Search Results (67)

Primary amoebic meningoencephalitis (PAM) is a rapidly progressive and fulminant disease that affects the central nervous system caused by the free-living amoeba Naegleria fowleri. The adhesion to extracellular matrix (ECM) proteins is considered as one of the key steps in the success of the infection and could represent an interesting target to be explored in the prevention and treatment of the disease. In this work, the effect of two sesquiterpenes with proven anti-Naegleria activity on the adhesion of the parasite was evaluated using an in vitro ECM-based model, compared with the reference drugs amphotericin B and staurosporine. Both laurinterol and (+)-elatol inhibited the adhesion of the N. fowleri trophozoites to the main proteins of the ECM when treating them at different concentrations and exposure times. This work not only reinforces the therapeutic potential of laurinterol and (+)-elatol against N. fowleri infection but also introduces the application of ECM-based adhesion assays as a novel and valuable tool for screening candidate compounds that disrupt host–pathogen interactions critical to PAM pathogenesis. 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

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

Balamuthia mandrillaris is a free-living amoeba pathogenic to humans, causing amoebic granulomatous encephalitis (GAE). Due to the associated mortality rates of <95%, the absence of treatments, and a clear understanding of the pathogenesis of this amoeba, Lippia graveolens could be an interesting alternative since it has been used against bacteria, fungi, and other pathogenic protozoa. This study employed RNA sequencing to analyze differentially expressed genes (DEGs), following treatment with two fractionated L. graveolens extracts (concentration: 150 µg/mL) at 48, 96, and 120 h. The DEGs identified are associated with several functions such as stress responses (Prohibitin domain-containing protein), and oxidative damage repair and cell stability (Peroxiredoxin). Genes implicated in virulence and host interaction also showed significant expression changes, such as the ADP ribosylation factor (Arf) GTPase and ephrin type-A receptor, alongside transcription factors involved in the phagocytosis of amoebas. Additionally, the analysis of Gene Ontology categories revealed terms including transmembrane signaling receptor and protein tyrosine activity, DNA replication initiation, the mitotic M phase, and membrane integrity. These results provide valuable insights into the molecular mechanisms utilized by B. mandrillaris to respond to environmental stressors and the repression of genes related to essential functions, which could serve as potential targets for developing novel strategies. 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

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

The Tajogaite Volcano erupted on the western slope of the Cumbre Vieja mountain range on La Palma Island in the Canary Islands, Spain, in 2021. As one of the multiple consequences of this eruption, a layer of tephra was deposited, to a variable extent, over a large part of the island. Tephra deposits affect all aspects of vegetation recovery, the water cycle, and the long-term availability of volcanic nutrients. Protozoa, including free-living amoeba (FLA), are known to be among the first microorganisms capable of colonizing harsh environments. In the present study, the presence of FLA has been evaluated in the Tajogaite Volcano deposits. Samples of the tephra were collected and incubated at 26 °C on 2% non-nutrient agar plates with a layer of heat-killed E. coli. Morphological features, as well as the DF3 region sequence of the 18S rDNA, confirmed the presence of a T4 genotype strain of Acanthamoeba. Thermotolerance and osmotolerance assays were used to evaluate the strain’s pathogenic potential. This strain was considered thermotolerant but poorly osmotolerant. To the best of our knowledge, this is the first report of Acanthamoeba being isolated from a recently erupted volcano. Full article

Balamuthia mandrillaris is a free-living amoeba (FLA) that causes granulomatous amebic encephalitis (GAE) and skin lesions. Transcriptomic analysis is a powerful tool used to study B. mandrillaris pathogenic infections. However, preliminary tests of RNA extraction showed poor results, so it has become essential to standardize a protocol for high-quality RNA. The present study evaluated 11 RNA extraction protocols based on three commercial kits by making modifications to the temperature and centrifugation times, and by combining kits. Four protocols, namely Q3 (based on QIAGEN RNeasy Mini Kit, with modifications in temperature and centrifugation times), T1 (Invitrogen TRIzol Reagent), T2 (combination of TRIzol and QIAGEN modified protocols) and T3 (combination of TRIzol and PROMEGA SV Total RNA Isolation protocols), presented RNA with good integrity and purity, except for the T1 protocol, which obtained an A260/230 value below the acceptable threshold. High RNA integrity (RIN) values were obtained with the Q3 (9.8), T2 (9.2), and T3 (8.9) protocols, while the T1 protocol obtained a lower RIN value (7.1). The Q3, T2, and T3 protocols obtained high-quality RNA from B. mandrillaris based on the criteria of integrity, purity, and concentration, where the implemented modifications and combinations raised the quality; thus, their use is recommended to obtain accurate results when performing transcriptomic analysis. Full article

Acanthamoeba, a widely distributed free-living amoeba found in various environments, is an opportunistic pathogen responsible for causing Acanthamoeba keratitis, a condition that may lead to blindness. However, identifying the pathogenicity of Acanthamoeba is challenging due to its complex life cycle, ability to adapt to different environments, variable virulence factors, and intricate interactions with the host immune system. Additionally, the development of an effective model for studying Acanthamoeba pathogenicity is limited, hindering a comprehensive understanding of the mechanisms underlying its virulence and host interactions. The aim of this study was to develop an ex vivo model for Acanthamoeba infection using porcine eyeballs and to evaluate the pathogenicity of the Acanthamoeba isolates. Based on slit lamp and biopsy analysis, the developed ex vivo model is capable of successfully infecting Acanthamoeba within 3 days. Histopathological staining revealed that clinical isolates of Acanthamoeba exhibited greater corneal stroma destruction and invasion in this model than environmental isolates. Our results highlight the importance of an ex vivo porcine eye model in elucidating the pathogenesis of Acanthamoeba infection and its potential implications for understanding and managing Acanthamoeba-related ocular diseases. Full article

Legionella pneumophila is a freshwater opportunistic pathogen and the leading cause of severe pneumonia known as Legionnaires’ disease. It can be found in all water systems and survives in biofilms, free-living amoebae, and a wide variety of facilities, such as air conditioning and showers in hospitals, hotels and spas. The reference cultural method allows for the isolation and identification in many days, and in addition, it does not detect viable but rather non-culturable bacteria, increasing the risk of infection. In this context, a new LAMP-based (loop-mediated isothermal amplification) kit was developed, allowing for the rapid, sensitive, and labor-saving detection of L. pneumophila. The kit, “Legionella pneumophila Glow”, was validated according to ISO/TS 12869:2012, testing sensitivity, inclusivity and exclusivity, and kit robustness. Sensitivity showed that the “Legionella pneumophila Glow” kit can detect up to 28 plasmid copies/µL. Robustness tests showed consistent results, with both contamination levels and the matrices used giving reproducible results. Furthermore, real samples were evaluated to compare the performance of the two methods. The LAMP kit “Legionella pneumophila Glow” proved a useful option for the rapid, efficient, and labor-saving screening of different typologies of water samples, offering significant advantages over the traditional method, as it is characterized by a high sensitivity, ease of use for laboratory testing, and a large reduction in analysis time, making it an asset to official controls. Full article

In contrast to “frank” pathogens, like Salmonella entrocolitica, Shigella dysenteriae, and Vibrio cholerae, that always have a probability of disease, “opportunistic” pathogens are organisms that cause an infectious disease in a host with a weakened immune system and rarely in a healthy host. Historically, drinking water treatment has focused on control of frank pathogens, particularly those from human or animal sources (like Giardia lamblia, Cryptosporidium parvum, or Hepatitis A virus), but in recent years outbreaks from drinking water have increasingly been due to opportunistic pathogens. Characteristics of opportunistic pathogens that make them problematic for water treatment include: (1) they are normally present in aquatic environments, (2) they grow in biofilms that protect the bacteria from disinfectants, and (3) under appropriate conditions in drinking water systems (e.g., warm water, stagnation, low disinfectant levels, etc.), these bacteria can amplify to levels that can pose a public health risk. The three most common opportunistic pathogens in drinking water systems are Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa. This report focuses on these organisms to provide information on their public health risk, occurrence in drinking water systems, susceptibility to various disinfectants, and other operational practices (like flushing and cleaning of pipes and storage tanks). In addition, information is provided on a group of nine other opportunistic pathogens that are less commonly found in drinking water systems, including Aeromonas hydrophila, Klebsiella pneumoniae, Serratia marcescens, Burkholderia pseudomallei, Acinetobacter baumannii, Stenotrophomonas maltophilia, Arcobacter butzleri, and several free-living amoebae including Naegleria fowleri and species of Acanthamoeba. The public health risk for these microbes in drinking water is still unclear, but in most cases, efforts to manage Legionella, mycobacteria, and Pseudomonas risks will also be effective for these other opportunistic pathogens. The approach to managing opportunistic pathogens in drinking water supplies focuses on controlling the growth of these organisms. Many of these microbes are normal inhabitants in biofilms in water, so the attention is less on eliminating these organisms from entering the system and more on managing their occurrence and concentrations in the pipe network. With anticipated warming trends associated with climate change, the factors that drive the growth of opportunistic pathogens in drinking water systems will likely increase. It is important, therefore, to evaluate treatment barriers and management activities for control of opportunistic pathogen risks. Controls for primary treatment, particularly for turbidity management and disinfection, should be reviewed to ensure adequacy for opportunistic pathogen control. However, the major focus for the utility’s opportunistic pathogen risk reduction plan is the management of biological activity and biofilms in the distribution system. Factors that influence the growth of microbes (primarily in biofilms) in the distribution system include, temperature, disinfectant type and concentration, nutrient levels (measured as AOC or BDOC), stagnation, flushing of pipes and cleaning of storage tank sediments, and corrosion control. Pressure management and distribution system integrity are also important to the microbial quality of water but are related more to the intrusion of contaminants into the distribution system rather than directly related to microbial growth. Summarizing the identified risk from drinking water, the availability and quality of disinfection data for treatment, and guidelines or standards for control showed that adequate information is best available for management of L. pneumophila. For L. pneumophila, the risk for this organism has been clearly established from drinking water, cases have increased worldwide, and it is one of the most identified causes of drinking water outbreaks. Water management best practices (e.g., maintenance of a disinfectant residual throughout the distribution system, flushing and cleaning of sediments in pipelines and storage tanks, among others) have been shown to be effective for control of L. pneumophila in water supplies. In addition, there are well documented management guidelines available for the control of the organism in drinking water distribution systems. By comparison, management of risks for Mycobacteria from water are less clear than for L. pneumophila. Treatment of M. avium is difficult due to its resistance to disinfection, the tendency to form clumps, and attachment to surfaces in biofilms. Additionally, there are no guidelines for management of M. avium in drinking water, and one risk assessment study suggested a low risk of infection. The role of tap water in the transmission of the other opportunistic pathogens is less clear and, in many cases, actions to manage L. pneumophila (e.g., maintenance of a disinfectant residual, flushing, cleaning of storage tanks, etc.) will also be beneficial in helping to manage these organisms as well. Full article

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. Full article

Chlorine and solar disinfection are widely used disinfectants in water treatment. However, certain potential pathogens can resist these methods, posing a public health risk. One such case is Acanthamoeba, a resistant free-living amoeba that protects pathogens inside from disinfection, thus endangering the health of water users. This work is the first evaluation of the inactivation efficiency achieved by combining NaClO (Cl₂) and solar radiation (SR) against two Acanthamoeba strains from different sources (freshwater and pool water) and their endosymbiont bacteria (EB). Amoebae were exposed to different Cl₂ doses (0–500 mg/L), SR wavelength ranges (280–800 nm and 320–800 nm), used as gold standards, and their combinations. The EB exhibited resistance to conventional Cl₂ and SR treatments, requiring up to 20 times higher disinfectant doses than those needed to inactivate their protective Acanthamoeba. The pool strain and its EB demonstrated greater resistance to all treatments compared to the freshwater strain. Treatments with Cl₂ (5 mg/L)/SR_280–800nm completely inactivated both Acanthamoeba and EB of the freshwater strain, reducing up to 100 times the necessary Cl₂ doses, suggesting that chlorine photolysis is an attractive treatment for disinfecting freshwater and preventing waterborne diseases associated with Acanthamoebae and its EB. Full article