Gut microbiota modulation induced by Zika virus infection in immunocompetent mice.

Gut microbiota composition can modulate neuroendocrine function, inflammation, and cellular and immunological responses against different pathogens, including viruses. Zika virus (ZIKV) can infect adult immunocompetent individuals and trigger brain damage and antiviral responses. However, it is not known whether ZIKV infection could impact the gut microbiome from adult immunocompetent mice. Here, we investigated modifications induced by ZIKV infection in the gut microbiome of immunocompetent C57BL/6J mice. Adult C57BL/6J mice were infected with ZIKV and the gut microbiota composition was analyzed by next-generation sequencing of the V4 hypervariable region present in the bacterial 16S rDNA gene. Our data showed that ZIKV infection triggered a significant decrease in the bacteria belonging to Actinobacteria and Firmicutes phyla, and increased Deferribacteres and Spirochaetes phyla components compared to uninfected mice. Interestingly, ZIKV infection triggered a significant increase in the abundance of bacteria from the Spirochaetaceae family in the gut microbiota. Lastly, we demonstrated that modulation of microbiota induced by ZIKV infection may lead to intestinal epithelium damage and intense leukocyte recruitment to the intestinal mucosa. Taken together, our data demonstrate that ZIKV infection can impact the gut microbiota composition and colon tissue homeostasis in adult immunocompetent mice.

Description of Oceanispirochaeta crateris sp. nov. and reclassification of Spirochaeta perfilievii as Thiospirochaeta perfilievii gen. nov., comb. nov.

A novel obligately anaerobic spirochete strain K2T was isolated from bottom marine sediments at Crater Bay of Yankicha Island (Kuril Islands, Russia). Strain K2T had helical shape and Gram-negatively stained. The optimal growth conditions were as follows: the optimum temperature was 28-30 °C with range 5-34 °C; optimal pH at 7.0-7.5 with range of 6.8-8.5; NaCl optimum at 3-3.5 % (w/v) and range of 1-7 % (w/v). Strain K2T was catalase- and oxidase-negative. Glucose fermentation products were acetate, lactate, ethanol, CO2, H2. The major fatty acids were C14 : 0, iso-C13 : 0, iso-C15:0, C14 : 0 DMA, iso-C15 : 0 DMA. The G+C content of genomic DNA was 43.2 mol%. Phylogenetic analyses of 16S rRNA genes showed that strain K2T belonged to the genus Oceanispirochaeta of the family Spirochaetaceae. The 16S rRNA gene sequence similarity of strain K2T and O. litoralis DSM 2029T and O. sediminicola DSM 104770T was 96 and 94 %, respectively. Based on the results of our study, we propose the name Oceanispirochaeta crateris sp. nov.; type strain K2T (=DSM 16308T=VKM B-3266T). Also, the taxonomic status of Spirochaeta perfilevii was revised: 16S rRNA genes sequence showed less than 89 % similarity to nearest phylogenetic neighbours. Therefore, we proposed to separate this species into a novel genus Thiospirochaeta - T. perfilievii gen. nov., comb. nov.

Sphaerochaeta halotolerans sp. nov., a novel spherical halotolerant spirochete from a Russian heavy oil reservoir, emended description of the genus Sphaerochaeta, reclassification of Sphaerochaeta coccoides to a new genus Parasphaerochaeta gen. nov. as Parasphaerochaeta coccoides comb. nov. and proposal of Sphaerochaetaceae fam. nov.

Anaerobic, fermentative, halotolerant bacteria, strains 4-11T and 585, were isolated from production water of two low-temperature petroleum reservoirs (Russia) and were characterized by using a polyphasic approach. Cells of the strains were spherical, non-motile and 0.30-2.5 µm in diameter. Strain 4-11T grew optimally at 35 °C, pH 6.0 and 1.0-2.0% (w/v) NaCl. Both strains grew chemoorganotrophically with mono-, di- and trisaccharides. The major cellular fatty acids of both strains were C14:0, C16:0, C16:1 ω9 and C18:0 3-OH. Major polar lipids were glycolipids and phospholipids. The 16S rRNA gene sequences of the strains 4-11T and 585 had 99.9% similarity and were most closely related to the sequence of Sphaerochaeta associata GLS2T (96.9, and 97.0% similarity, respectively). The G+C content of the genomic DNA of strains 4-11T and 585 were 46.8 and 46.9%, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain 4-11T and S. associata GLS2T were 73.0 and 16.9%, respectively. Results of phylogenomic metrics analysis of the genomes and 120 core proteins of strains 4-11T and 585 and their physiological and biochemical characteristics confirmed that the strains represented a novel species of the genus Sphaerochaeta, for which the name Sphaerochaeta halotolerans sp. nov. is proposed, with the type strain 4-11T (=VKM B-3269T=KCTC 15833T). Based on the results of phylogenetic analysis, Sphaerochaeta coccoides was reclassified as member of a new genus Parasphaerochaeta gen. nov., Parasphaerochaeta coccoides comb. nov. The genera Sphaerochaeta and Parasphaerochaeta form a separate clade, for which a novel family, Sphaerochaetaceae fam. nov., is proposed.

Rectinema subterraneum sp. nov, a chemotrophic spirochaete isolated from the deep terrestrial subsurface.

A novel, obligately anaerobic bacterium (strain SURF-ANA1T) was isolated from deep continental subsurface fluids at a depth of 1500 m below surface in the former Homestake Gold Mine (now Sanford Underground Research Facility, in Lead, South Dakota, USA). Cells of strain SURF-ANA1T were Gram-negative, helical, non-spore-forming and were 0.25-0.55×5.0-75.0 µm with a wavelength of 0.5-0.62 µm. Strain SURF-ANA1T grew at 15-50 °C (optimally at 40 °C), at pH 4.8-9.0 (pH 7.2) and in 1.0-40.0 g l-1 NaCl (10 g l-1 NaCl). The strain grew chemoheterotrophically with hydrogen or mono-, di- and polysaccharides as electron donors. The major cellular fatty acids in order of decreasing abundance (comprising >5% of total) were 10-methyl C16:0, iso-C15:0, C18:2 and C18:0 dimethyl acetal (DMA) and C20:0 methylene-nonadecanoic acid. Phylogenetic analysis based on the 16S rRNA gene sequence of strain SURF-ANA1T indicated a closest relationship with the recently characterized Rectinema cohabitans (99%). Despite high sequence identity, because of its distinct physiology, morphology and fatty acid profile, strain SURF-ANA1T is considered to represent a novel species within the genus Rectinema, for which the name Rectinema subterraneum sp. nov. is proposed. To our knowledge, this is the first report of an isolate within the phylum Spirochaetes from the deep (>100 m) terrestrial subsurface. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and genomic sequences of strain SURF-ANA1T are KU359248 and GCF 009768935.1, respectively. The type strain of Rectinema subterraneum is SURF-ANA1T (=ATCC TSD-67=JCM 32656).

Pleomorphochaeta naphthae sp. nov., a new anaerobic fermentative bacterium isolated from an oil field.

A novel anaerobic fermentative bacterium, strain SEBR 4209T, was isolated from a water sample of a Congolese oil field. Strain SEBR 4209T is phylogenetically related to the genus Pleomorphochaeta, in the family Spirochaetaceae. Its closest relatives are Pleomorphochaeta caudata SEBR 4223T (94.5 % 16S rRNA gene sequence similarity) and Pleomorphochaeta multiformis MO-SPC2T (94.3 % similarity). Like the other members of this genus, cells have a pleomorphic morphology, in particular an annular shape and long stalks. Optimal growth was observed at 37 °C, at pH between 6.8 and 7.0, and with 40 g l-1 NaCl. This strain was only able to grow by fermentation of carbohydrates. The fermentation products from glucose utilization were acetate, ethanol, CO2 and H2. Predominant fatty acids were C14 : 0, C14 : 0 DMA, C16 : 0 and C16 : 1ω7c. The major polar lipids were phosphoglycolipids, phospholipids and glycolipids. The G+C content of the DNA was 29.6 mol%. Based on phenotypic characteristics and phylogenetic traits, strain SEBR 4209T is considered to represent a novel species of the genus Pleomorphochaeta, for which the name Pleomorphochaetanaphthae sp. nov. is proposed. The type strain is SEBR 4209T (=DSM 104684T=JCM 31871T).

Description of Oceanispirochaeta sediminicola gen. nov., sp. nov., an obligately anaerobic bacterium isolated from coastal marine sediments, and reclassification of Spirochaeta litoralis as Oceanispirochaeta litoralis comb. nov.

An obligately anaerobic spirochaete (strain SY2T) was isolated from coastal marine sediments of Tongyeong-Si, South Korea. Strain SY2T was helical-shaped and Gram-stain-negative. Strain SY2T was able to grow at 10-40 °C (optima, 25-30 °C), pH 6.3-8.8 (optima, pH 7.0-8.0) and with 1-7 % (optimum, 2-3 %) NaCl concentration. Strain SY2T was negative for catalase and oxidase activity. The major end-products of glucose fermentation were acetate, ethanol, hydrogen and carbon dioxide. C14 : 0, C16 : 0, iso-C15 : 0, iso-C14 : 0 3-OH, iso-C15 : 1 H/C13 : 0 3-OH and iso-C17 : 1ω9c were predominant fatty acids (>5 %) with minor amounts (<5 %) of C18 : 0, iso-C13 : 0, iso-C17 : 0, iso-C17 : 1/anteiso-C17 : 1 B and C16 : 1ω6c/C16 : 1ω7c. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were major polar lipids. The genomic DNA G+C content was 53.5 mol%. 16S rRNA gene sequence comparisons indicated that strain SY2T represents a member of the family Spirochaetaceae in the phylum Spirochaetes. Strain SY2T has a sequence similarity of 95.1 % with Spirochaeta litoralis R1T and <90.1 % with other members of the genus Spirochaeta. Distinct morphological, physiological and genotypic differences from the previously described taxa support the classification of strain SY2T as a representative of a novel genus and species in the family Spirochaetaceae, for which the name Oceanispirochaeta sediminicola gen. nov., sp. nov. is proposed. The type strain is SY2T (=KEMB 3001-381T=DSM 104770T=KCTC 15593T). Reclassification of Spirochaeta litoralis as Oceanispirochaeta litoralis comb. nov. is also proposed based on polyphasic taxonomic analyses.

Characterisation of a newly isolated member of a candidatus lineage, Marispirochaeta aestuarii gen. nov., sp. nov.

Metagenome analysis of coastal marine habitats of Gujarat, India indicated the presence of twelve novel putative lineages of spirochaetes. Out of which a strain designated JC444T representing a novel putative lineage seven was isolated and characterized based on a polyphasic taxonomic approach. Strain JC444T was helical, Gram-stain-negative, obligate anaerobe, catalase and oxidase negative. Strain JC444T was able to grow at 15-45 °C (optimum at 30-35 °C), pH 6.5-8.6 (optimum at 7.5-8.0) and 0.6-5 % (optimum at 1.5-2.0 %) of NaCl concentration. The major end products of glucose fermentation were acetate, formate, hydrogen and carbon dioxide. C14 : 0, iso-C15 : 0, C16 : 0, C18 : 0, iso-C15 : 1H/C13 : 03OH (summed feature 1), iso-C13 : 0, anteiso-C15 : 0 and iso-C17 : 0 were present as fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unidentified lipids (L1-4) were the polar lipids. G+C mol% of strain JC444T was 53.6 %. 16S rRNA gene sequence comparisons indicated that strain JC444T represents a member of the family Spirochaetaceae in the order Spirochaetales. Strain JC444T has a sequence similarity of 97.1 % with 'Candidatus Marispirochaeta associata' JC231 and <90.1 % with other members of the family Spirochaetaceae. Distinct morphological, physiological and genotypic differences from the previously described taxa support the classification of strain JC444T as a representative of a new genus and species in the family Spirochaetaceae, for which the name Marispirochaeta aestuarii gen. nov., sp. nov. is proposed. Type strain is JC444T (=KCTC 15554T=DSM 103365T).

Pleomorphochaeta caudata gen. nov., sp. nov., an anaerobic bacterium isolated from an offshore oil well, reclassification of Sphaerochaeta multiformis MO-SPC2T as Pleomorphochaeta multiformis MO-SPC2T comb. nov. as the type strain of this novel genus and emended description of the genus Sphaerochaeta.

A strictly anaerobic Gram-stain-negative bacterium, designated strain SEBR 4223T, was isolated from the production water of an offshore Congolese oil field. Cells were non-motile, pleomorphic and had spherical, annular or budding shapes, often exhibiting long stalks. Strain SEBR 4223T grew on a range of carbohydrates, optimally at 37 °C and pH 7, in a medium containing 40 g l-1 NaCl. Predominant fatty acids were C14 : 0, C14 : 0 DMA, C16 : 0 and C16 : 1ω7c and the major polar lipids were phosphoglycolipids, phospholipids, glycolipids and diphosphatidylglycerol. The G+C content of the DNA was 28.7 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain SEBR 4223T and Sphaerochaeta multiformis MO-SPC2T formed a cluster with similarity to other species of the genus Sphaerochaeta of of less than 86 %. On the basis of the phenotypic characteristics and taxonomic analyses, we propose a novel genus, Pleomorphochaeta gen. nov., to accommodate the novel species Pleomorphochaeta caudata sp. nov., with SEBR 4223T (=DSM 103077T=JCM 31 475T) as the type strain. We also propose the reclassification of Sphaerochaeta multiformis MO SPC2T as Pleomorphochaeta multiformis MO-SPC2T comb. nov., the type strain of this novel genus and emend description of the genus Sphaerochaeta.

Sphaerochaeta associata sp. nov., a spherical spirochaete isolated from cultures of Methanosarcina mazei JL01.

An anaerobic, saccharolytic bacterial strain designated GLS2T was isolated from aggregates of the psychrotolerant archaeon Methanosarcina mazei strain JL01 isolated from arctic permafrost. Bacterial cells were non-motile, spherical, ovoid and annular with diameter 0.2-4 µm. They were chemoorganoheterotrophs using a wide range of mono-, di- and trisaccharides as carbon and energy sources. The novel isolate required yeast extract and vitamins for growth. The bacteria exhibited resistance to a number of ß-lactam antibiotics, rifampicin, streptomycin and vancomycin. Optimum growth was observed between 30 and 34 °C, at pH 6.8-7.5 and with 1-2 g NaCl l- 1. Isolate GLS2T was a strict anaerobe but it tolerated oxygen exposure. On the basis of 16S rRNA gene sequence similarity, strain GLS2T was shown to belong to the genus Sphaerochaeta within the family Spirochaetaceae. Its closest relatives were Sphaerochaeta globosa BuddyT (99.3 % 16S rRNA gene sequence similarity) and Sphaerochaeta pleomorpha GrapesT (95.4 % similarity). The G+C content of DNA was 47.2 mol%. The level of DNA-DNA hybridization between strains GLS2T and BuddyT was 34.7 ± 8.8 %. Major polar lipids were phosphoglycolipids, phospholipids and glycolipids; major fatty acids were C14 : 0, C16 : 0, C16 : 0 3-OH, C16 : 0 dimethyl acetal (DMA), C16 : 1n8 and C16 : 1 DMA; respiratory quinones were not detected. The results of DNA-DNA hybridization, physiological and biochemical tests demonstrated genotypic and phenotypic differentiation of strain GLS2T from the four species of the genus Sphaerochaeta with validly published names that allowed its separation into a new lineage at the species level. Strain GLS2T therefore represents a novel species, for which the name Sphaerochaeta associata sp. nov. is proposed, with the type strain GLS2T ( = DSM 26261T = VKM B-2742T).

Sphaerochaeta multiformis sp. nov., an anaerobic, psychrophilic bacterium isolated from subseafloor sediment, and emended description of the genus Sphaerochaeta.

An anaerobic, psychrophilic bacterium, strain MO-SPC2(T), was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediments collected from off the Shimokita Peninsula of Japan in the north-western Pacific Ocean. Cells were pleomorphic: spherical, annular, curved rod, helical and coccoid cell morphologies were observed. Motility only occurred in helical cells. Strain MO-SPC2(T) grew at 0-17 °C (optimally at 9 °C), at pH 6.0-8.0 (optimally at pH 6.8-7.2) and in 20-40 g NaCl l(-1) (optimally at 20-30 NaCl l(-1)). The strain grew chemo-organotrophically with mono-, di- and polysaccharides. The major end products of glucose fermentation were acetate, ethanol, hydrogen and carbon dioxide. The abundant polar lipids of strain MO-SPC2(T) were phosphatidylglycolipids, phospholipids and glycolipids. The major cellular fatty acids were C14 : 0, C16 : 0 and C16 : 1ω9. Isoprenoid quinones were not detected. The G+C content of the DNA was 32.3 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-SPC2(T) was affiliated with the genus Sphaerochaeta within the phylum Spirochaetes, and its closest relatives were Sphaerochaeta pleomorpha Grapes(T) (88.4 % sequence identity), Sphaerochaeta globosa Buddy(T) (86.7 %) and Sphaerochaeta coccoides SPN1(T) (85.4 %). Based on phenotypic characteristics and phylogenetic traits, strain MO-SPC2(T) is considered to represent a novel species of the genus Sphaerochaeta, for which the name Sphaerochaeta multiformis sp. nov. is proposed. The type strain is MO-SPC2(T) ( = JCM 17281(T) = DSM 23952(T)). An emended description of the genus Sphaerochaeta is also proposed.

Selected phenotypic features of BR91, a unique spirochaetal strain isolated from the Culex pipiens mosquito.

Growth temperature range, resistance to selective antibiotics, activities of 23 enzymes, protein fingerprints and fatty acids composition of the spirochaetal strain BR91, isolated from the Culex pipiens mosquito, were tested. The spirochaetes were grown in BSK-H Complete liquid medium. The optimal in vitro growth temperature of the strain was 33 °C. Strain BR91 was sensitive to trimethoprim, nalidixic acid, 5-fluorouracil, and tolerated phosphomycin. The strain produced acid and alkaline phosphatase, esterase (C4), esterase-lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and α-fucosidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay revealed several major proteins in the size range of 13-16 kDa, 22-30 kDa and 37-131 kDa. Fatty acid methyl ester (FAME) analysis showed that C14:0, C16:0, C18:1 ω9c and summed feature 5 (C18:2 ω6,9c and/or C18:0 anteiso) are major fatty acids. This study highlights certain phenotypic differences between strain BR91 and the Lyme disease spirochaete Borrelia burgdorferi, and supports the hypothesis that strain BR91 represents a unique taxonomical entity in a system of spirochaetal species.

Community and gene composition of a human dental plaque microbiota obtained by metagenomic sequencing.

Human dental plaque is a complex microbial community containing an estimated 700 to 19,000 species/phylotypes. Despite numerous studies analysing species richness in healthy and diseased human subjects, the true genomic composition of the human dental plaque microbiota remains unknown. Here we report a metagenomic analysis of a healthy human plaque sample using a combination of second-generation sequencing platforms. A total of 860 million base pairs of non-human sequences were generated. Various analysis tools revealed the presence of 12 well-characterized phyla, members of the TM-7 and BRC1 clade, and sequences that could not be classified. Both pathogens and opportunistic pathogens were identified, supporting the ecological plaque hypothesis for oral diseases. Mapping the metagenomic reads to sequenced reference genomes demonstrated that 4% of the reads could be assigned to the sequenced species. Preliminary annotation identified genes belonging to all known functional categories. Interestingly, although 73% of the total assembled contig sequences were predicted to code for proteins, only 51% of them could be assigned a functional role. Furthermore, ~2.8% of the total predicted genes coded for proteins involved in resistance to antibiotics and toxic compounds, suggesting that the oral cavity is an important reservoir for antimicrobial resistance.

Analysis of genes of tetrahydrofolate-dependent metabolism from cultivated spirochaetes and the gut community of the termite Zootermopsis angusticollis.

The hindguts of wood-feeding termites are the sites of intense, CO2-reductive acetogenesis. This activity profoundly influences host nutrition and methane emissions. Homoacetogens previously isolated from diverse termites comprised novel taxa belonging to two distinct bacterial phyla, Firmicutes and Spirochates. Little else is known about either the diversity or abundance of homoacetogenic species present in any given termite or the genetic details underlying CO2-reductive acetogenesis by Spirochaetes. A key enzyme of CO2-reductive acetogenesis is formyltetrahydrofolate synthetase (FTHFS). A previously designed primer set was used to amplify FTHFS genes from three isolated termite-gut spirochaetes. Sequencing DNA flanking the FTHFS gene of Treponema strain ZAS-2 revealed genes encoding two acetogenesis-related enzymes, methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase. Although termite-gut spirochaetes are only distantly related to clostridia at the ribosomal level, their tetrahydrofolate-dependent enzymes appear to be closely related. In contrast, homologous proteins identified in the non-homoacetogenic oral spirochaete Treponema denticola were only distantly related to those from clostridia and the termite-gut treponemes. Having demonstrated their utility with spirochaete pure cultures, the FTHFS primers were used to construct a 91-clone library from the termite-gut community DNA. From this, 19 DNA and eight amino acid FTHFS types were identified. Over 75 % of the retrieved clones formed a novel, coherent cluster with the FTHFS homologues obtained from the termite-gut treponemes. Thus, FTHFS gene diversity in the gut of the termite Zootermopsis angusticollis appears to be dominated by spirochaetes. The homoacetogenic capacity of termite-gut spirochaetes may have been acquired via lateral gene transfer from clostridia.

Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut.

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.

The impact of fermentative organisms on carbon flow in methanogenic systems under constant low-substrate conditions.

We compared carbon flow under constant low-substrate conditions (below 20 microM glucose in situ) in laboratory-scale glucose-fed methanogenic bioreactors containing two very different microbial communities that removed chemical oxygen demand at similar rates. One community contained approximately equal proportions of spiral and cocci morphologies, while the other community was dominated by cocci. In the former bioreactor, over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Spirochaetaceae-related sequences, while in the latter bioreactor over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Streptococcus-related sequences. Carbon flow was assessed by measuring 14C-labeled metabolites derived from a feeding of [U-14C]glucose that did not alter the concentration of glucose in the bioreactors. Acetate and ethanol were detected in the Spirochaetaceae-dominated reactor, whereas acetate and propionate were detected in the Streptococcus-dominated reactor. A spirochete isolated from a Spirochaetaceae-dominated reactor fermented glucose to acetate, ethanol, and small amounts of lactate. Maximum substrate utilization assays carried out on fluid from the same reactor indicated that acetate and ethanol were rapidly utilized by this community. These data indicate that an acetate- and ethanol-based food chain was present in the Spirochaetaceae-dominated bioreactor, while the typical acetate- and propionate-based food chain was prevalent in the Streptococcus-dominated bioreactor.

Brachyspira aalborgi infection diagnosed by culture and 16S ribosomal DNA sequencing using human colonic biopsy specimens.

In this study we report on the isolation and characterization of the intestinal spirochete Brachyspira aalborgi using human mucosal biopsy specimens taken from the colon of a young adult male with intestinal spirochetosis. A selective medium, containing 400 microg of spectinomycin/ml and 5 microg of polymyxin/ml was used for the isolation procedure. A high degree of similarity, in terms of phenotypic properties and 16S ribosomal DNA sequence, was observed between the isolated strain, named W1, and the type strain, 513A, of B. aalborgi. A similarity of 99.7% in the nucleotide sequence was found between W1 and 513A(T), based on the almost-complete gene. A short segment of the 16S rRNA gene was amplified by PCR using genetic material enriched from paraffin-embedded biopsy specimens, which were taken from the patient on two occasions. The products showed 16S rRNA gene sequences virtually identical to that of strain 513A(T) in the actual region. Immunohistochemistry was performed on the colonic biopsy specimens with a polyclonal antibody raised against an intestinal spirochete isolated in a previous case of human intestinal spirochetosis. The antibody reacted strongly with the spirochete on the luminal epithelium. No immune reaction was seen within or below the surface epithelium. Routine histology did not reveal signs of colitis. Electron microscopy showed spirochetes attached end-on to the colonic mucosal surface. The isolate grew poorly on a commonly used selective medium for intestinal spirochetes, which may explain previous failures to isolate B. aalborgi.

Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA.

The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.

Phylogenetic relationships of symbiotic spirochetes in the gut of diverse termites.

Phylogenetic relationships of symbiotic spirochetes in the gut of diverse termites were analyzed without cultivation of these microorganisms. A portion of the 16S rDNA (ca. 850 bp) was amplified directly from DNA of the mixed population in the gut by PCR and cloned. A total of 30 spirochetal phylotypes affiliated with the treponemes were identified from four termite species and they were compared with those already reported from other termites. They represented separate lines of descent from any known species of Treponema, and they were divided into two discrete clusters; one was related to Spirochaeta stenostrepta and S. caldaria, and the other was grouped together with members of the Treponema bryantii subgroup. Although some sequences from evolutionarily related termites showed close similarity, most of the sequences of spirochetes were dissimilar among different termite species, and spirochetal sequences from a single termite species occurred in several distinct phylogenetic positions. These findings suggest that termites constitute a rich reservoir of novel spirochetal diversity and that evolution of the symbiosis is not simple.

Phenotypic and genotypic heterogeneity among cultivable pathogen-related oral spirochetes and Treponema vincentii.

Recent findings challenge the assumption that pathogen-related oral spirochetes (PROS) are related to Treponema pallidum. Treponema vincentii, grown in OMIZ-Pat media, cross-reacted with monoclonal antibody H9-2 against T. pallidum, and cultivable PROS had 16S rRNA gene sequences similar to those of T. vincentii (C.-B. Choi, C. Wyss, and U. B. Göbel. J. Clin. Microbiol. 34:1922-1925, 1996). Aims of the present study were to determine whether antigen phenotypes of oral treponemas were influenced by growth conditions and to evaluate the genetic relatedness of cultivable PROS to T. pallidum and T. vincentii. Results show that three T. pallidum monoclonal antibodies (H9-1, H9-2, and F5) cross-reacted with whole cells from four Treponema species grown in modified OMIZ-Pat medium, but not with treponemas grown in NOS medium. Only H9-2 reacted in immunoblots with reduced proteins from cultivable PROS and T. vincentii. Three of five PROS isolates were amplified by T. vincentii-specific PCR, and one was amplified by Treponema medium-specific PCR. None were amplified by T. pallidum-specific PCR. Three of five PROS isolates had 16S ribosomal DNA restriction fragment length polymorphism patterns identical to that of T. vincentii, and the patterns of two isolates resembled that of T. medium. Arbitrarily primed-PCR profiles from whole genomic DNA were distinct among five PROS isolates and two T. vincentii strains. Thus, PROS isolates represent a heterogeneous group of treponemas that share some 16S rRNA gene sequences with T. vincentii and T. medium, but not with T. pallidum. It is proposed that the PROS nomenclature be dropped.