Whole Genome Sequence Typing to Investigate the Apophysomyces Outbreak following a Tornado in Joplin,Missouri, 2011

Case reports of Apophysomyces spp. in immunocompetent hosts have been a result of traumatic deep implantation of Apophysomyces spp. spore-contaminated soil or debris. On May 22, 2011 a tornado occurred in Joplin, MO, leaving 13 tornado victims with Apophysomyces trapeziformis infections as a result of lacerations from airborne material. We used whole genome sequence typing (WGST) for high-resolution phylogenetic SNP analysis of 17 outbreak Apophysomyces isolates and five additional temporally and spatially diverse Apophysomyces control isolates (three A. trapeziformis and two A. variabilis isolates). Whole genome SNP phylogenetic analysis revealed three clusters of genotypically related or identical A. trapeziformis isolates and multiple distinct isolates among the Joplin group; this indicated multiple genotypes from a single or multiple sources. Though no linkage between genotype and location of exposure was observed, WGST analysis determined that the Joplin isolates were more closely related to each other than to the control isolates, suggesting local population structure. Additionally, species delineation based on WGST demonstrated the need to reassess currently accepted taxonomic classifications of phylogenetic species within the genus Apophysomyces.     

Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit

BackgroundThe prevalence of opportunistic yeast infections has increased in recent decades as the result of an increasing immunocompromised patient population.AimsTo evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, to investigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypic method.MethodsBoth regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, were amplified using PCR and then sequenced. The sequences were compared to reference data available from the GenBank database of the National Center for Biotechnology Information using the BLASTn tool.ResultsUsing ID32C, 88% (44/50) of all strains were identified accurately at the species level, although 6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Candida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the four isolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITS region, but the remaining two were not distinguishable from closely related species. Based on the D1/D2 region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Trichosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions.ConclusionsIdentifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapid and reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest a two-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agar with Tween® 80 and API ID32C®) and sequence analysis of the ITS and D1/D2 region.     

Yamadazyma ubonensis f.a., sp. nov., a novel xylitol-producing yeast species isolated in Thailand

Three hundred and thirty-seven xylose-utilizing yeast strains were isolated from various natural samples. Among these, 68 strains produced xylitol in the range of 0.1–0.69 g xylitol/g xylose. Thirty-nine xylitol-producing strains were identified to be Candida tropicalis. Ten strains were found belonging to 14 known species in the genus Candida, Cyberlindnera, Meyerozyma, Pichia, Wickerhamomyces, Yamadazyma and Cryptococcus. Two strains were identified to be two Candida species and two strains (DMKU-XE142^T and DMKU-XE332) were found to be a novel species. Strain DMKU-XE142^T was isolated from tree bark and DMKU-XE332 was obtained from decaying plant leaf collected in Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics and sequence analysis of the D1/D2 region of the large subunit rRNA gene (LSU) and the internal transcribed spacer (ITS) region, the two strains were determined to represent a novel Yamadazyma species although formation of ascospores was not observed. The sequences of the D1/D2 region of the LSU rRNA gene and the ITS region of the two strains were identical but differed from Yamadazyma phyllophila, the closest species in terms of pairwise sequence similarity of the D1/D2 region, by 1.7 % nucleotide substitutions and 3.5 % nucleotide substitutions in the ITS region. The name Yamadazyma ubonensis f.a., sp. nov. is proposed (type strain is DMKU-XE142^T = BCC 61020^T = CBS 12859^T).     

Candida sanyaensis sp. nov., an ascomycetous yeast species isolated from soil

Strains representing a novel ascomycetous yeast species, Candida sanyaensis, were isolated from soil samples collected on Hainan Island and Taiwan Island in China. Analysis of the D1/D2 domains of the large subunit (LUS) rRNA gene and internal transcribed spacer (ITS) regions of these strains showed that this species was related to Candida tropicalis and Candida sojae, their closest relatives. C. sanyaensis differed by three substitutions and one gap from C. tropicalis, and by four substitutions and one gap from C. sojae, in the D1/D2 domain sequences. However, the ITS sequences of C. sanyaensis were quite divergent from the latter two species, showing that it is a genetically separate species. The novel strains were also found to have very similar PCR-fingerprinting profiles which were quite distinct from those of C. tropicalis and C. sojae strains. The type strain of C. sanyaensis is HN-26^T (= CICC 1979^T = CBS 12637^T).     

海南西海岸真红树内生细菌多样性及其延缓衰老活性研究

该文设计9种分离培养基,采用稀释涂布法从14份真红树植物的46份组织样品中分离纯化内生细菌。并基于菌株形态学特征和16S rRNA基因序列确定分离菌株的种属及分析其物种多样性,采用秀丽隐杆线虫模型筛选菌株延缓衰老活性。结果表明:(1)通过基因序列去重复后从46份真红树植物组织样品中获得32株海洋细菌,基于菌株16S rRNA基因序列信息分析,覆盖12科17属,其中芽孢杆菌属(Bacillus)为优势菌属,并获得1株疑似橙单胞菌属(Aurantimonas)新种,16S rRNA基因序列相似性低于97%;(2)经过秀丽隐杆线虫粗筛发现3株海洋细菌具有显著延缓线虫衰老的活性(P<0.05)。以上结果表明海南西海岸真红树内生细菌具有物种多样性,部分菌株具有延缓线虫衰老活性。     

Komagataella kurtzmanii sp. nov., a new sibling species of Komagataella (Pichia) pastoris based on multigene sequence analysis

A novel methanol assimilating yeast species Komagataella kurtzmanii is described using the type strain VKPM Y-727 (=KBP Y-2878 = UCD-FST 76-20 = Starmer #75-208.2 = CBS 12817 = NRRL Y-63667) isolated by W.T. Starmer from a fir flux in the Catalina Mountains, Southern AZ, USA. The new species is registered in MycoBank under MB 803919. The species was differentiated by divergence in gene sequences for D1/D2 LSU rRNA, ITS1-5.8S-ITS2, RNA polymerase subunit I, translation elongation factor-1α and mitochondrial small subunit rRNA. K. kurtzmanii differs from its phenotypically similar sibling species Komagataella pastoris, Komagataella pseudopastoris, Komagataella phaffii, Komagataella populi and Komagataella ulmi by absence of growth at 35 °C and inability to assimilate trehalose.     

Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt

The taxonomic diversity of forty-two Rhizobium strains, isolated from nodules of faba bean grown in Egypt, was studied using 16S rRNA sequencing, multilocus sequence analyses (MLSA) of three chromosomal housekeeping loci and one nodulation gene (nodA). Based on the 16S rRNA gene sequences, most of the strains were related to Rhizobium leguminosarum, Rhizobium etli, and Rhizobium radiobacter (syn. Agrobacterium tumefaciens). A maximum likelihood (ML) tree built from the concatenated sequences of housekeeping proteins encoded by glnA, gyrB and recA, revealed the existence of three distinct genospecies (I, II and III) affiliated to the defined species within the genus Rhizobium/Agrobacterium. Seventeen strains in genospecies I could be classified as R. leguminosarum sv. viciae. Whereas, a single strain of genospecies II was linked to R. etli. Interestingly, twenty-four strains of genospecies III were identified as A. tumefaciens. Strains of R. etli and A. tumefaciens have been shown to harbor the nodA gene and formed effective symbioses with faba bean plants in Leonard jar assemblies. In the nodA tree, strains belonging to the putative genospecies were closely related to each other and were clustered tightly to R. leguminosarum sv. viciae, supporting the hypothesis that symbiotic and core genome of the species have different evolutionary histories and indicative of horizontal gene transfer among these rhizobia.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for <Emphasis Type="Italic">Streptococcus</Emphasis>

The use of rrs (16S rRNA) gene is widely regarded as the “gold standard” for identifying bacteria and determining their phylogenetic relationships. Nevertheless, multiple copies of this gene in a genome is likely to give an overestimation of the bacterial diversity. In each of the 50 Streptococcus genomes (16 species, 50 strains), 4–7 copies of rrs are present. The nucleotide sequences of these rrs genes show high similarity within and among genomes, which did not allow unambiguous identification. A genome-wide search revealed the presence of 27 gene sequences common to all the Streptococcus species. Digestion of these 27 gene sequences with 10 type II restriction endonucleases (REs) showed that unique RE digestion in purH gene is sufficient for clear cut identification of 30 genomes belonging to 16 species. Additional gene-RE combinations allowed identification of another 15 strains belonging to S. pneumoniae, S. pyogenes, and S. suis. For the rest 5 strains, a combination of 2 genes was required for identifying them. The proposed strategy is likely to prove helpful in proper detection of pathogens like Streptococcus.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0561-5) contains supplementary material, which is available to authorized users.     

青藏高原察尔汗盐湖地区可培养中度嗜盐菌的群落结构与多样性

【目的】研究青海察尔汗盐湖地区的可培养中度嗜盐菌的群落结构及多样性。【方法】采用多种选择性培养基进行中度嗜盐菌的分离、培养;通过16S r RNA基因序列扩增、测定,根据序列信息,进行系统进化树构建、群落结构组成分析及多样性指数计算。【结果】从察尔汗盐湖卤水及湖泥中分离到中度嗜盐菌421株,合并重复菌株后共83株中度嗜盐菌。菌株16S rRNA基因序列信息显示,4株中度嗜盐菌为潜在的新分类单元。83株嗜盐细菌分布于3个门的6个科16个属。其中,Bacillus属、Oceanobacillus属和Halomonas属为优势属。多样性结果显示,水样中的菌株多样性高于泥样,而泥样中的菌株优势度高于水样。【结论】察尔汗盐湖中度嗜盐菌具有丰富的遗传多样性,种群种类丰富,优势菌群集中,该盐湖地区存在可分离培养的中度嗜盐菌的疑似新物种。     

Genomics of Sponge-Associated Streptomyces spp. Closely Related to Streptomyces albus J1074: Insights into Marine Adaptation and Secondary Metabolite Biosynthesis Potential

A total of 74 actinomycete isolates were cultivated from two marine sponges, Geodia barretti and Phakellia ventilabrum collected at the same spot at the bottom of the Trondheim fjord (Norway). Phylogenetic analyses of sponge-associated actinomycetes based on the 16S rRNA gene sequences demonstrated the presence of species belonging to the genera Streptomyces, Nocardiopsis, Rhodococcus, Pseudonocardia and Micromonospora. Most isolates required sea water for growth, suggesting them being adapted to the marine environment. Phylogenetic analysis of Streptomyces spp. revealed two isolates that originated from different sponges and had 99.7% identity in their 16S rRNA gene sequences, indicating that they represent very closely related strains. Sequencing, annotation, and analyses of the genomes of these Streptomyces isolates demonstrated that they are sister organisms closely related to terrestrial Streptomyces albus J1074. Unlike S. albus J1074, the two sponge streptomycetes grew and differentiated faster on the medium containing sea water. Comparative genomics revealed several genes presumably responsible for partial marine adaptation of these isolates. Genome mining targeted to secondary metabolite biosynthesis gene clusters identified several of those, which were not present in S. albus J1074, and likely to have been retained from a common ancestor, or acquired from other actinomycetes. Certain genes and gene clusters were shown to be differentially acquired or lost, supporting the hypothesis of divergent evolution of the two Streptomyces species in different sponge hosts.     

Prototheca tumulicola sp. nov., a novel achlorophyllous,yeast-like microalga isolated from the stone chamber interior of the Takamatsuzuka Tumulus

Two achlorophyllous microalgal strains were isolated from the soil and white moldy colony collected inside the stone chamber of the Takamatsuzuka Tumulus in Japan. Phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) and Dl/D2 large subunit ribosomal RNA (LSU rRNA) gene sequences, and concatenated gene sequences of the SSU and D1/D2 LSU rRNA genes indicated that our two isolates were the members of the non-photosynthetic, yeast-like microalgal Chlorellaceous genus Prototheca (Chlorellales, Trebouxiophyceae, Chlorophyta) but well distinguished from known species. Based on phenotypic and genotypic characteristics, isolates T6713-13-10^T and T61213-7-11 are proposed to represent a novel species in Prototheca, P. tumulicola, with the type strain JCM 31123^T (isolate T6713-13-10^T).     

New lipid-dependent Malassezia species from parrots

BackgroundAll the currently recognized Malassezia species have been isolated from mammals. However, only a few of them have been isolated from birds. In fact, birds have been less frequently studied as carriers of Malassezia yeasts than mammals.AimIn this study we describe two new taxa, Malassezia brasiliensis sp. nov. and Malassezia psittaci sp. nov.MethodsThe isolates studied in this publication were isolated from pet parrots from Brazil. They were characterized using the current morphological and physiological identification scheme. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene, the ITS-5.8S rRNA gene sequences and the β-tubulin gene were also performed.ResultsThe strains proposed as new species did not completely fit the phenotypic profiles of any the described species. The validation of these new species was supported by analysis of the genes studied. The multilocus sequence analysis of the three loci provides robust support to delineate these species.ConclusionsThese studies confirm the separation of these two new species from the other species of the genus Malassezia, as well as the presence of lipid-dependent Malassezia yeasts on parrots.     

Identification and nematicidal activity of bacteria isolated from cow dung

Two hundred and nineteen bacterial strains were isolated from cow dung. Among these, 59 isolates displayed nematicidal activity against the model nematode Caenorhabditis elegans. Of the 59 bacterial strains, 17 killed >90 % of the tested nematodes within 1 h. Based on their 16S rRNA sequences, these 17 strains were identified as Alcaligenes faecalis, Bacillus cereus, Proteus penneri, Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas otitidis, Staphylococcus sciuri, Staphylococcus xylosus, Microbacterium aerolatum, Pseudomonas beteli. Among these 17 strains, 14 produced volatile organic compound(s) that inhibited the mobility of the C. elegans nematodes. These 14 strains also showed nematicidal activity against a plant pathogenic nematode Meloidogyne incognita. This is the first report demonstrating nematicidal activity for strains in genera Proteus, Providencia and Staphylococcus.     

Differentiation of non-pylori Helicobacter species based on PCR–restriction fragment length polymorphism of the 23S rRNA gene

Phenotypic identification of non-pylori Helicobacter species has always been problematic and time-consuming in comparison with many other bacteria. We developed a rapid two-step identification assay based on PCR–restriction fragment length polymorphism (PCR–RFLP) analysis of the 23S rRNA gene for differentiating between non-pylori Helicobacter species. A new genus-specific primer pair based on all available complete and partial 23S rRNA sequences of Helicobacter species was designed. In silico restriction analysis of variable regions of the 23S rRNA gene suggested SmaI and HindIII endonucleases would provide a good level of differentiation. Analysis of the obtained 23S rRNA RFLP patterns divided all Helicobacter study strains into three species groups (groups A–C) and 12 unique restriction patterns. Wolinella succinogenes also gave a unique pattern. Our proposed PCR–RFLP method was found to be as a valuable tool for routine identification of non-pylori Helicobacter species from human or animal samples.     

Rhizobium sophorae is the dominant rhizobial symbiont of Vicia faba L. In North China

Faba bean (Vicia faba L.) is a major introduced grain-legume crop cultivated in China. In this study, rhizobia that nodulated faba bean grown in soils from three sites in North China (Hebei Province) were isolated and characterized. Firstly, isolates were categorized into genotypes by ribosomal IGS PCR-RFLP analysis, then representatives of the different IGS genotypes were further identified by phylogenetic analyses of 16S rRNA, housekeeping (atpD, recA) and nodulation (nodC) gene sequences. Rhizobial distribution based on the IGS genotype was related to the different soil physicochemical features by redundancy analysis. IGS typing and phylogenetic analyses of 16S rRNA and concatenated housekeeping gene sequences affiliated the 103 rhizobial strains isolated into four Rhizobium species/genospecies. A total of 69 strains of 3 IGS types were assigned to R. sophorae, 20 isolates of 5 IGS types to R. changzhiense and 9 isolates of 3 IGS types to R. indicum. The representative strain of the five remaining isolates (1 IGS type) was clearly separated from all Rhizobium type strains and was most closely related to defined genospecies according to the recently described R. leguminosarum species complex. Rhizobium sophorae strains (67% of total isolates) were common in all sites and shared an identical nodC sequence typical of faba bean symbionts belonging to symbiovar viciae. In this first study of rhizobia nodulating faba bean in Hebei Province, China, R. sophorae was found to be the dominant symbiont in contrast to other countries.     

Isolation and Identification of Myxobacteria from Saline-Alkaline Soils in Xinjiang,China

Fifty-eight terrestrial and salt-tolerant myxobacteria were isolated from the saline-alkaline soils collected from Xinjiang, China. Based on the morphologies and the 16S rRNA gene sequences, these isolates were assigned into 6 genera, Myxococcus, Cystobacter, Corallococcus, Sorangium, Nannocystis and Polyangium. All the strains grew better with 1% NaCl than without NaCl. Some Myxococcus strains were able to grow at 2% NaCl concentration, suggesting that these strains may be particular type of terrestrial myxobacteria.     

Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA–DNA hybridization,and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens

The Streptomyces albidoflavus 16S rRNA gene clade contains 10 species and subspecies with identical 16S rRNA gene sequences and very similar numerical taxonomic data, including Streptomyces griseus subsp. solvifaciens. Type strains of this clade, as well as three CGMCC strains which were received as Streptomyces galilaeus, Streptomyces sioyaensis and Streptomyces vinaceus, respectively, that shared the same 16S rRNA gene sequences with the clade, were subjected to multilocus sequence analysis (MLSA), DNA–DNA hybridization (DDH) and phenotypic characterization for a comprehensive reevaluation. The 13 strains still formed a distinct, albeit loosely related, clade in the phylogenetic tree based on concatenated sequences of aptD, gyrB, recA, rpoB and trpB genes, supported by a high bootstrap value and different tree-making algorithms, with MLSA evolutionary distances ranging from 0 to 0.003. DDH values among these strains were well above the 70% cut-off point for species delineation. Based on the genotypic data of MLSA and DDH, combined with key phenotypic properties in common, it is proposed that the 10 species and subspecies of the S. albidoflavus clade, namely S. albidoflavus, S. canescens, S. champavatii, S. coelicolor, S. felleus, S. globisporus subsp. caucasicus, S. griseus subsp. solvifaciens, S. limosus, S. odorifer and S. sampsonii, should be merged into a single genomic species, for which the name S. albidoflavus is retained, and that the three strains S. galilaeus CGMCC 4.1320, S. sioyaensis CGMCC 4.1306 and S. vinaceus CGMCC 4.1305 should be assigned to S. albidoflavus as well. The results also indicated that MLSA could be the procedure of choice for distinguishing between species within Streptomyces 16S rRNA gene clades.     

Nodulation in Dimorphandra wilsonii Rizz. (Caesalpinioideae), a Threatened Species Native to the Brazilian Cerrado

The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N₂-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N₂ fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N₂-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii.