Free-Living Amoebae Keratitis.

PURPOSE: To describe the diagnostic and clinical features and treatment results in 43 consecutive patients with microbiologically proven free-living amoebae (FLA) keratitis. METHODS: In this hospital-based, prospective case series, corneal scrapings from 43 patients with presumed amoebic keratitis were plated on nonnutrient agar. Amoebic isolates were identified morphologically and by the polymerase chain reaction. All patients with culture-proven FLA keratitis were treated with polyhexamethylene biguanide (PHMB) 0.02% eye drops. RESULTS: Forty-three corneal scrapings from 43 patients were found to be culture positive for FLA; 41 (95%) were from contact lens wearers and 2 (5%) were from noncontact lens wearers. Microscopic examination identified 4 Acanthamoeba spp, 24 Hartmannella spp, 12 vahlkampfiid amoebae, and 3 mixed infections with Hartmannella/vahlkampfiid amoebae. Morphological results were confirmed by the polymerase chain reaction. Patients with Acanthamoeba, Hartmannella, and vahlkampfiid keratitis had indistinguishable clinical features. In 38 eyes with keratitis at an early stage, treatment with PHMB 0.02% eye drops was fully successful. In 5 patients with advanced keratitis, topical PHMB 0.02% controlled the infection, but all of them developed a central corneal scar with visual deterioration. CONCLUSIONS: Acanthamoeba is not the only cause of amoebic keratitis, because this condition may also be caused by other FLA, such as Hartmannella and vahlkampfiid amoebae. This finding is epidemiologically interesting, suggesting a possible different geographical prevalence of the different FLA responsible for keratitis. Early diagnosis and proper antiamoebic treatment are crucial to yielding a cure.

Molecular detection of Acanthamoeba spp., Naegleria fowleri and Vermamoeba (Hartmannella) vermiformis as vectors for Legionella spp. in untreated and solar pasteurized harvested rainwater.

BACKGROUND: Legionella spp. employ multiple strategies to adapt to stressful environments including the proliferation in protective biofilms and the ability to form associations with free-living amoeba (FLA). The aim of the current study was to identify Legionella spp., Acanthamoeba spp., Vermamoeba (Hartmannella) vermiformis and Naegleria fowleri that persist in a harvested rainwater and solar pasteurization treatment system. METHODS: Pasteurized (45 °C, 65 °C, 68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples were screened for Legionella spp. and the heterotrophic plate count was enumerated. Additionally, ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) was utilized for the quantification of viable Legionella spp., Acanthamoeba spp., V. vermiformis and N. fowleri in pasteurized (68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples, respectively. RESULTS: Of the 82 Legionella spp. isolated from unpasteurized tank water samples, Legionella longbeachae (35 %) was the most frequently isolated, followed by Legionella norrlandica (27 %) and Legionella rowbothamii (4 %). Additionally, a positive correlation was recorded between the heterotrophic plate count vs. the number of Legionella spp. detected (ρ = 0.710, P = 0.048) and the heterotrophic plate count vs. the number of Legionella spp. isolated (ρ = 0.779, P = 0.0028) from the tank water samples collected. Solar pasteurization was effective in reducing the gene copies of viable V. vermiformis (3-log) and N. fowleri (5-log) to below the lower limit of detection at temperatures of 68-93 °C and 74-93 °C, respectively. Conversely, while the gene copies of viable Legionella and Acanthamoeba were significantly reduced by 2-logs (P = 0.0024) and 1-log (P = 0.0015) overall, respectively, both organisms were still detected after pasteurization at 93 °C. CONCLUSIONS: Results from this study indicate that Acanthamoeba spp. primarily acts as the vector and aids in the survival of Legionella spp. in the solar pasteurized rainwater as both organisms were detected and were viable at high temperatures (68-93 °C).

[The Investigation of Acanthamoeba and Other Free Living Amoeba in Swab Samples Obtained from Conjunctiva and Eye Lid]. / Göz Kapaklarindan ve Konjunktivadan Alinan Sürüntü Örneklerinde Acanthamoeba ve Diger Serbest Yasayan Amiplerin Arastirilmasi.

OBJECTIVE: In the study, it is aimed to determine the prevalence of Acanthamoeba and other free-living amoeba (FLA) species in the swab samples obtained from conjunctiva and lower eye lid. METHODS: For this purpose, swab samples from the 500 patients'eye lid and conjunctiva were obtained who admitted to Cumhuriyet University, Research and Application Hospital, Department of Ophthalmology with variety of reasons. Swab samples were carried out using sterile cotton swab in steril tubes. The swab samples were inoculated onto non-nutrient agar (NNA). Live Escherichia coli was used as food source for the growth of the FLA. The NNA plates were incubated at 300C and examined daily using ligth microscope for two weeks. For morphotyping of the trophozoites and cysts of the FLA were used taxonomic keys. RESULTS: Two of the 500 swab samples (0.4%) were positive for FLA. One of them (0.2%) were identified as Acanthamoeba spp. and other was identified as Hartmannella spp. However, these patients did not reveal any complaints yet. CONCLUSION: FLA both themselves and bacteria carrying in their body as reservoirs are potential pathogen. The rapid spread of Acanthamoeba keratitis in recent years reveal that these microorganisms are in contact with the eyes.

Occurrence and molecular characterization of free-living amoeba species (Acanthamoeba, Hartmannella, and Saccamoeba limax) in various surface water resources of Iran.

This study was conducted to determine the presence and molecular identity of Acanthamoeba species in the surface water resources of four provinces in Iran, namely Guilan, Mazandaran (North of Iran), Alborz, and Tehran (capital city), using culture- and molecular-based methods. During March to November 2014, 49 surface water samples were collected from environmental water sources-the distinct surface waters of Guilan, Mazandaran, Alborz, and Tehran provinces, in Iran. For the isolation of Acanthamoeba species, approximately 500 ml of the water samples were filtered through a cellulose nitrate membrane with a pore size of 0.45 µ. The filter was transferred onto non-nutrient agar plates seeded with Gram-negative bacteria (Escherichia coli) as a food source. The presence of Acanthamoeba was confirmed by the genus-specific primer pair JDP1 and 2, and/or NA primers were used to identify Acanthamoeba and certain other free-living amoebae. In total, 38 out of 49 samples were positive by culture and/or PCR for Acanthamoeba and other free-living amoebae from all three provinces. By sequencing the positive isolates, the strains were shown to belong to Acanthamoeba (16 isolates belonged to T4 and 2 isolates belonged to T5), Hartmannella vermiformis (3/24), and Saccamoeba limax (2/24). The T4 and T5 genotypes were detected in Guilan and Mazandaran provinces. Two isolates from Guilan and Tehran provinces belonged to S. limax, and H. vermiformis was detected in Guilan province. The results of this study highlight the need to pay more attention to free-living amoebae, as human activity was observed in all of the localities from which these samples were taken. These surface waters can be potential sources for the distribution and transmission of pathogenic Acanthamoeba in the study areas, and free-living amoebas (FLA) (particularly the Acanthamoeba species) can serve as hosts for and vehicles of various microorganisms.

Legionella spp., amoebae and not-fermenting Gram negative bacteria in an Italian university hospital water system.

INTRODUCTION: In hospital and other health care facilities, contamination of water systems by potentially infectious microorganisms, such as bacteria, viruses and protozoa, is a source of nosocomial infections, which may originate fromcolonization of water pipes, cooling towers, spa pools, taps, showers and water supplies. Objective. The study focuses on the occurrence of Legionella spp., free-living amoebae and non-fermenting Gram-negative microorganisms in a University hospital water system located in the town of Messina (Sicily, Italy), which had never been examined previously. MATERIALS AND METHODS: From January 2008 - March 2009, hot tap water samples were collected from 10 wards. Legionella spp. recovered on selective culture medium were identified by microagglutination latex test; free-living amoebae were cultured using Escherichia coli as a food source. Non-fermenting Gram negative microorganisms were identified by API 20 NE strips. RESULTS: Legionella spp. were found in 33.33% of the samples. L. pneumophila serogroup 1 was recovered from the Laboratory Diagnostic and Anaesthesia-Neurology Wards, with a peak of 3.5 × 10(4) cfu/L in May 2008. L. pneumophila serogroups 2-14 were found in the Othorhinolaryngology, Pathologic Anatomy, Paediatrics and Surgery Wards, and peaked (4 × 10(4) cfu/L) in April 2008. Pseudomonadaceae and Hyphomycetes were also detected. Legionella spp. were recovered from samples positive for non-pathogenic amoebae Hartmannella spp. CONCLUSION: This first study of a Messina hospital water system suggested potential health risks related to the detection of Hartmannella spp., as reservoirs for Legionella spp., and Pseudomonas aeruginosa, a Gram negative non-fermenting bacterium frequently causing nosocomial pneumonia. The urgent need for monitoring programmes and prevention measures to ensure hospital water safety is stressed.

Thermophilic potentially pathogenic amoebae isolated from natural water bodies in Poland and their molecular characterization.

The free-living amoebae (FLA) may live in the environment and also within other organisms as parasites and then they are called amphizoic. They are potentially pathogenic for humans and animals and are found in water that is a source of infection. The aim of this study was molecular detection and identification of these FLA in natural water bodies in North-Western Poland to evaluate the risk of the pathogenic amoebae infections. We examined surface water samples collected from 50 sites and first, the tolerance thermic test was performed in order to select thermophilic, potentially pathogenic strains. For molecular identification of FLA, regions of 18S rDNA, 16S rDNA and intergenic spacers were amplified. Acanthamoeba T4 and T16 genotypes of 18S rDNA gene and 18S rDNA of H. vermiformis were detected. We identified two variants of Acanthamoeba T4 genotype, two variants of Acanthamoeba T16 genotype and one variant of H. vermiformis. Identification of the T16 genotype and H. vermiformis in water was for the first time in Poland. Additionally, we made attempts to adapt the RLB method for detection and differentiation of FLA species and strains. PCR seems to be more sensitive than RLB hybridization, though.

Endosymbiotic Mycobacterium chelonae in a Vermamoeba vermiformis strain isolated from the nasal mucosa of an HIV patient in Lima, Peru.

In March 2010, a 35 year-old HIV/AIDS female patient was admitted to hospital to start treatment with Highly Active Antiretroviral Therapy (HAART) since during a routine control a dramatic decrease in the CD4(+) levels was detected. At this stage, a nasal swab from each nostril was collected from the patient to include it in the samples for the case study mentioned above. Moreover, it is important to mention that the patient was diagnosed in 2009 with invasive pneumococcal disease, acute cholecystitis, pancreatitis and pulmonary tuberculosis. The collected nasal swabs from both nostrils were positive for Vermamoeba vermiformis species which was identified using morphological and PCR/DNA sequencing approaches. Basic Local Alignment Search Tool (BLAST) homology and phylogenetic analysis confirmed the amoebic strain to belong to V.vermiformis species. Molecular identification of the Mycobacterium strain was carried out using a bacterial universal primer pair for the 16S rDNA gene at the genus level and the rpoB gene was amplified and sequenced as previously described to identify the Mycobacterium species (Shin et al., 2008; Sheen et al., 2013). Homology and phylogenetic analyses of the rpoB gene confirmed the species as Mycobacterium chelonae. In parallel, collected swabs were tested by PCR and were positive for the presence of V.vermiformis and M.chelonae. This work describes the identification of an emerging bacterial pathogen,M.chelonae from a Free-Living Amoebae (FLA) strain belonging to the species V.vermiformis that colonized the nasal cavities of an HIV/AIDS patient, previously diagnosed with TB. Awareness within clinicians and public health professionals should be raised, as pathogenic agents such as M.chelonae may be using FLA to propagate and survive in the environment.

Microbiome of free-living amoebae isolated from drinking water.

Free-living amoebae (FLA) are protozoa that can be found in water networks where they prey on bacteria within biofilms. Most bacteria are digested rapidly by phagocytosis, however some are able to survive within amoebae and some are even able to multiply, as it is the case for Legionella pneumophila. These resisting bacteria are a potential health problem as they could also resist to macrophage phagocytosis. Several publications already reported intra-amoebal bacteria but the methods of identification did not allow metagenomic analysis and are partly based on co-culture with one selected amoebal strain. The aim of our study was to conduct a rRNA-targeted metagenomic analysis on amoebae and intra-amoebal bacteria found in drinking water network, to provide the first FLA microbiome in environmental strains. Three sites of a water network were sampled during four months. Culturable FLA were isolated and total DNA was prepared, allowing purification of both amoebal and bacterial DNA. Metagenomic studies were then conducted through 18S or 16S amplicons sequencing. Hartmannella was by far the most represented genus of FLA. Regarding intra-amoebal bacteria, 54 genera were identified, among which 21 were newly described intra-amoebal bacteria, underlying the power of our approach. There were high differences in bacterial diversity between the three sites. Several genera were highly represented and/or found at least in two sites, underlying that these bacteria could be able to multiply within FLA. Our method is therefore useful to identify FLA microbiome and could be applied to other networks to have a more comprehensive view of intra-amoebal diversity.

Effect of GAC pre-treatment and disinfectant on microbial community structure and opportunistic pathogen occurrence.

Opportunistic pathogens in potable water systems are an emerging health concern; however, the factors influencing their proliferation are poorly understood. Here we investigated the effects of prior granular activated carbon (GAC) biofiltration [GAC-filtered water, unfiltered water, and a blend (30% GAC filtered and 70% unfiltered water)] and disinfectant type (chlorine, chloramine) on opportunistic pathogen occurrence using five annular reactors (ARs) to simulate water distribution systems, particularly premise plumbing. GAC pre-treatment effectively reduced total organic carbon (TOC), resulting in three levels of influent TOC investigated. Quantitative polymerase chain reaction (q-PCR) provided molecular evidence of natural colonization of Legionella spp., Mycobacterium spp., Acanthamoeba spp., Hartmannella vermiformis and Mycobacterium avium on AR coupons. Cultivable mycobacteria and amoeba, including pathogenic species, were also found in bulk water and biofilm samples. While q-PCR tends to overestimate live cells, it provided a quantitative comparison of target organisms colonizing the AR biofilms in terms of gene copy numbers. In most cases, total bacteria and opportunistic pathogens were higher in the three undisinfected ARs, but the levels were not proportional to the level of GAC pre-treatment/TOC. Chlorine was more effective for controlling mycobacteria and Acanthamoeba, whereas chloramine was more effective for controlling Legionella. Both chlorine and chloramine effectively inhibited M. avium and H. vermiformis colonization. Pyrosequencing of 16S rRNA genes in coupon biofilms revealed a significant effect of GAC pre-treatment and disinfectant type on the microbial community structure. Overall, this study provides insights into the potential of different disinfectants and GAC biofilters at the treatment plant and in buildings to control downstream opportunistic pathogens and broader drinking water microbial communities.

Cooccurrence of free-living amoebae and nontuberculous Mycobacteria in hospital water networks, and preferential growth of Mycobacterium avium in Acanthamoeba lenticulata.

The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.

Distribution of Legionella pneumophila bacteria and Naegleria and Hartmannella amoebae in thermal saline baths used in balneotherapy.

The present study was aimed at investigating the coexistence and interactions between free living amoebae of Naegleria and Hartmannella genera and pathogenic Legionella pneumophila bacteria in thermal saline baths used in balneotherapy in central Poland. Water samples were collected from November 2010 to May 2011 at intervals longer than 1 month. The microorganisms were detected with the use of a very sensitive fluorescence in situ hybridisation method. In addition, the morphology of the amoebae was studied. Despite relatively high salinity level, ranging from 1.5 to 5.0 %, L. pneumophila were found in all investigated baths, although their number never exceeded 10(6) cells dm(-3). Hartmannella were not detected, while Naegleria fowleri were found in one bath. The observation that N. fowleri and L. pneumophila may coexist in thermal saline baths is the first observation emphasising potential threat from these microorganisms in balneotherapy.

Differential Legionella spp. survival between intracellular and extracellular forms in thermal spring environments.

Legionella are commonly found in natural and man-made aquatic environments and are able to inhabit various species of protozoa. The relationship between the occurrence of Legionella spp. within protozoa and human legionellosis has been demonstrated; however, the proportions of intracellular and extracellular Legionella spp. in the aquatic environment were rarely reported. In this study, we developed a new method to differentiate intracellular and extracellular Legionella spp. in the aquatic environment. Water samples from three thermal spring recreational areas in southeastern Taiwan were collected and analyzed. For each water sample, concurrent measurements were performed for Legionella spp. and their free-living amoebae hosts. The overall detection rate was 32 % (16/50) for intracellular Legionella spp. and 12 % (6/50) for extracellular Legionella spp. The most prevalent host of Legionella spp. was Hartmannella vermiformis. The identified Legionella spp. differed substantially between intracellular and extracellular forms. The results showed that it may be necessary to differentiate intracellular and extracellular forms of Legionella spp.

The prevalence, isolation and morphotyping of potentially pathogenic free-living amoebae from tap water and environmental water sources in Sivas.

OBJECTIVE: To our knowledge, there is no study dealing with the prevalence of free-living amoebas (FLA) in water sources in Turkey, previous studies were mostly case presentations. The aim of the present study was to investigate the prevalence of FLA from tap water and natural water sources in different parts of the city. METHODS: In the study, 250 samples were collected from the city centre, districts and villages. Two litres of water was collected from each source and filtered through a vacuum filtration system. The filter papers were washed in "Page's Amoeba Saline (PAS)" solution and incubated overnight. Filter papers were removed from the tubes and centrifuged; the final pellet was inoculated on non-nutrient agar (NNA) plates. The growth rate of FLA was checked after three days of inoculation and the flagellation test was performed to determine the presence of Naegleria spp. Heat tolerance of isolated strains was checked at 37, 42 and 52°C for the presence of pathogenic Acanthamoeba species. The cyst and trophozoite morphology of amoebas were examined under a light microscope and the genera was identified according to morphotyping keys. RESULTS: FLA were found in 75 (30.0%) of examined water samples. Eleven (4.4%) were identified as Acanthamoeba spp., 25 (10.0%) as Naegleria spp. and 39 (15.6%) as Hartmannella spp. after microscopic examination. CONCLUSION: Our study revealed that FLA are common inhabitants of household water as they are in the environment, so their own potential risks as well as transferring bacteria as other pathogens is important for human health.

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.

Potentially pathogenic free-living amoebae isolated from hospital wards with immunodeficient patients in Tehran, Iran.

This study investigated the occurrence of free-living amoebae (FLA) in immunodeficiency wards of hospitals in Tehran, Iran. A total of 70 dust and biofilm samples from wards serving transplant, pediatric (malignancies), HIV, leukemia and oncology patients of five university hospitals were collected and examined for the presence of FLA using culturing and molecular approaches. Based on the morphology of the amoebae in plate cultures, primer sets were applied for molecular identification of Acanthamoeba, vahlkampfiid amoebae and Hartmannella. Out of 70 samples, 37 (52.9%) were positive for FLA. Acanthamoeba belonged to the T4 genotype was the most prevalent isolate. Presence of the T4 genotype on medical instruments, including an oxygen mask in an isolation room of an immunodeficiency pediatric ward, should be of concern for health authorities. Acanthamoeba T5 genotypes, Hartmannella vermiformis, and Vahlkampfia avara were also present. These results highlight a clear need for greater attention to improved disinfection, especially where susceptible patients, such as those who are immune-suppressed, are served. To our knowledge, this is the first report of these FLA in immunodeficiency wards in Iran, and also the first to identify Acanthamoeba T5, Hartmannella, and Vahlkampfia in moist habitats, such as biofilms, in this country.

Prevalence of Acanthamoeba spp. and other free-living amoebae in household water, Ohio, USA--1990-1992.

Knowledge of the prevalence of free-living amoebae (FLA) in US household water can provide a focus for prevention of amoeba-associated illnesses. Household water samples from two Ohio counties, collected and examined for amoebae during 1990-1992, were used to describe the prevalence of Acanthamoeba and other FLA in a household setting. Amoebae were isolated and identified by morphologic features. A total of 2,454 samples from 467 households were examined. Amoebae were found in water samples of 371 (79%) households. Sites most likely to contain amoeba were shower heads (52%) and kitchen sprayers (50%). Species of Hartmannella, Acanthamoeba, or Vahlkampfia were most common. Detection was higher in biofilm swab samples than in water samples. Detection of FLA and Acanthamoeba, at 79% and 51%, respectively, exceed estimates that have been published in previous surveys of household sources. We believe FLA are commonplace inhabitants of household water in this sample as they are in the environment.

Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area.

Acanthamoeba, Hartmannella, and Naegleria are free-living amoebae, ubiquitous in aquatic environments. Several species within these genera are recognized as potential human pathogens. These free-living amoebae may facilitate the proliferation of their parasitical bacteria, such as Legionella. In this study, we identified Acanthamoeba, Hartmannella, Naegleria, and Legionella using various analytical procedures and investigated their occurrence at a mud spring recreation area in Taiwan. We investigated factors potentially associated with the prevalence of the pathogens, including various water types, and physical and microbiological water quality parameters. Spring water was collected from 34 sites and Acanthamoeba, Hartmannella, Naegleria, and Legionella were detected in 8.8%, 35.3%, 14.7%, and 47.1%, respectively. The identified species of Acanthamoeba included Acanthamoeba castellanii and Acanthamoeba polyphaga. Nearly all the Hartmannella isolates are identified as Hartmannella vermiformis. The Naegleria species included Naegleria australiensis and its sister groups, and two other isolates referred to a new clade of Naegleria genotypes. The Legionella species identified included unnamed Legionella genotypes, Legionella pneumophila serotype 6, uncultured Legionella spp., Legionella lytica, Legionella drancourtii, and Legionella waltersii. Significant differences (Mann-Whitney U test, P<0.05) were observed between the presence/absence of Hartmannella and total coliforms, between the presence/absence of Naegleria and heterotrophic plate counts, and between the presence/absence of Legionella and heterotrophic plate counts. This survey confirms that pathogenic free-living amoebae and Legionella are prevalent in this Taiwanese mud spring recreation area. The presence of pathogens should be considered a potential health threat when associated with human activities in spring water.

Relationships between free living amoebae and Exophiala dermatitidis: a preliminary study.

Free living amoebae can play a role as reservoirs for pathogens isolated from hospital water. We have investigated the potential interactions between two protozoa (Acanthamoeba castellanii and Hartmanella vermiformis) that may be recovered from hospital water tips and Exophiala dermatitidis, a black yeast often recovered from water sources. We showed that the presence of trophozoites or supernatants of culture of H. vermiformis increased fungal growth, whereas the same phenomenon was observed only with the supernatant of A. castellanii cultures. These preliminary results highlight the fact that the recovering of free-living amoebae in hospital water taps could lead to the development of fungal nosocomial pathogens.

Early diagnosis of amoebic keratitis due to a mixed infection with Acanthamoeba and Hartmannella.

A mixed keratitis due to Acanthamoeba and Hartmannella species is reported. The patient was a soft contact lens wearer. Early diagnosis was achieved by polymerase chain reaction and culture. The pathogenic potential of the isolated amoebae was proven using cytotoxicity assays. The reported case underlines the difficulties in identifying a corneal amoebic infection. In our case, the early diagnosis of a mixed infection allowed a proper antiamoebic treatment in an early stage of infection. This may have been the reason of a successful outcome after therapy.

Differential Salmonella survival against communities of intestinal amoebae.

Predation from intestinal amoebae may provide selective pressure for the maintenance of high genetic diversity at the Salmonella enterica rfb locus, whereby serovars better escape predators in particular environments depending on the O-antigens they express. Here, the hypothesis that amoebae from a particular intestinal environment collectively prefer one serovar over another is tested. Collections of Acanthamoeba, Tetramitus, Naegleria and Hartmannella were isolated from the intestinal tracts of several vertebrate hosts, including bullfrog tadpoles, goldfish, turtles and bearded dragons, and their feeding preferences were determined. Congeneric amoebae from the same environment had significantly similar feeding preferences. Strikingly, even unrelated amoebae - such as Naegleria and Tetramitus from goldfish - also had significantly similar feeding preferences. Yet amoebae isolated from different environments showed no similarity in prey choice. Thus, feeding preferences of amoebae appear to reflect their environment, not their taxonomic relationships. A mechanism mediating this phenotypic convergence is discussed.