Search Results (72)

In this study, we investigated the presence and diversity of FLA in 62 environmental samples collected across Tenerife, Canary Islands, Spain including agricultural and playground soils, and on double treated water from public refrigerated fountains. Amoebae were isolated by culturing processed samples onto 2% Non-Nutrient Agar plates (NNA) which were checked daily for further processing up to molecular characterization. In this case, two approaches for molecular identification were assessed: direct multiplex qPCR targeting four potentially pathogenic FLA (Acanthamoeba spp., Vermamoeba vermiformis, Naegleria fowleri, and Balamuthia mandrillaris) DNA, and culture-based isolation followed by standard PCR and sequence analysis. Regarding qPCR results, 72.6% (45/62) of the samples were positive for at least one FLA, with V. vermiformis (37/62) and Acanthamoeba spp. (34/62) being the most frequent. Moreover, B. mandrillaris was detected for the first time in the Canary Islands in 6 out of 62 samples. Results from standard PCR from cultured isolates confirmed the presence of Acanthamoeba (mainly genotype T4) and Vermamoeba and also allowed the identification of Vahlkampfia and Vannella genera, as well as the genus Rhogostoma—its first report in the Canary Islands. Thermotolerance and osmotolerance assays were performed on Acanthamoeba spp. and, innovatively, on V. vermiformis isolates. Both were capable of surviving at 37 °C and during incubation with 0.5 M mannitol, suggesting potential pathogenicity. However, growth was significantly impaired under harsher conditions (42 °C and 1 M mannitol). These findings underscore the widespread occurrence of FLA in public and agricultural environments in Tenerife and highlight their potential risk to public health. Their ability to act as carriers of pathogenic bacteria/viruses further reinforces the need for routine surveillance and preventive measures in the environment. Full article

Free-living amoebae (FLA) are protozoa ubiquitous in nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study, FLA presence was evaluated and characterized at the molecular level from different water and soil samples in Fuerteventura Island, Canary Islands, Spain. A total of 31 samples were analyzed by culture and molecular assays (q-PCR and PCR). Moreover, the microbiological quality of the water samples was examined as required by current legislation and international standards. The obtained data revealed that the genus Acanthamoeba was the most prevalent genus of FLA in soil samples and the species Vermamoeba vermiformis was the most isolated in water samples collected from Fuerteventura by culture and molecular assays, q-PCR, and conventional PCR/Sanger sequencing. On the other hand, a microbiological analysis revealed heterogeneous contamination patterns. Escherichia coli was detected in several samples, with some exhibiting high counts while others showed no presence. Salmonella spp. appeared in multiple samples, particularly FTVW1, FTVW9, and FTVW13, whereas Shigella spp. was only found in one sample (FTVW1). Moreover, q-PCR detection offers advantages such as reduced detection time and cost. In addition, culture was proven to be more effective for confirming FLA viability and isolating a greater variety of FLA. Overall, the occurrence of potentially pathogenic free-living amoebae in habitats related to the human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans. Full article

Phytochemical investigation of Radula voluta, a liverwort species collected in the Ecuadorian Amazon, led to the isolation of four known bibenzyl derivatives: 2-prenyl-3,5-dihydroxy-bibenzyl (1), 2-geranyl-3,5-dihydroxybibenzyl (2), 2,2-dimethyl-5-phenethyl-2H-chromen-7-ol (3), and radulanin L (4). Structural elucidation was achieved through extensive NMR and MS analyses, supported by comparison with previously reported data. Compounds 1 and 4 are reported for the first time in R. voluta. The crude extract and isolated compounds were evaluated for their in vitro antiprotozoal activity against Trypanosoma cruzi, Leishmania amazonensis, Leishmania donovani, Naegleria fowleri, and Acanthamoeba castellanii Neff. Among the isolated compounds, bibenzyls 2 and 4 exhibited the most potent activity across multiple protozoan strains. Cytotoxicity was assessed against murine macrophages (J774A.1), obtaining moderate–low toxicities against compounds 1 and 3. These findings highlight the pharmacological value of liverwort-derived bibenzyls and support further research on R. voluta as a promising source of antiparasitic leads. Full article

The increasing global incidence of infections caused by free-living amoebae (FLA) and the lack of effective, safe, and approved treatments highlight the urgent need for novel amoebicidal compounds with pharmacological potential. Despite a growing body of literature on the anti-FLA properties of various compounds, comprehensive reviews summarizing this progress remain scarce. This study aimed to identify the most promising compounds tested in vitro and/or in vivo for anti-FLA activity. A systematic review was conducted, analyzing 108 studies published between 1986 and 2024, selected from an initial pool of 23,653 database results. A total of 537 compounds were evaluated for their in vitro anti-FLA activity. Compounds exhibiting ≥50% reduction in amoeba viability relative to untreated controls were classified as promising if they showed low toxicity in mammalian cell models, particularly when active at concentrations ≤ 10 µM, consistent with predicted favorable pharmacokinetic and pharmacodynamic profiles. The most promising compounds for drug and disinfectant development include ten trophocidal agents against B. mandrillaris, thirty-two trophocidal and four cysticidal agents against N. fowleri, and sixty-two trophocidal and nineteen cysticidal agents against Acanthamoeba spp. Compounds active at low concentrations (≤10 µM or <0.014 mg/mL) prioritized for in vivo drug development studies include: against Balamuthia mandrillaris, trophocidal 515, 531, 533; against Naegleria fowleri, trophocidal 421, 416, 518, 46, 254, 522, 111–120 and cysticidal 16; and against Acanthamoeba spp., trophocidal 498, 499, 500, 535, 107, 347, 348, and 340. Future studies should evaluate their efficacy, safety, pharmacokinetics, and pharmacodynamics toward developing effective drugs, antiseptics, and disinfectants. Full article

Nile tilapia (Oreochromis niloticus) is an indispensable source of high-quality protein worldwide. Along with the exponential expansion of tilapia aquaculture, several novel pathogenic viruses have emerged, and some cause significant economic losses. Unfortunately, there is scarce information on the biology and epidemiology of these viruses. This exploratory metagenomic study used Oxford Nanopore Technology (ONT) sequencing to profile the virome compositions of both wild and farmed Nile tilapia across five regions in Egypt. The Nile tilapia virome was dominated by two double-stranded DNA bacteriophages, Muvirus mu and M. sfmu, which constituted 79.8% of the detected sequences. Eukaryotic viruses, including members of the families Amnoonviridae, Peribunyaviridae, and Baculoviridae, were also identified. Two giant DNA viruses known to infect Acanthamoeba spp., Mollivirus sp., and Pandoravirus sp. were identified in the spleen virome of tilapia from a single sampling site. The diversity analysis showed no significant differences among tissue types or sampling sites. Phylogenetic analyses were performed on a single virus detected of potential pathogenicity, an amnoonvirus. The analyses demonstrated that the detected virus is a member of the family Amnoonviridae and placed it alongside members of the Tilapinevirus genus. The virus, however, was distinct from the other two members in the genus: T. tilapae and T. poikilos. This study underscores the usefulness of ONT in providing a foundational understanding of the Nile tilapia virome. Full article

The sustainable production of plant bioactive compounds is increasingly important as natural habitats decline. This study investigates whether in vitro cell cultures of Eryngium planum, Lychnis flos-cuculi, and Kickxia elatine can serve as alternative sources of biologically active biomass with antimicrobial and anti-Acanthamoeba properties. Callus cultures were established under optimized and controlled conditions, and metabolomic profiling was completed using UPLC-HRMS/MS. In silico analysis, using a molecular docking approach, was applied to understand the interaction between target compounds and Acanthamoeba profilin and identify possible targets for antimicrobial properties. Untargeted metabolomic analysis confirmed the presence of valuable compounds in the callus cultures of the studied species. Biological activity was assessed through anti-Acanthamoeba and antimicrobial assays. Lychnis flos-cuculi and Kickxia elatine callus extracts showed significant inhibitory effects on Acanthamoeba trophozoites, with 87.5% and 80.1% inhibition at 10 mg/mL, respectively. In contrast, E. planum extract stimulated amoebic growth. The anti-Acanthamoeba activity correlated with the presence of ferulic acid and p-coumaric acid in L. flos-cuculi extract, and acteoside in K. elatine extract. Antibacterial testing revealed moderate activity of E. planum and K. elatine extracts against Staphylococcus spp., while Gram-negative bacteria and fungi were largely resistant. These findings highlight the potential of in vitro cultures—particularly those from L. flos-cuculi and K. elatine—as promising, sustainable sources of anti-Acanthamoeba and antimicrobial agents, warranting further investigation into their pharmacologically active constituents. Full article

Free-living amoebae (FLA) such as Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, Sappinia pedata, Vermamoeba vermiformis and Vahlkampfia spp. are causal agents of deadly and/or disabling infections in humans. Despite recent data showing an increase in infection cases worldwide, studies on the prevalence of these emerging pathogens in water sources are scarce. Moreover, climate change is believed to facilitate the expansion and persistence of these environmental pathogens, further emphasizing the need for comprehensive surveillance. Therefore, the current study investigates the variety and abundance of free-living amoebae in different water sources in the autonomous community of Castilla y León, Spain, during different seasons of the year. Vermamoeba vermiformis was the most prevalent species and was detected in rivers, swamps, irrigation waters, swimming pools and recreational fountains. Moreover, genera such as Acanthamoeba and Naegleria and Vahlkampfia were also identified. This study highlights the diversity of FLA in the region and their relationship with local water characteristics. Given that certain FLA species are opportunistic pathogens, these results emphasize the necessity of monitoring this area and water sources. Full article

Free-living protozoa (FLP) can create biofilms in water supply systems and can harbor bacteria, which potentially can be pathogenic, such as Legionella spp. Each year there are more cases of legionellosis in Latvia, so this problem is actual: in 2019 there were 42 cases, but in 2024—88 cases. In this study, the investigated question of the coexistence of FLP and Legionella spp. and bacterial diversity in the drinking water supply systems of Riga, Salaspils, and Jurmala multiapartment buildings and hotels situated in Riga and Jurmala, identify the main FLP genus, and study factors associated with FLP and Legionella spp. occurrence. With microscopy, microbiological, and molecular biology methods, FLP and, specifically, free-living amoeba (FLA) were detected and identified, and Legionella spp. bacteria were isolated. Three FLP genera were identified, including Acanthamoeba, Vahlkampfia, and Hartmanella (Vermamoeba). In hot water, more FLP and Legionella co-existence occurrences were detected. In 64.7% of FLP-positive samples, Hartmanella (Vermamoeba) spp. was detected. Various potentially pathogenic bacteria, such as Coxiella, Leptospira, and Mycobacterium, were detected in the water sample DNA sequences. The average hot water temperature in Riga was lower than 50 °C, which is not enough to minimize the risk of the Legionella bacteria proliferation. The Shannon’s index values showed that bacterial diversity was higher in cold water samples, and the Pearson test showed that the correlation between building floor and Legionella quantity is positive. In this study, we also discovered that differences in bacterial diversity between water samples from two Daugava River banks’ water sources are not significant, but the biggest exception was a much higher percentage of Chaetonotida (hairybellies) in the left river bank samples. Noticeably, there are more Legionella and FLP-positive samples from the kitchen than from the apartment shower. Each hotel building from this study has its own similar bacterial diversity in its water supply system. Full article

Free-living amoebae (FLA) such as Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, Sappinia spp., Vahlkampfia spp., and Vermamoeba vermiformis are naturally widespread in the environment, causing rare but fatal and debilitating infections in humans. In the present study, a total of 87 soil samples were collected from four provinces in the autonomous community of Castilla y León, Spain. These samples were collected in three different seasons during 2022–2023 (t1–t3) and were analysed by culture and molecular techniques (conventional PCR/sanger sequencing and qPCR). The obtained data revealed that the genus Acanthamoeba and the species Vermamoeba vermiformis were the most prevalent FLA. Furthermore, other genera/species of FLA were identified in the tested soil sources, suggesting a rich microbial biodiversity in Castilla y León soils. In addition, this study provides an important basis for future research on the ecology of these organisms and their potential impact on public health and the environment. Full article

Until recently, bacterial species were the primary etiological factor of periodontal disease, but recent studies have shown that their effective removal from tooth surfaces does not necessarily prevent the progression of the disease. A logical hypothesis leads to the conclusion that various etiological factors contribute to the etiopathogenesis of this disease. Recent evidence also indicates protozoa as potential pathogens. In this review, the role of Entamoeba gingivalis, Acanthamoeba spp., and Trichomonas tenax in periodontal disease was analyzed, and the various aspects of the role of protozoa in the etiopathogenesis of periodontal disease, the bacterial–protozoa model of the disease, and the therapeutic implications were categorized. The multifactorial nature of periodontal disorders requires further research to better identify individuals at risk and prescribe earlier and more definitive treatments. The evidence supporting the role of protozoa in periodontal disease is compelling. These organisms are essential contributors to this complex disease. The key to developing more effective prevention and treatment strategies lies in understanding the intricate interactions between protozoa, bacteria, and the host. A better understanding of the protozoa aspect of periodontal disease progression could significantly change the future perspective of diagnosing, preventing, and treating periodontal disease. Full article

Microbial keratitis, a vision-threatening infection commonly linked to contact lens use, poses a significant challenge, particularly when caused by Acanthamoeba species. Acanthamoeba keratitis (AK) is difficult to treat due to the organism’s ability to form resilient cysts, necessitating prolonged and complex therapeutic interventions. This study evaluated novel amidopropyl dimethylamines (APDs) and amidopropyl quaternary trimethylammoniums (APTs) for their antimicrobial efficacy against Acanthamoeba castellanii and Acanthamoeba polyphaga cysts. Minimum effective concentrations were determined, and time–kill assays assessed microbial inactivation over 24 h. The results indicated that certain APTs, particularly elaidamidopropyl trimethylammonium (EAPT) and oleamidopropyl trimethylammonium (OAPT), demonstrated superior cysticidal activity compared to the commercially used MAPD, achieving greater log reductions within 24 h (p < 0.0001) at a concentration of 25 µM. The enhanced efficacy of these compounds is potentially attributed to their unsaturated alkyl chains and positive charge, improving antimicrobial activity through the greater disruption of the Acanthamoeba cell membrane. These findings highlight the potential of APTs as alternative agents for incorporation into multipurpose lens disinfectants and AK treatment, offering improved disinfection efficacy. Further investigation is justified to optimise formulations for clinical and commercial applications. Full article

The free-living amoeba Acanthamoeba castellanii is a unicellular eukaryote distributed in a wide range of soil or aquatic environments, either natural or human-made, such as rivers, lakes, drinking water, or swimming pools. Besides its capacity to transport potential pathogens, such as bacteria or viruses, Acanthamoeba spp. can have intrinsic pathogenic properties by causing severe infections at the ocular and cerebral level, named granulomatous amoebic encephalitis and amoebic keratitis, respectively. During its life cycle, A. castellanii alternates between a vegetative and mobile form, named the trophozoite, and a resistant, latent, and non-mobile form, named the cyst. The cyst wall of Acanthamoeba is double-layered, with an inner endocyst and an outer ectocyst, and is mainly composed of cellulose and proteins. The resistance of cysts to many environmental stresses and disinfection treatments has been assigned to the presence of cellulose. The current review aims to present the importance of this glycopolymer in Acanthamoeba cysts and to further report the pathways involved in encystment and excystment. Full article

Background: Diseases in humans caused by amphizoic amoebae that can result in visual impairment and even blindness, have recently been identified more frequently worldwide. Etiologically complex incidents of keratitis, including those connected with Acanthamoeba strains detected in Poland, were evaluated in this study. Methods: Corneal samples from cases resistant to antimicrobial therapy assessed for epidemiological, microbiological and parasitological aspects were investigated by phase-contrast microscope, slit lamp and by confocal microscopy. In vitro techniques were applied for detection of bacteria and fungi, and corneal isolates cultured under axenic condition using BSC medium—for detection of Acanthamoeba spp.; molecular techniques were applied for amoeba species identification. Results: Most etiologically complicated keratitis cases, detected in ~84% of incidents, was due to exposure of contact lenses to tap water or pool water; trophozoites and cysts of Acanthamoeba, concomitant bacteriae, e.g., Pseudomonas aeruginosa, fungi and microfilariae were identified in contact lens users. Conclusions: In samples from contact lens wearers where microbial keratitis is identified along with some connection with the patient’s exposure to contaminated water environments, a risk of Acanthamoeba spp. infections should be considered. Understanding the complicated relationship between Acanthamoeba spp., co-occurring pathogens including associated endosymbionts is needed. In vivo confocal microscopy and in vitro cultivation were necessary to identify potentially contagious concomitant factors affecting the complex course of the keratitis. Full article

(1) Background: Microbial keratitis is a serious eye infection that carries a significant risk of vision loss. Acanthamoeba spp. are known to cause keratitis and their bacterial endosymbionts can increase virulence and/or treatment resistance and thus significantly worsen the course of the disease. (2) Methods and Results: In a suspected case of Acanthamoeba keratitis, in addition to Acanthamoeba spp., an endosymbiont of acanthamoebae belonging to the taxonomic order of Holosporales was detected by chance in a bacterial 16S rDNA-based pan-PCR and subsequently classified as Candidatus Paracaedibacter symbiosus through an analysis of an enlarged 16S rDNA region. We used Oxford Nanopore Technology to evaluate the usefulness of whole-genome sequencing (WGS) as a one-step diagnostics method. Here, Acanthamoeba castellanii and the endosymbiont Candidatus Paracaedibacter symbiosus could be directly detected at the species level. No other microbes were identified in the specimen. (3) Conclusions: We recommend the introduction of WGS as a diagnostic approach for keratitis to replace the need for multiple species-specific qPCRs in future routine diagnostics and to enable an all-encompassing characterisation of the polymicrobial community in one step. Full article

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. Full article