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Thecate amoebae play important roles in terrestrial and aquatic ecosystems. This study introduces a novel thecofilosean amoeba from Arctic and Antarctic sea sediments. Phylogenetic analysis based on the 18S rDNA sequence places it in the family Chlamydophryidae (order Tectofilosida, class Thecofilosea). However, the novel organism exhibits a significant genetic divergence and distinct morphology from its closest relatives, prompting us to erect the novel genus Katarium with its type species Katarium polorum. K. polorum is a consumer of diatoms and prokaryotes, indicating an important role in nutrient cycling in the polar marine food webs. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are isolated from the hospital environments and known as Trojan horses for medical essential microorganisms. This study aimed to investigate the prevalence and the presence of FLA and two critical agents of nosocomial infections, in the hospital wards. Sixty samples were collected from four communities and cultured onto non-nutrient agar (NNA). After total DNA extraction, FLA were characterized using PCR and sequencing. The presence of Candida albicans and Staphylococcus aureus was evaluated using real-time and conventional PCR, respectively. Acanthamoeba sp. was characterized in 30 (50%) samples. Two (6.6%) and one (3.3%) samples were positive for Vahlkampfiidae and Vermamoeba vermiformis, respectively. S. aureus was detected in 13 (43.3%) of samples, while none of them were positive for methicillin-resistant gene. C. albicans DNA was detected in one (3.3%) FLA-positive sample. The isolation of FLA from hospital suggests an essential role these eukaryotes in the inter-ward circulation of nosocomial infections. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are prevalent in diverse environments, representing various genera and species with different pathogenicity. FLA-induced infections, such as the highly fatal amoebic encephalitis, with a mortality rate of 99%, primarily affect immunocompromised individuals while others such as Acanthamoeba keratitis (AK) and cutaneous amebiasis may affect immunocompetent individuals. Despite the prevalence of FLA, there is a lack of standardized guidelines for their detection near human habitats. To date, no studies on the isolation and identification of FLA in environmental soil samples in Warsaw have been published. The aim of this study was to determine the presence of amoebae in soil samples collected from Warsaw parks and squares frequented by humans. The isolated protozoa were genotyped. Additionally, their pathogenic potential was determined through thermophilicity tests. A total of 23 soil samples were seeded on non-nutrient agar plates (NNA) at 26 °C and monitored daily for FLA presence. From the total of 23 samples, 18 were positive for FLA growth in NNA and PCR (78.2%). Acanthamoeba spp. was the most frequently isolated genus, with a total of 13 positive samples (13/18; 72.2%), and the T4 genotype being the most common. Moreover, Platyamoeba placida (3/18; 16.7%), Stenamoeba berchidia (1/18; 5.6%) and Allovahlkampfia sp. (1/18; 5.6%), also potentially pathogenic amoebae, were isolated. To our knowledge, this is the first report of FLA presence and characterization in the Warsaw area. [ABSTRACT FROM AUTHOR]

Acanthamoebae spp. are considered the most commonly occurring free-living amoebae (FLA) in the environment. Their high resilience enables them to thrive in different types of environments. Using purposive sampling, 80 surface water samples were collected from identified coastal sites in Mariveles, Bataan, and Lingayen Gulf (40 water samples for each). Nineteen (23.75%) of the 80 water samples yielded positive amoebic growth during the 14-day culture and microscopic examination. The polymerase chain reaction confirmed Acanthamoeba spp. DNA in isolates MB1, A3, A4, A7, C5, and D3 using JDP1 and JDP2 primer sets. Further sequencing revealed that the isolates belonged to Acantha- moeba sp., Acanthamoeba culbertsoni, Acanthamoeba castellani, and Acanthamoeba genotype T4. The sequences were deposited in GenBank and registered under accession numbers PP741651, PP767364, PP741728, PP741729, PP767365, and PP767366, respectively. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs. [ABSTRACT FROM AUTHOR]

Natural hot springs are ideal places and environmental matrices that offer relaxation to people and microorganisms of different types. A total of 40 surface water samples were collected from the five identified collection sites, eight water samples for each site. Collection sites are designated 200 m apart to cover the entire study site. Surface water samples were collected approximately 10–20 cm from the surface. Water samples were filtered, cultured, and microscopically observed for 14 days. After 14 days of cultivation, eight (20%) water samples revealed cystic and trophozoite stages. Polymerase chain reaction using JDP1 and JDP2 specific primers confirmed the presence of Acanthamoeba spp. from two of our isolates in the hot spring, isolates 1.1 and 5.1. Further sequencing revealed that the isolates are Acanthamoeba T20 and Acanthamoeba genotype T7. Sequences were deposited to GenBank and were assigned accession numbers PP741726 and PP741727, respectively. The isolation of Acanthamoeba spp. in hot springs has significant health implications, especially for those who use it for recreational activity. Private resort owners are highly encouraged to regularly monitor and maintain hot spring resorts to avoid future infections. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are common in both natural and engineered freshwater ecosystems. They play important roles in biofilm control and contaminant removal through the predation of bacteria and other taxa. Bacterial predation by FLA is also thought to contribute to pathogen dispersal and infectious disease transmission in freshwater environments via the egestion of viable bacteria. Despite their importance in shaping freshwater microbial communities, the diversity and function of FLA in many freshwater ecosystems are poorly understood. In this study, we isolated and characterized FLA from two groundwater sites in Canterbury, New Zealand using microbiological, microscopic, and molecular techniques. Different methods for groundwater FLA isolation and enrichment were trialed and optimized. The ability of these isolated FLA to predate on human pathogen Legionella pneumophila was assessed. FLA were identified by 18S metagenomic amplicon sequencing. Our study showed that Acanthamoeba spp. (including A. polyphaga) and Vermamoeba veriformis were the main FLA species present in both groundwater sites examined. While most of the isolated FLA co-existed with L. pneumophila, the FLA populations in the L. pneumophila co-culture experiments predominantly consisted of A. polyphaga, Acanthamoeba spp., Naegleria spp., V. vermiformis, Paravahlkampfia spp., and Echinamoeba spp. These observations suggest that FLA may have the potential to act as reservoirs for L. pneumophila in Canterbury, New Zealand groundwater systems and could be introduced into the local drinking water infrastructure, where they may promote the survival, multiplication, and dissemination of Legionella. This research addresses an important gap in our understanding of FLA-mediated pathogen dispersal in freshwater ecosystems. [ABSTRACT FROM AUTHOR]

Introduction: Often associated with contact lens wear, Acanthamoeba keratitis is an extremely painful blinding infection that has remained significant despite advances in drug discovery and chemotherapies. Current treatment is intricate and demands early diagnosis, aggressive and prolonged application, and may result in infection recurrence. The latter is due to the ability of the parasite to undergo cellular differentiation and transform into a resistant cyst form or infective trophozoite form. Areas covered: Here, we present a review of our current understanding of emerging therapies and priorities tackling Acanthamoeba keratitis. Literature searches were carried out using bibliographic databases and results sifted and reviewed to present recent findings, challenges and novel opportunities in the development of therapeutic interventions and/or preventative measures against Acanthamoeba keratitis. Expert opinion: Nanomedicine-based theranostics to combine diagnosis and treatment of Acanthamoeba keratitis offer remarkable opportunity in the development of clinical applications in the management of Acanthamoeba keratitis. [ABSTRACT FROM AUTHOR]

Recent advancements in membrane technologies and disinfection methods have enhanced drinking water quality significantly. However, microorganisms, including free-living amoebae (FLA), persist and pose potential threats to humans. FLA are linked to severe neuro-ophthalmic infections and serve as hosts of pathogenic bacteria. This study examined FLA presence in chlorinated and ultrafiltration drinking water and evaluated chlorine's disinfectant. Of 115 water samples, 21 tested positive for Acanthamoeba sp., Allovahlkampfia sp., and Vermamoeba vermiformis, originating from chlorinated sources. FLA trophozoites withstand temperatures up to 37 °C, while the cysts tolerate heat shocks of 60–70 °C. Trophozoites are susceptible to 5 mg L−1 chlorine, but cysts remain viable at concentrations up to 10 mg L−1. FLAs' survival in chlorinated waters is attributed to high cyst tolerance and lower residual chlorine concentrations. These findings highlight the need for ultrafiltration or enhanced chlorination protocols to ensure safer drinking water. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are prevalent in nature and man-made environments, and they can survive in harsh conditions by forming cysts. Studies have discovered that some FLA species are able to show pathogenicity to human health, leading to severe infections of central nervous systems, eyes, etc. with an extremely low rate of recovery. Therefore, it is imperative to establish a surveillance framework for FLA in environmental habitats. While many studies investigated the risks of independent FLA, interactions between FLA and surrounding microorganisms determined microbial communities in ecosystems and further largely influenced public health. Here we systematically discussed the interactions between FLA and different types of microorganisms and corresponding influences on behaviors and health risks of FLA in the environment. Specifically, bacteria, viruses, and eukaryotes can interact with FLA and cause either enhanced or inhibited effects on FLA infectivity, along with microorganism community changes. Therefore, considering the co-existence of FLA and other microorganisms in the environment is of great importance for reducing environmental health risks. [ABSTRACT FROM AUTHOR]

Acanthamoeba castellanii are opportunistic pathogens known to cause infection of the central nervous system termed: granulomatous amoebic encephalitis, that mostly effects immunocompromised individuals, and a sight threatening keratitis, known as Acanthamoeba keratitis, which mostly affects contact lens wearers. The current treatment available is problematic, and is toxic. Herein, an amphiphilic star polymer with AB2 miktoarms [A = hydrophobic poly(ℇ-Caprolacton) and B = hydrophilic poly (ethylene glycol)] was synthesized by ring opening polymerization and CuI catalyzed azide-alkyne cycloaddition. Characterization by 1H and 13C NMR spectroscopy, size-exclusion chromatography and fluorescence spectroscopy was accomplished. The hydrophobic drug itraconazole (ITZ) was incorporated in self-assembled micellar structure of AB2 miktoarms through co-solvent evaporation. The properties of ITZ loaded (ITZ-PCL-PEG2) and blank micelles (PCL-PEG2) were investigated through zeta sizer, scanning electron microscopy and Fourier-transform infrared spectroscopy. Itraconazole alone (ITZ), polymer (DPB-PCL), empty polymeric micelles (PCL-PEG2) alone, and itraconazole loaded in polymeric micelles (ITZ-PCL-PEG2) were tested for anti-amoebic potential against Acanthamoeba, and the cytotoxicity on human cells were determined. The polymer was able to self-assemble in aqueous conditions and exhibited low value for critical micelle concentration (CMC) 0.05–0.06 µg/mL. The maximum entrapment efficiency of ITZ was 68%. Of note, ITZ, DPB, PCL-PEG2 and ITZ-PCL-PEG2 inhibited amoebae trophozoites by 37.34%, 36.30%, 35.77%, and 68.24%, respectively, as compared to controls. Moreover, ITZ-PCL-PEG2 revealed limited cytotoxicity against human keratinocyte cells. These results are indicative that ITZ-PCL-PEG2 micelle show significantly better anti-amoebic effects as compared to ITZ alone and thus should be investigated further in vivo to determine its clinical potential. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are prevalent in diverse environments, representing various genera and species with different pathogenicity. FLA-induced infections, such as the highly fatal amoebic encephalitis, with a mortality rate of 99%, primarily affect immunocompromised individuals while others such as Acanthamoeba keratitis (AK) and cutaneous amebiasis may affect immunocompetent individuals. Despite the prevalence of FLA, there is a lack of standardized guidelines for their detection near human habitats. To date, no studies on the isolation and identification of FLA in environmental soil samples in Warsaw have been published. The aim of this study was to determine the presence of amoebae in soil samples collected from Warsaw parks and squares frequented by humans. The isolated protozoa were genotyped. Additionally, their pathogenic potential was determined through thermophilicity tests. A total of 23 soil samples were seeded on non-nutrient agar plates (NNA) at 26 °C and monitored daily for FLA presence. From the total of 23 samples, 18 were positive for FLA growth in NNA and PCR (78.2%). Acanthamoeba spp. was the most frequently isolated genus, with a total of 13 positive samples (13/18; 72.2%), and the T4 genotype being the most common. Moreover, Platyamoeba placida (3/18; 16.7%), Stenamoeba berchidia (1/18; 5.6%) and Allovahlkampfia sp. (1/18; 5.6%), also potentially pathogenic amoebae, were isolated. To our knowledge, this is the first report of FLA presence and characterization in the Warsaw area.

Sappinia is a free-living amoeba isolated from soil, dead plant material, freshwater, ponds, water supplies, surface water and the faecal material of various organisms such as cows, bats, reptiles, King penguin, and even humans. Of note, Sappinia was reported to be the causative agent in a case of non-granulomatous amoebic encephalitis, in an immunocompetent male. Since only one case has been described, there is not much information regarding the pathogenesis of Sappinia. Nonetheless, the rise in temperatures accompanying global warming is predicted to increase numbers of infections caused by free-living amoebae, and this may include Sappinia. Worryingly, Sappinia has also been reported to harbour fungal and bacterial endosymbionts. Since little is known about the pathogenesis of Sappinia and its species, prospective studies should focus on deducing its pathology. Additionally, Sappinia should be inspected for harbouring any bacteria or fungi of medical relevance to humans. Herein, we review the taxonomy, biology, ecological distribution, etiology, and pathogenesis of Sappinia. Prospective studies should be focused on Sappinia as well as other free-living amoebae, comprising their pathogenesis, and in developing preventative methods as well as treatments. This is especially important, given the rise in water shortages globally and reliance of storing water in tanks, where such microorganisms may propagate.

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Free-living amoebae (FLA) are protozoa dispersed in different environments and are responsible for different infections caused to humans and other animals. Microorganisms such as Acanthamoeba spp., Vermamoeba sp., and Naegleria sp. are associated with diseases that affect the central nervous system, in addition to skin infections and keratitis, as occurs in the genus Acanthamoeba and with Vermamoeba vermiformis. Due to the concerns of these FLA in anthropogenic aquatic environments, this work aimed to identify these microorganisms present in waters of Porto Alegre, Brazil. One litre sample was collected in two watercourses during the summer of 2022 and inoculated onto non-nutrient agar plates containing heat-inactivated Escherichia coli. Polymerase chain reaction results indicated the presence of FLA of the genera Acanthamoeba, Vermamoeba, and Naegleria in the study areas. Genetic sequencing indicated the presence of V. vermiformis and Naegleria gruberi. These aquatic and anthropogenic environments can serve as a means of spread and contamination by FLA, which gives valuable information on public health in the city. HIGHLIGHTS Identify microorganisms present in anthropogenic aquatic environments.; First report of free-living amoebae in southern Brazil.; Detection of Acanthamoeba spp., Naegleria gruberi, and Vermamoeba vermiformis in watercourses.; These microorganisms are responsible for causing fatal diseases that affect the central nervous system.; These environments can serve as a means of spread and contamination.;

Free-living amoebae (FLA) are common in both natural and engineered freshwater ecosystems. They play important roles in biofilm control and contaminant removal through the predation of bacteria and other taxa. Bacterial predation by FLA is also thought to contribute to pathogen dispersal and infectious disease transmission in freshwater environments via the egestion of viable bacteria. Despite their importance in shaping freshwater microbial communities, the diversity and function of FLA in many freshwater ecosystems are poorly understood. In this study, we isolated and characterized FLA from two groundwater sites in Canterbury, New Zealand using microbiological, microscopic, and molecular techniques. Different methods for groundwater FLA isolation and enrichment were trialed and optimized. The ability of these isolated FLA to predate on human pathogen Legionella pneumophila was assessed. FLA were identified by 18S metagenomic amplicon sequencing. Our study showed that Acanthamoeba spp. (including A. polyphaga) and Vermamoeba veriformis were the main FLA species present in both groundwater sites examined. While most of the isolated FLA co-existed with L. pneumophila, the FLA populations in the L. pneumophila co-culture experiments predominantly consisted of A. polyphaga, Acanthamoeba spp., Naegleria spp., V. vermiformis, Paravahlkampfia spp., and Echinamoeba spp. These observations suggest that FLA may have the potential to act as reservoirs for L. pneumophila in Canterbury, New Zealand groundwater systems and could be introduced into the local drinking water infrastructure, where they may promote the survival, multiplication, and dissemination of Legionella. This research addresses an important gap in our understanding of FLA-mediated pathogen dispersal in freshwater ecosystems.

This editorial published in the journal Frontiers in Microbiology explores recent advancements in the study of free-living amoebae, which are single-celled microorganisms found in water and soil. While most of these amoebae are harmless, some species can cause opportunistic infections. The editorial highlights new research on topics such as extracellular vesicles, zoonotic bacteria, drug development, and epidemiology. It also discusses the potential impact of artificial intelligence on amoebae research. One study mentioned in the article used machine learning algorithms to identify potential compounds for treating acanthamoeba infections, and researchers discovered a marine microbial metabolite that showed promise in this regard. The article provides detailed information on the study's methodology and findings. [Extracted from the article]

Acanthamoeba is a free-living amoeba (FLA) that is ubiquitous in nature and can cause serious pathologies in humans. This protozoan has been detected in several environmental sources, such as soil, water, and swimming pools. The aim of this study was to evaluate the occurrence and molecular diversity of Acanthamoeba spp. in aquatic environments of the state of Sergipe, northeastern Brazil, and to determine the pathogenic potential of the isolated samples. A total of 138 samples were collected from 69 aquatic environments and, after cell culture, 74% of the samples were positive for FLA, 47% belonging to the genus Acanthamoeba. Genotypic analysis was performed using the primers JDP1 and JDP2, confirming distinct Acanthamoeba genotypes: 18 (75%) isolates belonging to genotype T4, two (8%) to T3, and one isolate (4%) to genotype T5. Tests carried out to analyze the pathogenic potential showed that 11 isolates could grow at 0.5 M mannitol concentration and seven isolates supported hyperosmolarity. In the thermotolerance test, two isolates grew at 37°C. These results confirm the presence and the pathogenic potential of FLA of the genus Acanthamoeba in aquatic environments of the municipalities of Sergipe. [ABSTRACT FROM AUTHOR]

Amoebae of the genus Vannella isolated from an ornamental fish aquarium were found to be infected with fungi. Upon plate culture, amoeba-trapping hyphal filaments were developed, and the amoeba trophozoites were found to harbour yeast-like parasites in their cytoplasm. Transfection of hyphae to a laboratory strain of Vannella resulted in the formation of conidia indicating the possible presence of zygomycetes of the genus Acaulopage, while efforts to culture the endoparasite remained unsuccessful. Biomolecular analysis based on rDNA revealed the presence of two distinct types of fungi, confirming the filamentous form as Acaulopage sp. (Zoopagomycota, Zoopagales) and identifying the yeast-like endoparasite as Cladosporium sp. (Ascomycota, Cladosporiales). To our knowledge, this is the first report of amoebae infected with Cladosporium. [ABSTRACT FROM AUTHOR]

Due to frequent variations in environmental conditions, free-living amoebae adapt through differentiation into different states. Hence, favorable conditions enable the formation of a feeding and proliferative form named “Trophozoïte” whereas unfavorable situations drive differentiation into resting and resistant single forms such as cysts, spores, or multicellular structures. Transformation into cyst, named “encystment” or “encystation”, is a common feature found in testate, naked, or flagellated free-living amoebae. Although much effort has been made to understand encystment, several blind spots are still present. This short opinion paper highlights some difficulties impeding a better understanding of encystment.

In response to growing global interest in organic agriculture, this study delves into the microbial landscape of organically grown raw produce with a focus on food safety. Vegetables that are consumed raw are potential vehicles for the transmission of any type of microorganism capable of causing human disease. Free-living amoebae (FLA) are ubiquitous protozoa found in many ecosystems and can serve as hosts to pathogenic bacteria. So far, data regarding the FLA bacterial microbiome in fresh produce remain scarce and are non-existent for those of organic origin. Thus, the aim of this preliminary work is to characterize the microbiome of FLA in commonly consumed raw vegetables to know their possible implications for consumers. A total of 40 organic cabbage, lettuce, spinach, and strawberry samples were analyzed. FLA were found in all samples, and their bacterial microbiome was obtained via amplicon sequencing using the Illumina MiSeq platform and pair-end protocol. Acanthamoeba spp. and Vermamoeba vermiformis were identified via qPCR in 65.0% and 25.0% of the samples, respectively. Regarding the bacterial microbiome of FLA, the most abundant genera were Pseudomonas (1.8–17.8%) and Flavobacterium (1.7–12.6%). Bacteria not previously related to FLA, such as Prosthecobacter or Cellvibrio, are described in this work. Importantly, several bacterial genera found within the FLA microbiome were identified as potential human pathogens, including Pseudomonas, Flavobacterium, Arcobacter, Klebsiella, Mycobacterium, Salmonella and Legionella. This is the first work in which FLA microbiome isolated from organic products has been characterized, underscoring the significance of understanding FLA's role as carriers of pathogenic bacteria in the context of organic food safety. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are protozoa dispersed in different environments and are responsible for different infections caused to humans and other animals. Microorganisms such as Acanthamoeba spp., Vermamoeba sp., and Naegleria sp. are associated with diseases that affect the central nervous system, in addition to skin infections and keratitis, as occurs in the genus Acanthamoeba and with Vermamoeba vermiformis. Due to the concerns of these FLA in anthropogenic aquatic environments, this work aimed to identify these microorganisms present in waters of Porto Alegre, Brazil. One litre sample was collected in two watercourses during the summer of 2022 and inoculated onto non-nutrient agar plates containing heat-inactivated Escherichia coli. Polymerase chain reaction results indicated the presence of FLA of the genera Acanthamoeba, Vermamoeba, and Naegleria in the study areas. Genetic sequencing indicated the presence of V. vermiformis and Naegleria gruberi. These aquatic and anthropogenic environments can serve as a means of spread and contamination by FLA, which gives valuable information on public health in the city. [ABSTRACT FROM AUTHOR]

In this study, the presence of Acanthamoeba spp., Blastocystis sp., Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia sp., Toxoplasma gondii and Vermamoeba vermiformis was assessed in organic leafy green vegetables (lettuce, spinach, cabbage) and fruits (strawberry), which are usually consumed raw. A total of 110 organic samples were collected in Valencia (Spain). Protozoa were concentrated before detection by immunofluorescence (Cryptosporidium spp. and Giardia sp.) or real-time qPCR (Acanthamoeba spp., Blastocystis sp., C. cayetanensis, E. histolytica, T. gondii and V. vermiformis). The most abundant protozoa in organic vegetables and berry fruits were Acanthamoeba (65.5%), followed by T. gondii (37.2%), V. vermiformis (17.3%), C. cayetanensis (12.7%), Cryptosporidium spp. (6.8%), Blastocystis sp. (1.8%) and Giardia sp. (1.7%). E. histolytica was not found in any of the organic samples. Thus, results showed that consumers can be exposed to protozoan parasites by consuming organic vegetables and berry fruits. This is the first report in Spain describing the presence of the protozoan pathogens Acanthamoeba spp., Blastocystis sp., C. cayetanensis, T. gondii and V. vermiformis, Cryptosporidium spp. and Giardia sp. in organic fresh produce. The results of this research will help determine the risk of foodborne protozoan parasites on organic leafy greens and strawberries that are available at local markets.

Hospital water systems are a significant source of Legionella, resulting in the potentially fatal Legionnaires' disease. One of the biggest challenges for Legionella management within these systems is that under unfavorable conditions Legionella transforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, on Legionella spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and Vermamoeba vermiformis and Acanthamoeba specific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers sampled was measured using the Enware Smart Flow® monitoring system. qPCR analysis demonstrated that 21.8% samples were positive for Legionella spp., 21% for L. pneumophila, 40.9% for V. vermiformis and 4.2% for Acanthamoeba. All samples that were Legionella spp. positive using qPCR (22%) were also positive for VBNC Legionella spp.; however, only 2.5% of samples were positive for culturable Legionella spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for Legionella spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 10³ CFU/L) were also significantly associated with high concentrations of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila (p < 0.01) and V. vermiformis (p < 0.05). Temporary water stagnation arising through intermittent usage (< 2 hours of usage per month) significantly (p < 0.01) increased the amount of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila, and V. vermiformis; however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion, Legionella spp. (DNA and VBNC) was associated with V. vermiformis, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella spp. within a hospital water system. The high percentage of false negative Legionella spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella spp. contamination within hospital water systems. [ABSTRACT FROM AUTHOR]

Naegleria fowleri causes primary amoebic meningoencephalitis, a deadly infection that occurs when free‐living amoebae enter the nose via freshwater and travel to the brain. N. fowleri naturally thrives in freshwater and soil and is thought to be associated with elevated water temperatures. While environmental and laboratory studies have sought to identify what environmental factors influence its presence, many questions remain. This study investigated the interactive effects of temperature, pH, and salinity on N. fowleri in deionized and environmental waters. Three temperatures (15, 25, 35°C), pH values (6.5, 7.5, 8.5), and salinity concentrations (0.5%, 1.5%, 2.5% NaCl) were used to evaluate the growth of N. fowleri via ATP luminescent assays. Results indicated N. fowleri grew best at 25°C, and multiple interactive effects occurred between abiotic factors. Interactions varied slightly by water type but were largely driven by temperature and salinity. Lower temperature increased N. fowleri persistence at higher salinity levels, while low salinity (0.5% NaCl) supported N. fowleri growth at all temperatures. This research provided an experimental approach to assess interactive effects influencing the persistence of N. fowleri. As climate change impacts water temperatures and conditions, understanding the microbial ecology of N. fowleri will be needed minimize pathogen exposure. [ABSTRACT FROM AUTHOR]

Aim Herein, the anti-parasitic activity of azoles (fluconazole and itraconazole) and 5-nitroimdazole (metronidazole) against the brain-eating amoebae: Naegleria fowleri and Balamuthia mandrillaris was elucidated. Methods and results Azoles and 5-nitroimidazole based nanoformulations were synthesized and characterized using a UV-visible spectrophotometer, atomic force microscopy, and fourier transform infrared spectroscopy. H1-NMR, EI-MS, and ESI-MS were performed to determine their molecular mass and elucidate their structures. Their size, zeta potential, size distribution, and polydispersity index (PDI) were assessed. Amoebicidal assays revealed that all the drugs and their nanoformulations, (except itraconazole) presented significant anti-amoebic effects against B. mandrillaris , while all the treatments indicated notable amoebicidal properties against N. fowleri. Amoebicidal effects were radically enhanced upon conjugating the drugs with nanoparticles. The IC50 values for KM-38-AgNPs-F, KM-20-AgNPs-M, and KM-IF were 65.09, 91.27, and 72.19 µg.mL-1, respectively, against B. mandrillaris. Whereas against N. fowleri , the IC50 values were: 71.85, 73.95, and 63.01 µg.mL-1, respectively. Additionally, nanoformulations significantly reduced N. fowleri -mediated host cell death, while nanoformulations along with fluconazole and metronidazole considerably reduced Balamuthia -mediated human cell damage. Finally, all the tested drugs and their nanoformulations revealed limited cytotoxic activity against human cerebral microvascular endothelial cell (HBEC-5i) cells. Conclusion These compounds should be developed into novel chemotherapeutic options for use against these distressing infections due to free-living amoebae, as currently there are no effective treatments. [ABSTRACT FROM AUTHOR]

Hospital water systems are a significant source of Legionella, resulting in the potentially fatal Legionnaires’ disease. One of the biggest challenges for Legionella management within these systems is that under unfavorable conditions Legionella transforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, on Legionella spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and Vermamoeba vermiformis and Acanthamoeba specific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers sampled was measured using the Enware Smart Flow® monitoring system. qPCR analysis demonstrated that 21.8% samples were positive for Legionella spp., 21% for L. pneumophila, 40.9% for V. vermiformis and 4.2% for Acanthamoeba. All samples that were Legionella spp. positive using qPCR (22%) were also positive for VBNC Legionella spp.; however, only 2.5% of samples were positive for culturable Legionella spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for Legionella spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 103 CFU/L) were also significantly associated with high concentrations of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila (p < 0.01) and V. vermiformis (p < 0.05). Temporary water stagnation arising through intermittent usage (< 2 hours of usage per month) significantly (p < 0.01) increased the amount of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila, and V. vermiformis; however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion, Legionella spp. (DNA and VBNC) was associated with V. vermiformis, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella spp. within a hospital water system. The high percentage of false negative Legionella spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella spp. contamination within hospital water systems.

Due to frequent variations in environmental conditions, free-living amoebae adapt through differentiation into different states. Hence, favorable conditions enable the formation of a feeding and proliferative form named "Trophozoïte" whereas unfavorable situations drive differentiation into resting and resistant single forms such as cysts, spores, or multicellular structures. Transformation into cyst, named "encystment" or "encystation", is a common feature found in testate, naked, or flagellated free-living amoebae. Although much effort has been made to understand encystment, several blind spots are still present. This short opinion paper highlights some difficulties impeding a better understanding of encystment. [ABSTRACT FROM AUTHOR]

Pathogenic Acanthamoeba produce keratitis and fatal granulomatous amoebic encephalitis. Treatment remains problematic and often ineffective, suggesting the need for the discovery of novel compounds. For the first time, here we evaluated the effects of the anticancer drugs Irosustat and STX140 alone, as well as their nanoformulations, against A. castellanii via amoebicidal, excystment, cytopathogenicity, and cytotoxicity assays. Nanoformulations of the compounds were successfully synthesized with high encapsulation efficiency of 94% and 82% for Irosustat and STX140, respectively. Nanoparticles formed were spherical in shape and had a unimodal narrow particle size distribution, mean of 145 and 244 nm with a polydispersity index of 0.3, and surface charge of −14 and −15 mV, respectively. Irosustat and STX140 exhibited a biphasic release profile with almost 100% drug released after 48 h. Notably, Irosustat significantly inhibited A. castellanii viability and amoebae-mediated cytopathogenicity and inhibited the phenotypic transformation of amoebae cysts into the trophozoite form, however their nanoformulations depicted limited effects against amoebae but exhibited minimal cytotoxicity when tested against human cells using lactate dehydrogenase release assays. Accordingly, both compounds have potential for further studies, with the hope of discovering novel anti-Acanthamoeba compounds, and potentially developing targeted therapy against infections of the central nervous system. [ABSTRACT FROM AUTHOR]

Introduction: Free-living amoebae of the Naegleria genus belong to the major protist clade Heterolobosea and are ubiquitously distributed in soil and freshwater habitats. Of the 47 Naegleria species described, N. fowleri is the only one being pathogenic to humans, causing a rare but fulminant primary amoebic meningoencephalitis. Some Naegleria genome sequences are publicly available, but the genetic basis for Naegleria diversity and ability to thrive in diverse environments (including human brain) remains unclear. Methods: Herein, we constructed a high-quality Naegleria genus pangenome to obtain a comprehensive catalog of genes encoded by these amoebae. For this, we first sequenced, assembled, and annotated six new Naegleria genomes. Results and Discussion: Genome architecture analyses revealed that Naegleria may use genome plasticity features such as ploidy/aneuploidy to modulate their behavior in different environments. When comparing 14 near-to-complete genome sequences, our results estimated the theoretical Naegleria pangenome as a closed genome, with 13,943 genes, including 3,563 core and 10,380 accessory genes. The functional annotations revealed that a large fraction of Naegleria genes show significant sequence similarity with those already described in other kingdoms, namely Animalia and Plantae. Comparative analyses highlighted a remarkable genomic heterogeneity, even for closely related strains and demonstrate that Naegleria harbors extensive genome variability, reflected in different metabolic repertoires. If Naegleria core genome was enriched in conserved genes essential for metabolic, regulatory and survival processes, the accessory genome revealed the presence of genes involved in stress response, macromolecule modifications, cell signaling and immune response. Commonly reported N. fowleri virulence-associated genes were present in both core and accessory genomes, suggesting that N. fowleri’s ability to infect human brain could be related to its unique species-specific genes (mostly of unknown function) and/or to differential gene expression. The construction of Naegleria first pangenome allowed us to move away from a single reference genome (that does not necessarily represent each species as a whole) and to identify essential and dispensable genes in Naegleria evolution, diversity and biology, paving the way for further genomic and postgenomic studies. [ABSTRACT FROM AUTHOR]

Legionella pneumophila is an opportunistic pathogen responsible for Legionnaires' disease or Legionellosis. This bacterium is found in the environment interacting with free-living amoebae such as Acanthamoeba castellanii. Until now, proteomic analyses have been done in amoebae infected with L. pneumophila but focused on the Legionella -containing vacuole. In this study, we propose a global proteomic analysis of the A. castellanii proteome following infection with L. pneumophila wild-type (WT) or with an isogenic Δ dotA mutant strain, which is unable to replicate intracellularly. We found that infection with L. pneumophila WT leads to reduced levels of A. castellanii proteins associated with lipid homeostasis/metabolism, GTPase regulation, and kinase. The levels of organelle-associated proteins were also decreased during infection. Legionellapneumophila WT infection leads to increased levels of proteins associated with polyubiquitination, folding or degradation, and antioxidant activities. This study reinforces our knowledge of this too little explored but so fundamental interaction between L. pneumophila and A. castellanii , to understand how the bacterium could resist amoeba digestion. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. In addition to their natural distribution, some species have been documented as pathogenic to humans. The main aim of the current study was the molecular identification, sequencing and phylogenetic analysis of morphologically detected FLA in water sources in El-Qalyubia, Egypt. A total of 96 water samples were collected from different water sources. Each water sample was filtrated and cultured on non-nutrient agar (NNA). Morphologically positive FLA were subjected to PCR, PCR products were sequenced and the obtained sequences were phylogenetically analysed. FLA were found in 41 water samples examined (42.7%). Nile water and groundwater were the sources with the highest prevalence rates (83.3 and 62.5%, respectively). Naegleria italica was first identified in Egypt from the waters of the Nile. In addition, Vahlkampfia spp. and Hartmannella spp. were also detected. However, other FLA species, including Acanthamoeba spp. and the pathogenic Naegleria fowleri, previously reported in Egypt, were not included in this study. The recent identification of these FLA in the Egyptian waters related to human populations indicates the need for more phylogenetic studies using larger sample sizes to investigate their potential threat to human health. HIGHLIGHTS This study documented the presence of FLA in different water sources in El-Qalyubia, Egypt.; Naegleria italica was identified for the first time in Egypt from the waters of the Nile.; Besides, Vahlkampfia spp. and Hartmannella spp. were also detected.; This study presents knowledge of the prior prevalence of FLA that should be considered by the clinicians and the environmental professionals in the region.;

The reported persistence of SARS-CoV-2 virions in aquatic environments highlights the need to better understand potential mechanisms that may prolong its dissemination. We evaluated the possibility that amoebae might serve as transport hosts by studying the interaction of the enveloped bacteriophage Phi6, as a potential surrogated along with one of the most common amoebae in engineered aquatic environments, Vermamoeba vermiformis. Using microscopy, imaging flow cytometry and bacteriophage cell culture, our results imply that the SARS-CoV-2 surrogate triggers amoebic mitochondria and induced apoptosis to promote viral persistence in trophozoites. Furthermore, virus-infected amoebae were still infectious after 2 months within FLA cysts. These results suggest that amoebae could contribute to the environmental persistence of SARS-CoV-2, including disinfection processes. In addition, amoebae could be a successful model system for understanding respiratory virus-eukaryotic biology at the cellular and molecular levels. HIGHLIGHTS Amoebic mitochondria serve as a support organelle platform for the SARS-CoV-2 surrogate persistence.; Long-term stability of RNA enveloped viruses in amoeba-forming cysts.; Free-living amoebae as a potential vector and environmental reservoir for RNA enveloped viruses.;

The present study identifies the Acanthamoeba genotypes and their pathogenic potential in five marine waters in Malaysia. Fifty water samples were collected between January and May 2019. Physical parameters of water quality were measured in situ, whereas chemical and microbiological analyses were conducted in the laboratory. All samples had undergone filtration using nitrocellulose membrane and were tested for Acanthamoeba using cultivation and polymerase chain reaction by targeting the 18S ribosomal RNA gene. The pathogenic potential of all positive isolates was identified using physiological tolerance tests. Thirty-six (72.0%) samples were positive for Acanthamoeba. Total coliforms (p = 0.013) and pH level (p = 0.023) displayed significant correlation with Acanthamoeba presence. Phylogenetic analysis showed that 27 samples belonged to genotype T4, four (T11), two (T18) and one from each genotype T5, T15 and T20. Thermo- and osmo-tolerance tests signified that three (8.3%) Acanthamoeba strains displayed highly pathogenic attributes. This study is the first investigation in Malaysia describing Acanthamoeba detection in marine water with molecular techniques and genotyping. The study outcomes revealed that the marine water in Malaysia could be an integral source of Acanthamoeba strains potentially pathogenic in humans. Thus, the potential risk of this water should be monitored routinely in each region. HIGHLIGHTS First report on Acanthamoeba genotypic distribution in marine water, Malaysia.; Highly pathogenic Acanthamoeba strain T4 was attributed.; Total coliforms and pH level displayed a correlation with Acanthamoeba presence.;

IntroductionFree-living amoebae of the Naegleria genus belong to the major protist clade Heterolobosea and are ubiquitously distributed in soil and freshwater habitats. Of the 47 Naegleria species described, N. fowleri is the only one being pathogenic to humans, causing a rare but fulminant primary amoebic meningoencephalitis. Some Naegleria genome sequences are publicly available, but the genetic basis for Naegleria diversity and ability to thrive in diverse environments (including human brain) remains unclear.MethodsHerein, we constructed a high-quality Naegleria genus pangenome to obtain a comprehensive catalog of genes encoded by these amoebae. For this, we first sequenced, assembled, and annotated six new Naegleria genomes.Results and DiscussionGenome architecture analyses revealed that Naegleria may use genome plasticity features such as ploidy/aneuploidy to modulate their behavior in different environments. When comparing 14 near-to-complete genome sequences, our results estimated the theoretical Naegleria pangenome as a closed genome, with 13,943 genes, including 3,563 core and 10,380 accessory genes. The functional annotations revealed that a large fraction of Naegleria genes show significant sequence similarity with those already described in other kingdoms, namely Animalia and Plantae. Comparative analyses highlighted a remarkable genomic heterogeneity, even for closely related strains and demonstrate that Naegleria harbors extensive genome variability, reflected in different metabolic repertoires. If Naegleria core genome was enriched in conserved genes essential for metabolic, regulatory and survival processes, the accessory genome revealed the presence of genes involved in stress response, macromolecule modifications, cell signaling and immune response. Commonly reported N. fowleri virulence-associated genes were present in both core and accessory genomes, suggesting that N. fowleri’s ability to infect human brain could be related to its unique species-specific genes (mostly of unknown function) and/or to differential gene expression. The construction of Naegleria first pangenome allowed us to move away from a single reference genome (that does not necessarily represent each species as a whole) and to identify essential and dispensable genes in Naegleria evolution, diversity and biology, paving the way for further genomic and post-genomic studies.

The quality of many freshwater environments is impacted by human activities, so that many rivers may represent a vehicle for the transmission of health-related microorganisms. This work aimed to isolate and identify genetically free-living amoeba (FLA) of the genus Acanthamoeba from a recreational river in Salta, Argentina, and isolate, if possible, an endocytobiont. Sampling took place at four points (P1–P4) throughout the river in the winter and the summer seasons. Free-living amoebae and Acanthamoeba were recovered from 20-L water concentrated through an ultrafiltration system. Isolation was performed in agar plates, confirmation of Acanthamoeba genus by PCR, and fellow identification and classification based on their sequence analyses. High concentrations of indicator bacteria were found especially in P2, which is intensively used for recreation. Out of a total of 29 FLA isolations, 9 were identified as Acanthamoeba genotype T4 subtype A, the most frequent genotype found in nature and associated with causing human disease. From an axenic culture of Acanthamoeba spp. (KY751412), a bacterial endocytobiont was isolated, and identified as Stenotrophomonas maltophilia. The endocytobiont showed resistance and intermediate resistance to a wide range of widely used antibiotics. Results were in concordance with the cosmopolitan behavior of Acanthamoeba, and showed the importance of studying this group of amoebae and related microorganisms in recreational environments. [ABSTRACT FROM AUTHOR]

We report the first case of Balamuthia mandrillaris granulomatous amoebic encephalitis definitively acquired in Africa. Our case emphasizes initial nonspecific dermatological features, delays in confirmation of the diagnosis, difficulties accessing recommended medication, and uncertainty about optimal treatment of a disease with a frequently fatal outcome. [ABSTRACT FROM AUTHOR]

Acanthamoeba castellanii is a ubiquitous free-living amoeba capable of instigating keratitis and granulomatous amoebic encephalitis in humans. Treatment remains limited and inconsistent. Accordingly, there is a pressing need for novel compounds. Nanotechnology has been gaining attention for enhancing drug delivery and reducing toxicity. Previous work has shown that various antibiotic classes displayed antiamoebic activity. Herein, we employed two antibiotics: ampicillin and ceftriaxone, conjugated with the nanocarrier zinc oxide and β-cyclodextrin, and tested them against A. castellanii via amoebicidal, amoebistatic, encystment, excystment, cytopathogenicity, and cytotoxicity assays at a concentration of 100 μg/mL. Notably, zinc oxide β-cyclodextrin ceftriaxone significantly inhibited A. castellanii growth and cytopathogenicity. Additionally, both zinc oxide β-cyclodextrin ceftriaxone and ceftriaxone markedly inhibited A. castellanii encystment. Furthermore, all the tested compounds displayed negligible cytotoxicity. However, minimal anti-excystment or amoebicidal effects were observed for the compounds. Accordingly, this novel nanoconjugation should be employed in further studies in hope of discovering novel anti-Acanthamoeba compounds. [ABSTRACT FROM AUTHOR]

Substantial knowledge has accumulated on the microbiome of the hyperarid Atacama Desert during the last two decades; however, information on Atacama free-living amoebae (FLA) is limited and increasing efforts are required. FLA are polyphyletic heterotrophic naked or testate protists that feed on organic matter, fungi, protozoa, and bacteria and may disseminate infections. Amoebae in Chile are represented by 416 taxa and 64 genera, and 29 taxa have been identified in arid shrub lands at the southern limit of the Atacama Desert, and Acanthamoeba are present in all the country's regions. To expand our knowledge and to contribute to the biogeographic distribution of Atacama FLA, we report the dominant presence of members of the genus Acanthamoeba in water and sediment sampled at the Loa and Salado rivers in the pre-Andean zone of the Antofagasta Region, northern Chile, at sites 2500 m above sea level. We expect these observations and preliminary evidence of FLA presence in other wetlands (Chiuchiu, Tebenquiche) in this region to be incentive for further exploration of Atacama amoebae. [ABSTRACT FROM AUTHOR]

Background and Objectives Acanthamoeba can cause serious diseases in humans such as amoebic keratitis and granulomatous amebic encephalitis which are most common in immunocompromised individuals. The present study aims to investigate the genotype of Acanthamoeba strains isolated from the oral and nasal cavities of immunocompromised individuals. Subjects and Methods In this study, the samples from the oral and nasal cavities of 179 patients with immunodeficiency were first collected by swap and then transferred to the laboratory of the department of parasitology at Ahvaz Jundishapur University of Medical Sciences. They were cultured in basic agar medium and positive samples were used for molecular studies. Results Of 179 patients, the samples of 6 people were positive for Acanthamoeba. They were sequenced and belonged to the T4 genotype. There was a significant difference between positive samples obtained from oral (4%) and nasal (1.5%) cavities. Conclusion The prevalence of infection with Acanthamoeba parasite is higher in immunocompromised individuals. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are cosmopolitan microorganisms known to be pathogenic to humans who often have a history of contact with contaminated water. Swimming pools and recreational waters are among the environments where the greatest human exposure to FLA occurs. This study aimed to determine the prevalence of FLA in swimming pools and recreational waters, through a systematic review and meta-analysis that included studies published between 1977 and 2022. A total of 106 studies were included and an overall prevalence of FLA in swimming pools and recreational waters of 44.34% (95% CI = 38.57–50.18) was found. Considering the studies published up to 2010 (1977–2010), between 2010 and 2015, and those published after 2010 (> 2010–2022), the prevalence was 53.09% (95% CI = 43.33–62.73) and 37.07% (95% CI = 28.87–45.66) and 45.40% (95% CI = 35.48–55.51), respectively. The highest prevalence was found in the American continent (63.99%), in Mexico (98.35%), and in indoor hot swimming pools (52.27%). The prevalence varied with the variation of FLA detection methods, morphology (57.21%), PCR (25.78%), and simultaneously morphology and PCR (43.16%). The global prevalence by genera was Vahlkampfia spp. (54.20%), Acanthamoeba spp. (33.47%), Naegleria spp. (30.95%), Hartmannella spp./Vermamoeba spp. (20.73%), Stenamoeba spp. (12.05%), and Vannella spp. (10.75%). There is considerable risk of FLA infection in swimming pools and recreational waters. Recreational water safety needs to be routinely monitored and, in case of risk, locations need to be identified with warning signs and users need to be educated. Swimming pools and artificial recreational water should be properly disinfected. Photolysis of NaOCl or NaCl in water by UV-C radiation is a promising alternative to disinfect swimming pools and artificial recreational waters. [ABSTRACT FROM AUTHOR]

Naegleria fowleri and Balamuthia mandrillaris are opportunistic protists, responsible for fatal central nervous system infections such as primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE) with mortality rates higher than 90%. Threatening a rise in cases is the increase in temperature due to global warming. No effective treatment is currently available. Herein, nanotechnology was used to conjugate Zinc oxide with Ampicillin, Ceftrixon, Naringin, Amphotericin B, and Quericitin, and the amoebicidal activity and host cell cytotoxicity of these resulting compounds were investigated. The compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-Nar, ZnO-CD-AMB, and ZnO-CD-QT were found to reduce N. fowleri viability to 35.5%, 39.6%, 52.0%, 50.8%, 35.9%, and 69.9%, respectively, and B. mandrillaris viability to 40.9%, 48.2%, 51.6%, 43.8%, and 62.4%, respectively, when compared with their corresponding controls. Furthermore, the compounds reduced N. fowleri-mediated and B. mandrillaris-mediated host cell death significantly. Additionally, the compounds showed limited cytotoxicity against human cells; cell toxicity was 35.5%, 36.4%, 30.9%, 36.6%, and 35.6%, respectively, for the compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-Nar, ZnO-CD-AMB, and ZnO-CD-QT. Furthermore, the minimum inhibitory concentrations to inhibit amoeba growth by 50% were determined for N. fowleri and B. mandrillaris. The MIC50 for N. fowleri were determined to be 69.52 µg/mL, 82.05 µg/mL, 88.16 µg/mL, 95.61 µg/mL, and 85.69 µg/mL, respectively; the MIC50 of the compounds for B. mandrillaris were determined to be 113.9 µg/mL, 102.3 µg/mL, 106.9 µg/mL, 146.4 µg/mL, and 129.6 µg/mL, respectively. Translational research to further develop therapies based on these compounds is urgently warranted, given the lack of effective therapies currently available against these devastating infections. [ABSTRACT FROM AUTHOR]

In response to growing global interest in organic agriculture, this study delves into the microbial landscape of organically grown raw produce with a focus on food safety. Vegetables that are consumed raw are potential vehicles for the transmission of any type of microorganism capable of causing human disease. Free-living amoebae (FLA) are ubiquitous protozoa found in many ecosystems and can serve as hosts to pathogenic bacteria. So far, data regarding the FLA bacterial microbiome in fresh produce remain scarce and are non-existent for those of organic origin. Thus, the aim of this preliminary work is to characterize the microbiome of FLA in commonly consumed raw vegetables to know their possible implications for consumers. A total of 40 organic cabbage, lettuce, spinach, and strawberry samples were analyzed. FLA were found in all samples, and their bacterial microbiome was obtained via amplicon sequencing using the Illumina MiSeq platform and pair-end protocol. Acanthamoeba spp. and Vermamoeba vermiformis were identified via qPCR in 65.0% and 25.0% of the samples, respectively. Regarding the bacterial microbiome of FLA, the most abundant genera were Pseudomonas (1.8–17.8%) and Flavobacterium (1.7–12.6%). Bacteria not previously related to FLA, such as Prosthecobacter or Cellvibrio, are described in this work. Importantly, several bacterial genera found within the FLA microbiome were identified as potential human pathogens, including Pseudomonas, Flavobacterium, Arcobacter, Klebsiella, Mycobacterium, Salmonella and Legionella. This is the first work in which FLA microbiome isolated from organic products has been characterized, underscoring the significance of understanding FLA’s role as carriers of pathogenic bacteria in the context of organic food safety.

Vermamoeba vermiformis is a widespread free-living amoeba, playing a major role as a host of pathogens and probably acting as an opportunistic parasite. Identification is usually done by targeting the 18S rRNA gene which is highly conserved between different strains. In this study, the main sublines and subtypes of the amoeba were identified by molecular phylogeny of nearly complete sequences and by analysis of secondary structure. Complete analysis of the available sequences showed the presence of two heterologous introns in seven strains of Vermamoeba belonging to three distinct sublines. One strain has a conventional S788 intron, the others harbor an S943 intron with degenerated endonucleases. The two introns are phylogenetically related and show a common origin with an intron identified in Clandestinovirus, a giant virus infecting Vermamoeba. Current data suggests that these elements are part of a more diverse group that spreads horizontally in various hosts. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate an effective, sustainable and accessible way to inactivate chlorine-resistant microorganisms, such as Acanthamoeba castellanii, through the photolysis of sodium chloride (NaCl) and sodium hypochlorite (NaOCl) in the water matrix. The trophozoites and cysts (2 × 107 per 8 mL) were exposed for 30, 60, 90, 120 and 150 min to the photolysis effect of NaOCl (1.0, 2.0, 4.0 and 8.0 mg/L) or NaCl (5.0, 10, 20 and 40 g/L) by ultraviolet light C (243 μW·cm2), then the viability was analyzed. The inactivation of all trophozoites was achieved by exposure to the photolysis effect of 2.0 mg/L of NaOCl or 20 g/L of NaCl, in 150 or 120 min, respectively. Inactivation of all cysts was achieved by double exposure to the photolysis effect of 1.0 mg/L NaOCl or 5.0 g/L NaCl from 90 min of each exposure round. The exposure time was a strong determinant in the inactivation of A. castellanii trophozoites or cysts. The photolysis of NaOCl or NaCl is an effective method to eliminate A. castellanii in water. These findings expand the list of chlorine-resistant microorganisms that can be inactivated by NaOCl photolysis and show that NaCl photolysis is a new and promising method for treating swimming pool water and wastewater.