size shrinkage. Further experiments are needed to better understand the mode of action of CLORICAN and to evaluate its in situ efﬁcacy in a scaled-up process, e.g., in a swimming pool.

Author Contributions: Conceptualization, I.S., M.R.-B., J.E.P., J.L.-M.; methodology, I.S., M.R.-B., J.E.P., J.L.-M.; software, I.S., A.R.-L., I.A.-J., R.L.R.-E.; validation, I.S., M.R.-B., J.E.P., and J.L.-M.; formal analysis, I.S., A.R.-L., I.A.-J., R.L.R.-E.; investigation, I.S., A.R.-L., I.A.-J., R.L.R.-E., M.R.-B.; resources, I.S., M.R.-B.; data curation, A.R.-L., I.A.-J., R.L.R.-E.; writing—original draft preparation, I.S., A.R.-L., I.A.-J., R.L.R.-E.; writing—review and editing, All the authors.; visualization, I.S., M.R.-B., J.E.P., J.L.-M.; supervision, J.E.P., J.L.-M.; project administration, J.E.P. and J.L.-M.; funding acquisition, J.E.P. and J.L.-M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the grants PI18/01380, Fondo Europeo de Desarollo Regional (FEDER) and RICET (project no. RD16/0027/0001 of the program of Redes Temáticas de Investigación Cooperativa, FIS), Spanish Ministry of Science, Innovation and Universities, Madrid, Spain. IS and MRB was also funded by RICET. ARL (TESIS2020010054), IAJ (TESIS2020010063) and RLRE (TESIS2020010117) were funded by the Agencia Canaria De Investigación Innovación Y Sociedad De La Información Gobierno De Canarias. (ACIISI), y Fondo Social Europeo (FSE). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We would like to thank Octavio Santana from VADEAGUAS for providing the CLORICAN and for their support. Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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