Seasonal Distribution of Thermotolerant Free-Living Amoebae. II. Lake Issaqueena1

A quantitative study of the seasonal distribution of thermotolerant (37°C and 45°C), small free-living amoebae (FLA) was conducted in Lake Issaqueena, a warm, monomictic lake with steep, sloping banks and a maximum basin depth of 10 m in the Piedmont region of South Carolina. Naegleria and Vahlkampfia were the most frequently encountered FLA in littoral sediment and surface water samples whereas Acanthamoeba was most commonly isolated from profundal sediment, especially during late summer. In the water column, FLA populations were highest in a persistent detrital layer; however, few amoebae were isolated from a massive (～1.5 m thick) layer of Oscillatoria. The only N. fowleri isolated in this study was from the detrital layer. Discussion of the influence of differences in watershed and basin morphology on variations in the size and generic composition of FLA populations for the aquatic ecosystems of Lake Issaqueena and Willard's Pond is included.     

Seasonal distribution of thermotolerant free-living amoebae. I. Willard's Pond

A quantitative study of the seasonal distribution of thermotolerant (37 degrees C and 45 degrees C), small free-living amoebae (FLA) was conducted in Willard's Pond, a warm, monomictic lake in the Piedmont region of South Carolina. Correlation of physical and chemical parameters with the seasonal distribution was facilitated by partitioning the aquatic ecosystem into benthic, planktonic, and neustonic habitats. Population densities of FLA peaked in late summer in each habitat; however, species composition varied between habitats. Littoral sediment appeared to be the major habitat for FLA, with peaks in populations of Acanthamoeba and Naegleria in August, Hartmannella in July, and Vahlkampfia in May. Populations in profundal sediment underwent dramatic seasonal shifts, apparently in response to the seasonal chemical changes in the hypolimnion. Acanthamoeba was most prevalent in late summer, representing as much as 82% of the FLA in profundal sediment. Distribution patterns and species composition of FLA from surface water were similar to those from littoral sediment; however, a greater percentage of Naegleria was found in surface water. Numerous FLA were isolated from the neustonic community (surface film), and the number of FLA isolated in the surface film at the deep water station was found to be significantly (P less than 0.05) greater than the number from subsurface (5-10 cm) samples. In the water column, FLA populations consistently were highest in the detrital layer, which persisted at a depth of 3.0-3.4 m throughout the summer period. The large percentage of Naegleria contributing to FLA in the detrital layer suggests that Naegleria amoeboflagellates sink through the layer, flagellate, and swim back up, such migrations possibly being triggered by a reduction of nutrients below the layer or by the presence of anoxic, reducing conditions in the hypolimnion. In addition, weather events were found to play a major role in the redistribution of FLA between various habitats in the aquatic ecosystem, with such changes probably due to resuspension of FLA from littoral sediment by wind action and input from the watershed via runoff.     

Vertical Distribution of Potentially Pathogenic Free-Living Amoebae in Freshwater Lakes1

The vertical distribution of thermotolerant (37°C and 45°C) free-living amoebae (FLA) in warm monomictic lakes was determined in relation to the onset of thermal stratification and associated physical and chemical changes. The position of abiotic or biotic paniculate layers in the water column was located by using a submersible horizontal beam transmissometer that measures attenuance, or the absorption and scattering of light by participates in the water column. During mixis, the vertical distribution of amoebae was sporadic with significant numbers of FLA only occurring in clay layers caused by runoff after heavy rains. With the onset of thermal stratification in the lakes, phytoplankton layers began to form. Few amoebae were isolated from layers containing flagellated phytoplankton; however, significant (P < 0.005) numbers of FLA were isolated from two paniculate layers dominated by the filamentous blue-green algae Aphanizomenon and Lyngbya, respectively. By late June, a persistent detrital or decomposition layer formed in the lower metalimnion, as well as a hypolimnetic iron layer where the Fe²⁺ state was predominant. In this midsummer period, 13 Naegieria fowleri were isolated, with three from the detrital layer and seven from the iron layer. The presence of attenuation zones was found to be the best indicator of the vertical distribution of FLA in the water column, and such layers represent an important, previously undescribed habitat for potentially pathogenic FLA.     

Interaction of Pasteurella multocida with Free-Living Amoebae

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.     

Processing of Free-Living Amoebae for Transmission Electron Microscopy

Ultrastructural features have proved useful in identifying and classifying free-living amoebae (Page 1985). A simple technic which facilitates embedding amoebae for transmission electron microscoov is described here.     

Presence of Legionella and Free-Living Amoebae in Composts and Bioaerosols from Composting Facilities

Several species of Legionella cause Legionnaires’ disease (LD). Infection may occur through inhalation of Legionella or amoebal vesicles. The reservoirs of Legionella are water, soil, potting soil and compost. Some species of free-living amoebae (FLA) that are naturally present in water and soil were described as hosts for Legionella. This study aimed to understand whether or not the composting facilities could be sources of community-acquired Legionella infections after development of bioaerosols containing Legionella or FLA. We looked for the presence of Legionella (by co-culture) and FLA (by culture) in composts and bioaerosols collected at four composting facilities located in southern Switzerland. We investigated the association between the presence of Legionella and compost and air parameters and presence of FLA. Legionella spp. (including L. pneumophila) were detected in 69.3% (61/88) of the composts and FLA (mainly Acanthamoeba, Vermamoeba, Naegleria and Stenamoeba) in 92.0% (81/88). L. pneumophila and L. bozemanii were most frequently isolated. FLA as potential host for Legionella spp. were isolated from 40.9% (36/88) of the composts in all facilities. In Legionella-positive samples the temperature of compost was significantly lower (P = 0.012) than in Legionella-negative samples. Of 47 bioaerosol samples, 19.1% (9/47) were positive for FLA and 10.6% (5/47) for L. pneumophila. Composts (62.8%) were positive for Legionella and FLA contemporaneously, but both microorganisms were never detected simultaneously in bioaerosols. Compost can release bioaerosol containing FLA or Legionella and could represent a source of infection of community-acquired Legionella infections for workers and nearby residents.     

Occurrence of Free-Living Amoebae in Communities of Low and High Endemicity for Buruli Ulcer in Southern Benin

Buruli ulcer or Mycobacterium ulcerans disease occurs mainly in areas in proximity to standing or slowly running freshwater, habitats in which free-living amoebae occur. For this reason, a possible link between the habitat of M. ulcerans and free-living amoebae was investigated. Free-living amoebae and mycobacteria were isolated from water and biofilm specimens taken from protected and unprotected sources of water in villages known to have either high or low endemicity for Buruli ulcer in Benin. Amoebae were isolated from 78.8% of samples. A greater proportion of water bodies in areas of high endemicity had amoebae than in areas of low endemicity (83.3% versus 66.7%). Protected sources of water were significantly more likely to contain amoebae in areas of high endemicity than in areas of low endemicity (88.0% versus 11.1%). Several pathogenic free-living amoebae and mycobacteria were isolated. However, no M. ulcerans was isolated and no specimen was positive for IS2404 PCR. Our results show that the study area has a water hygiene problem, which is greater in areas of high Buruli ulcer endemicity than in areas of low endemicity. Our observations indicate that additional studies are required to explore the possible link between free-living amoebae and mycobacteria.     

Serological Comparison of Selected Parasitic and Free-Living Amoebae in vitro,Using Diffusion-Precipitation and Fluorescent-Antibody Technics*

SYNOPSIS. The present study has been directed to a serological comparison of three closely similar species of Entamoeba: E. histolytica, E. invadens and E. moshkovskii, and two free-living soil amoebae: Hartmannella rhysodes and Mayorella palestinensis. Except for E. histolytica and E. moshkovskii, the other amoebae used here were grown axenically; this is the first report of the use of antigenic extracts from axenic cultures of parasitic amoebae. The method described here provides a potent antigen that elicits a good antibody titer and is generally applicable to both parasitic and free-living amoebae. Amoebae pooled from well-grown cultures were thoroughly washed, sonicated and mixed with Freund's Adjuvant; this antigenic preparation was injected into rabbits. Two subcutaneous injections were given at three-week intervals, and 2-3 weeks thereafter blood was withdrawn to obtain antiserum. Agar-gel diffusion, cellulose acetate paper and fluorescent antibody technics were used to test the antigen-antibody (Ag-Ab) reactions. Results of the Ag-Ab reactions can be summarized as follows: 1) The homologous Ag-Ab reaction was obtained in all cases tested. 2) There was no serological reaction between the parasitic and free-living amoebae tested. 3) There was a definite serological reaction between H. rhysodes and M. palestinensis. 4) Multiple antigens were found in E. invadens (PZ strain) and E. histolytica (DKB strain) when they were tested against anti-PZ serum and anti-DKB serum, respectively, and no reaction was found when the other test antigens were exposed to these two antisera in gel-diffusion tests. 5) With the fluorescent antibody technic, E. histolytica (Laredo strain), E. moshkovskii (DSR strain) and E. histolytica (DKB strain) showed some degree of serological reaction in descending order when they were stained with conjugated anti-E. invadens serum.     

Epidemiology of Free-Living Ameba Infections1

Small free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats including fresh water, thermal discharges of power plants, soil, sewage and also from the nose and throats of patients with respiratory illness as well as healthy persons. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system infections due to these amebas have occurred world-wide. A majority (144) of these cases have been due to Naegleria fowleri which causes an acute, fulminating disease, primary amebic meningoencephalitis. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba which causes a subacute and/or chronic infection called granulomatous amebic encephalitis (GAE). Acanthamoeba, in addition to causing GAE, also causes nonfatal, but nevertheless painful, vision-threatening infections of the human cornea, Acanthamoeba keratitis. Infections due to Acanthamoeba have also been reported in a variety of animals. These observations, together with the fact that Acanthamoeba spp., Naegleria fowleri, and Hartmannella sp. can harbor pathogenic microorganisms such as Legionella and or mycobacteria indicate the public health importance of these amebas.     

The Biochemical Identification of Vahlkampfiid Amoebae1

ABSTRACT. Isoenyme electrophoresis of three different enzymes was used to compare 16 strains of vahlkampfiid amoebae and a strain identified as a slime mold. The strain designated as an Echinostelium sp. was found to be an isolate of Naegleria fowleri on the basis of zymogram type and other characters, confirming Cursons & Brown's similar conclusion drawn in 1975. The N. fowleri strains examined expressed the typical zymograms of the species. The N. gruberi strains in this study presented two distinctive groups of patterns that were different from the two previously reported types for N. gruberi. Each of the remaining species studied formed single distinctive groups by which they could be identified.     

Effects of Grazing by the Free-Living Soil Amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis on Various Bacteria

Cultures of 10 different bacteria were used to serve as food sources for axenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis. The nonpigmented enterobacteriaceae Escherichia coli K-12 and Klebsiella aerogenes appeared to be excellent feed to all three amoebae. Hardly any growth or ammonium production was observed in tests with Chromatium vinosum and Serratia marcescens, which share the presence of pigmented compounds. Distinct differences in net ammonium production were detected and were correlated to the amoebal growth yield. In general, growth of amoebae and ammonium production increased in the order A. polyphaga, A. castellanii, and H. vermiformis.     

Concordance of Species Composition Patterns among Microcrustaceans,Rotifers and Testate Amoebae in a Shallow Pond

In this study, spatial concordance among microcrustaceans, rotifers and testate amoebae was examined in a small pond in Central Brazil. The main goal was to test how consistently different taxonomic groups ordinate 9 sites distributed along the pond's main axis. Microcrustaceans were more abundant during the dry season, characterized by waters with high transparency and dissolved oxygen concentration. An increase in species richness and densities of rotifers and testate amoebae was detected during rainy season, with low values of Secchi disk and concentrations of dissolved oxygen. However, the ordination patterns generated by these groups were different. Concordant patterns across sites were found only between cladocerans and copepods. These results suggest that great care should be taken when ordination patterns based on some taxonomic groups are extrapolated to other groups for detecting environmental changes. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

The Spatial Distribution and Ecology of Zoochlorellae-Bearing Ciliates in a Productive Pond1

ABSTRACT. The spatial distributions of five zoochlorellae-bearing ciliate species (Euplotes daidaleos, Frontonia vernalis, Acaryophrya sp., Disematostoma bütschlii, and Stokesia vernalis) were investigated in a productive freshwater pond. The vertical profiles of all species were compared to the levels of O₂, CO₂, light, temperature, dissolved inorganic nitrogen, and the availability of particulate food sources. In the stratified water column, Stokesia remained close to the water surface: all other species reached peak abundance close to the oxic-anoxic boundary. The latter behavior probably accommodated the ciliates’requirements for aerobic respiration and particulate food and their dependance on the essential resources of light and dissolved nutrients, which came from opposite directions. With the collapse of stratification, Euplotes and Frontonia returned to the sediment where they reverted to a heterotrophic nutrition although they retained some zoochlorellae. Acaryophrya and Disematostoma also became heterotrophic, but they remained evenly distributed in the water column and they lost their zoochlorellae. Stokesia disappeared, presumably because it encysted. There was some evidence for vertical spatial separation of the five species in the water column.     

Seasonal Changes in the Chemical Composition of Ceratophyllum demersum L. in a Small Pond

Seasonal content of total soluble carbohydrate, total soluble protein, free fatty acids and alkaloids were examined in a population of Ceratophyllum demersum growing in a pond on the southwestern edge of the Canadian Shield. Total soluble carbohydrate and total soluble protein were inversely correlated. Free fatty acids were dominated by (16:0 + 16:1), 18:2 and 18:3. Relative proportions of the different free fatty acids remained roughly constant throughout the season. Alkaloid concentrations showed large fluctuations, with the highest values found during the first half of the season. Carbohydrate and protein contents of associated periphyton were not correlated with host macrophyte metabolism.     

Comparison of Free-Living Amoebae in Hot Water Systems of Hospitals with Isolates from Moist Sanitary Areas by Identifying Genera and Determining Temperature Tolerance

Legionella-contaminated hot water systems and moist sanitary areas in six hospitals were sampled for amoebae by following a standardized collection protocol. Genus identifications and temperature tolerance determinations were made. Amoebae identified as Hartmannella vermiformis (65%), Echinamoebae spp. (15%), Saccamoebae spp. (12%), and Vahlkampfia spp. (9%) were detected in 29 of 56 (52%) hot water samples. Twenty-three of 49 (47%) swabs obtained from moist areas were amoeba positive. The following genera were identified: Acanthamoeba (22%), Naegleria (22%), Vahlkampfia (20%), Hartmannella (15%), and Vanella (7%). The temperature tolerance of amoebae from hot water systems was strikingly different from that of amoebae from moist areas. At 44°C on agar, 59% of amoebic isolates sampled from hot water systems showed growth. The corresponding value for isolates from moist areas was only 17%. Six Acanthamoeba isolates from the moist areas were considered potential pathogens. Four Hartmannella and two Saccamoeba isolates from hot water could be cultured at 53°C.     

Long-Term Characterization of Free-Living and Particle-Associated Bacterial Communities in Lake Tiefwaren Reveals Distinct Seasonal Patterns

Seasonal changes in environmental conditions have a strong impact on microbial community structure and dynamics in aquatic habitats. To better elucidate the response of bacterial communities to environmental changes, we have measured a large variety of limnetic variables and investigated bacterial community composition (BCC) and dynamics over seven consecutive years between 2003 and 2009 in mesotrophic Lake Tiefwaren (NE Germany). We separated between free-living (FL, >0.2, <5.0?μm) and particle-associated (PA, >5.0?μm) bacteria to account for different bacterial lifestyles and to obtain a higher resolution of the microbial diversity. Changes in BCC were studied by DGGE based on PCR-amplified 16S rRNA gene fragments. Sequencing of DGGE bands revealed that ca. 70?% of all FL bacteria belonged to the Actinobacteria, whereas PA bacteria were dominated by Cyanobacteria (43?%). FL communities were generally less diverse and rather stable over time compared to their PA counterpart. Annual changes in reoccurring seasonal patterns of dominant freshwater bacteria were supported by statistical analyses, which revealed several significant correlations between DGGE profiles and various environmental variables, e.g. temperature and nutrients. Overall, FL bacteria were generally less affected by environmental changes than members of the PA fraction. Close association of PA bacteria with phytoplankton and zooplankton suggests a tight coupling of PA bacteria to organisms of higher trophic levels. Our results indicate substantial differences in bacterial lifestyle of pelagic freshwater bacteria, which are reflected by contrasting seasonal dynamics and relationships to a number of environmental variables.     

Seasonal Variation in Population Density and Heterotrophic Activity of Attached and Free-Living Bacteria in Coastal Waters

The abundance and heterotrophic activity of attached and free-living bacteria were examined seasonally in coastal water. Heterotrophic activity was determined by the uptake of [¹⁴C]glucose. The density of attached bacteria was always minor, not showing a seasonal variation, whereas the free-living bacteria were more numerous and showed a marked seasonal variation, their density being higher under warmer conditions. The contribution of the attached bacteria to the total assimilation of [¹⁴C]glucose (from 10 to 38%) was lower than that of the free-living bacteria, neither of them showing a seasonal variation. On a cellular basis, attached bacteria were more active, since they assimilated more [¹⁴C]glucose and showed, under warmer conditions, a higher cellular volume (0.102 versus 0.047 μm³). We consider that the factors responsible for these observations were the amount and quality of the particulate material, the different availability of organic matter for the two types of bacteria, and in a fundamental way, the variation in water temperature.