Importance of the icmN gene in the growth of Legionella pneumophila in amoebic cells at low temperature

Legionella pneumophila grows in amoebae and has achieved the ability to grow at various temperatures, although the mechanisms controlling this ability remain poorly understood. The Icm/Dot type IVB secretion system is composed of more than 25 proteins and is known to be essential for intracellular growth. The role of the icmN gene in intracellular multiplication and the effects of culture temperatures on it are not precisely understood. We conducted our investigation using an icmN mutant made by gene replacement mutagenesis. Intracellular growth of the mutant was impaired both in mammalian macrophages and amoeba at 37?°C. In particular, intracellular growth in amoebae was completely impaired at 25?°C. It was found that genes from icmN to icmC formed an operon, i.e., icmN, -M, -L, -E, -G, -C,, and the promoter activity of the icmN operon was stronger at 25 than at 37?°C. It was suggested that icmM and its downstream genes had a secondary promoter that enables icmN mutant grow in amoebae at lower temperatures and macrophages at 37?°C. These results show that the icmN promoter has a low temperature inducible nature, and gene products of the icmN operon require high expression for bacterial proliferation at low temperatures within amoeba.     

The pathogenic amoeboflagellate Naegleria fowleri: environmental isolations, competitors, ecologic interactions, and the flagellate-empty habitat hypothesis

From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C. Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling. The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45 degrees C. Heat tolerant competitors were much more common than N. fowleri. Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient. Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45-49 degrees C) amoebae, and one thermophilic (52 degrees C) Acanthamoeba. Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N. fowleri from almost all other amoebae on agar plates. The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing.     

Isolation and molecular identification of free-living amoebae of the genus Naegleria from Arctic and sub-Antarctic regions

Twenty-three freshwater samples with sediment taken from two regions in the Arctic, Spitzbergen and Greenland, and one region in sub-Antarctica, Ile de la Possession, were cultured for amoebae at 37 degrees C and room temperature (RT). Only two samples yielded amoebae at 37 degrees C and the two isolates were identified from their morphological features to belong to the genus Acanthamoeba. Vahlkampfiid amoebae were isolated from 11 samples at RT. Morphological analysis of the cysts identified all 11 isolates as belonging to the genus Naegleria, although only about half of them (45%) transformed into flagellates. Ribosomal DNA sequence analysis demonstrated that these isolates represent novel species and that N. antarctica, N. dobsoni and N. chilensis are their closest relatives. Not surprisingly, these three species also grow at lower temperatures (<37 degrees C) than the majority of described Naegleria spp. Two of the eight new species were found in both Arctic and sub-Antarctic regions, and other new species from the Arctic are closely related to new species from the sub-Antarctic. Therefore, it seems the Naegleria gene pool present in the polar regions is different from that found in temperate and tropical regions.     

Paravahlkampfia francinae n. sp. Masquerading as an Agent of Primary Amoebic Meningoencephalitis

ABSTRACT. Paravahlkampfia francinae n. sp., a new species of the free-living amoeba genus Paravahlkampfia , designated as CDC:V595, was isolated from the cerebrospinal fluid of a patient with headache, sore throat, and vomiting, typical symptoms of primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri . The isolate grew at 33 °C, 37 °C, 40 °C, and 42 °C and destroyed mammalian cell cultures. However, it did not kill young mice upon intranasal inoculation. P. francinae does not produce flagellates and does not grow on agar plates coated with Gram-negative bacteria such as Escherichia coli , the usual food source of Paravahlkampfia ustiana , the type species of the genus. The trophozoite at light microscopy exhibited eruptive locomotion and possessed a single vesicular nucleus. Ultrastructurally, the trophozoites had numerous mitochondria with discoidal cristae but did not have a Golgi apparatus. The trophozoites differentiated into cysts after consuming most of the monolayer. The cyst had an inner well-differentiated endocyst and an outer thin, wrinkled, and wavy ectocyst with no pores. During excystation trophozoites ruptured the cyst wall and emerged from the cysts. A unique feature seen in the cysts was the presence of bacterial endosymbionts, both in the endoplasm and within the cyst wall. Full-length sequencing analysis of the 18S and 5.8S RNA genes of P. francinae showed that they were distinct from those of other Paravahlkampfia species. The patient recovered within a few days indicating that some of the previously reported cases of PAM that survived may have been due to P. francinae .     

Characterization of a Deep‐Branching Heterolobosean,Pharyngomonas turkanaensis n. sp., Isolated from a Non‐Hypersaline Habitat,and Ultrastructural Comparison of Cysts and Amoebae Among Pharyngomonas Strains

An unusual heterolobosean amoeba, isolate LO, was isolated recently from a sample with a salinity of ~4‰, from Lake Turkana in East Africa. 18S rDNA phylogenies confirm that isolate LO branches among halophilic amoeboflagellates assigned to Pharyngomonas. We examined the ultrastructure of the amoeba and cyst stages of isolate LO, as well as the amoebae and cysts of Pharyngomonas kirbyi (isolates AS12B and SD1A). The amoebae of all three isolates lacked discrete dictyosomes and had discoidal/flattened mitochondrial cristae, but the mitochondria were not enrobed by rough endoplasmic reticulum. The cysts of all three isolates showed a thick, bipartite cyst wall, and lacked cyst pores. The cysts of isolate LO were distinct in that the ectocyst was very loose‐fitting, and could contain “crypts”. No flagellate form of isolate LO has been observed to date, and a salinity‐for‐growth experiment showed that isolate LO can grow at 15–100‰ salinity, indicating that it is halotolerant. By contrast, other studied Pharyngomonas isolates are amoeboflagellates and true halophiles. Therefore, we propose isolate LO as a new species, Pharyngomonas turkanaensis n. sp. It is possible that P. turkanaensis descended from halophilic ancestors, and represents a secondary reestablishment of a physiology adapted for moderate salinity.     

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 °C, subsequently displayed a high thermotolerance, being able to grow at 42 °C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.     

Viability of Listeria monocytogenes in co-culture with Acanthamoeba spp.

Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and can grow and survive under a wide range of environmental conditions. It contaminates foods via raw materials or food-processing environments. However, the current knowledge of its ecology and, in particular, the mode of environmental survival and transmission of this intracellular pathogen remains limited. Research has shown that several intracellular pathogens are able to survive or replicate within free-living amoebae. To examine the viability of L. monocytogenes in interaction with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba lenticulata . The survival of bacteria and amoebae was determined using culture techniques and microscopy. Under the experimental conditions used, all amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew saprophytically on materials released from amoebae, which may play an important role in the survival of bacteria under extreme environmental conditions.     

Evaluation of the Indirect Fluorescent-Antibody Technique for Identification of Naegleria Species

The indirect fluorescent-antibody technique was used to assess a rapid method for identification of amoebae belonging to the genus Naegleria. Thirty-eight Naegleria and eight other limax amoeba strains were examined by using one N. gruberi and two N. fowleri antisera. All pathogenic Naegleriae, most of which originated from fatal cases of primary amoebic meningo-encephalitis, were identified as belonging to the fowleri species. Most of the N. gruberi strains showed irregular fluorescence. Other limax amoebae, such as Vahlkampfia, Acanthamoeba, Hartmannella, and Schizopyrenus sp. gave negative responses with the prepared antisera. The indirect fluorescent-antibody technique allows the identification of N. fowleri in a mixed culture of both N. fowleri and N. gruberi strains. Twenty-two Naegleria isolated from a suspected stream, other surface waters, and muddy soil could be excluded from the fowleri species with the indirect fluorescent-antibody technique. The results obtained demonstrate that this immunological technique is a valid method for the rapid identification of N. fowleri trophozoites.     

Acanthamoeba isolates from the Houston Coastal Center: some characteristics and establishment in pure culture

Acanthamoeba species were isolated from soil samples taken at the University of Houston Coastal Center. Five pure culture isolates (clones) were established starting from single Acanthamoeba cysts. All five isolates were able to grow and encyst at 37 degrees C in synthetic media. The isolates also were able to infect, cause cytopathogenic effects and death of African green monkey kidney tissue culture cells.     

Stimulatory effect of cooling tower biocides on amoebae.

Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds, another quaternary ammonium compound alone, and an isothiazolin derivative. The amoebae isolated were Acanthamoeba hatchetti and a Cochliopodium species. Two other amoeba cultures, an A. hatchetti culture and Cochliopodium bilimbosum, were obtained from the American Type Culture Collection (ATCC) and were also tested. The cooling tower isolates were more resistant to most of the biocides than the ATCC isolates were. The isothiazolin derivative was the least inhibitory to all four amoeba isolates, and tributyltin neodecanoate mixed with quaternary ammonium compounds was the most inhibitory to three of the four isolates. After exposure to lower concentrations of the biocides, including for one strain the manufacturer's recommended concentration of one biocide, the cooling tower amoeba populations increased significantly compared with unexposed controls, whereas the ATCC isolates were not stimulated at any of the concentrations tested. In some cases, concentrations which stimulated cooling tower amoebae inhibited the growth of the ATCC isolates. These results suggest that cooling tower amoebae may adapt to biocides, underscoring the need to use freshly isolated cooling tower organisms rather than organisms from culture collections for testing the efficacy of such biocides. The stimulatory effect of biocides on amoeba populations is an alarming observation, since these organisms may be reservoirs for legionellae. Biocides used to control microbial growth may actually enhance populations of host organisms for pathogenic bacteria.     

Relationship of the DNA content in the nuclei of Amoeba proteus to the food regimen]

The relative DNA content of isolated Amoeba proteus nuclei has been measured by cytofluorometry. With the amoeba strain studied, the generation time is roughly equal to 48 hours at 25 degrees C, and with the presence of food in the medium. After the synchronous divisions, amoebae were maintained in the medium either with or without food organisms (Tetrahymena pyriformis). DNA contents in the nuclei of both the amoebae groups were measured within 4 and 48 hours after division. Before 16 hours, the nuclear DNA contents did not differ in either group. Starting from 20 hours, the DNA amount in fed amoebae exceeded that in starved animals. On the whole, the differences in DNA quantity increased by a 48th hour after division, when the nuclei of the former contained 145% DNA of the latter. The results obtained suggest that the DNA synthesis in amoeba nuclei may proceed during the whole interphase, and that during the second half of interphase the content of DNA may depend on the feeding intensity in amoebae. After refeeding the starved animals, DNA contents in their nuclei increased to reach the same level as in the constantly fed amoebae seen in the end of interphase.     

A Description of a New "Amoebozoan" Isolated from the American Lobster, Homarus americanus

ABSTRACT. Our knowledge of the diversity of amoeboid protists is rapidly expanding as new and old habitats are more fully explored. In 2003, while investigating the cause of an amoeboid disease afflicting lobsters on the East Coast, samples were examined for the presence of amoebae from the carapace washings of the American lobster, Homarus americanus . During this survey a unique community of gymnamoebae was discovered. Among the new taxa discovered was a small Thecamoeba -like organism with a single posteriorly directed pseudopodium. Although resembling Parvamoeba rugata , this amoeba displayed distinctive morphology from that isolate or any other amoebozoan. Phylogenetic analysis shows this amoeba is distantly related to the Thecamoebidae. In this paper we describe the unique morphology of a second species of Parvamoeba and discuss its phylogenetic position with respect to the "Amoebozoa."     

Incubation of water samples containing amoebae improves detection of legionellae by the culture method.

Some protozoans isolated from aquatic habitats, including domestic water supplies, can support the intracellular replication of autochthonous legionellae in vitro. We studied the effect of incubating water samples containing amoebae on the sensitivity of culture for legionellae. Samples collected during investigations of legionellosis epidemics and shown by conventional culture procedures to contain amoebae, but not legionellae, were incubated at 35 degrees C and replated. Legionellae were recovered from 59 of 144 such samples. Species isolated included L. pneumophila, L. anisa, L. bozemanii, L. gormanii, L. micdadei, L. rubrilucens, L. sainthelensi, L. steigerwaltii, and an unnamed species. Acanthamoeba polyphaga, Acanthamoeba hatchetti, a Rosculus sp., Hartmannella vermiformis, and Vahlkampfia spp. were among the autochthonous amoebae identified. Legionellae were recovered by this procedure from only 3 of 63 samples that were negative for amoebae by conventional culture procedures. These results show that water samples negative for legionellae, but positive for amoebae, by standard culture techniques should be incubated and replated to maximize the sensitivity of culture for legionellae.     

A new heterolobosean amoeba Solumitrus palustris n. g., n. sp. isolated from freshwater marsh soil

During the course of research on the bacterial feeding behavior and resistance of amoebae to virulent pathogens, we isolated a new strain of amoeba from organic rich soil at the margin of freshwater swamp in the northeastern United States. Light microscopic morphology is characteristically heterolobosean, resembling vahlkampfiids, including a broadened, limax shape, and eruptive locomotion, but occasionally becoming more contracted and less elongated with lateral or anterior bulges and somewhat branching sparse, uroidal filaments. Electron microscopic evidence, including mitochondria with flattened cristae surrounded by rough endoplasmic reticulum, further indicates a heterolobosean affinity. The solitary nucleus contains a centrally located nucleolus. Cysts are rounded with occasionally an eccentrically located nucleus. The cyst walls are relatively thin, becoming crenated, and loosely enclosing the cyst when mature. Molecular genetic evidence places this isolate among the Heterolobosea, branching most closely in a clade including Allovahlkampfia spelaea and previously isolated, un-named strains of soil amoebae. Based on differentiated features, including morphology of the uroid, cyst wall structure, and molecular genetic evidence that distinguish it from A. spelaea, a new genus and species, Solumitrus palustris, is proposed for this new heterolobosean.     

"Slime molds" among the Tubulinea (Amoebozoa): molecular systematics and taxonomy of Copromyxa

Copromyxa protea is a dung-inhabiting amoeboid organism that aggregates to form simple macroscopic fruiting structures, sorocarps, which are composed of a single cell type. In a recent effort to find the phylogenetic positions of the less well-known sorocarpic protists considered to be "cellular slime molds," or aggregatively fruiting amoebae, we isolated C. protea and sequenced the nuclear-encoded small subunit ribosomal RNA gene from four samples collected from cattle farms in the central USA. Phylogenetic analyses of these data place C. protea in the eukaryotic supergroup Amoebozoa together with the Tubulinea, in which there has been no previous report of an aggregative fruiting habit. This is consistent with the morphology of the trophozoites. In fact, Copromyxa protea is found to be very closely related to Hartmannella cantabrigiensis and to a since lost amoeba isolate, Hartmannella sp. 4/3Da/10. This new grouping of Copromyxa+H. cantabrigiensis is sister to Glaeseria, which together are sister to the Amoebidae (Amoeba+Chaos). We suggest renaming, H. cantabrigiensis as C. cantabrigiensis and designate isolate 4/3Da/10 as C. protea. Future work is needed to see if these newly assigned members of the genus Copromyxa also show evidence of an ability to fruit.     

Incubation of water samples containing amoebae improves detection of legionellae by the culture method.

Some protozoans isolated from aquatic habitats, including domestic water supplies, can support the intracellular replication of autochthonous legionellae in vitro. We studied the effect of incubating water samples containing amoebae on the sensitivity of culture for legionellae. Samples collected during investigations of legionellosis epidemics and shown by conventional culture procedures to contain amoebae, but not legionellae, were incubated at 35 degrees C and replated. Legionellae were recovered from 59 of 144 such samples. Species isolated included L. pneumophila, L. anisa, L. bozemanii, L. gormanii, L. micdadei, L. rubrilucens, L. sainthelensi, L. steigerwaltii, and an unnamed species. Acanthamoeba polyphaga, Acanthamoeba hatchetti, a Rosculus sp., Hartmannella vermiformis, and Vahlkampfia spp. were among the autochthonous amoebae identified. Legionellae were recovered by this procedure from only 3 of 63 samples that were negative for amoebae by conventional culture procedures. These results show that water samples negative for legionellae, but positive for amoebae, by standard culture techniques should be incubated and replated to maximize the sensitivity of culture for legionellae.     

Fossil amoebae (Hemiarcherellidae fam. nov.) from Albian (Cretaceous) amber of France

Abstract: Two extraordinarily well‐preserved testate amoebae are described from Late Albian age amber from south‐western France. The specimens are attributed to a new family, the Hemiarcherellidae fam. nov., and are described as Hemiarcherella christellae gen. et sp. nov. The amoebae described herein originate from highly fossiliferous amber pieces. Based on syninclusions, Hemiarcherella christellae was a soil‐dwelling organism, probably an active bacterivore. This taxon represents the third species of testate amoebae described from mid‐Cretaceous French amber. Analysis of this fossil amoeba fauna illustrates the uniqueness of mid‐Cretaceous French amber deposits. Indeed, most amoebae found in amber have been assigned to modern species, corroborating the hypothesis of morphological stasis in different microbial lineages. However, the well‐preserved amoebae fauna found in French amber can be distinguished clearly from modern species and help us to better understand the fossil record of these organisms.     

Growth and Bacteriolytic Activity of a Soil Amoeba, Hartmannella glebae

A soil amoeba, Hartmannella glebae, could grow on a variety of gram-positive and gram-negative bacteria, although the rate of growth was faster in the presence of gram-negative bacteria. The amoeba, however, could not use yeasts, molds, or a green alga as a nutritional source. The extract prepared from amoebae grown in the presence of Aerobacter aerogenes and Alcaligenes faecalis could lyse intact cells and cell walls of many gram-positive bacteria at different rates. The spectrum of lytic activity was similar to that of egg-white lysozyme, with the exception that several species and strains of Bacillus, Micrococcus, and Staphylococcus were resistant to lysozyme and susceptible to the extract. The gram-negative bacteria tested were resistant.     

An Analysis of Clumping in the Soil Amoeba Mayorella palestinensis*

SYNOPSIS. Mayorella palestinensis, a small soil amoeba, clumped during active growth in axenic, fluid culture media. Clumping was not associated with starvation (as in the case of cellular slime molds) or with encystation Hartmannella rhysodes was used as a control amoeba which did not clump to the same extent, but otherwise resembled M. palestinensis. Mixtures of both amoebae formed mixed clumps, without subsequent segregation of amoebae in the clumps. Clumping by M. palestinensis was temperature dependent, localized to the surface of the amoeba and required surface conformation. The requirement for a living system could be separated from the surface specificity of M. palestinensis and satisfied with living amoebae that did not clump by themselves, i.e., H. rhysodes, but did clump with dead M. palestinensis. It is proposed that amoeba-to-amoeba adhesion is similar to foreign substrate adhesion, and that M. palestinensis adheres to itself because its surface is more suitable for adhesion than other surfaces present in the culture. A competition for adhesion exists between surfaces so that clumps can be dissociated by providing a foreign surface that is more suitable for adhesion than the surface of the amoeba.