Activity of water biocide chemicals and contact lens disinfectants on pathogenic free-living amoebae

Free-living amoebae (FLA) are found in most soil and aquatic environments. They claim our attention not only because they are intrinsically interesting microbes about which little is known but also because of the capacity for certain species to cause severe and often fatal disease in man. Naegleria fowleri produces acute meningoencephalitis, whilst Acanthamoeba spp. cause chronic encephalitis in immunocompromised individuals and also a destructive ocular infection associated with contact lens wear. FLA can also support the intracellular growth of Legionella pneumophila, the bacterium causing Legionnaires' disease in man. The ability to regulate the presence of pathogenic and nonpathogenic FLA in the environment throufh the use of chemical disinfection is therefore relevant in the prevention of human disease. In this paper the activity of water biocide chemicals and contact lens disinfectants against pathogenic FLA is presented.     

Application of flow cytometry to studies of pathogenic free-living amoebae

Species of small, free-living amoebae of the genera Naegleria and Acanthamoeba can cause fatal amoebic meningoencephalitis. Previous investigations have shown that pathogenic amoebae are associated with thermally altered water. Flow cytometric techniques for identifying species of pathogenic and nonpathogenic amoebae from such water have been developed, using immunofluorescence and fluorescein-bound concanavalin A. Flow cytometry is accomplished with a cytofluorograph, in which cells are dispersed in a suspended carrier liquid and passed in front of a focused argon ion laser beam. Cells are then distinguished by the degree of scattered light (size) or fluorescence. Flow cytometry techniques have proven efficient for environmental samples, as indicated by the identification of pathogenic Naegleria fowleri and nonpathogenic Naegleri gruberi and Acanthamoeba castellanii isolated from the Savannah River Plant in South Carolina. Cytofluorographic analysis of environmental samples has several advantages over the current methods of isolation and classification of free-living amoebae. With this system, it is possible to rapidly identify species and quantitate mixtures of pathogenic amoebae in environmental samples. Cytofluorographic analysis of amoebic isolates reduces the time presently required to screen environmental sites for pathogenic amoebae. The cytofluorograph permits detection and species identification of nonthermophilic Naegleria spp. and Acanthamoeba spp. that could not easily be isolated for species identification by conventional methods. Other advantages of flow cytometry over fluorescent microscopy include a high degree of statistical precision due to the large numbers measured, high immunofluorescent titers, and elimination of subjectivity and fluorescence fading.     

Polymerase chain reaction detection of potentially pathogenic free-living amoebae in dental units

Several genera of amoebae can be found in water from dental units and on the inner surface of waterlines. The presence of bacterial biofilms on these surfaces is thought to favor the proliferation of amoebae. Potentially pathogenic Acanthamoeba and Naegleria spp. may be an infection risk for patients through contact with open surgical sites or aerosolization. A polymerase chain reaction of DNA extracted from pelleted samples showed that Acanthamoeba spp. and Naegleria spp. were present in water from dental units, suction lines, and suction filters at the dental clinic of the Université de Montréal. Acanthamoeba spp. were detected in 24.2% of 66 samples and Naegleria spp. in 3.0%. We discuss the infection risk associated with these results.     

Viability of pathogenic and nonpathogenic free-living amoebae in long-term storage at a range of temperatures.

The long-term storage of pathogenic and nonpathogenic strains of both Naegleria and Acanthamoeba spp. were tested on Page amoeba saline agar slopes for 24 months at room temperature and for 8 months at -10, 4, 10, and 15 degrees C. Acanthamoeba strains showed better survival potential than Naegleria strains, particularly when they were stored at temperatures equal to or lower than room temperature.     

Pathogenic free-living amoebae in Korea

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.     

Nutritional factors and conditions for the axenic culture of free-living nematodes

• 1.1. Soy-peptone has been fractionated to yield a series of increasingly purified components which sharply increase the populations of Caenorhabditis briggsae and Caenorhabditis elegans when added to the basal medium. The nutritionally active material appears to be a small polypeptide.
• 2.2. C. briggsae and C. elegans routinely reach populations of 150,000/ml or greater in 9 days in still culture, starting from an inoculum of only 500 organisms per ml. C. elegans is particularly sensitive to the depth of the medium. However, large populations can be achieved in deep cultures if continuous shaking is carried out.
• 3.3. Panagrellus silusiae shows improved populations if the basal medium is supplemented with the nutritional factor from soy-peptone. However, 0.5% acetic acid or 1% ethanol added to the medium serves equally well. There is no additive effect of ethanol and the factor.

     

Bacterial endosymbionts of free-living amoebae

The occurrence of bacterial endosymbionts in free-living amoebae has been known for decades, but their obligate intracellular lifestyle hampered their identification. Application of the full cycle rRNA approach, including 16S rRNA gene sequencing and fluorescence in-situ hybridization with 16S rRNA-targeted oligonucleotide probes, assigned the symbionts of Acanthamoeba spp. and Hartmannella sp. to five different evolutionary lineages within the Proteobacteria, the Bacteroidetes, and the Chlamydiae, respectively. Some of these bacterial symbionts are most closely related to bacterial pathogens of humans, and it has been suggested that they should be considered potential emerging pathogens. Complete genome sequence analysis of a chlamydia-related symbiont of Acanthamoeba sp. showed that this endosymbiont uses similar mechanisms for interaction with its eukaryotic host cell as do the well-known bacterial pathogens of humans. Furthermore, phylogenetic analysis suggested that these mechanisms have been evolved by the ancestor of these amoeba symbionts in interplay with ancient unicellular eukaryotes.     

The maintenance of free-living amoebae by cryopreservation.

We have successfully cryopreserved free-living amoebae in order to maintain them feasibly under the conditions in our laboratory. The viability of trophozoites was higher when frozen by slow cooling (overall 0.7 degree C/min) than by fast cooling (overall 1.3 degrees C/min). Glycerol and dimethylsulfoxide at the final concentration of 7.5% each was used for cryopreservation of free-living amoebae trophozoites. The survival rate was 2-39% after storage in the liquid nitrogen for 60 days. Gross cultural or morphological changes were not noted in trophozoites thawed from frozen suspensions.     

Cocultivation of Legionella pneumophila and free-living amoebae.

Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophila as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. royreba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth.     

Viability of pathogenic and nonpathogenic free-living amoebae in long-term storage at a range of temperatures

The long-term storage of pathogenic and nonpathogenic strains of both Naegleria and Acanthamoeba spp. were tested on Page amoeba saline agar slopes for 24 months at room temperature and for 8 months at -10, 4, 10, and 15 degrees C. Acanthamoeba strains showed better survival potential than Naegleria strains, particularly when they were stored at temperatures equal to or lower than room temperature.     

Polyamines biosynthesis and oxidation in free-living amoebae

Summary. In this paper we describe the polyamine biosynthesis and oxidation processes, giving an overview about recent results in free-living Amoebae.The protozoa polyamine levels are different in comparison with mammalian cells. Also, the polyamine levels in protozoa cells change if these species are pathological or not for the human beings. All the amoeba strains show high concentrations of 1,3-diaminopropane (DAP), spermidine and acetylspermidine while spermine is absent. In these amoeba a considerable polyamine oxidase activity has been found, which acts on N⁸-acetylspermidine, but not on free polyamines. This enzyme is responsible, together with polyamine acetylase, of DAP synthesis whose function is not well known.     

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.     

Seasonal distribution of thermotolerant free-living amoebae. II. Lake Issaqueena

A quantitative study of the seasonal distribution of thermotolerant (37 degrees C and 45 degrees 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 (approximately 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.     

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.     

Toxoplasma gondii: uptake and survival of oocysts in free-living amoebae

Waterborne transmission of the oocyst stage of Toxoplasma gondii can cause outbreaks of clinical toxoplasmosis in humans and infection of marine mammals. In water-related environments and soil, free-living amoebae are considered potential carriers of various pathogens, but knowledge on interactions with parasitic protozoa remains elusive. In the present study, we assessed whether the free-living Acanthamoebacastellanii, due to its phagocytic activity, can interact with T. gondii oocysts. We report that amoebae can internalize T. gondii oocysts by active uptake. Intracellular oocysts in amoebae rarely underwent phagocytic lysis, retained viability and established infection in mice. Interaction of T. gondii with amoebae did not reduce the infectivity and pathogenicity of oocysts even after prolonged co-cultivation. Our results show that uptake of oocysts by A. castellanii does not restrain the transmission of T. gondii in a murine infection model.     

Free-living pathogenic and nonpathogenic amoebae in Maryland soils.

Tests for potentially pathogenic amoebae were carried out on soil samples from the following sites: (i) farmlands fertilized with municipal sewage wastes, (ii) a stream receiving sewage effluent from a sludge lagoon, (iii) a ravine receiving storm runoff from a cattle farm, (iv) farmlands not fertilized with sewage wastes, and (v) a vegetated shoreline of a waterfront estate not used for farming or livestock production. Study sites were located on the eastern shore of Maryland, bordered to the north by Delaware and to the south by Virginia. Twenty-four species of soil amoebae, including five potentially pathogenic Acanthamoeba species (members of the family Acanthamoebidae), were identified. All of the sites yielded two or more of the potential pathogens.