Characterization of an Axenic Strain of Hartmannella vermiformis Obtained from an Investigation of Nosocomial Legionellosis

ABSTRACT. A free-living amoeba identified as Hartmannella vermiformis was isolated from a water sample obtained during an investigation of nosocomial legionellosis. Hartmannella vermiformis is known to support the intracellular multiplication of Legionella pneumophila . This strain of H. vermiformis , designated CDC-19, was cloned and established in axenic culture to develop a model for the study of the pathogenicity of legionellae. Isoenzyme patterns of axenically-cultivated strain CDC-19 were compared with two strains of H. vermiformis derived from the type strain, one axenic (ATCC 50236) and the other grown in the presence of bacteria (ATCC 30966). Enzyme patterns suggested that all three strains are assignable to the species H. vermiformis. Axenic H. vermiformis strain CDC-19 has been deposited with the American Type Culture Collection (ATCC 50237) and should prove useful in the study of protozoan-bacterial interaction.     

Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis

A free-living amoeba identified as Hartmannella vermiformis was isolated from a water sample obtained during an investigation of nosocomial legionellosis. Hartmannella vermiformis is known to support the intracellular multiplication of Legionella pneumophila. This strain of H. vermiformis, designated CDC-19, was cloned and established in axenic culture to develop a model for the study of the pathogenicity of legionellae. Isoenzyme patterns of axenically-cultivated strain CDC-19 were compared with two strains of H. vermiformis derived from the type strain, one axenic (ATCC 50236) and the other grown in the presence of bacteria (ATCC 30966). Enzyme patterns suggested that all three strains are assignable to the species H. vermiformis. Axenic H. vermiformis strain CDC-19 has been deposited with the American Type Culture Collection (ATCC 50237) and should prove useful in the study of protozoan-bacterial interaction.     

Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 x 10(-1) and 1.14 x 10(4) cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 +/- 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (> or =98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.     

Differences in isoenzyme patterns of axenically and monoxenically grown Acanthamoeba and Hartmannella

Axenically and monoxenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga and different isolates of Hartmannella vermiformis strains were examined by polyacrylamide isoelectric focusing in the pH range 3–10. Isoenzyme patterns of acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were compared. Zymograms were used to reveal differences in typical isoenzyme patterns between axenically and monoxenically grown amoebae and to compare axenically grown A. castellanii, A. polyphaga and H. vermiformis. Comparison of zymograms for AP, PE and MDH between axenically grown Acanthamoeba and Hartmannella strains revealed different isoenzyme patterns. Acanthamoeba showed strong bands for ADH and extremely weak bands for GPI and PGM, while Hartmannella lacked ADH but possessed bands for GPI and PGM.\par Comparison of zymograms from axenically and monoxenically grown amoebae revealed a lower intensity and even lack of typical isoenzyme bands in lysates from monoxenic cultures. The observed changes in typical isoenzyme patterns induced by the bacterial substrate can influence the correct isoenzymatic typing of different strains in clinical and phylogenetic studies.     

The Occurrence of Amphizoic Amebae in Domestic Animals

SYNOPSIS Random examination of domestic animals revealed the frequent presence of free-living amebae in their bodies. In diseased or dead cows, dogs, pigs, rabbits, pigeons, and turkeys 15 strains of amebae were found, belonging to the genera Acanthamoeba (A. polyphaga), Hartmannella (H. vermiformis) , and Vahlkampfia (V. avara, V. enterica, V. inornata). , They were usually accompanied by other infectious agents in different parts of the host bodies. Pathogenicity of 3 isolates could not be demonstrated by inoculation of laboratory animals.
Some features of the isolates differed from those previously known for members of these genera. These strains may be considered amphizoic amebae according to Page (1974).     

Free-living freshwater amoebae differ in their susceptibility to the pathogenic bacterium Legionella pneumophila

Legionella pneumophila is known as a facultative intracellular parasite of free-living soil and freshwater amoebae, of which several species have been shown to support the growth of the pathogenic bacteria. We report for the first time the behaviour of two strains (c2c and Z503) of the amoeba Willaertia magna towards different strains of L. pneumophila serogroup 1 and compared it with Acanthamoeba castellanii and Hartmannella vermiformis , known to be L. pneumophila permissive. In contrast to the results seen with other amoebae, W. magna c2c inhibited the growth of one strain of Legionella ( L. pneumophila , Paris), but not of others belonging to the same serogroup ( L. pneumophila , Philadelphia and L. pneumophila , Lens). Also, the different L. pneumophila inhibited cell growth and induced cell death in A. castellanii, H. vermiformis and W. magna Z503 within 3–4 days while W. magna c2c strain remained unaffected even up to 7 days. Electron microscopy demonstrated that the formation of numerous replicative phagosomes observed within Acanthamoeba and Hartmannella is rarely seen in W. magna c2c cocultured with L. pneumophila . Moreover, the morphological differences were observed between L. pneumophila cultured either with Willaertia or other amoebae. These observations show that amoebae are not all equally permissive to L. pneumophila and highlight W. magna c2c as particularly resistant towards some strains of this bacterium.     

Gentamicin-Containing Peptone-Yeast Extract Medium for Cocultivation of Hartmannella vermiformis ATCC 50256 and Virulent Strains of Legionella pneumophila

We evaluated the use of peptone-yeast extract (PY) medium, different strains of Hartmannella vermiformis, and gentamicin in a coculture system to improve the discrimination of virulent and avirulent strains of Legionella pneumophila. H. vermiformis ATCC 50256 was unique among four strains of H. vermiformis, in that it multiplied equally well in Medium 1034 and PY medium (Medium 1034 without fetal calf serum, folic acid, hemin, and yeast nucleic acid and with a 50% reduction of peptone). However, both a virulent strain of L. pneumophila and its avirulent derivative strain multiplied in cocultures when PY medium was used. The multiplication of this avirulent strain was greatly reduced by incorporating gentamicin (1 (mu)g/ml) into the cocultivation system. Five virulent-avirulent sets of L. pneumophila strains were then tested for multiplication in cocultures with H. vermiformis ATCC 50256 and the gentamicin-containing PY medium. Only the virulent strains multiplied. The modified cocultivation system can discriminate between virulent and avirulent strains of L. pneumophila.     

18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa

Cochliopodium is a very distinctive genus of discoid amoebae covered by a dorsal tectum of carbohydrate microscales. Its phylogenetic position is unclear, since although sharing many features with naked "gymnamoebae", the tectum sets it apart. We sequenced 18S ribosomal RNA genes from three Cochliopodium species (minus, spiniferum and Cochliopodium sp., a new species resembling C. minutum). Phylogenetic analysis shows Cochliopodium as robustly holophyletic and within Amoebozoa, in full accord with morphological data. Cochliopodium is always one of the basal branches within Amoebozoa but its precise position is unstable. In Bayesian analysis it is sister to holophyletic Glycostylida, but distance trees mostly place it between Dermamoeba and a possibly artifactual long-branch cluster including Thecamoeba. These positions are poorly supported and basal amoebozoan branching ill-resolved, making it unclear whether Discosea (Glycostylida, Himatismenida, Dermamoebida) is holophyletic; however, Thecamoeba seems not specifically related to Dermamoeba. We also sequenced the small-subunit rRNA gene of Vannella persistens, which constantly grouped with other Vannella species, and two Hartmannella strains. Our trees suggest that Vexilliferidae, Variosea and Hartmannella are polyphyletic, confirming the existence of two very distinct Hartmannella clades: that comprising H. cantabrigiensis and another divergent species is sister to Glaeseria, whilst Hartmannella vermiformis branches more deeply.     

农村生活污水处理厂中阿米巴原虫的定量检测

【目的】自由生活的棘阿米巴属(Acanthamoeba spp.)和哈曼属原虫(Hartmannella vermiformis)普遍存在于自然界的土壤和各种水体中,这两个属中的某些种类被认为对人和动物具有潜在的致病性,应用染料法实时荧光定量PCR技术建立特异性强、灵敏度高及重复性好的快速检测阿米巴虫的方法具有实际意义。【方法】采用非培养方法选择适合低拷贝基因检测的荧光染料BRYT Green? dye用于农村生活污水处理厂不同工艺阶段水样中Acanthamoeba spp.和H. Vermiformis 18S rRNA基因的检测和定量分析。【结果】在整个处理工艺流程中均检测到Acanthamoeba spp.和H. Vermiformis,并呈现出不同的变化趋势,进水中分别达到8.70×105拷贝/L和1.84×106拷贝/L。与进水相比,调节池、好氧池和膜池中阿米巴原虫的数量均降低了1?2个数量级,但是出水中Acanthamoeba spp. 则出现增加趋势。【结论】对阿米巴原虫可能造成的潜在健康危害应引起重视,并有必要作为污水处理达标的补充标准。     

rRNA and nifD phylogeny of Bradyrhizobium from sites across the Pacific Basin

Many undomesticated legumes harbor nodule bacteria related to the soybean symbiont Bradyrhizobium elkanii, but little is known about their phylogenetic relationships or geographic distribution. Sequences of ribosomal genes (16S rRNA and partial 23S rRNA) and the nitrogenase alpha-subunit gene (nifD) were analyzed in 22 isolates of this group sampled from diverse legumes in Korea, Japan, the USA, Mexico, Costa Rica and Panama. Some strains from Asia and North America shared identical sequences for both ribosomal genes. However, pairs of strains with closely related nifD sequences were almost never found in different regions. The major exceptions involved North American isolates B. elkanii USDA 76 and USDA 94, which had nifD sequences highly similar to certain Korean strains. However, 16S rRNA sequences of USDA 76 and USDA 94 were closely related to Central American rather than Asian bradyrhizobia, implying that these strains are genetic mosaics combining sequences from distinct ancestral areas. Several other conflicts between rRNA and nifD tree topologies indicated that the genealogical histories of these loci have been influenced by recurrent lateral gene transfer events.     

Comparative genomics of 2009 seasonal plague (Yersinia pestis) in New Mexico

Plague disease caused by the gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19(th) century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen.     

Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis.

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.     

Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.     

Knowledge of morphology is still required when identifying new amoeba isolates by molecular techniques

We have isolated several free-living amoeba strains from the environment in Ghana, which have internal transcribed spacers, including the 5.8S rDNA, sequences similar to sequences attributed to Vahlkampfiidae (Heterolobosea) in databases. However, morphological examination shows that the isolates belong to the Hartmannellidae (Amoebozoa). We provide evidence that the sequences in the databases are wrongly classified as belonging to a genus or species of the Vahlkampfiidae, but rather belong to strains of the genus Hartmannella.     

Rapid recovery of nuclear and mitochondrial genes by genome skimming from Northern Hemisphere freshwater crayfish

Molecular phylogenetics has benefited tremendously from the advent of next‐generation sequencing, enabling quick and cost‐effective recovery of whole mitogenomes via an approach referred to as ‘genome skimming’. Recently, genome skimming has been utilised to recover highly repetitive nuclear genes such as 18S and 28S ribosomal RNA genes that are useful for inferring deeper evolutionary relationships. To address some outstanding issues in the relationships among Northern Hemisphere freshwater crayfish (Astacoidea), we sequenced the partial genome of crayfish species from Asian, North American and European genera and report the successful recovery of whole mitogenome sequences in addition to three highly repetitive nuclear genes, namely histone H3, 18S and 28S ribosomal RNA. Consistent with some previous studies using short mtDNA and nuclear gene fragments, phylogenetic analyses based on the concatenation of recovered mitochondrial and/or nuclear sequences recovered the Asian cambarid lineage as basal to all astacids and North American cambarids, which conflicts with the current taxonomic classification based on morphological and reproduction‐related characters. Lastly, we show that complete H3, 18S and 28S ribosomal RNA genes can also be consistently recovered from a diverse range of animal taxa, demonstrating the potential wide utility of genome skimming for nuclear markers.     

Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean

The study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivable Legionella spp. in drinking water supplies in a tropical region. Treated and distributed water (±30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan host Hartmannella vermiformis and cultivable Legionella pneumophila were observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hosts Acanthamoeba spp., Echinamoeba exundans, E. thermarum, and an Neoparamoeba sp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration of H. vermiformis correlated with the concentration of Legionella spp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity (<0.2 nephelometric turbidity units [NTU]), and the varying ATP concentrations (1 to 12 ng liter(-1)) suggest that biofilms promoted protozoan growth in this supply. Ciliophora represented 25% of the protozoan OTUs in supply CA-2 with elevated ATP concentrations (maximum, 55 ng liter(-1)) correlating with turbidity (maximum, 62 NTU) caused by corroding iron pipes. Cercozoan types represented 70% of the protozoan clones in supply CA-3 with ATP concentrations of <1 ng liter(-1) and turbidity of <0.5 NTU in most samples of distributed water. The absence of H. vermiformis in most samples from supply CA-3 suggests that growth of this protozoan is limited at ATP concentrations of <1 ng liter(-1).     

Assessment of the relationships of geographic variation in species richness to climate and landscape variables within and among lineages of North American freshwater fishes

Aim Geographic variation in species richness is a well‐studied phenomenon. However, the unique response of individual lineages to environmental gradients in the context of general patterns of biodiversity across broad spatial scales has received limited attention. The focus of this research is to examine relationships between species richness and climate, topographic heterogeneity and stream channel characteristics within and among families of North American freshwater fishes. Location The United States and Canada. Methods Distribution maps of 828 native species of freshwater fishes were used to generate species richness estimates across the United States and Canada. Variation in species richness was predicted using spatially explicit models incorporating variation in climate, topography and/or stream channel length and stream channel diversity for all 828 species as well as for the seven largest families of freshwater fishes. Results The overall gradient of species richness in North American freshwater fishes is best predicted by a model incorporating variables describing climate and topography. However, the response of species richness to particular climate or landscape variables differed among families, with models possessing the highest predictive ability incorporating data on climate, topography and/or stream channel characteristics within a region. Main conclusions The correlations between species richness and abiotic variables suggest a strong influence of climate and physical habitat on the structuring of regional assemblages of North American freshwater fishes. However, the relationship between these variables and species richness varies among families, suggesting the importance of phylogenetic constraints on the regulation of geographic distributions of species.     

赤潮棕囊藻(Phaeocystis globosa)rDNA ITS区序列变异与二级结构分析

测定了南海球形棕囊藻香港株P1、P2和湛江株ZhJ1的rDNAITS区序列(含5.8srDNA),结合Gen Bank的13条同源序列,比对长度为904bp,变异位点271个,简约信息位点221个,平均(A+T)(34.5%)<(G+C)(65.4%).藻株P1、P2和ZhJ1序列存在变异位点20个,序列间相似性为97.9%～98.5%.ITS序列在种间和种内的解析度高于18srDNA和28srDNA基因;构建的NJ树、MP树、贝叶斯推断系统树的结构是一致的,不同种类的棕囊藻单独聚类,不同地理来源的球形棕囊藻混杂分布但相同地理来源的藻株多聚类在一起.RNA二级结构显示,不同藻种间5.8srDNA区结构基本一致,表现出属的特异性;ITS1、2区结构表现较大的种间差异,表明ITS区RNA二级结构可为棕囊藻分类鉴定提供有用的分子结构信息.     

Nitrogen Metabolizing Enzyme Activities In the Free-Living Soil Amoebae Acanthamoeba Castellanii, Acanthamoeba Polyphaga and Hartmannella Vermiformis

ABSTRACT. Free-living soil amoebae consume a wide variety of food, including algae, yeast, small protozoa and especially bacteria, which they digest to fulfil their nutritional requirements. Amoebae play an active role in the nitrogen mineralization in soils due to their nitrogen metabolizing capacities. However, little is known about nitrogen metabolizing enzyme activities in these free-living soil amoebae. In this study a number of key enzymes involved in the nitrogen metabolism of the axeaically cultivated free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga and two different strains of Hartmannella vermiformis were determined. the specific enzyme activities for exponential growth phase ceils were calculated and it appeared that the species tested possessed urate oxidase, glutamine synthetase, NADH-dependent glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activity. Glutamate synthase activity could not be detected in any of these species. the levels of specific activities varied depending on the enzymes tested. For all species the highest activities were detected for the transaminase reactions yielding glutamate, and for glutamate dehydrogenase. A general conclusion is that the pathway of nitrogen assimilation in free-living soil amoebae is similar to the one observed for other eukaryotes. Differences in specific activities were detected between the species.     

Sampling gene diversity across the supergroup Amoebozoa: large EST data sets from Acanthamoeba castellanii, Hartmannella vermiformis, Physarum polycephalum, Hyperamoeba dachnaya and Hyperamoeba sp

From comparative analysis of EST data for five taxa within the eukaryotic supergroup Amoebozoa, including two free-living amoebae (Acanthamoeba castellanii, Hartmannella vermiformis) and three slime molds (Physarum polycephalum, Hyperamoeba dachnaya and Hyperamoeba sp.), we obtained new broad-range perspectives on the evolution and biosynthetic capacity of this assemblage. Together with genome sequences for the amoebozoans Dictyostelium discoideum and Entamoeba histolytica, and including partial genome sequence available for A. castellanii, we used the EST data to identify genes that appear to be exclusive to the supergroup, and to specific clades therein. Many of these genes are likely involved in cell-cell communication or differentiation. In examining on a broad scale a number of characters that previously have been considered in simpler cross-species comparisons, typically between Dictyostelium and Entamoeba, we find that Amoebozoa as a whole exhibits striking variation in the number and distribution of biosynthetic pathways, for example, ones for certain critical stress-response molecules, including trehalose and mannitol. Finally, we report additional compelling cases of lateral gene transfer within Amoebozoa, further emphasizing that although this process has influenced genome evolution in all examined amoebozoan taxa, it has done so to a variable extent.