Search Results (133)

The increasing consumption of fresh organic produce has given rise to concerns regarding the microbiological safety of minimally processed foods. Organic cultivation may be associated with increased exposure to environmental microorganisms due to soil-based inputs and reduced chemical interventions, including both beneficial taxa and potential foodborne pathogens. Fresh produce is known to harbour complex microbial ecosystems, which are shaped by farming practices, plant physiology, handling, packaging and storage, particularly in raw-consumed products such as leafy greens and strawberries. In this study, bacterial (16S rRNA) and eukaryotic (18S rRNA) communities were characterized by amplicon sequencing. In parallel, an amoeba-associated bacterial microbiome was analyzed and DVC-FISH was used to assess the viability and metabolic activity of pathogenic bacteria internalized within free-living amoebae (FLA). No significant differences in alpha or beta diversity were observed between organic and conventional products, suggesting microbiome convergence at the retail stage driven by post-harvest handling and processing. Potentially pathogenic genera, including Pseudomonas, Stenotrophomonas, and Acinetobacter (bacterial), as well as Tilletiopsis, Candida, and Naegleria (eukaryotic), were identified in both organic and non-organic microbiomes. The viability of FLA-internalized Pseudomonas spp. was confirmed by DVC-FISH, demonstrating that FLA act as reservoirs, enhancing pathogen persistence in fresh produce. This integrated assessment of organic and conventional fruits and vegetables at the retail stage highlights the importance of post-harvest handling and retail conditions in shaping microbiological safety. The integration of microbiome profiling with targeted viability analyses demonstrates that downstream stages are critical control points for food safety and consumer exposure, beyond the influence of the production system alone. Full article

For Entamoeba histolytica to establish an infection, it must employ several mechanisms of pathogenicity to produce and secrete virulence factors that allow the parasite to adhere to and finally colonize and invade the host. However, in the intestinal epithelium, trophozoites (amoebae) encounter lactoferrin (Lf), a glycoprotein of the first line of defense, together with immunoglobulins and other molecules. We previously reported that iron-free bovine Lf (bLf) could kill amoebae both in vitro and in animal models of intestinal and hepatic amoebiasis. In this work, selected pathogenic mechanisms were evaluated in trophozoites by exposing cultures to sublethal concentrations of bLf to determine which amoebic functions could be altered. At a sublethal bLf concentration, this glycoprotein was removed from the trophozoite. In the presence of erythrocytes, bLf colocalized with erythrocytes at the capping site; this was observed by confocal microscopy of living cells. In addition, the erythrophagocytosis rate, proteolytic activity, adhesion, and cytotoxic properties towards Caco2 colonic cancer cells were reduced in the presence of bLf. Lactoferrin could be a particularly important protein that naturally protects colonic epithelial cells from E. histolytica infection. Full article

The genus Naegleria comprises free-living amoebae characterized as amphizoic and ubiquitous microorganisms. Naegleria fowleri is the only species pathogenic to humans, causing primary amebic meningoencephalitis. The 26S proteasome represents the principal catalytic complex responsible for the degradation and recycling of intracellular proteins in eukaryotic cells. This complex consists of the 20S and 19S proteasome subunits, with the latter involved in the recognition and processing of ubiquitinated proteins and their delivery to the degradation site. Although the 26S proteasome has been characterized in various pathogenic protozoa, only the 20S proteasome has been studied within the genus Naegleria. The objective of this study was to demonstrate the presence of 19S subunits in N. fowleri. Bioinformatics analyses were employed to evaluate the presence and homology of non-ATPase subunits (Rpn10, Rpn11, and Rpn13) and ATPase subunits (Rpt2, Rpt3, and Rpt5). Additionally, the presence, localization, and correlation of 19S proteasome proteins with the 20S proteasome were assessed using experimental approaches. The results indicate that N. fowleri possesses proteins corresponding to the 19S proteasome, which, together with the 20S core particle, contribute to the formation of the 26S proteasome. Full article

Acanthamoeba is a free-living amoeba (FLA) that causes the majority of human infections. It is found predominantly in aquatic environments and is classified according to morphology or genotype (T1-T23). Research on this FLA aims to monitor its distribution, identify existing genotypes, assess its infectious potential, and identify factors that contribute to its recurrence. This study performed a molecular characterisation of Acanthamoeba strains isolated from tap water in Cajeme, Sonora, Mexico, to classify their genotypes. This was complemented by whole-genome sequencing and mapping of the 18S rRNA region in a divergent strain, LUDO1, to obtain higher-resolution data for more reliable assessment of its divergence from known genotypes. Genotypes T4, T5, T11, and T15 were identified in the Acanthamoeba-specific amplimer S1 (ASA.S1) region using the maximum-likelihood method. The inclusion of the 18S rRNA region from strain LUDO1 enabled its classification as a new genotype (T24), with a dissimilarity exceeding 5% compared with the 23 known genotypes. Additionally, culture analysis revealed notable variation in trophozoite size among strains that correlated with phylogenetic sub-branching. This analysis contributed to the epidemiological understanding of Acanthamoeba’s high resistance to treatments and infection systems and demonstrated a broadening of the phylogenetic distribution within the genus. Full article

Background/Objectives: Primary amoebic meningoencephalitis (PAM) is a rare, fulminant, and often fatal central nervous system infection caused by the opportunistic free-living amoeba Naegleria fowleri. Although Naegleria species are widely present in freshwater and soil worldwide, human disease is associated specifically with pathogenic N. fowleri rather than the many nonpathogenic environmental species, and virulence may vary across N. fowleri isolates. This systematic review aimed to synthesize contemporary global data from 2000 to 2024 to identify recent trends in epidemiology, clinical presentation, diagnosis, treatment, and outcomes. Methods: A systematic literature search was conducted across PubMed, Scopus, and the Cochrane Library, identifying 58 eligible publications encompassing 66 individual cases. Results: Most reports originated from the United States, India, and China. The median patient age was 14 years, with 78% of cases occurring in males. Annual case reports increased from one per year (2000–2005) to over four per year (2020–2024), reflecting either a true rise in incidence or improved detection. Common presenting symptoms included fever, headache, and altered mental status. Diagnosis was confirmed via polymerase chain reaction (PCR) testing or post-mortem biopsy in nearly one-third of cases. Treatment regimens varied, with amphotericin B and miltefosine being the most frequently used agents. Overall mortality was 83%, with survival strongly associated with early initiation of combination therapy. Pediatric patients had a higher survival rate (22%) compared to adults (7.1%). Conclusions: The findings highlight the need for heightened clinical awareness, especially in the context of climate-driven ecological changes that may expand N. fowleri’s geographic range. This review underscores critical gaps in surveillance and diagnostics and emphasizes the importance of a One Health approach to addressing emerging threats like PAM. Further research into novel therapeutics, rapid diagnostics, and global case reporting systems is urgently needed. Full article

Devastating or nearly invariably fatal infections caused by free-living amoebae (FLA), including Acanthamoeba keratitis (AK), granulomatous amoebic encephalitis (GAE), and primary amoebic meningoencephalitis (PAM), remain a significant public health concern, driven by increasing case numbers, geographic expansion, and the lack of approved, effective, and safe treatments. Despite decades of research, no new drugs have been successfully approved, highlighting the severe limitations of de novo drug development for these infections, particularly for GAE and PAM, largely due to the challenges of conducting clinical trials for these rare and rapidly lethal diseases. In this context, drug repurposing represents a cost-effective and promising strategy to accelerate therapeutic advances and overcome key bottlenecks of conventional drug development. Accordingly, we conducted a systematic review of in vitro studies and animal models of AK, GAE, and PAM reported in indexed databases to identify promising drug repurposing candidates against FLA infections. After screening 23,624 records, 112 studies were included in the analysis. Overall, 2726 drugs and drug combinations, spanning 865 pharmacological classes and approved for 565 therapeutic indications, were assessed for their repurposing potential. Among these, 166 compounds showed substantial trophocidal activity (≥IC₅₀) at potentially translatable concentrations (≤10 µM), including six with additional cysticidal activity. In vitro, four compounds were active against Balamuthia mandrillaris, 44 against Acanthamoeba spp. (three cysticidal), and 115 against Naegleria spp. (three cysticidal). In in vivo studies, sulfadiazine and rifampicin were effective as preventive or early monotherapies for GAE. For AK, the combination of polyhexamethylene biguanide, neomycin, and atropine, as well as voriconazole and nitazoxanide monotherapies, showed the greatest promise. In PAM, azithromycin alone or in combination with amphotericin B emerged as the most promising therapeutic options. Further studies are required to advance the clinical translatability of these findings. To the best of our knowledge, this work provides the first comprehensive and integrated synthesis of repurposable drug candidates against FLA infections. Full article

Acanthamoeba castellanii, an opportunistic free-living amoeba, causes severe infections including Acanthamoeba keratitis. This exploratory study evaluated whether three non-steroidal anti-inflammatory drugs (NSAIDs)—acetylsalicylic acid, ibuprofen, and diclofenac (100 µM)—modulate pathogenicity-related processes in A. castellanii and explored the involvement of a gp63-like protein during encystment and adhesion. Trophozoites were continuously exposed to each drug and analyzed for adhesion, migration on host-derived discontinuous brain micropatterns, encystment efficiency, and parasite-induced cytoskeletal remodeling in MDCK epithelial cells. In silico docking was performed to assess potential drug–protein interactions. Drug exposure reduced adhesion with maximal inhibition at 60 min. After 1 h, migration decreased by 49%, 64%, and 38%, and encystment was reduced by 50%, 85%, and up to 90%, respectively, in cultures treated with acetylsalicylic acid, ibuprofen, and diclofenac. Co-incubation with untreated trophozoites lowered actin fluorescence to approximately 50%, whereas drug-treated co-cultures preserved fluorescence near control levels. Colocalization analysis showed increased spatial overlap between gp63-like protein and F-actin in cysts (~40%) and migrating trophozoites (~20%) compared with non-stimulated forms (~3.8%). Collectively, these findings suggest that NSAID-sensitive pathways influence host interaction, migration, and encystment in A. castellanii and allow for the proposal of gp63-like protein as a putative molecular component of the NSAIDs sensitive pathways. Full article

Objectives: Free-living amoeba Naegleria fowleri causes primary amoebic meningoencephalitis (PAM). While infection is rare, PAM’s fatality rate exceeds 97%. The recommended treatment includes combination therapy, which does not result in uniform survival. Thus, there is a critical unmet need for finding better therapy for PAM. Drug repurposing can expedite the discovery of effective treatment for PAM. Isavuconazonium is approved for the treatment of fungal infections. Given that isavuconazole is the major metabolite of isavuconazonium and isavuconazole penetrates into the brain with high efficiency, our objective was to determine the activity of both isavuconazonium and isavuconazole on N. fowleri trophozoites. Methods: To test the effect of both compounds, we determined their dose–responses against N. fowleri and two mammalian cells. To establish how fast the prodrug and the metabolite kill the trophozoites, we measured potency at different time points. Finally, we investigated the effect of combining isavuconazonium or isavuconazole with amphotericin B on both N. fowleri and mammalian cells. Results: Both isavuconazonium and the metabolite isavuconazole were active against multiple strains, with clinically relevant isavuconazole exhibiting potency ranging between 0.1 and 0.6 µM. They were less toxic on mammalian cells. Isavuconazonium and isavuconazole required 24 h to achieve nanomolar potency. Combination with amphotericin B was synergistic without eliciting toxicity on mammalian cells. Conclusions: Our findings, together with the use of intravenous and oral formulations of isavuconazonium to treat pediatric and adult patients, support further in vivo efficacy study of isavuconazonium for its potential use for the treatment of PAM. Full article

Background: Naegleria fowleri is a free-living amoeba that inhabits warm freshwater and causes primary amoebic meningoencephalitis (PAM), a rapidly fatal infection with >95% mortality. Due to the lack of early diagnosis and effective therapy, preventive vaccination represents a promising strategy. Methods: This study evaluated short- and long-term immune protection in BALB/c mice (20 mice per group) immunized intranasally with total N. fowleri extract co-administered with cholera toxin (CT). Mice were challenged with a lethal dose of trophozoites either 24 h (short-term) or three months (long-term) after the fourth immunization; the latter group received a booster 24 h before challenge. Serum and nasal washes were analyzed for IgA and IgG antibodies by immunoblot, and lymphocyte subsets from nasal-associated lymphoid tissue (NALT) and nasal passages (NPs) were characterized by flow cytometry. Results: Immunization conferred complete (100%) survival in the 24 h group and 60% protection in the 3-month group, whereas all control mice died. Immunoblotting showed that IgA and IgG antibodies recognized major N. fowleri antigens of 37, 45, 48 and 19, 37, and 100 kDa, respectively. Flow cytometry revealed increased activated and memory B lymphocytes, dendritic cells, and expression of CCR10, integrin α4β1, and FcγRIIB receptors, particularly in the 24 h group. Conclusions: Intranasal immunization with N. fowleri extract plus CT elicited both systemic and mucosal immune responses capable of short- and long-term protection. These findings highlight the potential of this immunization strategy as a foundation for developing effective vaccines against PAM. Full article

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

The absence of molecular tools for manipulation of gene expression in the pathogenic free-living amoeba Naegleria fowleri has historically limited our understanding of gene function in the organism and has coincidently impacted the identification of potential druggable pathways and proteins. Here, we describe the development of approaches for the generation of transgenic amoebae using polyethyleneimine nanoparticles to deliver plasmids designed to confer antibiotic resistance and fluorescence to the cells. Through a series of optimization steps, we found that transfection of plasmids encoding the fluorescent protein mCherry fused by a T2A self-cleaving peptide to a codon-optimized puromycin acetyltransferase selectable marker yielded fluorescent cells that were resistant up to 100 µg/mL puromycin. Transfected trophozoites harbored between 45 and 65 copies of the transgene per cell and both fluorescence and resistance were persistent in the presence of selector through continued passages. The development of these approaches is anticipated to enable application of an array of genetic manipulation techniques including forward and reverse genetics to the study of this important pathogen. 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

Parasitic infections remain a major global health challenge, particularly in resource-limited settings where they are closely tied to poverty and inadequate sanitation. The increasing emergence of drug resistance and the limited accessibility of current therapies highlight the urgent need for novel, safe, and affordable alternatives. Mangifera indica L. (mango), a widely cultivated fruit tree deeply rooted in traditional medicine, has long been used to treat conditions symptomatic of parasitic diseases, including fever, diarrhea, and dysentery. Phytochemical investigations have revealed a rich spectrum of bioactive compounds, notably mangiferin, phenolic compounds and terpenoids, which exhibit antimicrobial, antioxidant, and immunomodulatory activities. This review critically synthesizes evidence on the antiparasitic potential of M. indica against protozoa, such as Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii, Entamoeba histolytica, and free-living amoebae, as well as helminths. Strongest evidence exists for malaria and helminth infections, where both crude extracts and isolated compounds demonstrated significant activity in vitro and in vivo. Encouraging but limited findings are available for leishmaniasis and trypanosomiasis, while data on toxoplasmosis and amoebiasis remain largely speculative. Variations in efficacy across studies are influenced by plant parts and extraction methods, with ethanolic extracts and mangiferin often showing superior results. Despite promising findings, mechanistic studies, standardized methodologies, toxicological evaluations, and clinical trials are scarce. Future research should focus on elucidating molecular mechanisms, exploring synergistic interactions with existing drugs, and leveraging advanced delivery systems to enhance bioavailability. Full article

Acanthamoeba is a widespread free-living amoeba known as an opportunistic parasite of humans and other animals. It comprises several species, whose characterisation relies currently on the analysis of 18S rDNA sequences, recognising more than twenty genotypes; however, the distinction between closely related lineages remains unclear. In this study, the spacer region between the mitochondrial large and small subunits of rRNA genes was analysed for its usefulness as a marker for molecular typing. Previous studies have shown that the mitochondrial spacer contains a group of five transfer RNA (tRNA) genes, and that its length and sequence vary considerably between strains. A total of forty-two mitochondrial spacers were examined here, including twenty-five newly recovered sequences, from ten genotypes covering the three morphological groups of Acanthamoeba. The results showed that lineage-specific profiles can be defined for morphological groups 2 and 3 species (MG2 and MG3), with phylogenetic analysis consistent with that of rDNA, allowing for strain identification at the subtype level. In addition, morphological group 1 (MG1) species have a different tRNA gene arrangement distinguishing them from the others. Mitochondrial spacers therefore appear to be promising phylogenetic markers for the molecular typing of Acanthamoeba. Full article