Naegleria Australiensis Research Articles

The presence of yeasts and bacteria in free-living amoebae isolated from COVID-19 patients: concern for secondary infections

ABSTRACT This study aimed to investigate the presence of SARS-CoV-2, yeasts, and bacteria in isolated free-living amoeba (FLA) from COVID-19 patients. Nasopharyngeal swabs (n = 60) were obtained from COVID-19 patients. After cultivation, morphological characterization, and RNA/DNA extraction, the presence of selected microorganisms was investigated. From 60 COVID-19 samples, 18 (30%) were positive for FLA. Acanthamoeba sp. Naegleria australiensis, Tetramitus sp. and Vermamoeba vermiformis were characterized in 12 (80%), 1 (6.66%), 2 (13.33%), and 7 (38.88%) of samples, respectively. SARS-CoV-2 RNA was not detected in FLA. Candida albicans, C. tropicalis, and C. parapsilosis were detected in (11/18; 61.11%), (3/18; 16.67%), and (3/18; 16.67%) of samples, respectively. Geotrichum candidum was detected in 10/18 (55.55%) of samples. Streptococcus spp. and Staphylococcus spp. were identified in 16/18 (88.88%) and 3/18 (16.67%), respectively. The presence of yeasts and bacteria signifies the possible role of FLA in distribution of secondary infections in susceptible patients.

Fatal gastric amebiasis in a Linnaeus's two-toed sloth associated with Naegleria australiensis infection.

Here we describe a case of fatal amebic gastritis associated with Naegleria australiensis infection in an 11-mo-old Linnaeus's two-toed sloth (Choloepus didactylus). The sloth had a history of weight loss and intermittent diarrhea for 18 d, and subsequently died despite empirical treatment. Postmortem findings included emaciation, gastric dilation with fluid content, and fibrinonecrotic gastritis with intralesional amebic trophozoites and cysts in the glandular region of the fundus. Transmission electron microscopy ruled out Amoebozoa of the family Entamoebidae based on the presence of mitochondria in the amoeboid organisms. PCR for pan-free-living amebae followed by next-generation sequencing of the PCR product revealed 99% identity with Naegleria australiensis. Gastric amebiasis has been reported sporadically in macropods and in leaf-eating monkeys with a sacculated stomach. To our knowledge, gastric amebiasis has not been reported previously in a sloth, which also has a sacculated and multi-chambered stomach.

Naegleria australiensis isolated from a wastewater treatment station in Santiago Island, Cape Verde.

Despite the Naegleria genus being isolated from different natural environments such as water, soil, and air, not all Naegleria species are capable of causing infections in humans, and they are capable of completing their life cycle in environmental niches. However, the presence of this genus may suggest the existence of one of the highly pathogenic free-living amoeba (FLA) species: Naegleria fowleri or the brain-eating amoeba. This facultative parasitic protozoon represents a risk to public health, mainly related to domestic and agricultural waters. In this research, our main objective was to determine the existence of pathogenic protozoa in the Santa Cruz wastewater treatment plant, Santiago Island. Using 5 L of water we confirmed the presence of potentially pathogenic Naegleria australiensis, being the first report on Naegleria species in Cape Verde. This fact demonstrates the low efficiency in the treatment of wastewater and, consequently, a potential threat to public health. Nevertheless, more studies will be needed for the prevention and control of possible infections in this Macaronesian country.

Environmental water surveillance for free-living amoeba in North India

PurposeFree-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 methodsSurface 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. ResultsFLA 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. ConclusionsThe 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.

Isolation and identification of potentially pathogenic free-living amoeba in dialysis fluid samples of hydraulic systems in hemodialysis units.

Free-living amoeba (FLA), including Acanthamoeba, Naegleria, Balamuthia and Vermamoeba, have been isolated from water, sand, soil, dust and air. Numerous studies considered that FLA are a significant cause of neurological and ocular complications in high-risk groups, including immunocompromised individuals. The present study aimed to identify morphological and molecular characteristics of FLA isolates in dialysis fluid samples of hydraulic systems in hemodialysis units in Iran. A total of 328 dialysis fluid samples were collected from 16 dialysis machines, including 164 samples before hemodialysis sessions (after cleaning) and 164 samples after hemodialysis sessions (before cleaning). Filtration and cultivation were performed on non-nutrient agar medium. Also, PCR and sequencing were applied by using the genus-specific primers along with a common primer set on positive samples. Both morphology and molecular investigations showed that 22.5% (74/328) of dialysis fluid samples were positive for FLA. There was a positive relationship between the high frequency of FLA after hemodialysis sessions (before cleaning) compared with before hemodialysis sessions (after cleaning) (OR=2.86; 95% CI 1.5 to 5.45). Considering the PCR assay, 16.46% (54/328) samples were identified as Acanthamoeba spp. (belonging to T3 and T4 genotypes), 5.18% (17/328) as Vermamoeba vermiformis and 0.91% (3/328) as Vahlkampfiidae family (Naegleria australiensis, Naegleria pagei and Allovahlkampfia). The present results support a need to improve filtration and purification methods for dialysis fluid of hydraulic systems in hemodialysis units. They also highlight the relevance of periodic screenings for FLA-related diseases in hemodialysis patients.

Free living amoebae isolation in irrigation waters and soils of an insular arid agroecosystem

The use of freshwater in agricultural systems represents a high percentage of total water consumption worldwide. Therefore, alternative sources of water for irrigation will need to be developed, particularly in arid and semi-arid areas, in order to meet the growing demand for food in the future. The use of recycled wastewater (RWW), brackish water (BW) or desalinated brackish water (DBW) are among the different non-conventional water resources proposed. However, it is necessary to evaluate the health risks for humans and animals associated with the microbiological load of these waters. Protozoa such as free-living amoebae (FLA) are considered an emerging group of opportunistic pathogens capable to cause several diseases in humans (e.g. cutaneous and ocular infections, lung, bone or adrenal gland conditions or fatal encephalitis). In the present study we evaluate FLA presence in three different irrigation water qualities (RWW, BW and DBW) and its survival in irrigated agricultural soils of an extremely arid insular ecosystem (Fuerteventura, Canary Islands, Spain). Samples were cultured on 2% Non-Nutrient Agar (NNA) plates covered with a thin layer of heat killed E. coli and checked daily for the presence of FLA. According to the prevalence of FLA, Vermamoeba vermiformis (53,8%), Acanthamoeba spp. (30,8%), Vahlkampfia avara (7,7%) and Naegleria australiensis (7,7%) were detected in the analysed water samples, while Acanthamoeba (83,3%), Cercozoa spp. (8,3%) and Vahlkampfia orchilla (8,3%) were isolated in irrigated soils. Only Acanthamoeba strains were isolated in no irrigated soils used as control, evidencing the capability of these protozoa to resist environmental harsh conditions. Additionally, all analysed water sources and the irrigated soils presented growth of several pathogenic bacteria. Therefore, the coexistence in water and soils of pathogenic bacteria and FLA, can mean an increased risk of infection in agroecosystems.

Metagenomic analysis of viruses, bacteria and protozoa in irrigation water.

Viruses (e.g., noroviruses and hepatitis A and E virus), bacteria (e.g., Salmonella spp. and pathogenic Escherichia coli) and protozoa (e.g., Cryptosporidium parvum and Giardia intestinalis) are well-known contributors to food-borne illnesses linked to contaminated fresh produce. As agricultural irrigation increases the total amount of water used annually, reclaimed water is a good alternative to reduce dependency on conventional irrigation water sources. European guidelines have established acceptable concentrations of certain pathogens and/or indicators in irrigation water, depending on the irrigation system used and the irrigated crop. However, the incidences of food-borne infections are known to be underestimated and all the different pathogens contributing to these infections are not known. Next-generation sequencing (NGS) enables the determination of the viral, bacterial and protozoan populations present in a water sample, providing an opportunity to detect emerging pathogens and develop improved tools for monitoring the quality of irrigation water. This is a descriptive study of the virome, bacteriome and parasitome present in different irrigation water sources. We applied the same concentration method for all the studied samples and specific metagenomic approaches to characterize both DNA and RNA viruses, bacteria and protozoa. In general, most of the known viral species corresponded to plant viruses and bacteriophages. Viral diversity in river water varied over the year, with higher bacteriophage prevalences during the autumn and winter. Reservoir water contained Enterobacter cloacae, an opportunistic human pathogen and an indicator of fecal contamination, as well as Naegleria australiensis and Naegleria clarki. Hepatitis E virus and Naegleria fowleri, emerging human pathogens, were detected in groundwater. Reclaimed water produced in a constructed wetland system presented a virome and bacteriome that resembled those of freshwater samples (river and reservoir water). Viral, bacterial and protozoan pathogens were occasionally detected in the different irrigation water sources included in this study, justifying the use of improved NGS techniques to get a comprehensive evaluation of microbial species and potential environmental health hazards associated to irrigation water.

Occurrence and the first report of Naegleria australiensis presence in a major lake in the Philippines.

Laguna de Bay or Laguna Lake is one of the six major lakes in the Philippines to be in close contact with population activities due to the expansion of urban settlements in the immediate cities surrounding the lake, thus pushing the population to settle upon its shores. To date, there are no data showing the biodiversity of free-living amoebae (FLA) present in this lake. The present study aims to isolate and identify the FLA present in Laguna de Bay, Philippines. Thirty subsurface water samples were taken from Laguna De Bay using random purposive sampling in May 2018 and were examined for amoebic growth under light microscopy (LM). Results show that 8 out of 30 (26.6%) water samples were positive for amoebic growth and were further tested for more advanced data and genetic variation of the species. Initial molecular analysis using polymerase chain reaction (PCR) and sequencing showed the presence of potentially pathogenic FLA Naegleria australiensis (MK418954). The detection of potential pathogenic FLA in lakes and dams may prove useful in preventing and controlling possible human infections in the country. More data from this study will aid in public awareness and establishing safety guidelines and control programs.

Isolation and Molecular Identification of Naegleria australiensis in Irrigation Water of Fuerteventura Island, Spain.

Saline groundwater desalination has recently emerged as an alternative source of irrigation water in arid and semiarid regions due to the gradual reduction in the quantity and quality of conventional water resources for agricultural use. In Fuerteventura Island (Spain), an extremely arid territory in the European Union, brackish water desalination is one of the few available water sources for agricultural production. Very little research has been conducted on the microbiological quality of this water mainly used for irrigation of vegetable crops. Free-living amoebae (FLA) are widely distributed protozoa in the environment and have been isolated from many environmental sources such as dust, soil and water. Among the pathogenic genera included in this group, Acanthamoeba spp., Naegleria fowleri and Balamuthia mandrillaris have been reported to be causative agents of lethal encephalitis, disseminated infections and keratitis. Particularly, Naegleria fowleri is a pathogenic FLA species which causes primary amoebic meningoencephalitis (PAM). In the present study, the presence of pathogenic FLA strains on desalinated brackish water samples for irrigation has been evaluated during 7months. From the analysed samples, only one was positive for Naegleria australiensis. This is the first report of Naegleria spp. in desalinated brackish water for irrigation in Spain.

Identification and phylogenetic position of Naegleria spp. from geothermal springs in Italy

Naegleria spp. are free-living amoebae belonging to the family Vahlkampfiidae, in the class Heterolobosea. Among the recognized species, Naegleria fowleri causes primary amoebic meningoencephalitis (PAM), while two other species, Naegleria australiensis and Naegleria italica, have been reported as pathogenic in experimental animals. Due to the thermotolerance properties of some species, geothermal water sources including hot springs represent suitable habitats for their proliferation. The main aim of this study was a year-round sampling in two geothermal springs in Central Italy, to investigate the presence of Naegleria spp. using PCR/DNA sequencing based methods. The affinities between the sequences generated here and others reported in the literature were explored by using POY, which implements the concept of dynamic homology. Naegleria australiensis, Naegleria italica, and Naegleria lovaniensis, plus an unassigned Naegleria spp. were detected. Indels in the rDNA ITS1 and ITS2 turned out to be critical to distinguish the three species and confirmed their phylogenetic relationships. This is the first molecular report on the Naegleria spp. occurrence in geothermal waters in Central Italy, coupled with a fine genetic characterization.

Isolation of Thermotolerant Pathogenic Naegleria australiensis from Diverse Water Resources Including Household Drinking Water in Pakistan

Naegleria is well recognized for primary amoebic meningoencephalitis which always results into death. To date there is not a single report demonstrating molecular identification of Naegleria from water resources of Pakistan thus the aim of the proposed study. Here, in total 135 various water samples (like domestic tap water, municipal water, sea water, well water, tube well water, canal water, boring water, sewage water, lake water and stream water) were collected across Pakistan and evaluated for Naegleria species. Naegleria australiensis (pathogenic) and Naegleria lovaniensis (non pathogenic) were isolated on non nutrient agar plates and were further identified by PCR and sequencing. To best of our knowledge we have described for the first time the isolation and molecular identification of thermotolerant pathogenic and non pathogenic Naegleria species from diverse water samples including drinking water across Pakistan. The presence of pathogenic Naegleria species in diverse water samples may add the health threat to the community.

Monitoring of Waterborne Parasites in Two Drinking Water Treatment Plants: A Study in Sarawak, Malaysia.

The occurrence of waterborne parasites coupled with water parameters at various processing sites of two drinking water treatment plants (A and B) and seven distribution system (DS) sites in Sarawak, Malaysia were studied. Ten liters of water underwent immunomagnetic separation (IMS) technique to detect the presence of Giardia and Cryptosporidium (oo)cysts. The remaining supernatant was used to detect other parasites whilst 50 mL of water sample was each used in the detection of free-living amoebae and fecal coliforms. Sampled water was positive for Giardia (32.9%; 28/85), Cryptosporidium (18.8%; 16/85) followed by Spirometra ova-like (25.9%; 22/85), Blastocystis-like (25.9%; 22/85), nematode larvae-like (8.2%; 7/85) and Taenia ova-like (1.2%; 1/85). Meanwhile, 90.2% (55/61) samples were positive for Acanthamoeba and Naegleria via cultivation and of these, 11 isolates were confirmed as Acanthamoeba genotype T3 (5/7) and T4 (2/7) followed by Naegleria sp. (4/11), Naegleria italica (2/11), Naegleria australiensis (1/11), Naegleria angularis (1/11) and Vahlkampfia sp. (3/11). Cryptosporidium, Acanthamoeba and Naegleria were also detected in one of the seven tested DS sites. Only Giardia and Cryptosporidium showed significant correlations with fluoride and fecal coliforms. These results describe the occurrence of waterborne parasites that will assist key stakeholders in mitigating contamination at the specific sites.

Ophthalmology hospital wards contamination to pathogenic free living Amoebae in Iran.

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.

Surveillance and evaluation of the infection risk of free-living amoebae and Legionella in different aquatic environments

Free-living amoebae (FLA) are ubiquitous in various aquatic environments. Several amoebae species are pathogenic and host other pathogens such as Legionella, but the presence of FLA and its parasites as well as the related infection risk are not well known. In this study, the presence of pathogenic FLA and Legionella in various water bodies was investigated. Water samples were collected from a river, intake areas of drinking water treatment plants, and recreational hot spring complexes in central and southern Taiwan. A total of 140 water samples were tested for the presence of Acanthamoeba spp., Naegleria spp., Vermamoeba vermiformis, and Legionella. In addition, phylogenetic characteristics and water quality parameters were also assessed. The pathogenic genotypes of FLA included Acanthamoeba T4 and Naegleria australiensis, and both were abundant in the hot spring water. In contrast, Legionella pneumophila was detected in different aquatic environments. Among the FLA assessed, V. vermiformis was most likely to coexist with Legionella spp. The total bacteria level was associated with the presence of FLA and Legionella especially in hot spring water. Taken together, FLA contamination in recreational hot springs and drinking water source warrants more attention on potential legionellosis and amoebae infections.

Identification and significance of Naegleria fowleri isolated from the hot spring which related to the first primary amebic meningoencephalitis (PAM) patient in Taiwan

Naegleria fowleri can cause primary amoebic meningoencephalitis, a rapidly developing and highly lethal infectious disease. The first confirmed case of primary amoebic meningoencephalitis in Taiwan was reported in November 2011, in which the patient visited a thermal spring recreational area 1week prior to hospitalisation. Water sampling was performed to verify the presence of Naegleria at the facility. According to our results, 32% and 20% of recreational water samples were contaminated with Naegleria spp. and Acanthamoeba spp., respectively. The genotypes of Naegleria identified at the hot spring included N. fowleri, Naegleria australiensis and Naegleria lovaniensis. Using PCR, it was determined that the strain of N. fowleri in one sample possessed the same genotype 2 as the clinical isolate. Thus, the thermal spring was suggested to be the likely source of infection. This is the first known instance of simultaneously isolating N. fowleri from both a patient as well as from a hot spring in Taiwan. Following this initial study, the pools at the thermal spring recreational area were drained, scrubbed and disinfected, and a follow-up study was performed 1month later. Naegleria fowleri was not detected in follow-up testing; however, other Naegleria spp. were identified. We postulate that the biofilm in the waterlines may have provided a reservoir for free-living amoebae. The presence/absence of Acanthamoeba and Naegleria spp. did not differ significantly with any measured parameters related to water quality; however, a high percentage of the thermal water pool samples were contaminated with Naegleria or Acanthamoeba. Thus, amoebic contamination may present a serious threat to the health of humans who engage in leisure activities at thermal springs.

Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri

Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

Occurrence and distribution of Naegleria species from thermal spring environments in Taiwan

Naegleria spp. is a free-living amoeba that can be found in the natural environment. A number of Naegleria spp. can cause fatal infections in the central nervous system in humans and animals, and the most important source of infection is through direct water contact. In this study, water samples from various thermal springs were taken from four thermal spring areas. Naegleria spp. was detected via culture confirmation and molecular taxonomic identification. Among the 60 samples obtained, Naegleria spp. was identified in 26 (43·3%) samples. The identified species included Naegleria australiensis, Naegleria gruberi, Naegleria lovaniensis and Naegleria mexicana. The presence of living Naegleria spp. was significantly associated with elevated pH value in the water sample. In this study, we examined the presence of living Naegleria spp. in thermal spring waters in south-eastern Taiwan. Naegleria spp. was isolated and culture-confirmed from thermal spring water. Naegleria fowleri was not found in all water samples, and Naegleria australiensis was the most common Naegleria genotype.

Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments

This study compares five genera of free-living amoebae (FLA) hosts by Legionella spp. in the fixed and floating biofilm samples from spring environments. Detection rate of Legionella spp. was 26.9% for the floating biofilms and 3.1% for the fixed biofilms. Acanthamoeba spp., Hartmanella vermiformis, and Naegleria spp. were more frequently detected in floating biofilm than in fixed biofilm samples. The percentage of pathogenic Acanthamoeba spp. among all the genus Acanthamoeba detected positive samples was 19.6%. The potential pathogenic Naegleria spp. (for example, Naegleria australiensis, Naegleria philippinensis, and Naegleria italica) was 54.2% to all the Naegleria detected positive samples. In the study, 12 serotypes of possible pneumonia causing Legionella spp. were detected, and their percentage in all the Legionella containing samples was 42.4%. The FLA parasitized by Legionella included unnamed Acanthamoeba genotype, Acanthamoeba griffini, Acanthamoeba jacobsi, H. vermiformis, and N. australiensis. Significant differences were also observed between the presence/absence of H. vermiformis and Legionella parasitism in FLA. Comparisons between the culture-confirmed method and the PCR-based detection method for detecting FLA and Legionella in biofilms showed great variation. Therefore, using these analysis methods together to detect FLA and Legionella is recommended.

Isolation and identification of Legionella and their host amoebae from weak alkaline carbonate spring water using a culture method combined with PCR

Legionella were detected with the direct DNA extraction method, Legionella culture method, and free-living amoebae (FLA) culture method from weak alkaline carbonate spring water in Taiwan. Moreover, we also investigated the existence of Acanthamoeba, Hartmannella, and Naegleria, ubiquitous FLA in aquatic environments, to identify the correlations between existing Legionella. This study reports detecting Legionella in 15 of the 51 weak alkaline carbonate spring water samples (29.4%). This work also found five of the 51 samples (9.8%) analyzed by the direct DNA extraction method, three of the 51 (5.9%) samples analyzed by the Legionella culture method, and 11 of the 51 samples (21.6%) evaluated using the FLA culture method to be positive for Legionella. The most frequently identified Legionella species was the Legionella-like amoebal pathogen (n=5), followed by unidentified Legionella spp. (n=4), and Legionella pneumophila (n=4), Legionella fairfieldensis (n=3), and then Legionella rubrilucens (n=2). Legionella waltersii was detected once. The occurrence of Acanthamoeba, Hartmannella, and Naegleria were 5.9% (3/51), 52.9% (27/51), and 5.9% (3/51), respectively. All Hartmannella isolates were identified as Hartmannella vermiformis, and Naegleria isolates were all identified as Naegleria australiensis. The three Acanthamoeba isolates were identified as one Acanthamoeba polyphaga and two Acanthamoeba jacobsi. H. vermiformis (40.7%) were Legionella hosts, including all of the amoebae-resistant Legionella detected in the present study. Therefore, the important correlations between Legionella and H. vermiformis require further clarification. The combined results of this survey confirm that Legionella and FLA are ubiquitous in weak alkaline carbonate spring water in Taiwan.

Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan

Here, we carried out a survey to determine the prevalence of free-living amoebae (FLA) in tap-water sources from rivers and water treatment plants located in Osaka Prefecture, Japan. A total of 374 raw water samples were collected from 113 sampling points. The samples were filtrated and transferred to non-nutrient agar plates seeded with a heat-killed suspension of Escherichia coli and incubated for 2 to 7 days at 30 degrees C or 42 degrees C. The plates were examined by microscopy to morphologically identify FLA families, and polymerase chain reaction and sequence analysis were then performed to define the species of the detected Naegleria and Acanthamoeba isolates. A total of 257 of 374 samples (68.7%) were positive for FLA by microscopy, and among these there were 800 FLA isolates, including Acanthamoeba and Naegleria species. Sequence analysis identified five Acanthamoeba spp. isolates of the known pathogenic T4 genotype and 43 Naegleria australiensis isolates, a reported pathogen to mice and also of concern as a potential pathogen to humans. Our results suggest a wide distribution of FLA, including potential pathogenic species, in tap-water sources of western Japan.