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

A special issue of Pathogens (ISSN 2076-0817).

Deadline for manuscript submissions: 31 July 2020.

Special Issue Editors

Dr. Jacob Lorenzo-Morales
Website
Guest Editor
Departamento de Obstetricia, Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Universidad de La Laguna (ULL), Avda. Astrofísico F. Sánchez, 2, 38206 La Laguna, Tenerife, Spain
Interests: free living amoebae; therapeutics; emerging parasitic protozoa; diagnosis; natural compounds; synthetic compounds
Special Issues and Collections in MDPI journals
Dr. Lissette Maria Retana Moreira

Guest Editor
Department of Parasitology, Faculty of Microbiology, University of Costa Rica, Pedro de Montes de Oca, San José 4652, Costa Rica
Dr. Elizabeth Abrahams-Sandí

Guest Editor
Department of Parasitology, Faculty of Microbiology, University of Costa Rica, San Pedro, San José, Costa Rica

Special Issue Information

Dear Colleagues,

The International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM2019), which occurs every two years, covers all aspects of amphizoic free-living amoebae with pathogenic potential in humans and animals. Scientists who are concerned with all aspects of the biology, pathogenicity, and related research fields of free-living amoebae gather in this meeting to share recent advances in the free-living amoebae field. In the 18th Edition of FLAM which was held in Puntarenas, Costa Rica in November 2019, novelties about the biology, diagnosis, and therapeutics were discussed and presented to the attendants. Moreover, it was the first time a FLAM meeting was associated with the presence of a foundation dedicated to amoebae infections awareness—the Jordan Smelski foundation.

Free-living amoebae belonging to Acanthamoeba, Balamuthia mandrillaris, and Naegleria fowleri species are causative agents of lethal encephalitis (and keratitis in the case of Acanthamoeba). The current lack of awareness about these infections as well as established diagnostic tools and fully effective therapies are key issues in this field, and thus this meeting has been gathering importance in recent years due to the increased number of cases of FLA infections worldwide.

This Special Issue is focused in the compilation of presented data in this last FLAM meeting, and authors wishing to publish their work presented at the congress or related work are welcome to submit.

Dr. Jacob Lorenzo-Morales
Dr. Lissette Maria Retana Moreira
Dr. Elizabeth Abrahams-Sandí
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Pathogens is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1400 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • free living amoebae
  • Naegleria
  • Balamuthia
  • Vermamoeba
  • Acanthamoeba
  • water
  • encephalitis
  • PAM
  • GAE
  • keratitis
  • therapy
  • pathogens
  • emerging

Published Papers (7 papers)

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Research

Open AccessArticle
Cartography of Free-Living Amoebae in Soil in Guadeloupe (French West Indies) Using DNA Metabarcoding
Pathogens 2020, 9(6), 440; https://doi.org/10.3390/pathogens9060440 - 04 Jun 2020
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
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Open AccessCommunication
An Optimized Most Probable Number (MPN) Method to Assess the Number of Thermophilic Free-Living Amoebae (FLA) in Water Samples
Pathogens 2020, 9(5), 409; https://doi.org/10.3390/pathogens9050409 - 24 May 2020
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
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Open AccessArticle
Silver Nanoparticles as a Novel Potential Preventive Agent against Acanthamoeba Keratitis
Pathogens 2020, 9(5), 350; https://doi.org/10.3390/pathogens9050350 - 05 May 2020
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
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Open AccessArticle
Encystment Induces Down-Regulation of an Acetyltransferase-Like Gene in Acanthamoeba castellanii
Pathogens 2020, 9(5), 321; https://doi.org/10.3390/pathogens9050321 - 26 Apr 2020
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
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Open AccessArticle
Combined Amoebicidal Effect of Atorvastatin and Commercial Eye Drops against Acanthamoeba castellanii Neff: In Vitro Assay Based on Mixture Design
Pathogens 2020, 9(3), 219; https://doi.org/10.3390/pathogens9030219 - 17 Mar 2020
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
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Open AccessArticle
Description of Virulent Factors and Horizontal Gene Transfers of Keratitis-Associated Amoeba Acanthamoeba Triangularis by Genome Analysis
Pathogens 2020, 9(3), 217; https://doi.org/10.3390/pathogens9030217 - 16 Mar 2020
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
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Open AccessArticle
Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles
Pathogens 2020, 9(2), 144; https://doi.org/10.3390/pathogens9020144 - 21 Feb 2020
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
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