Search Results (43)

Neuroinvasive free-living amoebae (FLA), particularly Naegleria fowleri and Acanthamoeba spp., are responsible for rare but devastating infections of the central nervous system (CNS). Approximately 480 cases of primary amoebic meningoencephalitis (PAM) and fewer than 200 well-documented cases of Acanthamoeba-associated granulomatous amoebic encephalitis (GAE) have been reported worldwide. Mortality rates frequently exceed 90%. PAM typically develops following exposure to warm freshwater contaminated with N. fowleri and progresses rapidly in otherwise healthy individuals. In contrast, GAE usually follows a more indolent course and occurs predominantly in immunocompromised hosts. Despite their distinct clinical courses, both infections are characterized by CNS invasion, amoeba-mediated tissue destruction, blood–brain barrier (BBB) disruption, and host inflammatory responses. These processes drive neuroinflammation, neuronal injury, and neurological deterioration. Early diagnosis remains challenging because clinical manifestations are nonspecific and disease progression can be either fulminant or initially subtle. Therapeutic management is hindered by poor CNS drug penetration, limited efficacy of currently available therapies, treatment-related toxicity, and the absence of standardized treatment protocols or controlled clinical trials. This narrative review critically synthesizes current evidence on CNS invasion, neuroinflammation, neuropathology, diagnostic challenges, and therapeutic strategies in neuroinvasive FLA infections. It also highlights key translational priorities, including earlier diagnosis, standardized treatment protocols, stronger clinical evidence, and improved CNS-targeted drug delivery. Full article

Access to safe drinking water remains a global public health concern due to its role in the transmission of infectious diseases. Despite the 20th-century achievement of chlorine-based disinfection, drinking water systems face threats from aging infrastructure, climate-induced stressors, and emerging pathogens that evade traditional treatment. This bibliometric review maps three decades of research on microbial contamination in drinking water systems to explain its historical developments, current knowledge, and important updates. Only original and review articles retrieved on 13 April 2026 were screened for inclusion, requiring a focus on detecting, monitoring, or mitigating microbial contamination in drinking water systems. Analysis of 93 records identified a linear growth pattern, shifting from acute enteric pathogen monitoring to the management of opportunistic pathogens (OPs), antimicrobial resistance (AMR), and disinfection by-products (DBPs). Additionally, traditional fecal indicator bacteria (FIB), such as Escherichia coli, may not fully predict the presence of resilient pathogens protected within biofilms or free-living amoebae (FLA), which serve as environmental reservoirs for infection. To address these limitations, this review presents a conceptualization of waterborne pathogens by proposing formal case definitions and diagnostic criteria for critical contamination events (CCE) and chronic low-level exposure (CLLE). Lastly, knowledge gaps and open research questions relevant to future studies on microbial contamination in drinking water systems were identified and discussed. Full article

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

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

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

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 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

Urban settings in developing countries present unique challenges such as high population density, inadequate water infrastructure and water supply, all factors that contribute to the growing threat of premise plumbing pathogens such as Legionella. Water droplets from showers and faucets aerosolise Legionella, which, when inhaled, invade the human respiratory tract to manifest as Legionnaires’ disease. Densely populated, high-rise buildings present an ideal case study for investigating the presence of Legionella. The aim of this study was to investigate the occurrence of Legionella pneumophila (L. pneumophila) in water systems of 15 high-rise buildings in Hillbrow, Johannesburg, South Africa. A total of 67 hot- and cold-water samples and 121 swab samples were collected and analysed for the presence of Legionella pneumophila. Samples were analysed using the Legiolert assay, the South African National Standard (SANS) 11731:2017 method, and the amoeba enrichment method for detecting amoeba-associated Legionella. Molecular confirmation of the pathogen was conducted using conventional PCR and quantitative real-time PCR targeting the mip gene. Legionella pneumophila was found in 93% (14/15) of the buildings that were sampled and was more prevalent in cold-water samples (65%) compared to warm-water (35%) samples. All buildings were positive (100%) for the growth of free-living amoeba (FLA) from water and swab samples. Of these samples, three were confirmed positive for L. pneumophila by PCR and the sequencing alignment results confirmed the identity and relatedness of the isolates to L. pneumophila. Full article

Free-living amoebae (FLAs) are widely present in the environment and may be pathogenic for animals and humans. Studies on the prevalence of FLAs in European soils are few in number. This study aimed to molecularly identify the species and genotypes of FLAs occurring in soil from Southern Poland. Forty soil samples were collected in June 2024 in the Babiogórski National Park. Amoebae cultures and a thermal-tolerance test were conducted, and all strains grew at 37 °C. Five PCR protocols were applied for the amplification of FLA SSU rRNA fragments. The following FLAs, including potentially pathogenic ones, were detected: Acanthamoeba T4 and T13 genotypes in 79.1% of positive samples, Naegleria gruberi and Naegleria galeacystis in 25%, Vermamoeba vermiformis in 12.5%, and Paravahlkampfia sp. and Ptolemeba bulliensis in 8.3%. Species and genotype identification were determined by sequence comparison and phylogenetic analysis. This study reports, for the first time, the isolation of N. galeacystis from soil and N. gruberi and V. vermiformis from soil collected in Europe. The used primer sets have different usefulness for Naegleria species identification and their phylogenetic analysis. The primers applied in this study may not reveal the full diversity of amoebae in soil; therefore, it is necessary to design new primers for this purpose. 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

Acanthamoeba, a free-living amoeba ubiquitous in environmental water, has been considered as the environmental reservoir of certain bacterial pathogens, including Campylobacter jejuni, an intracellular human pathogen causing self-limiting gastroenteritis. Acanthamoeba-C. jejuni interaction mechanisms may help clarify how the otherwise fastidious bacterium C. jejuni survives in environmental waters. In this study, we constructed single deletion mutants of C. jejuni strain 81–176 for the 10 selected genes (motAB, ciaB, kpsE, virB11, cheY, flaAB, cstII, docB, sodB, and cadF) previously shown to be important for the interaction (invasion and intracellular survival) of C. jejuni with mammalian hosts. We used a modified gentamicin protection assay to quantify the internalization and intracellular survival of these mutants and the wild type with the two species of Acanthamoeba (A. castellanii and A. polyphaga). Both internalization and intracellular survival were significantly lower for all mutants compared to the wild type with both amoeba strains, except for ΔcstII in the internalization assay with A. castellanii (p < 0.05). The results of this study highlight that the mechanisms used by C. jejuni to interact with mammalian hosts are conserved in its interactions with amoeba hosts. This understanding may be useful in developing effective strategies to reduce the transmission of C. jejuni to chickens through drinking water. 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

Among free-living amoebae (FLA), Acanthamoeba polyphaga is an important causal agent of Acanthamoeba keratitis (AK), a severe and potentially sight-threatening condition. The present study evaluated the “in vitro” efficiency of Melaleuca alternifolia Cheel (tea tree) (tea tree oil—TTO) and Eucalyptus globulus Labill. (Eucalyptus essential oil—EEO) essential oils against an Acanthamoeba strain isolated from human keratitis. The Minimum Inhibitory Concentration (MIC) of the EOs and the Fractional Inhibitory Concentration (FIC) Index were used to evaluate the decrease in viable cells of Acanthamoeba over time and at different concentrations of EOs, used alone or in association. A relevant amoebicidal effect emerged during the first hours of exposure for both compounds, and TTO was the most effective. The TTO/EEO association clearly indicated a synergistic effect in all tests, and at 2 days post-treatment, no viable A. polyphaga cells were observed at all tested concentrations. In conclusion, the potential therapeutic use of EOs represents a promising therapeutic strategy for the treatment of AK. 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