PCR Detection and Analysis of the Free-Living Amoeba Naegleria in Hot Springs in Yellowstone and Grand Teton National Parks

Free-living thermotolerant amoebae pose a significant health risk to people who soak and swim in habitats suitable for their growth, such as hot springs. In this survey of 23 different hot springs in Yellowstone and Grand Teton National Parks, we used PCR with primer sets specific for Naegleria to detect three sequence types that represent species not previously described, as well as a fourth sequence type identified as the pathogen Naegleria fowleri.     

Nitrification in terrestrial hot springs of Iceland and Kamchatka

Archaea have been detected recently as a major and often dominant component of the microbial communities performing ammonia oxidation in terrestrial and marine environments. In a molecular survey of archaeal ammonia monooxygenase (AMO) genes in terrestrial hot springs of Iceland and Kamchatka, the amoA gene encoding the alpha-subunit of AMO was detected in a total of 14 hot springs out of the 22 investigated. Most of these amoA-positive hot springs had temperatures between 82 and 97 degrees C and pH range between 2.5 and 7. In phylogenetic analyses, these amoA genes formed three independent lineages within the known sequence clusters of marine or soil origin. Furthermore, in situ gross nitrification rates in Icelandic hot springs were estimated by the pool dilution technique directly on site. At temperatures above 80 degrees C, between 56 and 159 mumol NO(3)(-) L(-1) mud per day was produced. Furthermore, addition of ammonium to the hot spring samples before incubation yielded a more than twofold higher potential nitrification rate, indicating that the process was limited by ammonia supply. Our data provide evidence for an active role of archaea in nitrification of hot springs in a wide range of pH values and at a high temperature.     

16S rRNA gene and lipid biomarker evidence for anaerobic ammonium-oxidizing bacteria (anammox) in California and Nevada hot springs

Anammox, the oxidation of ammonium with nitrite to dinitrogen gas under anoxic conditions, is an important process in mesophilic environments such as wastewaters, oceans and freshwater systems, but little is known of this process at elevated temperatures. In this study, we investigated anammox in microbial mats and sediments obtained from several hot springs in California and Nevada, using geochemical and molecular microbiological methods. Anammox bacteria-specific ladderane core lipids with concentrations ranging between 0.3 and 52 ng g⁻¹ sediment were detected in five hot springs analyzed with temperatures up to 65 °C. In addition, 16S rRNA gene analysis showed the presence of genes phylogenetically related to the known anammox bacteria Candidatus Brocadia fulgida, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis (96.5–99.8% sequence identity) in three hot springs with temperatures up to 52 °C. Our data indicate that anammox bacteria may be able to thrive at thermophilic temperatures and thus may play a significant role in the nitrogen cycle of hot spring environments.     

Linkage of high rates of sulfate reduction in Yellowstone hot springs to unique sequence types in the dissimilatory sulfate respiration pathway

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89 degrees C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.     

Shift in Cyanobacteria Community Diversity in Hot Springs of India

Two ecologically distinct tropical sulfur-rich alkaline hot springs, Taptapani at 48°C harboring mesophiles and Atri at 58°C comprising thermophiles situated in the Eastern Ghats foothills of India, differ in their geochemical conditions, and provide an interesting platform to unravel the eco-physiological reasons behind the differential cyanobacterial diversity. The predominance of mesophilic Arthronema (83.81%) in Taptapani and shifting predominance of thermophilic Leptolyngbya (96.25%) in Atri as discovered through 16S rRNA gene Illumina sequencing of their metagenomics DNA as a function of temperature are the intriguing features of the present study. Differential presence of the cyanobacterial community at the phylum level in these two hot springs was found to be correlated with the unequal coexistence of Chloroflexi, Taptapani the non-cyanobacteria members and the possible influence of physiochemical parameters including temperature. Variation in cyanobacterial diversity and composition of these hot springs as revealed through sequence analysis were also evinced by respective differences in richness, evenness, and Shannon diversity indices.     

srRNA evolution and phylogenetic relationships of the genus Naegleria (Protista: Rhizopoda)

A rapid RNA sequencing technique was used to partially sequence the small-subunit ribosomal RNA (srRNA) of four species of the amoeboid genus Naegleria. The extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. However, the pattern of variation among the Naegleria species was quite different from that found for those species of tetrapods characterized to date. A phylogenetic analysis of the consensus Naegleria sequence showed that Naegleria was not monophyletic with either Acanthamoeba castellanii or Dictyostelium discoideum, two other amoebas for which sequences were available. It was shown that the semiconserved regions of the srRNA molecule evolve in a clocklike fashion and that the clock is time dependent rather than generation dependent.     

Actinobacterial Diversity in Hot Springs in Tengchong (China), Kamchatka (Russia), and Nevada (USA)

Actinobacterial diversity was investigated in three hot springs [Tengchong Frog Mouth (TFM) hot spring (in Tengchong, China), Kamchatka Robb Flag hot spring (in Kamchatka, Russia), and Nevada Boiling Spring (in Nevada, USA)], a Tengchong Frog Mouth hot spring-derived pond and a soil sample near the TFM hot spring. Culture-independent techniques were employed including denaturing gradient gel electrophoresis and actinobacterial 16S rRNA gene phylogenetic analysis. The three hot springs and the hot spring-derived pond had pH of 6.8 to 7.9 and temperature of 50 to 81°C. Actinobacteria were for the first time found to be very diverse in hot springs with temperature up to 81°C, indicating that they have an unexpected capability of adapting to hot spring environments. A total of 346 actinobacterial clones were obtained from the three hot springs, and these clones were identified as 31 RFLP types, falling into the Actinomycetales (only limit to the Frankineae suborder), the Rubrobacterales, uncultured Candidatus Microthrix, unclassified Actinobacteria, and uncultured Candidate Division OP10. Our data have implications for a better understanding of the distribution of Actinobacteria in hot springs.     

Linkage of High Rates of Sulfate Reduction in Yellowstone Hot Springs to Unique Sequence Types in the Dissimilatory Sulfate Respiration Pathway

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89°C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.     

Bacterial and archaeal diversities in Yunnan and Tibetan hot springs,China

Thousands of hot springs are located in the north‐eastern part of the Yunnan–Tibet geothermal zone, which is one of the most active geothermal areas in the world. However, a comprehensive and detailed understanding of microbial diversity in these hot springs is still lacking. In this study, bacterial and archaeal diversities were investigated in 16 hot springs (pH 3.2–8.6; temperature 47–96°C) in Yunnan Province and Tibet, China by using a barcoded 16S rRNA gene‐pyrosequencing approach. Aquificae, Proteobacteria, Firmicutes, Deinococcus‐Thermus and Bacteroidetes comprised the large portion of the bacterial communities in acidic hot springs. Non‐acidic hot springs harboured more and variable bacterial phyla than acidic springs. Desulfurococcales and unclassified Crenarchaeota were the dominated groups in archaeal populations from most of the non‐acidic hot springs; whereas, the archaeal community structure in acidic hot springs was simpler and characterized by Sulfolobales and Thermoplasmata. The phylogenetic analyses showed that Aquificae and Crenarchaeota were predominant in the investigated springs and possessed many phylogenetic lineages that have never been detected in other hot springs in the world. Thus findings from this study significantly improve our understanding of microbial diversity in terrestrial hot springs.     

Phylogenetic analysis of the bacterial community in a geothermal spring,Rupi Basin,Bulgaria

Hot environments are between the supporting life extreme niches that appear to have maintained some degree of pristine quality and of special biotechnological interest. Knowledge on biodiversity in terrestrial hot springs is still scanty and has not been compared in the light of the specificity of those extreme ecological niches. Study on diversity of thermophilic bacteria inhabiting a hot spring located in Rupi Basin (RB), South-West Bulgaria, revealed a high phylogenetic richness in it (genotypic diversity is 0.37). A total of 120 clones were examined, and grouped in 28 phylogenetic types by their RFLP profile. 16S rRNA gene analysis allowed the identification of nine divisions from the domain Bacteria and one Candidate division. Ten of the retrieved bacterial sequences representing one third of the sequence types showed less than 97% similarity to the closest neighbor and were referred as new sequences. Four of them were distantly related to validly described bacteria (showed ≤90% similarity) suggesting new taxons on at least genus level. Comparison of biodiversity in the spring from Rupi Basin, Bulgaria with that described from other terrestrial hot springs revealed that Proteobacteria, Hydrogenobacter/Aquifex and Thermus are common bacterial groups for terrestrial hot springs. Simultaneously, specific bacterial taxons were observed in different springs.     

On the Identity of the Amoeboflagllates Didascalus throntoni and Adelphamoeba galeacystis

ABSTRACT. Didascalus thorntoni , Singh 1952 has been classified alternately as a separate genus or as a species of Naegleria. In the 18th edition of the American Type Culture Collection catalogue it is classified as Naegleria thorntoni. To resolve the question of its identity we have used riboprinting and sequencing of the small subunit ribosomal DNA. The results indicate that D. thorntoni does not belong to the genus Naegleria. The sequence of the small subunit ribosomal DNA differs only in 20 nucleotides (1%) from that of the Paratetramitus jugosus. The difference is much smaller than between some species of Naegleria. Therefore, it is not clear whether D. thorntoni should be considered as a species of Paratetramitus or as a separate genus. The strain used in different laboratories as the type strain of Adelophamoeba gleacystis has been identified as a Naegleria strain. We believe that the type strain of A. galeacystis was mislabeled prior to submission to the American Type Culture Collection and to the Culture Collection of Algae and Protozoa. A recent isolate, which on the basis of morphology was identified as a strain of A. Galeacystis , has the identical small subunit ribosomal DNA sequence as D. throntoni. Our results prove Page was right when he stated that Adelphamoeba might be a synonym of Didascalus.     

PCR-based identification of thermotolerant free-living amoebae in Italian hot springs

Several thermal areas, also used for leisure purposes, may represent suitable habitats for free-living amoebae (FLAs), but few studies have been carried out in search for these organisms. The aim of this study was to assess the presence and distribution of FLAs by culture detection and molecular identification, over a one year-round sampling of two sites in Central Italy. Two geothermal springs (Site A and Site B) were investigated for a total of 36 water samples. Four sets of primers were used to amplify FLA DNA from all cultures positive for amoebic growth at both 37 °C and 45 °C. Overall, 33 (91.6%) water samples produced PCR amplification. Eleven taxa were identified. The array of identified species varied over the sampling period, and differed between the two hot springs, Site A harbouring 11 taxa compared to 5 of site B. However, both sites were characterized by the most common species Vermamoeba vermiformis and Naegleria australiensis. Acanthamoeba genotypes T4 and T15 were found at low frequency. Differences in the composition between the two sites could reflect environmental changes in biotic and chemical/physical parameters. From a public health perspective, the detection of potentially pathogenic amoebae could unveil a potential risk for humans.     

Cancer Mortality Studies in Misara,a Radon Hot Spring in Japan: A Summary up to 2003

Misasa, which is well known for its radon hot springs, is a historical town located in Tottori Prefecture, Japan. The presence of the hot springs is noted in an 1164 entry in town records. The presence of radioactivity in the hot springs was recognized in 1914. Since ancient times, the spring water has been believed to be healthful. The town of Misasa has 90 radon hot spring sources, yielding a total volume of 3,000 tons of spring water a day. The concentration of ²²²Rn in the spring water ranges from 17.4 to 9,361 Bq per liter. A series of cancer mortality studies on the radon hot springs were conducted through the collaboration of scientists scattered throughout different Japanese cities. At present, no definite evidence has been discovered that indicates an increase of cancer mortality in the Misasa radon hot spring area. Even a lower risk was found for stomach cancers in both radon and no-radon hot springs.     

中国温泉微生物物种多样性及其酶活性研究进展

微生物作为生物群体的重要组成成员,其生长受外界物化条件(如温度、盐度、pH等)影响较大。温泉作为极端水生环境之一,属于相对稳定且较为特殊的生态系统,使生长于其中的微生物可能具有适应高温等特殊生境的独特生存生理机制,具体表现为微生物物种及其活性次级代谢产物呈现出一定的多样性与新颖性。本文从菌株物种多样性分析及其酶活性研究方面,综述了近5年来国内温泉微生物相关研究进展,以期为温泉等极端环境微生物资源开发与保护提供参考。     

Hydrogen sulfide concentrations in indoor air of thermal springs

This study aimed to assess hydrogen sulfide (H₂S) concentrations in the pool indoor air of hot springs and its link to some physicochemical properties of the springs in Ardabil province. Twenty-two hot springs from different regions were selected and monitored for H₂S concentrations using a portable gas meter. Respective mean concentrations of H₂S in hot springs indoor air were 9.11 ± 11. Ghotour Souei hot springs had the highest concentration of H₂S with a mean concentration of 29.4 ± 7.7 and 25.2 ± 8.16 ppm at the source and general pool areas, respectively. Oxidation-reduction potential and pH of the water were the most important factor influencing H₂S concentrations in the hot springs. H₂S concentrations in indoor air of Ardabil hot springs were noticeably higher than OEL-TWA and OEL-STEL, and therefore may pose important risks for human health on both short-and long-term exposures.     

Restriction endonuclease analysis of mitochondrial DNA as an aid in the taxonomy of Naegleria and Vahlkampfia

Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.     

Identification and epidemiological typing of Naegleria fowleri with DNA probes.

Naegleria fowleri is a small free-living amoeboflagellate found in warm water habitats worldwide. The organism is pathogenic to humans, causing fatal primary amoebic meningoencephalitis. When monitoring the environment for the presence of N. fowleri, it is important to reliably differentiate the organism from other closely related but nonpathogenic species. To this end, we have developed species-specific DNA probes for use in the rapid identification of N. fowleri from the environment. Samples were taken from the thermal springs in Bath, England, and cultured for amoebae. Of 84 isolates of thermophilic Naegleria spp., 10 were identified as N. fowleri by probe hybridization. The identity of these isolates was subsequently confirmed by their specific whole-cell DNA restriction fragment length polymorphisms (RFLPs). One DNA clone was found to contain a repeated element that detected chromosomal RFLPs that were not directly visible on agarose gels. This enabled the further differentiation of strains within geographically defined whole-cell DNA RFLP groups. N. fowleri DNA probes represent a specific and potentially rapid method for the identification of the organism soon after primary isolation from the environment.     

腾冲两热泉泉古菌多样性及系统发育的初步分析

通过构建16S rRNA基因片段的克隆文库对腾冲热海两温泉中泉古菌的多样性和系统发育关系进行了初步的研究.一共得到18个泉古茵克隆序列,可分为12个OTUs,两温泉的克隆序列与已知GenBank上关系最近序列的平均相似性较低,无名泉为92.56%,热爆区为93%.从基于16S rRNA基因片段序列构建的系统发育树来看,74℃的无名泉样点中既有属于超高温环境类群的泉古菌,同时又有属于和常温环境较接近的泉古菌;45℃的热爆区样点的泉古茵,相对来说则更接近于常温类群.本次研究表明,腾冲热泉与世界其它同类热泉之间的泉古茵类群存在着一定的差异;而且两实验样点代表了超高温和高温环境泉古菌逐渐向常温过度的两个重要环境.     

Three different group I introns in the nuclear large subunit ribosomal DNA of the amoeboflagellate Naegleria.

We have amplified the large subunit ribosomal DNA (LSUrDNA) of the 12 described Naegleria spp. and of 34 other Naegleria lineages that might be distinct species. Two strains yielded a product that is longer than 3 kb, which is the length of the LSUrDNA of all described Naegleria spp. Sequencing data revealed that the insert in one of these strains is a group I intron without an open reading frame (ORF), while the other strain contains two different group I introns, of which the second intron has an ORF of 175 amino acids. In the latter ORF there is a conserved His-Cys box, as in the homing endonucleases present in group I introns in the small subunit ribosomal DNA (SSUrDNA) of Naegleria spp. Although the group I introns in the LSUrDNA differ in sequence, they are more related to each other than they are to the group I introns in the SSUrDNA of Naegleria spp. The three group I introns in the LSUrDNA in Naegleria are at different locations and are probably acquired by horizontal transfer, contrary to the SSUrDNA group I introns in this genus which are of ancestral origin and are transmitted vertically.     

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts. International journal for Parasitology4: 139–142. Cysts of 4 strains of non-pathogenic Naegleria gruberi and 5 strains of pathogenic Naegleria fowleri were examined in the scanning electron microscope. Excystment of the Naegleria gruberi amoebae occurred via preformed exit pores in the cyst wall. Similar structures were not found in the cysts of Naegleria fowleri, and excystment occurred by rupture of the cyst wall. The sequence of cyst wall rupture is illustrated for one of the pathogenic strains.