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14 pages, 2611 KiB

Open AccessArticle

Isolation and Identification of Naegleria Species in Irrigation Channels for Recreational Use in Mexicali Valley, Mexico

by Patricia Bonilla-Lemus, Saúl Rojas-Hernández, Elizabeth Ramírez-Flores, Diego A. Castillo-Ramírez, Alejandro Cruz Monsalvo-Reyes, Miguel A. Ramírez-Flores, Karla Barrón-Graciano, María Reyes-Batlle, Jacob Lorenzo-Morales and María Maricela Carrasco-Yépez

Pathogens 2020, 9(10), 820; https://doi.org/10.3390/pathogens9100820 - 07 Oct 2020

Cited by 9 | Viewed by 3240

Abstract

Members of the genus Naegleria are free-living amoebae that are widely distributed in water and soil environments. Moreover, Naegleria fowleri is a pathogenic amoeba species that causes a fatal disease in the central nervous system known as primary amoebic meningoencephalitis (PAM) in humans. [...] Read more.

Members of the genus Naegleria are free-living amoebae that are widely distributed in water and soil environments. Moreover, Naegleria fowleri is a pathogenic amoeba species that causes a fatal disease in the central nervous system known as primary amoebic meningoencephalitis (PAM) in humans. Since most reported infections due to N. fowleri are reported in recreational waters worldwide, this study was aimed to describe the presence of these amoebic genus in Mexicali Valley irrigation channels of recreational use. A total of nine water samples were collected and processed by triplicate, in nine different sites of the Valley. After filtering and culturing the samples, plates were examined, and the observed amoebae were morphologically identified at the genus level. In addition, the pathogenicity of these amoebic isolates was checked, and molecular characterization was performed by PCR/sequencing. The results revealed the presence of Naegleria spp. in all the channels sampled. Finally, molecular identification confirmed the presence of five different species of Naegleria: N. fowleri, N. australiensis, N. gruberi, N. clarki and N. pagei. The presence of these protists, particularly N. fowleri, should be considered as a potential human health risk in the region. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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15 pages, 17333 KiB

Open AccessArticle

Morphological Description of the Early Events during the Invasion of Acanthamoeba castellanii Trophozoites in a Murine Model of Skin Irradiated under UV-B Light

by Mariana Hernández-Jasso, Dolores Hernández-Martínez, José Guillermo Avila-Acevedo, José del Carmen Benítez-Flores, Isis Amara Gallegos-Hernández, Ana María García-Bores, Adriana Montserrat Espinosa-González, Tomás Ernesto Villamar-Duque, Ismael Castelan-Ramírez, María del Rosario González-Valle and Maritza Omaña-Molina

Pathogens 2020, 9(10), 794; https://doi.org/10.3390/pathogens9100794 - 27 Sep 2020

Cited by 3 | Viewed by 3048

Abstract

Skin infections have been associated with Acanthamoeba, nevertheless the events during skin invasion and UV-B light effects on it are unknown. The early morphological events of Acanthamoeba castellanii skin invasion are shown in SKH-1 mice that were chronically UV-B light irradiated. Mice [...] Read more.

Skin infections have been associated with Acanthamoeba, nevertheless the events during skin invasion and UV-B light effects on it are unknown. The early morphological events of Acanthamoeba castellanii skin invasion are shown in SKH-1 mice that were chronically UV-B light irradiated. Mice that developed skin lesions (group 1) were topical and intradermally inoculated with A. castellanii trophozoites and sacrificed 48 h or 18 days later. Mice that showed no skin lesions (group 2) were intradermally inoculated and sacrificed 24, 48 or 72 h later. Mice ventral areas were considered controls with and without trophozoites intradermally inoculated. Skin samples were processed by histological and immunohistochemistry techniques. In group 1, trophozoites were immunolocalized in dermal areas, hair cysts, sebaceous glands, and blood vessels, and collagen degradation was observed. One of these mice shown trophozoites in the spleen, liver, and brain. In group 2, few trophozoites nearby collagenolytic activity zones were observed. In control samples, nor histological damage and no trophozoites were observed. Adherence and collagenolytic activity by A. castellanii were corroborated in vitro. We can infer that UV-B light irradiated skin could favor A. castellanii invasiveness causing damage in sites as far away as the brain, confirming the invasive capacity and pathogenic potential of these amphizoic amoebae. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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8 pages, 2396 KiB

Open AccessArticle

In Vitro Evaluation of Farnesyltransferase Inhibitor and its Effect in Combination with 3-Hydroxy-3-Methyl-Glutaryl-CoA Reductase Inhibitor against Naegleria fowleri

by Hye Jee Hahn and Anjan Debnath

Pathogens 2020, 9(9), 689; https://doi.org/10.3390/pathogens9090689 - 22 Aug 2020

Cited by 4 | Viewed by 2457

Abstract

Free-living amoeba Naegleria fowleri causes a rapidly fatal infection primary amebic meningoencephalitis (PAM) in children. The drug of choice in treating PAM is amphotericin B, but very few patients treated with amphotericin B have survived PAM. Therefore, development of efficient drugs is a [...] Read more.

Free-living amoeba Naegleria fowleri causes a rapidly fatal infection primary amebic meningoencephalitis (PAM) in children. The drug of choice in treating PAM is amphotericin B, but very few patients treated with amphotericin B have survived PAM. Therefore, development of efficient drugs is a critical unmet need. We identified that the FDA-approved pitavastatin, an inhibitor of HMG Co-A reductase involved in the mevalonate pathway, was equipotent to amphotericin B against N. fowleri trophozoites. The genome of N. fowleri contains a gene encoding protein farnesyltransferase (FT), the last common enzyme for products derived from the mevalonate pathway. Here, we show that a clinically advanced FT inhibitor lonafarnib is active against different strains of N. fowleri with EC₅₀ ranging from 1.5 to 9.2 µM. A combination of lonafarnib and pitavastatin at different ratios led to 95% growth inhibition of trophozoites and the combination achieved a dose reduction of about 2- to 28-fold for lonafarnib and 5- to 30-fold for pitavastatin. No trophozoite with normal morphology was found when trophozoites were treated for 48 h with a combination of 1.7 µM each of lonafarnib and pitavastatin. Combination of lonafarnib and pitavastatin may contribute to the development of a new drug regimen for the treatment of PAM. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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9 pages, 777 KiB

Open AccessArticle

In Vitro Effect of Pitavastatin and Its Synergistic Activity with Isavuconazole against Acanthamoeba castellanii

by Hye Jee Hahn, Jose Ignacio Escrig, Brian Shing and Anjan Debnath

Pathogens 2020, 9(9), 681; https://doi.org/10.3390/pathogens9090681 - 21 Aug 2020

Cited by 5 | Viewed by 2192

Abstract

Acanthamoeba keratitis (AK) can occur in healthy individuals wearing contact lenses and it is a painful, blinding infection of the cornea caused by a free-living ameba Acanthamoeba. Current treatment for AK relies on a combination of chlorhexidine, propamidine isethionate, and polyhexamethylene biguanide. [...] Read more.

Acanthamoeba keratitis (AK) can occur in healthy individuals wearing contact lenses and it is a painful, blinding infection of the cornea caused by a free-living ameba Acanthamoeba. Current treatment for AK relies on a combination of chlorhexidine, propamidine isethionate, and polyhexamethylene biguanide. However, the current regimen includes an aggressive disinfectant and in 10% of cases recurrent infection ensues. Therefore, development of efficient and safe drugs is a critical unmet need to avert blindness. Acanthamoeba sterol biosynthesis includes two essential enzymes HMG-CoA reductase (HMGR) and sterol 14-demethylase (CYP51), and we earlier identified a CYP51 inhibitor isavuconazole that demonstrated nanomolar potency against A. castellanii trophozoites. In this study, we investigated the effect of well-tolerated HMGR inhibitors and identified pitavastatin that is active against trophozoites of three different clinical strains of A.castellanii. Pitavastatin demonstrated an EC₅₀ of 0.5 to 1.9 µM, depending on strains. Combination of pitavastatin and isavuconazole is synergistic and led to 2- to 9-fold dose reduction for pitavastatin and 11- to 4000-fold dose reduction for isavuconazole to achieve 97% of growth inhibition. Pitavastatin, either alone or in combination with isavuconazole, may lead to repurposing for the treatment of Acanthamoeba keratitis. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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9 pages, 771 KiB

Open AccessArticle

Primary Amebic Meningoencephalitis Related to Groundwater in Costa Rica: Diagnostic Confirmation of Three Cases and Environmental Investigation

by Lissette Retana Moreira, Leidy Zamora Rojas, Muriel Grijalba Murillo, Silvia Elena Molina Castro and Elizabeth Abrahams Sandí

Pathogens 2020, 9(8), 629; https://doi.org/10.3390/pathogens9080629 - 01 Aug 2020

Cited by 6 | Viewed by 4337

Abstract

During the first trimester of 2020, the Ministry of Health of Costa Rica reported the first three cases of primary amebic meningoencephalitis (PAM). In two cases, laboratory personnel of the hospitals preliminarily identified amoeboid forms in cerebrospinal fluid (CSF) samples. For the molecular [...] Read more.

During the first trimester of 2020, the Ministry of Health of Costa Rica reported the first three cases of primary amebic meningoencephalitis (PAM). In two cases, laboratory personnel of the hospitals preliminarily identified amoeboid forms in cerebrospinal fluid (CSF) samples. For the molecular confirmation of species, CSF samples were sent to our laboratory. We carried out microscopic analyses and exflagellation assays. Besides, samples were cultured in 2% casein hydrolysate medium and in non-nutrient agar plates supplemented with Escherichia coli. Finally, PCR and sequencing were employed for the molecular diagnosis and species identification. In all cases, the presence of Naegleria fowleri was confirmed. An environmental investigation to identify the possible infection sources was also performed. Water samples from hot springs and groundwater from an artisan well were collected and after filtration and culture in non-nutrient agar plates supplemented with E. coli, thermotolerance and exflagellation assays were carried out. For the positive samples, PCR and sequencing were performed, confirming the presence of N. fowleri in several water samples. The report of these cases and the possible association with hot springs has had a significant impact on the population and health authorities of Costa Rica. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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13 pages, 1936 KiB

Open AccessArticle

Stenamoeba dejonckheerei sp. nov., a Free-Living Amoeba Isolated from a Thermal Spring

by Manuel Alejandro Borquez-Román, Luis Fernando Lares-Jiménez, Libia Zulema Rodriguez-Anaya, Jose Reyes Gonzalez-Galaviz, Paul A. Fuerst, José Cuauhtémoc Ibarra-Gámez, Ramón Casillas-Hernández and Fernando Lares-Villa

Pathogens 2020, 9(7), 586; https://doi.org/10.3390/pathogens9070586 - 17 Jul 2020

Cited by 6 | Viewed by 2505

Abstract

Two amoeboid organisms were obtained from water samples taken from a thermal spring, "Agua Caliente", in Northwestern Mexico. The isolates were obtained when samples were cultivated at 37 °C on non-nutrient agar coated with Escherichia coli. The initial identification of the isolates was [...] Read more.

Two amoeboid organisms were obtained from water samples taken from a thermal spring, "Agua Caliente", in Northwestern Mexico. The isolates were obtained when samples were cultivated at 37 °C on non-nutrient agar coated with Escherichia coli. The initial identification of the isolates was performed morphologically using light microscopy. The samples were found to have trophozoite morphology consistent with members of the genus Stenamoeba, a genus derived in 2007 from within the abolished polyphyletic genus Platyamoeba. Further analysis was performed by sequencing PCR products obtained using universal eukaryotic primers for the small subunit ribosomal ribonucleic acid (SSU rRNA) gene. Sequencing primers were designed to allow the comparison of the 18S rRNA gene sequences of the new isolates with previous sequences reported for Stenamoeba. Phylogenetic relationships among sequences from Stenamoeba were determined using Maximum Likelihood analysis. The results showed the two "Agua Caliente" sequences to be closely related, while clearly separating them from those of other Stenamoeba taxa. The degrees of sequence differentiation from other taxa were considered sufficient to allow us to propose that the Mexican isolates represent a new species. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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16 pages, 1056 KiB

Open AccessArticle

Discovery of Anti-Amoebic Inhibitors from Screening the MMV Pandemic Response Box on Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba castellanii

by Christopher A. Rice, Emma V. Troth, A. Cassiopeia Russell and Dennis E. Kyle

Pathogens 2020, 9(6), 476; https://doi.org/10.3390/pathogens9060476 - 16 Jun 2020

Cited by 31 | Viewed by 4414

Abstract

Pathogenic free-living amoebae, Balamuthia mandrillaris, Naegleria fowleri, and several Acanthamoeba species are the etiological agents of severe brain diseases, with case mortality rates > 90%. A number of constraints including misdiagnosis and partially effective treatments lead to these high fatality rates. [...] Read more.

Pathogenic free-living amoebae, Balamuthia mandrillaris, Naegleria fowleri, and several Acanthamoeba species are the etiological agents of severe brain diseases, with case mortality rates > 90%. A number of constraints including misdiagnosis and partially effective treatments lead to these high fatality rates. The unmet medical need is for rapidly acting, highly potent new drugs to reduce these alarming mortality rates. Herein, we report the discovery of new drugs as potential anti-amoebic agents. We used the CellTiter-Glo 2.0 high-throughput screening methods to screen the Medicines for Malaria Ventures (MMV) Pandemic Response Box in a search for new active chemical scaffolds. Initially, we screened the library as a single-point assay at 10 and 1 µM. From these data, we reconfirmed hits by conducting quantitative dose–response assays and identified 12 hits against B. mandrillaris, 29 against N. fowleri, and 14 against A. castellanii ranging from nanomolar to low micromolar potency. We further describe 11 novel molecules with activity against B. mandrillaris, 22 against N. fowleri, and 9 against A. castellanii. These structures serve as a starting point for medicinal chemistry studies and demonstrate the utility of phenotypic screening for drug discovery to treat diseases caused by free-living amoebae. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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16 pages, 1733 KiB

Open AccessArticle

Identification of Immunogenic Antigens of Naegleria fowleri Adjuvanted by Cholera Toxin

by Saúl Rojas-Hernández, Mara Gutiérrez-Sánchez, Diego Alexander Rojas-Ortega, Patricia Bonilla-Lemus, Arturo Contis-Montes de Oca, Jorge Herrera-Díaz, Israel López-Reyes and María Maricela Carrasco-Yépez

Pathogens 2020, 9(6), 460; https://doi.org/10.3390/pathogens9060460 - 10 Jun 2020

Cited by 5 | Viewed by 2815

Abstract

The intranasal administration of Naegleria fowleri lysates plus cholera toxin (CT) increases protection against N. fowleri meningoencephalitis in mice, suggesting that humoral immune response mediated by antibodies is crucial to induce protection against the infection. In the present study, we applied a protein [...] Read more.

The intranasal administration of Naegleria fowleri lysates plus cholera toxin (CT) increases protection against N. fowleri meningoencephalitis in mice, suggesting that humoral immune response mediated by antibodies is crucial to induce protection against the infection. In the present study, we applied a protein analysis to detect and identify immunogenic antigens from N. fowleri, which might be responsible for such protection. A Western blot assay of N. fowleri polypeptides was performed using the serum and nasal washes from mice immunized with N. fowleri lysates, either alone or with CT after one, two, three, or four weekly immunizations and challenged with trophozoites of N. fowleri. Immunized mice with N. fowleri plus CT, after four doses, had the highest survival rate (100%). Nasal or sera IgA and IgG antibody response was progressively stronger as the number of immunizations was increased, and that response was mainly directed to 250, 100, 70, 50, 37, and 19 kDa polypeptide bands, especially in the third and fourth immunization. Peptides present in these immunogenic bands were matched by nano-LC–ESI-MSMS with different proteins, which could serve as candidates for a vaccine against N. fowleri infection. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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14 pages, 6666 KiB

Open AccessArticle

Schwann Cell Autophagy and Necrosis as Mechanisms of Cell Death by Acanthamoeba

by Ismael Castelan-Ramírez, Lizbeth Salazar-Villatoro, Bibiana Chávez-Munguía, Citlaltepetl Salinas-Lara, Carlos Sánchez-Garibay, Catalina Flores-Maldonado, Dolores Hernández-Martínez, Verónica Anaya-Martínez, María Rosa Ávila-Costa, Adolfo René Méndez-Cruz and Maritza Omaña-Molina

Pathogens 2020, 9(6), 458; https://doi.org/10.3390/pathogens9060458 - 09 Jun 2020

Cited by 8 | Viewed by 2503

Abstract

Amoebae of the genus Acanthamoeba are etiological agents of granulomatous amoebic encephalitis (GAE). Recently, through an in vivo GAE model, Acanthamoeba trophozoites were immunolocalized in contact with the peripheral nervous system (PNS) cells—Schwann cells (SC). In this study, we analyzed in greater detail [...] Read more.

Amoebae of the genus Acanthamoeba are etiological agents of granulomatous amoebic encephalitis (GAE). Recently, through an in vivo GAE model, Acanthamoeba trophozoites were immunolocalized in contact with the peripheral nervous system (PNS) cells—Schwann cells (SC). In this study, we analyzed in greater detail the in vitro early morphological events (1, 2, 3, and 4 h) during the interaction of A. culbertsoni trophozoites (ATCC 30171) with SC from Rattus norvegicus (ATCC CRL-2941). Samples were processed for scanning and transmission electron microscopy as well as confocal microscopy. After 1 h of interaction, amoebae were observed to be adhered to the SC cultures, emitting sucker-like structures associated with micro-phagocytic channels. In addition, evidence of necrosis was identified since edematous organelles as well as multivesicular and multilamellar bodies characteristics of autophagy were detected. At 2 h, trophozoites migrated beneath the SC culture in which necrosis and autophagy persisted. By 3 and 4 h, extensive lytic zones were observed. SC necrosis was confirmed by confocal microscopy. We reported for the first time the induction of autophagic and necrotic processes in PNS cells, associated in part with the contact-dependent pathogenic mechanisms of A. culbertsoni trophozoites. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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13 pages, 1792 KiB

Open AccessArticle

Cartography of Free-Living Amoebae in Soil in Guadeloupe (French West Indies) Using DNA Metabarcoding

by Yann Reynaud, Célia Ducat, Antoine Talarmin and Isabel Marcelino

Pathogens 2020, 9(6), 440; https://doi.org/10.3390/pathogens9060440 - 04 Jun 2020

Cited by 5 | Viewed by 3446

Abstract

Free-living amoebae (FLA) are ubiquitous protists. Pathogenic FLA such as N. fowleri can be found in hot springs in Guadeloupe, soil being the origin of this contamination. Herein, we analyzed the diversity and distribution of FLA in soil using a targeted metataxonomic analysis. [...] Read more.

Free-living amoebae (FLA) are ubiquitous protists. Pathogenic FLA such as N. fowleri can be found in hot springs in Guadeloupe, soil being the origin of this contamination. Herein, we analyzed the diversity and distribution of FLA in soil using a targeted metataxonomic analysis. Soil samples (n = 107) were collected from 40 sites. DNA was extracted directly from soil samples or from FLA cultivated at different temperatures (30, 37 and 44 °C). Metabarcoding studies were then conducted through FLA 18SrDNA amplicons sequencing; amplicon sequence variants (ASV) were extracted from each sample and taxonomy assigned against SILVA database using QIIME2 and SHAMAN pipelines. Vermamoeba were detected in DNA extracted directly from the soil, but to detect other FLA an amoebal enrichment step was necessary. V. vermiformis was by far the most represented species of FLA, being detected throughout the islands. Although Naegleria were mainly found in Basse-Terre region, N. fowleri was also detected in Grand Terre and Les Saintes Islands. Acanthamoeba were mainly found in areas where temperature is approx. 30 °C. Vannella and Vahlkampfia were randomly found in Guadeloupe islands. FLA detected in Guadeloupe include both pathogenic genera and genera that can putatively harbor microbial pathogens, therefore posing a potential threat to human health. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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8 pages, 447 KiB

Open AccessCommunication

An Optimized Most Probable Number (MPN) Method to Assess the Number of Thermophilic Free-Living Amoebae (FLA) in Water Samples

by Mirna Moussa, Isabel Marcelino, Vincent Richard, Jérôme Guerlotté and Antoine Talarmin

Pathogens 2020, 9(5), 409; https://doi.org/10.3390/pathogens9050409 - 24 May 2020

Cited by 3 | Viewed by 4697

Abstract

Detection and quantification of pathogenic free-living amoebae (FLA) in water samples is critical for assessing water quality and for disease management issues. The most probable number (MPN) is commonly used to account for FLA in water. Nevertheless, this requires a high number of [...] Read more.

Detection and quantification of pathogenic free-living amoebae (FLA) in water samples is critical for assessing water quality and for disease management issues. The most probable number (MPN) is commonly used to account for FLA in water. Nevertheless, this requires a high number of water replicates and working volumes, and a consequent number of non-nutrient agar (NNA)-plates seeded with Escherichia coli. Herein, we aimed at optimizing this difficult method, taking also into account key factors such as (i) the counting method, (ii) the delay between sample collection and sample processing, and (iii) the temperature during water sample transportation. To simplify the MPN method, we filtrated 1 × 1000 and 1 × 100 mL water samples, and cellulose acetate filters were cut in 10 parts and inverted on NNA-plates overlaid with E. coli. The comparison between the classical and our optimized MPN method showed that the final counts were similar, therefore validating the use of the optimized method. Our results also showed that for thermophilic FLA (such as Naegleria fowleri), water samples can be kept at around +30°C and processed within 24 h. This improved MPN method is now routinely used in our laboratory to control Naegleria sp. in the water samples in Guadeloupe. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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15 pages, 5349 KiB

Open AccessEditor’s ChoiceArticle

Silver Nanoparticles as a Novel Potential Preventive Agent against Acanthamoeba Keratitis

by Edyta B. Hendiger, Marcin Padzik, Ines Sifaoui, María Reyes-Batlle, Atteneri López-Arencibia, Aitor Rizo-Liendo, Carlos J. Bethencourt-Estrella, Desirée San Nicolás-Hernández, Olfa Chiboub, Rubén L. Rodríguez-Expósito, Marta Grodzik, Anna Pietruczuk-Padzik, Karolina Stępień, Gabriela Olędzka, Lidia Chomicz, José E. Piñero and Jacob Lorenzo-Morales

Pathogens 2020, 9(5), 350; https://doi.org/10.3390/pathogens9050350 - 05 May 2020

Cited by 23 | Viewed by 3576

Abstract

Free living, cosmopolitan amoebae from Acanthamoeba genus present a serious risk to human health. As facultative human parasites, these amoebae may cause Acanthamoeba keratitis (AK). Acanthamoeba keratitis is a severe, vision-threatening corneal infection with non-specific symptoms. The number of reported AK cases worldwide [...] Read more.

Free living, cosmopolitan amoebae from Acanthamoeba genus present a serious risk to human health. As facultative human parasites, these amoebae may cause Acanthamoeba keratitis (AK). Acanthamoeba keratitis is a severe, vision-threatening corneal infection with non-specific symptoms. The number of reported AK cases worldwide has been increasing every year. Moreover, 90% of Acanthamoeba keratitis cases are related to contact lens use. Wearing and storage contact lenses not in accordance with the physicians and manufacturers recommendations are the primary key risk factors of this disease. Amoebae can easily adhere to the contact lens surface and transmit to the corneal epithelium. Preventing amoebae adhesion to the contact lens surface could significantly decrease the number of AK infections. Until now, the effective therapy against AK is still under development. Currently proposed therapies are mainly limited to the chlorhexidine digluconate combined with propamidine isethionate or hexamidine applications, which are insufficient and very toxic to the eye. Due to lack of effective treatment, looking for new potential preventive agents is crucial to decrease the number of Acanthamoeba keratitis infections, especially among contact lens users. Nanoparticles have been already included in several novel therapies against bacteria, viruses, fungi, and protist. However, their anti-amoebic potential has not been fully tested yet. The aim of this study was to assess silver nanoparticles (AgNPs) and platinum nanoparticles (PtNPs) anti-amoebic activity and influence on the amoebae adhesion to the surface of four different groups of contact lenses—classified according to the Food and Drugs Administration (FDA) guidelines. The obtained results show that both tested nanoparticles were effective against Acanthamoeba trophozoites and decreased the amoebae adhesion to the contact lens surface. AgNPs showed better anti-amoebic activity to cytotoxicity dependence and reduced amoebae adhesion in a wider spectrum of the tested contact lenses. Our studies also confirmed that ionization next to hydration of the contact lens material is a crucial parameter influencing the Acanthamoeba adhesion to the contact lens surface. In conclusion, silver nanoparticles might be considered as a novel preventive agent against Acanthamoeba keratitis infection. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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11 pages, 1100 KiB

Open AccessArticle

Encystment Induces Down-Regulation of an Acetyltransferase-Like Gene in Acanthamoeba castellanii

by Steven Rolland, Luce Mengue, Cyril Noël, Stéphanie Crapart, Anne Mercier, Willy Aucher, Yann Héchard and Ascel Samba-Louaka

Pathogens 2020, 9(5), 321; https://doi.org/10.3390/pathogens9050321 - 26 Apr 2020

Cited by 4 | Viewed by 2406

Abstract

Acanthamoeba castellanii is a ubiquitous free-living amoeba. Pathogenic strains are causative agents of Acanthamoeba keratitis and granulomatous amoebic encephalitis. In response to adverse conditions, A. castellanii differentiate into cysts, which are metabolically inactive and resistant cells. This process, also named encystment, involves biochemical [...] Read more.

Acanthamoeba castellanii is a ubiquitous free-living amoeba. Pathogenic strains are causative agents of Acanthamoeba keratitis and granulomatous amoebic encephalitis. In response to adverse conditions, A. castellanii differentiate into cysts, which are metabolically inactive and resistant cells. This process, also named encystment, involves biochemical and genetic modifications that remain largely unknown. This study characterizes the role of the ACA1_384820 Acanthamoeba gene during encystment. This gene encodes a putative N-acetyltransferase, belonging to the Gcn5-related N-acetyltransferase (GNAT) family. We showed that expression of the ACA1_384820 gene was down-regulated as early as two hours after induction of encystment in A. castellanii. Interestingly, overexpression of the ACA1_384820 gene affects formation of cysts. Unexpectedly, the search of homologs of ACA1_384820 in the Eukaryota gene datasets failed, except for some species in the Acanthamoeba genus. Bioinformatics analysis suggested a possible lateral acquisition of this gene from prokaryotic cells. This study enabled us to describe a new Acanthamoeba gene that is down-regulated during encystment. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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11 pages, 1668 KiB

Open AccessArticle

Combined Amoebicidal Effect of Atorvastatin and Commercial Eye Drops against Acanthamoeba castellanii Neff: In Vitro Assay Based on Mixture Design

by Ines Sifaoui, Eulalia Capote Yanes, María Reyes-Batlle, Rubén L. Rodríguez-Expósito, José E. Piñero and Jacob Lorenzo-Morales

Pathogens 2020, 9(3), 219; https://doi.org/10.3390/pathogens9030219 - 17 Mar 2020

Cited by 6 | Viewed by 2374

Abstract

The establishment of an effective therapeutic agent against Acanthamoeba keratitis (AK), remains until present, an issue to be solved due to the existence of a cyst stage in the life cycle of Acanthamoeba. Moreover, the effectiveness of the current standard therapeutic agents [...] Read more.

The establishment of an effective therapeutic agent against Acanthamoeba keratitis (AK), remains until present, an issue to be solved due to the existence of a cyst stage in the life cycle of Acanthamoeba. Moreover, the effectiveness of the current standard therapeutic agents varies depending on the tested Acanthamoeba strains and its resistance pattern. In the present study, two 10-point augmented simplex-centroid designs were used to formulate a three-component mixture system using water, atorvastatin, and Diclofenaco-lepori or Optiben. The amoebicidal effects and in vitro-induced toxicity in a eukaryotic cell line were determined for all experiments. The optimal mixture to inhibit the parasite without inducing toxicity was established in the first plan as 30% Optiben, 63.5% atorvastatin, and 3.1% water. As for the second experimental design, the optimal mixture to inhibit Acanthamoeba with lower toxicity effect was composed of 17.6% Diclofenaco-lepori and 82.4% atorvastatin. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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17 pages, 3243 KiB

Open AccessArticle

Description of Virulent Factors and Horizontal Gene Transfers of Keratitis-Associated Amoeba Acanthamoeba Triangularis by Genome Analysis

by Issam Hasni, Julien Andréani, Philippe Colson and Bernard La Scola

Pathogens 2020, 9(3), 217; https://doi.org/10.3390/pathogens9030217 - 16 Mar 2020

Cited by 15 | Viewed by 3383

Abstract

Acanthamoeba triangularis strain SH 621 is a free-living amoeba belonging to Acanthamoeba ribo-genotype T4. This ubiquitous protist is among the free-living amoebas responsible for Acanthamoeba keratitis, a severe infection of human cornea. Genome sequencing and genomic comparison were carried out to explore the [...] Read more.

Acanthamoeba triangularis strain SH 621 is a free-living amoeba belonging to Acanthamoeba ribo-genotype T4. This ubiquitous protist is among the free-living amoebas responsible for Acanthamoeba keratitis, a severe infection of human cornea. Genome sequencing and genomic comparison were carried out to explore the biological functions and to better understand the virulence mechanism related to the pathogenicity of Acanthamoeba keratitis. The genome assembly harbored a length of 66.43 Mb encompassing 13,849 scaffolds. The analysis of predicted proteins reported the presence of 37,062 ORFs. A complete annotation revealed 33,168 and 16,605 genes that matched with NCBI non-redundant protein sequence (nr) and Cluster of Orthologous Group of proteins (COG) databases, respectively. The Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) annotation reported a great number of genes related to carbohydrate, amino acid and lipid metabolic pathways. The pangenome performed with 8 available amoeba genomes belonging to genus Acanthamoeba revealed a core genome containing 843 clusters of orthologous genes with a ratio core genome/pangenome of less than 0.02. We detected 48 genes related to virulent factors of Acanthamoeba keratitis. Best hit analyses in nr database identified 99 homologous genes shared with amoeba-resisting microorganisms. This study allows the deciphering the genome of a free-living amoeba with medical interest and provides genomic data to better understand virulence-related Acanthamoeba keratitis. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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16 pages, 3026 KiB

Open AccessEditor’s ChoiceArticle

Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles

by Lissette Retana Moreira, Daniel Vargas Ramírez, Fátima Linares, Alexa Prescilla Ledezma, Annette Vaglio Garro, Antonio Osuna, Jacob Lorenzo Morales and Elizabeth Abrahams Sandí

Pathogens 2020, 9(2), 144; https://doi.org/10.3390/pathogens9020144 - 21 Feb 2020

Cited by 10 | Viewed by 4263

Abstract

Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases [...] Read more.

Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases in humans. The wide distribution of Acanthamoeba, the characteristic of being amphizoic and the severity of the disease motivate researchers to focus on the isolation of these organisms, but also in demonstrating direct and indirect factors that could indicate a possible pathogenic potential. Here, we performed the characterization of the pathogenic potential of an Acanthamoeba T5 isolate collected from a water source in a hospital. Osmo- and thermotolerance, the secretion of proteases and the effect of trophozoites over cell monolayers were analyzed by different methodologies. Additionally, we confirm the secretion of extracellular vesicles (EVs) of this isolate incubated at two different temperatures, and the presence of serine and cysteine proteases in these vesicles. Finally, using atomic force microscopy, we determined some nanomechanical properties of the secreted vesicles and found a higher value of adhesion in the EVs obtained at 37 °C, which could have implications in the parasite´s survival and damaging potential in two different biological environments. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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34 pages, 2525 KiB

Open AccessEditor’s ChoiceReview

Species, Sequence Types and Alleles: Dissecting Genetic Variation in Acanthamoeba

by Paul A. Fuerst and Gregory C. Booton

Pathogens 2020, 9(7), 534; https://doi.org/10.3390/pathogens9070534 - 02 Jul 2020

Cited by 25 | Viewed by 2980

Abstract

Species designations within Acanthamoeba are problematic because of pleomorphic morphology. Molecular approaches, including DNA sequencing, hinted at a resolution that has yet to be fully achieved. Alternative approaches were required. In 1996, the Byers/Fuerst lab introduced the concept of sequence types. Differences between [...] Read more.

Species designations within Acanthamoeba are problematic because of pleomorphic morphology. Molecular approaches, including DNA sequencing, hinted at a resolution that has yet to be fully achieved. Alternative approaches were required. In 1996, the Byers/Fuerst lab introduced the concept of sequence types. Differences between isolates of Acanthamoeba could be quantitatively assessed by comparing sequences of the nuclear 18S rRNA gene, ultimately producing 22 sequence types, designated T1 through T22. The concept of sequence types helps our understanding of Acanthamoeba evolution. Nevertheless, substantial variation in the 18S rRNA gene differentiates many isolates within each sequence type. Because the majority of isolates with sequences in the international DNA databases have been studied for only a small segment of the gene, designated ASA.S1, genetic variation within this hypervariable region of the 18S rRNA gene has been scrutinized. In 2002, we first categorized variation in this region in a sample of T3 and T4 isolates from Hong Kong, observing ten “alleles” within type T4 and five “alleles” within T3. Subsequently, confusion occurred when different labs applied redundant numerical labels to identify different alleles. A more unified approach was required. We have tabulated alleles occurring in the sequences submitted to the international DNA databases, and determined their frequencies. Over 150 alleles have occurred more than once within 3500+ isolates of sequence type T4. Results from smaller samples of other sequence types (T3, T5, T11 and T15, and supergroup T2/6) have also been obtained. Our results provide new insights into the evolutionary history of Acanthamoeba, further illuminating the degree of genetic separation between significant taxonomic units within the genus, perhaps eventually elucidating what constitutes a species of Acanthamoeba. Full article

(This article belongs to the Special Issue Selected Papers from the International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019))

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