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FLA-related conditions are a rare medical occurrence. Despite their rarity, they are considered a public health concern for two reasons: the absence of a regular treatment regimen in the case of central nervous system infections and the fast progression of the symptoms leading to fatal outcomes. A total of 358 articles were retrieved from different databases (91 from PubMed, 26 from NCBI, 138 from Academia, 102 from Science Direct, and one from IJMED). 7 (46.6%) clinical cases came from Egypt, 2 (13.3%) cases of FLA infection came from Nigeria, 3 (20%) cases came from the Gambia, and 1 (6.6%) case was reported from African countries like Algeria, Tunisia, South Africa, and Zambia. Medical conditions caused by free-living amoeba are considered significant public health concerns. These ubiquitous organisms can cause both fatal and debilitating health conditions. Immediate diagnosis of cases and proper hygienic practices are necessary to provide direct medical intervention. They may be the key to reducing the morbidity and mortality rates from FLA-acquired infections. Although several government-led initiatives have been implemented to mitigate a plethora of parasitic diseases, the case of FLA-related conditions in African countries has yet to be realized.

Introduction: Primary amoebic meningoencephalitis (PAM) is a rare but lethal infection of the brain caused by a eukaryote called Naegleria fowleri ( N. fowleri ). The aim of this review is to consolidate the recently published case reports of N. fowleri infection by describing its epidemiology and clinical features with the goal of ultimately disseminating this information to healthcare personnel., Methods: A comprehensive literature search was carried out using PubMed, Web of Science, Scopus, and OVID databases until 31 December 2022 by two independent reviewers. All studies from the year 2013 were extracted, and quality assessments were carried out meticulously prior to their inclusion in the final analysis., Results: A total of 21 studies were selected for qualitative analyses out of the 461 studies extracted. The cases were distributed globally, and 72.7% of the cases succumbed to mortality. The youngest case was an 11-day-old boy, while the eldest was a 75-year-old. Significant exposure to freshwater either from recreational activities or from a habit of irrigating the nostrils preceded onset. The symptoms at early presentation included fever, headache, and vomiting, while late sequalae showed neurological manifestation. An accurate diagnosis remains a challenge, as the symptoms mimic bacterial meningitis. Confirmatory tests include the direct visualisation of the amoeba or the use of the polymerase chain reaction method., Conclusions: N. fowleri infection is rare but leads to PAM. Its occurrence is worldwide with a significant risk of fatality. The suggested probable case definition based on the findings is the acute onset of fever, headache, and vomiting with meningeal symptoms following exposure to freshwater within the previous 14 days. Continuous health promotion and health education activities for the public can help to improve knowledge and awareness prior to engagement in freshwater activities.

Free Living Amoebae (FLA) are ubiquitous microorganisms reported from harsh environmental conditions. Oil refinery facilities consume vast volumes of water during their processes, generating a large amount of wastewater. The present study aimed to evaluate the wastewater treatment process in an oil refinery wastewater treatment facility (ORWWTF) for the presence of FLA. Water samples were collected from an oil refinery wastewater (ORWW) for nine months. After recording physical-chemical features, samples were cultivated onto non-nutrient agar (NNA). The discriminative fragments of the ribosomal RNA (rRNA) gene were amplified and sequenced to characterize the isolated FLA. Phylogenetic tree, and network analysis were employed to evaluate genetic relationships. The thermo- and osmotolerant tests were performed on the isolated FLA. Twenty-five (32.9%) samples were positive for FLA cultivation. Acanthamoeba spp., Vahlkampfiids, and Vermamoeba spp. were detected, of which Acanthamoeba species were predominant. There was no statistical correlation between pH, NH 3 , PO 4 , H 2 S, and TDS with the presence of FLA. A statistical correlation between the presence of FLA and the type of wastewater treatment plants (WWTPs) was significant (P-value = 0.011). All Acanthamoeba spp. isolates belonged to the genotypes T4 (17/21; 80.95%) and T11 (4/21; 19.05%). Vahlkampfiids were Naegleria spp., (7/10; 70%), Tetramitus aberdonicus (1/10; 10%), Learamoeba spp., (1/10; 10%), and Vahlkampfia spp., (1/10; 10%). All three Vermamoeba spp. were V. vermiformis. The ORWW contains toxic materials, and a few microorganisms can stay active in these environments. This is the first study which isolates FLA from such super harsh conditions. For the first time, T. aberdonicus, and Learamoeba spp., were isolated from oily wastewater. Our findings signify the concern due to the distribution of potentially pathogenic FLA to downstream lands via treated wastewater that may be released after treatment processing., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

Free-living amoebae (FLA) are considered environmental pathogens and thus pose a public health threat. Their ubiquity in natural sources may magnify the potential severity of health outcomes in the future. However, less attention was given despite several probable public health risks that arise from the presence of pathogenic strains in the environment. Here, we provide epidemiological data based on investigations involving the distribution and occurrence of free-living amoebae in the Republic of the Philippines. This aims to connect data of fragmented studies of these organisms and provide potential roadmaps in FLA research in the country. The majority of the reviewed articles (n = 19) focused on characterization studies (36.8%; 7/19) while environmental isolation and isolation from biological samples had an equal frequency of 31.6% (6/19) each. There is a great disparity between the established ubiquity in environmental sources and the number of cases of FLA infections in the country. FLA-related research in the Philippines is still in its inceptive stage with several gaps to fill, which can be used to formulate policy briefs in the future regarding its isolation, identification, diagnosis, therapeutic management, and control of FLA infections in the country.

Purpose: Free-living amoebae (FLA) exist in various environmental sources and are the causative agents of many serious human diseases. We surveyed various natural and artificial water bodies in areas of north India to detect the presence of FLA species., Materials and Methods: Surface water samples were collected from 29 water bodies and subjected to microscopy, non-nutrient agar (NNA) culture, and various polymerase chain reaction (PCR) assays for the detection of FLA., Results: FLA were found in 62.1% (18/29) of the natural and artificial water bodies by NNA culture. Based on PCR based detection, Acanthamoeba spp. was identified in 41.4% (12/29), Vannella spp./Vahlkampfia ovis in 17.2% (5/29) and Naegleria spp. in 20.7% (6/29) of the samples from different locations. PCR-sequencing of Naegleria positive samples identified them as Naegleria australiensis and Naegleria species., Conclusions: The present study reports widespread occurrence of medically important FLA species in various natural and artificial water bodies, and highlights the need for regular molecular surveillance of FLA in the environment for better control and prevention of the diseases caused by FLA., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase ( Nf Glck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii ( Ac Glck) and B. mandrillaris ( Bm Glck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, Ac Glck and Bm Glck have apparent K m values of 472.5 and 41.0 μM, while Homo sapiens Glck ( Hs Glck) has a value of 310 μM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent K m values of 45.9 μM ( Ac Glck) and 124 μM ( Bm Glck) compared to ~8 mM for Hs Glck. Additionally, Ac Glck and Bm Glck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.

Background: Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis (PAM), is a free-living amoeba. It is a water-borne infection usually detected in children and young people with healthy immune system who swim, dive and perform activities in fresh and hot springs., Purpose: In this study, it was aimed to raise awareness in the differential diagnosis of meningitis etiopathogenesis by showing that N. fowleri may also be the causative agent, albeit very rarely, in meningitis cases in Turkey., Methods: Our case was an 18-year-old male patient whose relatives stated that he has gone to the hot spring; his headache complaint started after 2 to 3 days after return from the hot spring. Cerebrospinal fluid (CSF) sample taken from the patient was investigated by direct microscopic examination, real-time PCR method and sequence analysis., Results: The CSF sample collected was taken into distilled water considering the possibility of transformation of trophozoites to intermediate form and incubated at 37 °C for 1 to 2 h, and pear-shaped non-permanent flagellated forms were observed in the direct microscopic examination, and molecular typing was performed to confirm the diagnosis. This study was a comprehensive case of N. fowleri whose etiological agent was isolated and confirmed by real-time PCR in Turkey., Conclusion: Clinician awareness would be the key factor in correctly diagnosing PAM. It is also recommended to investigate all likely environmental water sources in Turkey for more detailed information on the distribution and molecular identification of Naegleria species, ultimately to evaluate the potential pathogenic threat to human health and to develop strategies to combat such threats., (© 2022. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Purpose: Acanthamoeba spp. are free-living amoebas with worldwide distribution and play an important role as disease-causing agents in humans. Drug inability to completely eradicate these parasites along with their toxic effects suggest urgent need for new antimicrobials. Nisin is a natural antimicrobial peptide produced by Lactococcus lactis. Nisin is also the only bacteriocin approved for use in food preservation. In this work, we analyzed the effect of nisin on the growth of Acanthamoeba castellanii trophozoites., Methods: A total of 8 × 10 4 trophozoites were exposed to increasing concentrations of nisin to determine its activity. Changes in cell membrane and cellular cycle of trophozoites were investigated by flow cytometry, and nisin cytotoxicity in mammalian cells was evaluated in L929 cells by MTT method., Results: After 24 h exposure to increasing nisin concentrations, an IC 50 of 4493.2 IU mL -1 was obtained for A. castellanii trophozoites. However, after 72 h a recovery in amoebic growth was observed, and it was no longer possible to determine IC 50 . Flow cytometry analysis showed that nisin has no effect on the membrane integrity. Treatment with nisin induced cell-cycle arrest during G1 and S phases in A. castellanii trophozoites, which recovered their growth after 72 h., Conclusion: This is one of the first studies showing the effect of internationally approved nisin against A. castellanii trophozoites. Nisin caused cell-cycle arrest in trophozoites, momentarily interfering with the DNA replication process. The data highlight the amoebostatic activity of nisin, and suggest its use as an adjuvant for the treatment of infections caused by Acanthamoeba spp., (© 2021. Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Infections caused by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris result in a variety of clinical manifestations in humans. These amoebae are found in water and soil worldwide. Acanthamoeba spp. and B. mandrillaris cause granulomatous amoebic encephalitis (GAE), which usually presents as a mass, while N. fowleri causes primary amoebic meningoencephalitis (PAM). Acanthamoeba spp. can also cause keratitis, and both Acanthamoeba spp. and B. mandrillaris can cause lesions in skin and respiratory mucosa. These amoebae can be difficult to diagnose clinically as these infections are rare and, if not suspected, can be misdiagnosed with other more common diseases. Microscopy continues to be the key first step in diagnosis, but the amoeba can be confused with macrophages or other infectious agents if an expert in infectious disease pathology or clinical microbiology is not consulted. Although molecular methods can be helpful in establishing the diagnosis, these are only available in referral centers. Treatment requires combination of antibiotics and antifungals and, even with prompt diagnosis and treatment, the mortality for neurological disease is extremely high.

An alternative amoeba cloning technique is described. The amoebic cells were scraped from the agar and inoculated in a new agar medium separated in isolated squares, preventing a cell from invading the space of another cell, increasing the reliability of cloning, and providing an efficient quality control of technique., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Recently, indoor swimming pool activities have increased to promote health-enhancing physical activities, which require establishing suitable protocols for disinfection and water quality control. Normally, the assessment of the microbial quality of the water in the pools only considers the presence of different bacteria. However, other less frequent but more resistant pathogens, such as free-living amoebas (FLA), are not contemplated in both existing recommendation and research activities. FLA represent a relevant human health risk, not only due to their pathogenicity but also due to the ability to act as vehicles of other pathogens, such as bacteria. Therefore, this work aimed to study the physicochemical characteristics and the occurrence of potentially pathogenic FLA and bacteria in water samples from 20 public indoor swimming facilities in Northern Portugal. Our results showed that some swimming pools presented levels of pH, free chlorine, and conductivity out of the recommended limits. Pathogenic FLA species were detected in two of the facilities under study, where we also report the presence of both, FLA and pathogenic bacteria. Our findings evidence the need to assess the occurrence of FLA and their existence in the same environmental niche as pathogenic bacteria in swimming pool facilities worldwide and to establish recommendations to safeguard the health of the users., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Free-living amoebae (FLA) are ubiquitous and many isolates have been shown to be infected with amoeba-resisting bacteria, as the example of Acanthamoeba and Legionella interaction. Due to the high environmental prevalence of Acanthamoeba. in the Castilian Plateau (Spain), the aims of this work were to investigate the occurrence of Acanthamoeba and other FLA in water from several sampling points from four Drinking Water Treatment Plants (DWTP) and to investigate the presence of Legionella spp. and other amoeba-resisting bacteria in biofilms in raw and finished water, taking into account that no legislation exists for this protozoa control. Acanthamoeba was detected at different sampling points, and sand filters seemed to contribute to amoebic enrichment. After ozonation, a temporary decrease in viable amoebae was observed. The genotypes detected were T3, T4, and T5, revealing the first report of genotype T5 in waters from this region. Moreover, Balamuthia mandrillaris, Vermamoeba vermiformis and Paravahlkampfia sp. were detected. Regarding Legionella, PCR detection in raw and finished water was higher than by agar culture, but even higher after Acanthamoeba co-culture. Also, Legionella's presence was higher in raw water than in finished water. The decrease of free Legionella observed from raw (27.5%, by PCR) to finished water (3.4% by PCR) contrasted with the increase of Legionella-infected FLA from raw (30.7%) to finished water (52%). At biofilm, free Legionella was not detected, and the percentage of infected FLA was low (3.8%). Legionella species identified in these samples were L. drozanskii, L. donaldsonii and L. feeleii. Additionally, Acanthamoeba co-culture led to the isolation of Pseudomonas aeruginosa, P. stutzeri, P. fluorecens, Achromobacter xylosoxidans and Stenotrophomonas maltophilia. The highly disseminated presence of Acanthamoeba and the detection of amoeba-resisting bacteria inside amoebae highlight the importance of developing methods for controlling FLA in order to limit human pathogenic amoeba-resisting bacteria survival to the water purification processes., Competing Interests: Declaration of competing interest All authors declare no actual or potential conflict of interest including any financial, personal or other relationships with founding organizations., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Free-living amoebas (FLAs) can cause severe disease in humans and animals when they become infected. However, there are no accurate survey reports on the prevalence of FLAs in Korea. In this study, we collected 163 tap water samples from buildings, apartments, and restrooms of highway service areas in 7 Korean provinces with high population density. All these buildings and facilities have water storage tanks in common. The survey was separated into categories of buildings, apartments, and highway service areas. Five hundred milliliters of tap water from each building was collected and filtered with 0.2 µm pore filter paper. The filters were incubated in agar plates with heated E. coli at 25°C. After axenization, genomic DNA was collected from each FLA, and species classification was performed using partial 18S-rDNA PCR-sequencing analysis. We found that 12.9% of tap water from buildings with storage tanks in Korea was contaminated with FLAs. The highway service areas had the highest contamination rate at 33.3%. All of the FLAs, except one, were genetically similar to Vermamoeba vermiformis (Hartmannella vermiformis). The remaining FLA (KFA21) was very similar to Acanthamoeba lugdunensis (KA/E26). Although cases of human infection by V. vermiformis are very rare, we must pay attention to the fact that one-third of tap water supplies in highway service areas have been contaminated.

The presence of Legionella spp. in natural and man-made water systems is a great public health concern and heavily depends on the presence of free-living amoebae. Taking advantage of the development and affordability of next-generation sequencing technology, we present here a method to characterize the whole bacterial community directly from water samples, as well as from isolated free-living amoebae.

Despite advances in drug discovery and modifications in the chemotherapeutic regimens, human infections caused by free-living amoebae (FLA) have high mortality rates (~95%). The FLA that cause fatal human cerebral infections include Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba spp. Novel drug-target discovery remains the only viable option to tackle these central nervous system (CNS) infection in order to lower the mortality rates caused by the FLA. Of these FLA, N. fowleri causes primary amoebic meningoencephalitis (PAM), while the A. castellanii and B. Mandrillaris are known to cause granulomatous amoebic encephalitis (GAE). The infections caused by the FLA have been treated with drugs like Rifampin, Fluconazole, Amphotericin-B and Miltefosine. Miltefosine is an anti-leishmanial agent and an experimental anti-cancer drug. With only rare incidences of success, these drugs have remained unsuccessful to lower the mortality rates of the cerebral infection caused by FLA. Recently, with the help of bioinformatic computational tools and the discovered genomic data of the FLA, discovery of newer drug targets has become possible. These cellular targets are proteins that are either unique to the FLA or shared between the humans and these unicellular eukaryotes. The latter group of proteins has shown to be targets of some FDA approved drugs prescribed in non-infectious diseases. This review out-lines the bioinformatics methodologies that can be used in the discovery of such novel drug-targets, their chronicle by in-vitro assays done in the past and the translational value of such target discoveries in human diseases caused by FLA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Free-living amoebae (FLA) are ubiquitous organisms present in various natural and artificial environments, such as drinking water storage towers (DWST). Some FLA, such as Acanthamoeba sp., Naegleria fowleri, and Balamuthia mandrillaris, can cause severe infections at ocular or cerebral level in addition to being potential reservoirs of other pathogens. In this work, the abundance and diversity of FLA was evaluated in two sampling campaigns: one performed over five seasons in three DWST at three different levels (surface, middle and bottom) in water and biofilm using microscopy and PCR, and one based on the kinetics analysis in phase contrast and confocal microscopy of biofilm samples collected every two weeks during a 3-month period at the surface and at the bottom of a DWST. In the seasonal study, the FLA were detected in each DWST water in densities of ~20 to 25amoebaeL -1 . A seasonal variation of amoeba distribution was observed in water samples, with maximal densities in summer at ~30amoebaeL -1 and minimal densities in winter at ~16amoebaeL -1 . The FLA belonging to the genus Acanthamoeba were detected in two spring sampling campaigns, suggesting a possible seasonal appearance of this potentially pathogenic amoeba. Interestingly, a 1 log increase of amoebae density was observed in biofilm samples collected at the surface of all DWST compared to the middle and the bottom where FLA were at 0.1-0.2amoebae/cm 2 . In the kinetics study, an increase of amoebae density, total cell density, and biofilm thickness was observed as a function of time at the surface of the DWST, but not at the bottom. To our knowledge, this study describes for the first time a marked higher FLA density in biofilms collected at upper water levels in DWST, constituting a potential source of pathogenic micro-organisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections. The aim of our study was to detect FLA and Acanthamoeba spp. in hospital water circuits. Eighty-four water samples were collected over a period of 4 months (September-December 2011) from different wards of the Sfax University Hospital (surgical services, intensive care unit, operating theater, and water storage tanks). FLA were detected in 53.5 % of samples as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), water storage tanks (6.6 %). The predominant morphotype was the acanthopodial (89 %). The others morphotypes were as follows: monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Acanthamoeba was found in 40 samples (47.6 %). 64.2 % of isolates were identified as Acanthamoeba spp. by PCR, using primers to amplify a region of 18S rDNA which showed variation in the product length. Sequence analysis of five PCR products identified Acanthamoeba sp. These isolates belong to T4, T10, and T11 genotypes, and to our knowledge this is the first report of the T10 and T11 genotype in Tunisia.The occurrence of potentially pathogenic FLA in the hospital environment may represent a health risk for patients, since these organisms can cause severe opportunistic illness and also can harbor pathogenic agents. Thus, increased awareness regarding these parasites and recognition of their importance, particularly in immunocompromised patients is crucial.

Aims: To examine the occurrence and quantity of potential pathogens and an indicator of microbial contamination in the sediments of municipal drinking water storage tanks (MDWSTs), given the absence of such data across the United States., Methods and Results: Sediment samples (87 MDWST) from eighteen locations across ten states of the United States were collected and assayed by qPCR for a range of potential enteric and opportunistic microbial pathogens and a sewage-associated Bacteroides marker. Potential opportunistic pathogens dominated, with the highest detection of occurrence (per cent positive detection; average cell equivalence (CE)) being Mycobacterium spp. (88·9%; 6·7 ± 8·5 × 10(4) CE g(-1) ), followed by Legionella spp. (66·7%; 5·2 ± 5·9 × 10(3) CE g(-1) ), Pseudomonas aeruginosa (22·2%; 250 ± 880 CE g(-1) ) and Acanthamoeba spp. (38·9%; 53 ± 70 CE g(-1) ), with no detected Naegleria fowleri. Most enteric pathogens (Campylobacter jejuni, Escherichia coli 0157:H7, Salmonella enterica, Cryptosporidium parvum and Giardia duodenalis) were not detected, except for a trace signal for Campylobacter spp. There was significant correlation between the qPCR signals of Legionella spp. and Acanthamoeba spp. (R(2) = 0·61, n = 87, P = 0·0001). Diverse Legionella spp. including Leg. pneumophila, Leg. pneumophila sg1 and Leg. anisa were identified, each of which might cause legionellosis., Conclusions: These results imply that potential opportunistic pathogens are common within MDWST sediments and could act as a source of microbial contamination, but need downstream growth to be of potential concern., Significance and Impact of the Study: The results imply that opportunistic pathogen risks may need to be managed by regular tank cleaning or other management practices., (2015 The Society for Applied Microbiology.)

Free-living amoeba such as Acanthamoeba and Balamuthia mandrillaris can act as opportunistic parasites on a wide range of vertebrates and they are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices and their ubiquitous distribution. This work was carried out in order to study the presence of these free-living amoebae (FLA) and their possible seasonality in a continental-Mediterranean climate in different types of water. For this purpose, a total of 223 water samples were collected during one year from four drinking water treatment plants (DWTP), seven wastewater treatment plants (WWTP) and six locations of influence (LI) on four river basins from Spain. Water samples were concentrated using the IDEXX Filta-Max(®) system and analyzed by a triplex real time PCR that detects Acanthamoeba, B. mandrillaris and Naegleria fowleri. Agar plates were also seeded for Acanthamoeba culture. From the three FLA studied, N. fowleri was not detected in any sample while B. mandrillaris was found at the entrance of a DWTP; this being, to our knowledge, the first report of these protozoa in water worldwide. On the other hand, the presence of Acanthamoeba observed was higher, 94.6% of the studied points were positive by real time PCR and 85.2% by culture, resulting in 99.1% positive for Acanthamoeba with both methods. All genetically analyzed Acanthamoeba were genotype T4 but nine different T4/DF3 sequences were observed, three of them being described for the first time, assigning new codes. No seasonal distribution of Acanthamoeba was found. These facts should serve as a warning to contact lens wearers of the risk of a poor hygiene when handling their contact lenses. It should also serve as a signal to physicians to consider FLA as a possible causative agent of nervous system infections as well as Acanthamoeba keratitis due to their high environmental presence shown in this study., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mycobacterium leprae is an acid-fast bacterium that causes leprosy diseases, which remains a problem worldwide. Even though leprosy prevalence in the world has decreased significantly, many endemic pocket areas continue reporting new cases and harbor M. leprae in the environment, including water and soil. The presence of obligate intracellular bacteria-M. leprae in the environment raises a question on how it survives. Free-living amoeba has been proposed as its reservoir host in the environment. The study was conducted to give evidence that M. leprae can survive inside free-living amoeba isolated from water sources of leprosy endemic areas. M. leprae from leprosy patients was cultured together with Acanthamoeba sp. isolated from the water source of the leprosy endemic area. Viability and duplication of M. leprae inside amoeba then observed at day 14 and 28 using reverse transcriptase PCR and qPCR. The results showed that M. leprae survived inside the amoeba until day 28, but no bacterial replication was observed. The study reveals in vitro evidence of viable M. leprae inside free-living amoeba of leprosy endemic area environment. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are widely distributed protozoa in both natural and artificial environments such as drinking water. In addition to the ability of all FLA to transport various pathogenic microorganisms, certain species, such as Acanthamoeba spp. or Balamuthia mandrillaris, have intrinsic pathogenic abilities and cause severe cerebral infections. Previous work has shown an enrichment of FLA cysts in biofilm developed in upper levels of Drinking Water Storage Towers (DWSTs), suggesting that differences in densities of FLA cysts may play a role in their unequal distribution in the water column. To evaluate this hypothesis, a model of a water column was created for this study and used to analyze the vertical distribution of cysts of the FLA Acanthamoeba castellanii, Vermamoeba vermiformis, and Balamuthia mandrillaris from 0 to 23 weeks. Interestingly, our data showed that the cysts of both A. castellanii and V. vermiformis were enriched in upper water levels during their aging. However, B. mandrillaris cysts were equally distributed in the water column during the entire study. These results show that, in addition to the role of water level variation in the DWST, some FLA cysts can become less dense during their aging, which contributes to their enrichment in upper water and therefore biofilm levels. [ABSTRACT FROM AUTHOR]

Traditional cysticidal assays for Acanthamoeba species revolve around treating cysts with compounds and manually observing the culture for evidence of excystation. This method is time-consuming, labor-intensive, and low throughput. We adapted and trained a YOLOv3 machine learning, object detection neural network to recognize Acanthamoeba castellanii trophozoites and cysts in microscopy images to develop an automated cysticidal assay. This trained neural network was used to count trophozoites in wells treated with compounds of interest to determine if a compound treatment was cysticidal. We validated this new assay with known cysticidal and noncysticidal compounds. In addition, we undertook a large-scale bioluminescence-based screen of 9,286 structurally unique marine microbial metabolite fractions against the trophozoites of A. castellanii and identified 29 trophocidal hits. These hits were then subjected to this machine learning-based automated cysticidal assay. One marine microbial metabolite fraction was identified as both trophocidal and cysticidal. IMPORTANCE The free-living Acanthamoeba can exist as a trophozoite or cyst and both stages can cause painful blinding keratitis. Infection recurrence occurs in approximately 10% of cases due to the lack of efficient drugs that can kill both trophozoites and cysts. Therefore, the discovery of therapeutics that are effective against both stages is a critical unmet need to avert blindness. Current efforts to identify new anti-Acanthamoeba compounds rely primarily upon assays that target the trophozoite stage of the parasite. We adapted and trained a machine learning, object detection neural network to recognize Acanthamoeba trophozoites and cysts in microscopy images. Our machine learning-based cysticidal assay improved throughput, demonstrated high specificity, and had an exquisite ability to identify noncysticidal compounds. We combined this cysticidal assay with our bioluminescence-based trophocidal assay to screen about 9,000 structurally unique marine microbial metabolites against A. castellanii. Our screen identified a marine metabolite that was both trophocidal and cysticidal.

Opportunistic premise plumbing pathogens (OPPPs) have been detected in buildings' plumbing systems causing waterborne disease outbreaks in the United States. In this study, we monitored the occurrence of OPPPs along with free-living amoeba (FLA) and investigated the effects of residential activities in a simulated home plumbing system (HPS). Water samples were collected from various locations in the HPS and analyzed for three major OPPPs: Legionella pneumophila, nontuberculous mycobacterial species (e.g., Mycobacterium avium, M. intracellulare, and M. abscessus), and Pseudomonas aeruginosa along with two groups of amoebas (Acanthamoeba and Vermamoeba vermiformis). A metagenomic approach was also used to further characterize the microbial communities. Results show that the microbial community is highly diverse with evidence of spatial and temporal structuring influenced by environmental conditions. L. pneumophila was the most prevalent pathogen (86% of samples), followed by M. intracellulare (66%) and P. aeruginosa (21%). Interestingly, M. avium and M. abscessus were not detected in any samples. The data revealed a relatively low prevalence of Acanthamoeba spp. (4%), while V. vermiformis was widely detected (81%) across all the sampling locations within the HPS. Locations with a high concentration of L. pneumophila and M. intracellulare coincided with the highest detection of V. vermiformis, suggesting the potential growth of both populations within FLA and additional protection in drinking water. After a period of stagnation lasting at least 2-weeks, the concentrations of OPPPs and amoeba immediately increased and then decreased gradually back to the baseline. Furthermore, monitoring the microbial population after drainage of the hot water tank and partial drainage of the entire HPS demonstrated no significant mitigation of the selected OPPPs. This study demonstrates that these organisms can adjust to their environment during such events and may survive in biofilms and/or grow within FLA, protecting them from stressors in the supplied water. [ABSTRACT FROM AUTHOR]

Background: Pathogenic free-living amoeba (FLA) such as Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are causative agents of fatal amoebic encephalitis/meningoencephalitis. The diagnosis of such infections is challenging due to a lack of clinical suspicion and expertise in microscopic identification. We evaluated the performance of molecular assays for the timely and accurate detection of FLA-causing central nervous system (CNS) afflictions. Methods: This study included samples from 156 patients with suspected encephalitis/meningoencephalitis, including 149 cerebrospinal fluid (CSF) samples, 5 brain tissue biopsies, and 2 brain abscess samples. All the samples were subjected to PCR-based detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris. The diagnostic characteristics and the inter-rater reliability scores were evaluated for parasite-specific polymerase chain reaction (PCR) using culture on non-nutrient agar (NNA)/microscopy or histopathological examination as a confirmatory test for Acanthamoeba spp. and N. fowleri and histopathology for B. mandrillaris. Results: We detected 11 samples positive for FLA, including 6 Acanthamoeba spp., 3 B. mandrillaris, and 2 N. fowleri. Furthermore, all 11 samples were positive according to the confirmatory tests, i.e., culture on NNA/microscopy/histopathology in the case of Acanthamoeba spp. and N. fowleri and histopathology of tissue biopsies for B. mandrillaris. The inter-rater reliability between the PCRs and the confirmatory tests for the detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris was 100%. Conclusions: The PCR-based detection of FLA in patients suspected of encephalitis/meningoencephalitis was found to be fast, efficient, and reliable in our study. We suggest the use of these PCRs in laboratories to obtain additional data on their efficiency in diagnosing FLA infections of the CNS. The present study was conducted with a small sample size of 156 patient samples, and we found only six Acanthamoeba spp., three B. mandrillaris, and two N. fowleri. The present study should be conducted on a larger sample size for better evaluation of the primer pairs. [ABSTRACT FROM AUTHOR]

Although the presence of free-living amoebae (FLAs) in various water sources has been reported, few studies have been surveyed on their abundance in medical-unit and dental-unit water samples. The current study aimed to identify morphological and molecular characteristics of FLA isolates in the water samples of the dental unit in Iran. A total of 232 water samples were collected from 17 dental units. Then, filtration and cultivation were conducted on a non-nutrient agar (NNA) medium. Also, polymerase chain reaction (PCR) assay and sequencing were performed by using the genus/species-specific primers plus a common primer set on positive samples. One hundred and sixty-six samples were positive for FLA by the microscopic method, whereas 114 samples were positive by the molecular method with a common primer set. Considering the PCR assay with genus/species-specific primers, 23.27% (54/232) samples were identified as Acanthamoeba spp. (belonging to T4 genotype), 36.63% (85/232) as Vermamoeba vermiformis, and 1.72% (4/232) as Vahlkampfiidae family (Naegleria lovaniensis). These results highlight a need to improve filtration systems in dental units and periodic screenings for FLA in dental-unit water. [ABSTRACT FROM AUTHOR]

Free-living amoebas (FLAs) can cause neurological and ocular complications in humans. Water supplies play a critical role in transmitting FLAs to humans. The aim of the present study was to investigate the presence of FLAs in various aquatic sources including drinking water, stagnant water, and surface water in Alborz province, northern Iran, using morphological and molecular techniques. A total of 70 water samples were collected from 34 drinking waters, 23 surface waters, and 13 stagnant waters. Filtration and cultivation were employed to isolate FLAs. PCR assay was applied by using the genus-specific primers on positive samples. Pathogenicity tests (osmo-and thermo-tolerance properties) were performed for Acanthamoeba spp., positive sample. Considering the morphological criteria, four positive samples of Acanthamoeba sp., three Vermamoeba sp., two mixed Vermamoeba sp. with Vahlkamfiids, and one mixed Acanthamoeba sp. with Vahlkamfiids were isolated. Five Acanthamoeba sp. isolates were amplified using the JDP primer pairs. Among them, two genotypes, T4 (three isolates) and T5 (two isolates) corresponding to A. lenticulata, were identified. Four V. vermiformis samples were confirmed using the sequencing. This study highlighted the occurrence of potentially pathogenic waterborne FLAs in water habitats associated with high human activity. The results of such research on the prevalence of FLAs, as a human hazard, should be communicated to health policymakers. [ABSTRACT FROM AUTHOR]

Free-living amoebas (FLAs) can cause severe disease in humans and animals when they become infected. However, there are no accurate survey reports on the prevalence of FLAs in Korea. In this study, we collected 163 tap water samples from buildings, apartments, and restrooms of highway service areas in 7 Korean provinces with high population density. All these buildings and facilities have water storage tanks in common. The survey was separated into categories of buildings, apartments, and highway service areas. Five hundred milliliters of tap water from each building was collected and filtered with 0.2 µm pore filter paper. The filters were incubated in agar plates with heated E. coli at 25°C. After axenization, genomic DNA was collected from each FLA, and species classification was performed using partial 18S-rDNA PCR-sequencing analysis. We found that 12.9% of tap water from buildings with storage tanks in Korea was contaminated with FLAs. The highway service areas had the highest contamination rate at 33.3%. All of the FLAs, except one, were genetically similar to Vermamoeba vermiformis (Hartmannella vermiformis). The remaining FLA (KFA21) was very similar to Acanthamoeba lugdunensis (KA/E26). Although cases of human infection by V. vermiformis are very rare, we must pay attention to the fact that one-third of tap water supplies in highway service areas have been contaminated. [ABSTRACT FROM AUTHOR]

Free-living amoebas (FLAs) are facultative parasites, and some of them can be highly pathogenic for animals and humans. These protozoan are found in abundance in environmental resources such as freshwater and soil, and thus the possible colonization of such FLA is probable. Our previous studies showed that Acanthamoeba spp. can colonize nasal mucosa of immunosuppressed patients. However, there was no report of colonization of free-living amoebae in healthy individuals in Iran. The present study was conducted to investigate the possibility that FLAs are the normal flora of the nasal mucosa. Overall, 46 nasal swab samples were collected from healthy individuals and cultured on a 1.5% non-nutrient agar (NNA) plate enriched with a lawn of heatkilled Escherichia coli. The plates were incubated at room temperature. After one week, the plates were microscopically examined, and this was continued for one month. None of the samples were found positive for FLAs. Our findings may indicate that FLAs are not the normal flora of the nasal mucosa, or their number is so limited in the nasal mucosa that they cannot be isolated by the method used. However, supplementary and comprehensive studies are recommended. [ABSTRACT FROM AUTHOR]

Naegleria fowleri, a free‐living amoeba found in soil and freshwater environments, is the causative agent of Primary Amoebic Meningoencephalitis. Infection occurs when amoebae enter the nasal cavity, attach to the nasal mucosa and travel along olfactory neurons towards the olfactory bulb. Upon reaching the central nervous system, the amoebae replicate very rapidly and can cause death in 3–10 days. Little is known about the conditions in which the amoeba can survive in the environment. We have tested conditions beyond the known boundaries on the viability of amoebae by introducing them into moderate and extreme salinity, pH, and temperatures. Our data shows that although viability expectedly decreases towards each of these extreme conditions, their tolerance was much greater than anticipated, including viability in moderate salinity, a wide pH range, and temperatures higher than the previously reported 45 °C. [ABSTRACT FROM AUTHOR]

A free-living soil amoeba Thecamoeba quadrilineata (Carter, 1856) Lepşi, 1960 (Amoebozoa: Thecamoebidae) was isolated from endozoic conditions for the first time. Presence of amoebae was detected after 4 days following inoculation of the gut of the earthworm Lumbricus terrestris on agar plate with Escherichia coli. On the basis of our isolation, we consider T. quadrilineata as further amphizoic amoeba species. This study enlarges the range of amphizoic tendency in members of the genus Thecamoeba and stresses the need for further research on the pathogenic potential of Thecamoeba species. [ABSTRACT FROM AUTHOR]

Balamuthia mandrillaris is a free-living amoeba that lives in soil and water near human settlements. B. mandrillaris was first isolated from a mandrill baboon that died at the San Diego Zoo Wildlife Park in California in 1986, and the first human infection was reported in 1990. Although reported B. mandrillaris infections are often not properly characterized, it appears that B. mandrillaris invades the living body from the soil and water, either via a wound or the nasal cavity. Most confirmed infections have originated in South and North America. B. mandrillaris inhabits warm climates and is recognized as a pathogen in warm areas such as desert climates and tropical climates. B. mandrillaris has been isolated from environmental samples since 2000, most of which originated from warm areas such as step climates, tropical climates, and desert climates. However, B. mandrillaris may survive in diverse environments, although fewer granulomatous amebic encephalitis patients have been reported in colder Japanese and Northern European regions. In the present study, we conducted a survey of 13 soil samples in Aomori Prefecture located at the northernmost tip of Japan Honshu and successfully isolated one strain of B. mandrillaris from soil for the first time in Japan. In addition, B. mandrillaris gene was detected from several soils. This confirms that B. mandrillaris is capable of spreading to a wider climatic region. [ABSTRACT FROM AUTHOR]

The recent outbreak of primary meningoencephalitis caused by free-living amoebae (FLA), Naegleria fowleri, has gained increasing attention due to their confirmed fatality. It is caused by the entrance of contaminated water into nasal passage mainly by ablution practices. The symptoms include severe headache, nausea, vomiting along with fever finally leading to death. FLA other than N. fowleri such as Acanthamoeba and Balamuthia species are also harmful because they are vectors of many bacterial pathogens including Vibrio, Pseudomonas, Legionella, Enterobacter and Mycobacterium species which help them to feed and colonize in environments, thus contributing to their pathogenesis and transferability to their hosts. Pakistan, being an underdeveloped country, faces long-term shortfalls of electricity resulting in serious water scarcity leading to public dependence on stored water resources, which are breeding hubs for FLA. The rationale of the present review is to highlight the importance of N. fowleri and primary meningoencephalitis and to investigate the recent outbreak in Pakistan. [ABSTRACT FROM AUTHOR]

Acanthamoeba castellanii is a free-living amoeba commonly found in aquatic environment. It feeds on bacteria even if some bacteria resist amoebal digestion. Thus, A. castellanii is described as a Trojan horse able to harbor pathogenic bacteria. L. pneumophila is one of the amoeba-resisting bacteria able to avoid host degradation by phagocytosis and to multiply inside the amoeba. When infecting its host, L. pneumophila injects hundreds of effectors via a type IV secretion system that change physiology of the amoeba to its profit. In this study, we assess mobility of A. castellanii upon infection with L. pneumophila . Electron-microscopy analysis of amoebae revealed a reduction of acanthopodia on cells infected with L. pneumophila . Analysis of velocity showed that migration of A. castellanii infected with L. pneumophila was significantly impaired compare to uninfected cells. Taken together, infection with L. pneumophila could prevent formation of cytoplasmic extensions such as acanthopodia with consequences on the shape, adherence and mobility of A. castellanii . [ABSTRACT FROM AUTHOR]

Background: Free-living amoebae have various genera that are found in several environmental niches such as soil, freshwater, dust, seawater and hotsprings. Most of Free-living amoebae are normally harmless to humans. However, some ameoba such as Acanthamoeba and also Naegleria fowleri, Balamuthia manderillaris and Sappinia are identified as opportunistic and pathogenic amoebae that can cause eye diseases, encephalitis, and meningoencephalitis in human. Vannellidae are a family of free-living amoebae and exist mainly in soil, freshwater, and marine habitats. This amoeba is nonpathogenic for human, but can act as a Trojan horse for other pathogens such as Microsporidia. The present study reports the occurrence of Vannella spp. in a hotspring of Amol city. Materials and Methods: 22 samples were taken from hotsprings of Mazandaran province during our previous study. The plates were checked for the presence of Vannella spp. according to the specific morphological criteria. DNA extraction, PCR and sequencing was performed on the positive isolate. Results: The result showed that one plate contained fan-shaped amoebae suspected to Vannella spp. PCR analysis and sequencing was confirmed the occurrence of Vannella spp. in one sample of a hot spring of Amol, northern Iran. Conclusion: The result confirmed the presence of Vannella amoebae in the hotspring of Amol city. More studies are needed to clarify the real distribution of Vannella spp. in environmental niches and its pathogenic potential in Iran and worldwide. [ABSTRACT FROM AUTHOR]

We describe a new protosteloid amoeba, Luapeleamoeba hula. Protosteloid amoebae, sometimes called protostelids, are sporocarpic amoebae that make fruiting bodies that consist of a stalk and one to a few spores. This new taxon was cultured from dead leaves of mamaki (Pipturus albidus) from the Manuka Natural Area Reserve, Hawaii, USA. Light microscopic examination showed that this amoeba has a short, rigid stalk with a small apophysis and a spore that changes shape continuously until it is shed and crawls away from the stalk. In addition, this amoeba was initially observed to maintain a diurnal rhythm in which fruiting body formation occurred primarily in the late afternoon. This new species is unique in both its amoebal and fruiting body morphology. Spore deciduousness appears to be a result of shape changes in the spore itself. This is the fourth species of protosteloid amoeba described with a clearly described diurnal rhythm. In addition, previous molecular phylogenetic analyses suggested that this new species has SSU rRNA gene sequences that clearly separate it from any other protosteloid amoebae and place it as sister to Protacanthamoeba bohemica among the Acanthamoebidae family in Centramoebida of Amoebozoa. Because this new amoeba species does not fit into any of the centramoebid genera, we have proposed a new genus Luapeleamoeba. [ABSTRACT FROM AUTHOR]

The present study was conducted to determine the occurrence of potentially pathogenic free-living amoeba in ophthalmology wards in reference hospitals in Iran. Since an increasing number of Acanthamoeba Keratitis cases after eye surgery and eye trauma have been recently observed in this country, it could be possible that the disinfection procedures undertaken in the clinical setting may not have a good hygiene and disinfection procedures, hence the aim of this study. Therefore, 42 dust and biofilm samples were collected from different areas of ophthalmology wards and checked for the presence of FLA using morphological criteria, PCR based analysis and DNA sequencing. Of the 42 samples from dust and biofilm sources, 18(42.86%) isolates were found to contain FLA and 12(92.3%) isolates belonged to Acanthamoeba T4 genotype. Isolation of the pathogenic genotype T4 from medical instruments, including slit lamp in corneal wards, may be a threat for patients undergoing eye surgery in these wards. Other FLA isolated in this study included Acanthamoeba genotype T5, Vahlkampfia sp, Naegleria australiensis, Vermamoeba vermiformis and Echinamoeba exudans. To our knowledge, this is the first report of the presence of potentially pathogenic FLA in ophthalmology wards in Iran. Improved disinfection methods and monitoring of hospitals ward are thus necessary in this area in order to minimize the risk of infection in patients. [ABSTRACT FROM AUTHOR]

Vermamoeba vermiformis is a free-living amoeba (FLA) widely distributed in the environment, known to colonize hot water networks and to be the reservoir of pathogenic bacteria such as Legionella pneumophila. FLA are partly resistant to biocides, especially in their cyst form. The control of V. vermiformis in hot water networks represents an important health issue, but there are very few data on their resistance to disinfection treatments. The sensitivity of cysts of two strains of V. vermiformis to three disinfectants frequently used in hot water networks (chlorine, heat shock, peracetic acid (PAA) mixed with hydrogen peroxide (H2O2)) was investigated. In vitro, several concentrations of biocides, temperatures and exposure times according to the French regulation were tested. Cysts were fully inactivated by the following conditions: 15 mg/L of chlorine for 10 min; 60 °C for 30 min; and 0.5 g/L equivalent H2O2 of PAA mixed with H2O2 for 30 min. For the first time, the strong efficacy of subtilisin (0.625 U/mL for 24 h), a protease, to inactivate the V. vermiformis cysts has been demonstrated. It suggests that novel approaches may be efficient for disinfection processes. Finally, V. vermifomis cysts were sensitive to all the tested treatments and appeared to be more sensitive than Acanthamoeba cysts. [ABSTRACT FROM AUTHOR]

In addition to their role as parasites, free-living amoebae (FLA) can act as hosts of and vehicles for phylogentically diverse microorganisms while some of them replicate intracellularly. These microorganisms are adapted to the intracellular conditions in the amoeba, find suitable conditions and protection from negative environmental influences and take advantage of the dispersal in the environment by their amoebic host. It is expedient to call these organisms 'endocytobionts', at least during the initial steps of any studies. By doing so, it is not necessary to go into potential characteristics of these relationships such as parasitism, phoresy, zoochory, or mutualism at an early stage of study. Among those organisms resisting the lysis within their amoebic host, there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals. FLA-endocytobiont relationships are not only important for the tenacity of the involved microorganisms. Especially if FLA are present in biofilms and there are close ties with many other microorganisms, the odds are for some of these microorganisms to develop human pathogenic properties. Here, the amoebic passage seems to be a prerequisite for the development of virulence factors and it may have an impact on evolutionary processes. [ABSTRACT FROM AUTHOR]

Aims: Isolation and characterization of the clinically relevant amphizoic amoebas in vegetated farmlands, which may present a risk to farmers’ health. Methods and Results: Acanthamoeba species was isolated and characterized via morphological and molecular means in the rice field where the patient was exposed to rice paddy water which most probably was the point of infection. An Acanthamoeba sp. abundant in the rice field was identified. Genotyping showed the strain to be genotype T4, which was identical to the amoebic parasite found in patient’s cerebrospinal fluid. During the course of the study, three nonpathogenic free-living amoeba species were also isolated and characterized for the first time in Taiwan. Conclusions: This study successfully located a possible source of granulomatous amoebic encephalitis in a patient and provided the first evidence that Acanthamoeba genotype T4 may be a potential pathogen in Taiwan. Significance and Impact of the Study: The integration of field survey, clinical data and morphological and genetic examination represents a sound strategy for investigation of the possible role of free-living amoebae in causing human diseases. Future work should include investigating the potential contributory role of other nonpathogenic free-living protozoa in disease of livestock or even human. [ABSTRACT FROM AUTHOR]

Free-living amoebae of the genus Acanthamoeba are widely distributed in soil and water collections, where trophozoites (vegetative, multiplicative stages) feed mainly by phagocytosis and thus control bacterial populations in the environment. Here, we examined the growth, encystment and survival of Acanthamoeba castellanii receiving different bacteria ( Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, and Staphylococcus aureus) in nonnutrient saline. All bacteria assayed induced a dose-dependent proliferative response, in most cases maximized with a bacterial dose of 1 × 109 mL−1; except for M. luteus, trophozoites grew better with viable than with heat-killed bacteria. In addition, Acanthamoeba growth was improved by adding bacteria on alternate days. Single-dose experiments indicated a temporal association between the growth of trophozoite and bacterial consumption, and higher consumption of M. luteus, E. coli and P. aeruginosa, bacterial species that allowed the highest trophozoite yields. Long-term Acanthamoeba–bacteria incubation revealed that encystment was significantly delayed by almost all the bacteria assayed (including S. aureus, which elicited a poor growth response) and that the presence of bacteria markedly increased cyst yield; final cyst recovery clearly depended on both the dose and the type of the bacterium given, being much higher with E. coli, M. luteus and P. aeruginosa. [ABSTRACT FROM AUTHOR]

Free-living amoebae can serve a great variety of organisms, predominantly bacteria but to a certain extent also fungi, as a suitable host supplying them with nutrients and protecting them from adverse environmental conditions. In the current study 18S rDNA sequencing was performed to identify a fungal parasite in a Thecamoeba quadrilineata isolate. This parasite morphologically resembled Cochlonema euryblastum, a member of the order Zoopagales, which comprises parasitic species on fungi and invertebrates. Sequence analysis corroborated the morphological identification and the fungal parasite clearly can be assigned to the Zoopagales. Phylogenetic analysis revealed C. euryblastum clustering with two representatives of the mycoparasitic family Piptocephalidaceae. This zooparasitic-mycoparasitic clade represents a sister group of a clade including another member of the Piptocephalidaceae and two other zooparasitic families. Thus, the addition of C. euryblaslum to the zoopagalean tree further confirms the finding that molecular data do not support the traditional classification of the Zoopageles. [ABSTRACT FROM AUTHOR]

Free-living amoeba (FLA) are ubiquitously distributed in the environment. However, they are also the causative agents of opportunistic infections in humans and other animals. A biofilm comprises any syntrophic consortium of microorganisms in which cells stick to each other and often also to a surface. Moreover, FLA have been detected in various biofilms around the world. Therefore, the present study aimed to check for presence of FLA in samples from household biofilms in Iran and to characterize them at the molecular level. A total of 69 biofilm samples collected from showerheads, kitchen areas, and bathroom sinks were analyzed. Positive samples for FLA were characterized at the morphological and molecular levels. Furthermore, the results of morphology analysis indicated that 26.08% (18/69) of biofilm samples were positive for Acanthamoeba spp., Vermamoeba genus, and Vahlkampfiids. According to sequence analysis, five strains of Acanthamoeba isolates related to the T4 genotype and two strains belonged to the T2 genotype. In addition, the pathogenic potential of Acanthamoeba-positive isolates was conducted using the tolerance ability test. The results of BLASTn of Vermamoeba sequences were similar to what was expected for Vermamoeba vermiformis. The above-mentioned reasons revealed that the relative high contamination of household biofilm samples with FLA may pose a risk for people using soft contact lenses and for patients with traumatic cataract. Our finding proposes that filtration should be performed in shower heads and indicates the need to monitor people at increased risk of Acanthamoeba keratitis. [ABSTRACT FROM AUTHOR]

The genus Naegleria, of the free-living amoeba (FLA) group, has been investigated mainly due to its human health impact, resulting in deadly infections and their worldwide distribution on freshwater systems. Naegleria fowleri, colloquially known as the "brain-eating amoeba," is the most studied Naegleria species because it causes primary amoebic meningoencephalitis (PAM) of high lethality. The assessment of FLA biodiversity is fundamental to evaluate the presence of pathogenic species and the possibility of human contamination. However, the knowledge of FLA distribution in Brazil is unknown, and to rectify this situation, we present research on identifying Naegleria spp. in the Monjolinho River as a model study. The river is a public Brazilian freshwater source that crosses the city of São Carlos, in São Paulo state, Brazil. Five distinct sampling sites were examined through limnological features, trophozoites culturing, and PCR against internal transcribed spacer (ITS) regions and 5.8S rRNA sequences. The results identified N. philippinensis, N. canariensisi, N. australiensis, N. gruberi, N. dobsoni sequences, as well as a Hartmannella sequence. The methodology delineated here represents the first Brazilian Naegleria spp. study on a freshwater system. Our results stress the urgency of a large scale evaluation of the presence of free-living amoebas in Brazil. [ABSTRACT FROM AUTHOR]