Oral microbial profile variation during canine ligature-induced peri-implantitis development.

BACKGROUND: Dental implants have become well-established in oral rehabilitation for fully or partially edentulous patients. However, peri-implantitis often leads to the failure of dental implants. The aim of this study was to understand the core microbiome associated with peri-implantitis and evaluate potential peri-implantitis pathogens based on canine peri-implantitis model. RESULTS: In this study, three beagle dogs were used to build peri-implantitis models with ligature-induced strategy. The peri-implant sulcular fluids were collected at four different phases based on disease severity during the peri-implantitis development. Microbial compositions during peri-implantitis development were monitored and evaluated. The microbes were presented with operational taxonomic unit (OTU) classified at 97% identity of the high-throughput 16S rRNA gene fragments. Microbial diversity and richness varied during peri-implantitis. At the phylum-level, Firmicutes decreased and Bacteroides increased during peri-implantitis development. At the genus-level, Peptostreptococcus decreased and Porphyromonas increased, suggesting peri-implantitis pathogens might be assigned to these two genera. Further species-level and co-occurrence network analyses identified several potential keystone species during peri-implantitis development, and some OTUs were potential peri-implantitis pathogens. CONCLUSION: In summary, canine peri-implantitis models help to identify several potential keystone peri-implantitis associated species. The canine model can give insight into human peri-implantitis associated microbiota.

Rectinema cohabitans gen. nov., sp. nov., a rod-shaped spirochaete isolated from an anaerobic naphthalene-degrading enrichment culture.

The anaerobic, non-motile strain HMT was isolated from the naphthalene-degrading, sulfate-reducing enrichment culture N47. For 20 years, strain HMT has been a stable member of culture N47 although it is neither able to degrade naphthalene nor able to reduce sulfate in pure culture. The highest similarity of the 16S rRNA gene sequence of strain HMT (89 %) is with a cultivated member of the family Spirochaetaceae, Treponema caldariumstrain H1T (=DSM 7334T), an obligately anaerobic, thermophilic spirochaete isolated from cyanobacterial mat samples collected at a freshwater hot spring in Oregon, USA. In contrast to this strain and the majority of spirochaete species described, strain HMT showed a rod-shaped morphology. Growth occurred at temperatures between 12 and 50 °C (optimum 37 °C) but the isolate was not able to grow at 60 °C. The strain fermented various sugars including d-glucose, d-fructose, lactose and sucrose. Addition of 0.1 % (w/v) yeast extract or 0.1 % (w/v) tryptone to the culture medium was essential for growth and could not be replaced by either the vitamin solutions tested or by 0.1 % (w/v) peptone or 0.1 % (w/v) casamino acids. The DNA G+C content of the isolate was 51.5 mol%. The major fatty acids were C14 : 0, C18 : 1ω13c, C16 : 1ω9t, C16 : 1ω11c and C16 : 1ω9c. Based on the unique morphology and the phylogenetic distance from the closest cultivated relative, a novel genus and species, Rectinema cohabitans gen. nov., sp. nov., is proposed. The type strain is strain HMT (=DSM 100378T=JCM 30982T).

Influences of pH and Iron Concentration on the Salivary Microbiome in Individual Humans with and without Caries.

This study aimed to identify the differences in the oral microbial communities in saliva from patients with and without caries by performing sequencing with the Illumina MiSeq platform, as well as to further assess their relationships with environmental factors (salivary pH and iron concentration). Forty-three volunteers were selected, including 21 subjects with and 22 without caries, from one village in Gansu, China. Based on 966,255 trimmed sequences and clustering at the 97% similarity level, 1,303 species-level operational taxonomic units were generated. The sequencing data for the two groups revealed that (i) particular distribution patterns (synergistic effects or competition) existed in the subjects with and without caries at both the genus and species levels and (ii) both the salivary pH and iron concentration had significant influences on the microbial community structure. IMPORTANCE: The significant influences of the oral environment observed in this study increase the current understanding of the salivary microbiome in caries. These results will be useful for expanding research directions and for improving disease diagnosis, prognosis, and therapy.

Spirochaetes dominate the microbial community associated with the red coral Corallium rubrum on a broad geographic scale.

Mass mortality events in populations of the iconic red coral Corallium rubrum have been related to seawater temperature anomalies that may have triggered microbial disease development. However, very little is known about the bacterial community associated with the red coral. We therefore aimed to provide insight into this species' bacterial assemblages using Illumina MiSeq sequencing of 16S rRNA gene amplicons generated from samples collected at five locations distributed across the western Mediterranean Sea. Twelve bacterial species were found to be consistently associated with the red coral, forming a core microbiome that accounted for 94.6% of the overall bacterial community. This core microbiome was particularly dominated by bacteria of the orders Spirochaetales and Oceanospirillales, in particular the ME2 family. Bacteria belonging to these orders have been implicated in nutrient cycling, including nitrogen, carbon and sulfur. While Oceanospirillales are common symbionts of marine invertebrates, our results identify members of the Spirochaetales as other important dominant symbiotic bacterial associates within Anthozoans.

Alkalispirochaeta cellulosivorans gen. nov., sp. nov., a cellulose-hydrolysing, alkaliphilic, halotolerant bacterium isolated from the gut of a wood-eating cockroach (Cryptocercus punctulatus), and reclassification of four species of Spirochaeta as new combinations within Alkalispirochaeta gen. nov.

An obligately anaerobic spirochaete designated strain JC227T was isolated from the gut of a wood-eating cockroach, Cryptocercus punctulatus (Scudder), from the Rann of Kutch, Gujarat, India. Strain JC227T was Gram-stain-negative, mesophilic, halotolerant and alkaliphilic. Based on 16S rRNA gene sequence analysis, strain JC227T belongs to the genus Spirochaeta, with Spirochaeta sphaeroplastigenens JC133T (99.51%), S. odontotermitis JC202T (99.30%), S. alkalica Z-7491T (99.10%), S. americana (98.54%) and other members of the genus Spirochaeta (<92.7%) as its closest phylogenetic neighbours. However, DNA-DNA hybridization between strain JC227T and S. sphaeroplastigenens JC133T, S. odontotermitis JC202T, S. alkalica DSM 8900T and S. americana DSM 14872T was 62±2, 63, 58±2 and 48±4 %, respectively. Strain JC227T contained phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid and six unidentified lipids. Summed feature C18:1ω7c/C18:1ω6c was the predominant cellular fatty acid, with significant proportions of C16:0, C14:0, C12:0, C15:1ω6c, C16:1ω5c, C16:1ω6c/C16:1ω7c and C17:0 2-OH. The DNA G+C content of strain JC227T was 55.5 mol%. On the basis of physiological, biochemical, chemotaxonomic (including metabolomic) and genomic differences from previously described taxa, strain JC227T can be differentiated from members of the genus Spirochaeta and represents a novel species of a new genus, for which the name Alkalispirochaeta cellulosivorans gen. nov., sp. nov. is proposed. The type strain of Alkalispirochaeta cellulosivorans is JC227T (=KCTC 15343T=NBRC 110105T). We also propose the reclassification of Spirochaeta sphaeroplastigenens, Spirochaeta odontotermitis, Spirochaeta alkalica and Spirochaeta americana as Alkalispirochaeta sphaeroplastigenens comb. nov. (type strain JC133T=KCTC 15220T=NBRC 109056T), Alkalispirochaeta odontotermitis comb. nov. (type strain JC202T=KCTC 15324T=NBRC 110104T), Alkalispirochaeta alkalica comb. nov. (type strain Z-7491T=DSM 8900T=ATCC 700262T) and Alkalispirochaeta americana comb. nov. (type strain ASpG1T=ATCC BAA-392T=DSM 14872T). The type species of Alkalispirochaeta gen. nov. is Alkalispirochaeta alkalica comb. nov.

Spirochaeta odontotermitis sp. nov., an obligately anaerobic, cellulolytic, halotolerant, alkaliphilic spirochaete isolated from the termite Odontotermes obesus (Rambur) gut.

A Gram-stain-negative spirochaete (strain JC202T) was isolated from the gut of the termite Odontotermes obesus (Rambur) from Rann of Kutch, Gujarat, India. This strain was obligately anaerobic, mesophilic, halotolerant and required alkaline conditions for growth. Strain JC202T was resistant to rifampicin and kanamycin, but sensitive to gentamicin, tetracycline, ampicillin and chloramphenicol. Strain JC202T possessed phosphatidylglycerol, diphosphatidylglycerol, glycolipid and six unidentified lipids. C18 : 1ω7c was the predominant cellular fatty acid with significant proportions of C16 : 0, C18 : 1ω9c, C14 : 0, C18 : 0, C16 : 1ω5c, C18 : 1ω5c and C20 : 1ω9c. The DNA G+C content of strain JC202T was 59 mol%. Based on 16S rRNA gene sequence analysis, strain JC202T is considered to belong to the genus Spirochaeta with Spirochaeta sphaeroplastigenens JC133T (100 % similarity), Spirochaeta alkalica Z-7491T (99.92 %), Spirochaeta americana ATCC BAA-392T (99.47 %) and other members of the genus Spirochaeta ( < 93.83 %) as the closest phylogenetic neighbours. However, mean DNA-DNA hydridization values between strain JC202T and S. sphaeroplastigenens JC133T, S. alkalica DSM 8900T ( = Z-7491T) and S. americana DSM 14872T ( = ASpG1T) were 55 ± 2, 22 ± 3 and 32 ± 1 %, respectively. On the basis of physiological, biochemical, chemotaxonomic (including metabolome) and genomic differences from the previously described taxa, strain JC202T is differentiated from other members of the genus Spirochaeta and is considered to represent a novel species, for which the name Spirochaeta odontotermitis sp. nov. is proposed. The type strain is JC202T ( = KCTC 15324T = NBRC 110104T).

Spirochaeta sinaica sp. nov., a halophilic spirochaete isolated from a cyanobacterial mat.

A strain of free-living obligately anaerobic, halophilic spirochaete, SLT, was isolated from a sample of a cyanobacterial mat of the hypersaline Solar Lake, Sinai shore. The strain had motile helical cells, 0.35-0.40 × 6-10 µm. Strain SLT exhibited high resistance to NaCl among known halophilic spirochaetes growing at NaCl concentrations from 2 to 12% (optimum growth at 7%). The strain grew at temperatures from 10 to 32 °C (optimum at 28 °C) and pH from 6 to 8.5 (optimum at pH 7.0-7.5). Carbohydrates, but not alcohols, organic acids or nitrogenous compounds (peptone, yeast extract and amino acids), were used as energy substrates for growth. Ethanol, acetate, lactate, H2 and CO2 were the products of glucose fermentation. Sulfide was produced in the presence of S0 or thiosulfate in the medium. The DNA G+C content was 44.7 mol%. Based on 16S rRNA gene sequence analysis, strain SLT clustered within the genus Spirochaeta, exhibiting 94.2 and 93.7% similarity with its closest relatives, Spirochaeta bajacaliforniensis DSM 160554T and Spirochaeta smaragdinae DSM 11293T, respectively; similarity with other species did not exceed 86%. The phenotypic and chemotaxonomic characteristics of the strain, as well as the results of phylogenetic analysis support the classification of strain SLT as representing a novel species of the genus Spirochaeta, for which the name Spirochaeta sinaica sp. nov. is proposed. The type strain is SLT ( = DSM 14994 = NIQEM U 783).

Spirochaeta lutea sp. nov., isolated from marine habitats and emended description of the genus Spirochaeta.

Metagenome analysis of forty samples indicated a rich diversity of free living spirochaetes among Indian habitats. Only 2-3% of the total bacterial diversity was represented by spirochaetes with five distinct phylogenetic OTUs. A strain (JC230(T)) was brought to cultivation and its 16S rRNA gene sequence showed highest sequence similarity with that of Spirochaeta asiatica Z-7591(T) (90.78%). Genome sequence analysis of strain JC230(T) indicates the presence of 117 genes related to motility and chemotaxis. Strain JC230(T) is an obligate anaerobe. It is helical shaped, Gram-stain-negative and catalase and oxidase negative. Strain JC230(T) produces carotenoids and have all the genes related to carotenogenesis of spirilloxanthin series. Isoprenoid biosynthesis occurs through HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) pathway. Twelve bacterial haemoglobin and one flavohaemoglobin related genes are present in the genome of strain JC230(T). The genome of strain JC230(T) showed the presence of heavy metal resistance related genes of cobalt, zinc, cadmium and arsenic. Six genes related to colicin V and bacteriocin production cluster are present. Major (>2%) fatty acids were C12:0, C14:0, iso-C14:0, iso-C15:1H/C13:03OH, anteiso-C15:0, C16:0, and iso-C16:0. Strain JC230(T) contains diphosphatylglycerol, phosphatydilglycerol, phosphatydilethanolamine, unidentified phospholipid (PL1) and unidentified lipids (L1-7). G+C mol% of strain JC230(T) was 54.1%. Distinct morphological, physiological and genotypic differences from the previously described taxa support the classification of strain JC230(T) as a representative of a new species in the genus Spirochaeta, for which the name Spirochaeta lutea sp. nov. is proposed. The type strain is JC230(T) (=KCTC 15387(T)=DSM 29074(T)).

Spirochaeta psychrophila sp. nov., a psychrophilic spirochaete isolated from subseafloor sediment, and emended description of the genus Spirochaeta.

An obligately anaerobic, psychrophilic spirochaete, strain MO-SPC1(T), was isolated from a methanogenic microbial community grown in a continuous-flow bioreactor. Originally, this community was obtained from subseafloor sediments off the Shimokita Peninsula of Japan in the north-western Pacific Ocean. The cells were motile, Gram-stain-negative, helical, 0.25-0.55×3.6-15 µm, with a wavelength of approximately 0.5-0.6 µm. Strain MO-SPC1(T) grew at 0-18 °C (optimally at 15 °C), at pH 6.0-7.5 (optimally at pH 6.8-7.0) and in 20-70 g NaCl l(-1) (optimally at 30-40 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-SPC1(T) were diphosphatidylglycerol, phosphatidylglycerol, unknown phospholipids and an unknown lipid. The major cellular fatty acids (>5% of the total) were C(14 : 0), C(16 : 0), iso-C(13 : 0), iso-C(14 : 0), iso-C(15 : 0), anteiso-C(13 : 0) and anteiso-C(15 : 0). To the best of our knowledge, this is the first report of the fatty acids iso-C(13 : 0) and anteiso-C(13 : 0) from a species of the genus Spirochaeta. Isoprenoid quinones were not found. The G+C content of the genomic DNA was 39.8 mol%. 16S rRNA gene sequence-based phylogenetic analysis showed that strain MO-SPC1(T) was affiliated with the genus Spirochaeta, and its closest relatives were Spirochaeta isovalerica MA-2(T) (95.6% sequence identity) and Spirochaeta litoralis R1(T) (89.4%). Based on its phenotypic characteristics and phylogenetic traits, strain MO-SPC1(T) is placed in a separate taxon at the level of a novel species within the genus Spirochaeta, for which the name Spirochaeta psychrophila sp. nov. is proposed, reflecting its true psychrophilic physiology. The type strain is MO-SPC1(T) ( = JCM 17280(T) = DSM 23951(T)). To our knowledge, this is the first report of an isolate of the phylum Spirochaetes from a deep-sea sedimentary environment, and of an obligately psychrophilic nature.

The discovery of stromatolites developing at 3570 m above sea level in a high-altitude volcanic lake Socompa, Argentinean Andes.

We describe stromatolites forming at an altitude of 3570 m at the shore of a volcanic lake Socompa, Argentinean Andes. The water at the site of stromatolites formation is alkaline, hypersaline, rich in inorganic nutrients, very rich in arsenic, and warm (20-24°C) due to a hydrothermal input. The stromatolites do not lithify, but form broad, rounded and low-domed bioherms dominated by diatom frustules and aragonite micro-crystals agglutinated by extracellular substances. In comparison to other modern stromatolites, they harbour an atypical microbial community characterized by highly abundant representatives of Deinococcus-Thermus, Rhodobacteraceae, Desulfobacterales and Spirochaetes. Additionally, a high proportion of the sequences that could not be classified at phylum level showed less than 80% identity to the best hit in the NCBI database, suggesting the presence of novel distant lineages. The primary production in the stromatolites is generally high and likely dominated by Microcoleus sp. Through negative phototaxis, the location of these cyanobacteria in the stromatolites is controlled by UV light, which greatly influences their photosynthetic activity. Diatoms, dominated by Amphora sp., are abundant in the anoxic, sulfidic and essentially dark parts of the stromatolites. Although their origin in the stromatolites is unclear, they are possibly an important source of anaerobically degraded organic matter that induces in situ aragonite precipitation. To the best of our knowledge, this is so far the highest altitude with documented actively forming stromatolites. Their generally rich, diverse and to a large extent novel microbial community likely harbours valuable genetic and proteomic reserves, and thus deserves active protection. Furthermore, since the stromatolites flourish in an environment characterized by a multitude of extremes, including high exposure to UV radiation, they can be an excellent model system for studying microbial adaptations under conditions that, at least in part, resemble those during the early phase of life evolution on Earth.

Spirochaeta sphaeroplastigenens sp. nov., a halo-alkaliphilic, obligately anaerobic spirochaete isolated from soda lake Lonar.

Two helical-shaped bacteria (strains JC133(T) and JC143), which stain Gram-negative, were isolated from an alkaline soda lake, Lonar, India. Both strains were obligate anaerobes, mesophilic and required halo-alkaline conditions for growth. Both strains were resistant to rifampicin and kanamycin, but sensitive to gentamicin, tetracycline, ampicillin and chloramphenicol. Both strains had phosphatidylglycerol (PG), diphosphotidylglycerol (DPG), glycolipid (GL) and four unidentified lipids (L1-4) as the major polar lipids. C18:1ω7c was the predominant cellular fatty acid with significant proportions of C16:0, C18:1ω9c, C14:0, C18:0, C16:1ω5c, C18:1ω5c and C20:1ω9c. The DNA G+C contents of strain JC131(T) and JC143 were 58.2 and 58.5 mol%, respectively, and the two strains showed DNA reassociation >85% (based on DNA-DNA hybridization). Based on the 16S rRNA gene sequence analysis, both strains were identified as belonging to the genus Spirochaeta with Spirochaeta alkalica Z-7491(T) (99.6% sequence similarity), Spirochaeta americana ASpG1(T) (99 %) and other members of the genus Spirochaeta (<93%) as their closest phylogenetic neighbours. However, strain JC133(T) and JC143 displayed less than 53.5 % binding (based on DNA-DNA hybridization) with S. alkalica Z-7491(T) and S. americana ASpG1(T). On the basis of physiological, biochemical, chemotaxonomic and molecular properties, strains JC133(T) and JC143 can be differentiated from other members of the genus Spirochaeta and represent a novel species of the genus Spirochaeta, for which the name Spirochaeta sphaeroplastigenens sp. nov. is proposed. The type strain is JC133(T) ( = KCTC 15220(T) = NBRC 109056(T)).

Risk factors for noma disease: a 6-year, prospective, matched case-control study in Niger.

BACKGROUND: Noma is a poorly studied disease that leads to severe facial tissue destruction in children in developing countries, but the cause remains unknown. We aimed to identify the epidemiological and microbiological risk factors associated with noma disease. METHODS: We did a prospective, matched, case-control study in Niger between Aug 1, 2001, and Oct 31, 2006, in children younger than 12 years to assess risk factors for acute noma. All acute noma cases were included and four controls for each case were matched by age and home village. Epidemiological and clinical data were obtained at study inclusion. We undertook matched-paired analyses with conditional logistic regression models. FINDINGS: We included 82 cases and 327 controls. Independent risk factors associated with noma were: severe stunting (odds ratio [OR] 4·87, 95% CI 2·35-10·09) or wasting (2·45, 1·25-4·83); a high number of previous pregnancies in the mother (1·16, 1·04-1·31); the presence of respiratory disease, diarrhoea, or fever in the past 3 months (2·70, 1·35-5·40); and the absence of chickens at home (1·90, 0·93-3·88). After inclusion of microbiological data, a reduced proportion of Fusobacterium (4·63, 1·61-13·35), Capnocytophaga (3·69, 1·48-9·17), Neisseria (3·24, 1·10-9·55), and Spirochaeta in the mouth (7·77, 2·12-28·42), and an increased proportion of Prevotella (2·53, 1·07-5·98), were associated with noma. We identified no specific single bacterial or viral pathogen in cases. INTERPRETATION: Noma is associated with indicators of severe poverty and altered oral microbiota. The predominance of specific bacterial commensals is indicative of a modification of the oral microbiota associated with reduced bacterial diversity. FUNDING: Gertrude Hirzel Foundation.

The chimeric genome of Sphaerochaeta: nonspiral spirochetes that break with the prevalent dogma in spirochete biology.

UNLABELLED: Spirochaetes is one of a few bacterial phyla that are characterized by a unifying diagnostic feature, namely, the helical morphology and motility conferred by axial periplasmic flagella. Their unique morphology and mode of propulsion also represent major pathogenicity factors of clinical spirochetes. Here we describe the genome sequences of two coccoid isolates of the recently described genus Sphaerochaeta which are members of the phylum Spirochaetes based on 16S rRNA gene and whole-genome phylogenies. Interestingly, the Sphaerochaeta genomes completely lack the motility and associated signal transduction genes present in all sequenced spirochete genomes. Additional analyses revealed that the lack of flagella is associated with a unique, nonrigid cell wall structure hallmarked by a lack of transpeptidase and transglycosylase genes, which is also unprecedented in spirochetes. The Sphaerochaeta genomes are highly enriched in fermentation and carbohydrate metabolism genes relative to other spirochetes, indicating a fermentative lifestyle. Remarkably, most of the enriched genes appear to have been acquired from nonspirochetes, particularly clostridia, in several massive horizontal gene transfer events (>40% of the total number of genes in each genome). Such a high level of direct interphylum genetic exchange is extremely rare among mesophilic organisms and has important implications for the assembly of the prokaryotic tree of life. IMPORTANCE: Spiral shape and motility historically have been the unifying hallmarks of the phylum Spirochaetes. These features also represent important virulence factors of highly invasive pathogenic spirochetes such as the causative agents of syphilis and Lyme disease. Through the integration of genome sequencing, microscopy, and physiological studies, we conclusively show that the spiral morphology and motility of spirochetes are not universal morphological properties. In particular, we found that the genomes of the members of the recently described genus Sphaerochaeta lack the genes encoding the characteristic flagellar apparatus and, in contrast to most other spirochetes, have acquired many metabolic and fermentation genes from clostridia. These findings have major implications for the isolation and study of spirochetes, the diagnosis of spirochete-caused diseases, and the reconstruction of the evolutionary history of this important bacterial phylum. The Sphaerochaeta sp. genomes offer new avenues to link ecophysiology with the functionality and evolution of the spirochete flagellar apparatus.

Typhlocolitis associated with spirochaetes in duck flocks.

The aetiology of increased mortality observed in two breeder duck flocks (Flock A consisting of 3500 laying ducks and Flock B comprising 4300 laying ducks) during the first egg-laying season was studied. In Flocks A and B, 773 ducks and 715 ducks (18.4% and 16.6%) died within a 24-week and a 20-week period, respectively. Death was preceded by clinical signs including movement difficulties, lack of appetite and depression lasting for 1 to 2 days. Diarrhoea was not observed. On gross pathological examination, the ducks were found to have haemorrhagic to fibrinonecrotic typhlocolitis, renal degeneration accompanied by fibrosis and mineralization, hepatic and splenic amyloidosis, and swelling of some of the metatarsal and phalangeal joints. Histopathological and immunohistochemical examination consistently demonstrated spirochaetes in the mucous membrane of the affected large intestine. On the basis of their cultural and biochemical properties and polymerase chain reaction sequencing analysis, four out of seven spirochaete strains isolated from the ducks (Flock A) by culture on special media under anaerobic conditions were identified as Brachyspira hyodysenteriae, and five out of eight strains (Flock B) were identified as Brachyspira pilosicoli. This is the first report on the isolation of B. hyodysenteriae and B. pilosicoli from laying ducks affected by fibrinonecrotic typhlocolitis.

Spirochaeta perfilievii sp. nov., an oxygen-tolerant, sulfide-oxidizing, sulfur- and thiosulfate-reducing spirochaete isolated from a saline spring.

A novel strain of fermenting, aerotolerant, chemo-organoheterotrophic spirochaete designated P(T) was isolated from a sulfur 'Thiodendron' mat in a saline spring at the Staraya Russa resort (Novgorod Region, Russia). Cells of strain P(T) exhibited a helical shape. The spirochaete required sulfide in the growth medium and was able to oxidize it non-enzymically to elemental sulfur via the interaction of H(2)O(2) with sulfide and deposit it in the periplasmic space. Growth occurred at 4-32 °C (optimum at 28-30 °C), pH 6.0-8.5 (optimum pH 7.0-7.5), and in 0.1-1 M NaCl (optimum 0.35 M). The isolate used several sugars and polysaccharides as carbon or energy sources but did not use peptides, amino acids, organic acids or alcohols. The products of glucose fermentation were formate, acetate, ethanol, pyruvate, CO(2) and H(2). The genomic DNA G+C content was 41.7 mol%. 16S rRNA gene sequence analysis showed that strain P(T) fell within a group of species in the genus Spirochaeta, including Spirochaeta litoralis, S. isovalerica and S. cellobiosiphila, with which it shared less then 89 % sequence similarity. On the basis of its morphology, physiology and other phenotypic properties, as well as its phylogenetic position, the new isolate is considered to represent a novel species of the genus Spirochaeta, for which the name Spirochaeta perfilievii sp. nov. is proposed. The type strain is P(T) (=DSM 19205(T) =VKM B-2514(T)).

Spirochaeta dissipatitropha sp. nov., an alkaliphilic, obligately anaerobic bacterium, and emended description of the genus Spirochaeta Ehrenberg 1835.

A novel obligately anaerobic, mesophilic, alkaliphilic spirochaete, strain ASpC2(T), was isolated from an anaerobic sediment of alkaline, hypersaline Owens Lake in California, USA. The Gram-negative cells are motile, helical in shape and 0.23 x 8.0-18.0 mum. Growth occurs within the following ranges: 13-41 degrees C, with optimal growth at 35 degrees C; 1-3 % (w/v) NaCl, with optimal growth at 2 % (w/v) NaCl; and pH 7.8-10.5, with optimal growth at pH 10.0. The novel isolate is strictly alkaliphilic and requires high concentrations of carbonate ions in the medium. It utilizes some sugars, some organic acids, some amino acids, Casamino acids, yeast extract and peptone. The main end products of glucose fermentation are CO(2) and acetate. Strain ASpC2(T) is resistant to kanamycin and rifampicin, but sensitive to ampicillin, chloramphenicol, gentamicin and tetracycline. The DNA G+C content of the new isolate is 43.8 mol%, its genome size is 6 x 10(8) Da and the melting temperature of its genomic DNA is 71 degrees C. DNA-DNA hybridization experiments demonstrated 46 % similarity with the phylogenetically most closely related species, Spirochaeta asiatica Z-7591(T). On the basis of physiological and molecular properties, the new isolate belongs taxonomically to a novel species within the genus Spirochaeta, for which the name Spirochaeta dissipatitropha sp. nov. is proposed (type strain, ASpC2(T)=ATCC BAA-1083(T)=JCM 12856(T)). S. dissipatitropha ASpC2(T) is the second strain in the genus (after Spirochaeta smaragdinae SEBR 4228(T)) that is able to use proteolysis products as the sole energy source, and additional tests have shown that other halo-alkaliphilic spirochaetes (Spirochaeta americana, Spirochaeta alkalica and Spirochaeta africana) are also able to grow on yeast extract alone; therefore, an emended description for the genus Spirochaeta is given.

Phylogenetic diversity and temporal variation in the Spirochaeta populations from two Mediterranean microbial mats.

Spirochetes are among the bacterial groups often observed in hydrogen-sulfide-rich layers of coastal microbial mats. However, relatively few spirochetes from these microbial mats have been described and characterized. We used 16S rDNA phylogenetic analysis to investigate the spirochetal diversity of microbial mats from two locations in the western Mediterranean (Ebro Delta, Spain, and Camargue, France). Samples from each location were monitored in the spring and winter over a period of 1 to 2 years. In the sequence analysis of 332 clones derived from samples of both locations, 42 novel phylotypes of not-yet-cultivated spirochetes belonging to the genus Spirochaeta were detected. None of the phylotypes were identified as known culturable species of Spirochaeta or previously identified phylotypes cloned from other hypersaline microbial mat such as Guerrero Negro, Mexico. Eight of the phylotypes were common to Ebro and Camargue mats, and two (IF058 and LL066) were present both in spring and winter. Some phylotypes appeared to show seasonal variation, i.e., they were found only in the spring, but not in the winter. Ebro and Camargue phylotypes, like phylotypes from Guerrero Negro, grouped according to the vertical gradient of oxygen and sulfide in the mat. Some phylotypes, such as LH073, IE028, LH042, or LG013 were harbored in low H2S or H2S-O2 interface zone. In contrast, major phylotypes were detected in deeper layers and they were likely strict anaerobes and high tolerant to H2S. The presence of spirochetes in differently located microbial mats suggests that they constitute very diverse and stable populations involved in a well-integrated metabolic symbiosis (i.e., permanent physiological cooperation) with other guild populations in the mats, where they maintain a coordinated functional and stable community.