Megasphaera lornae sp. nov., Megasphaera hutchinsoni sp. nov., and Megasphaera vaginalis sp. nov.: novel bacteria isolated from the female genital tract.

Six strictly anaerobic Gram-negative bacteria representing three novel species were isolated from the female reproductive tract. The proposed type strains for each species were designated UPII 199-6T, KA00182T and BV3C16-1T. Phylogenetic analyses based on 16S rRNA gene sequencing indicated that the bacterial isolates were members of the genus Megasphaera. UPII 199-6T and KA00182T had 16S rRNA gene sequence identities of 99.9 % with 16S rRNA clone sequences previously amplified from the human vagina designated as Megasphaera type 1 and Megasphaera type 2, members of the human vaginal microbiota associated with bacterial vaginosis, preterm birth and HIV acquisition. UPII 199-6T exhibited sequence identities ranging from 92.9 to 93.6 % with validly named Megasphaera isolates and KA00182T had 16S rRNA gene sequence identities ranging from 92.6-94.2 %. BV3C16-1T was most closely related to Megasphaera cerevisiae with a 16S rRNA gene sequence identity of 95.4 %. Cells were coccoid or diplococcoid, non-motile and did not form spores. Genital tract isolates metabolized organic acids but were asaccharolytic. The isolates also metabolized amino acids. The DNA G+C content for the genome sequences of UPII 199-6T, KA00182T and BV3C16-1T were 46.4, 38.9 and 49.8 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity between the genital tract isolates and other validly named Megasphaera species suggest that each isolate type represents a new species. The major fatty acid methyl esters include the following: C12 : 0, C16 : 0, C16 : 0 dimethyl acetal (DMA) and summed feature 5 (C15 : 0 DMA and/or C14 : 0 3-OH) in UPII 199-6T; C16 : 0 and C16 : 1 cis 9 in KA00182T; C12 : 0; C14 : 0 3-OH; and summed feature 5 in BV3C16-1T. The isolates produced butyrate, isobutyrate, and isovalerate but there were specific differences including production of formate and propionate. Together, these data indicate that UPII 199-6T, KA00182T and BV3C16-1T represent novel species within the genus Megasphaera. We propose the following names: Megasphaera lornae sp. nov. for UPII 199-6T representing the type strain of this species (=DSM 111201T=ATCC TSD-205T), Megasphaera hutchinsoni sp. nov. for KA00182T representing the type strain of this species (=DSM 111202T=ATCC TSD-206T) and Megasphaera vaginalis sp. nov. for BV3C16-1T representing the type strain of this species (=DSM 111203T=ATCC TSD-207T).

More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

BACKGROUND: Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. METHODS: Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. RESULTS: We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. CONCLUSIONS: We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation.

Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov., obligately anaerobic bacteria from the human oral cavity, and emended description of the genus Oribacterium.

Three strictly anaerobic, Gram-positive, non-spore-forming, rod-shaped, motile bacteria, designated strains ACB1(T), ACB7(T) and ACB8, were isolated from human subgingival dental plaque. All strains required yeast extract for growth. Strains ACB1(T) and ACB8 were able to grow on glucose, lactose, maltose, maltodextrin and raffinose; strain ACB7(T) grew weakly on sucrose only. The growth temperature range was 30-42 °C with optimum growth at 37 °C. Major metabolic fermentation end products of strain ACB1(T) were acetate and lactate; the only product of strains ACB7(T) and ACB8 was acetate. Major fatty acids of strain ACB1(T) were C(14 : 0), C(16 : 0), C(16 : 1)ω7c dimethyl aldehyde (DMA) and C(18 : 1)ω7c DMA. Major fatty acids of strain ACB7(T) were C(12 : 0), C(14 : 0), C(16 : 0), C(16 : 1)ω7c and C(16 : 1)ω7c DMA. The hydrolysate of the peptidoglycan contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. Genomic DNA G+C content varied from 42 to 43.3% between strains. According to 16S rRNA gene sequence phylogeny, strains ACB1(T), ACB8 and ACB7(T) formed two separate branches within the genus Oribacterium, with 98.1-98.6% sequence similarity to the type strain of the type species, Oribacterium sinus. Predicted DNA-DNA hybridization values between strains ACB1(T), ACB8, ACB7(T) and O. sinus F0268 were <70%. Based on distinct genotypic and phenotypic characteristics, strains ACB1(T) and ACB8, and strain ACB7(T) are considered to represent two distinct species of the genus Oribacterium, for which the names Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov. are proposed. The type strains are ACB1(T) ( = DSM 24637(T) = HM-481(T) = ATCC BAA-2638(T)) and ACB7(T) ( = DSM 24638(T) = HM-482(T) = ATCC BAA-2639(T)), respectively.

Stomatobaculum longum gen. nov., sp. nov., an obligately anaerobic bacterium from the human oral cavity.

A strictly anaerobic Gram-stain-variable but positive by structure, non-spore-forming bacterium designated Lachnospiraceae bacterium ACC2 strain DSM 24645(T) was isolated from human subgingival dental plaque. Bacterial cells were 4-40 µm long non-motile rods, often swollen and forming curved filaments up to 200 µm. Cells contained intracellular, poorly crystalline, nanometre-sized iron- and sulfur-rich particles. The micro-organism was able to grow on yeast extract, trypticase peptone, milk, some sugars and organic acids. The major metabolic end-products of glucose fermentation were butyrate, lactate, isovalerate and acetate. The growth temperature and pH ranges were 30-42 °C and 4.9-7.5, respectively. Major fatty acids were C14 : 0, C14 : 0 DMA (dimethyl aldehyde), C16 : 0, C16 : 1ω7c DMA. The whole-cell hydrolysate contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. The DNA G+C content was calculated to be 55.05 mol% from the whole-genome sequence and 55.3 mol% as determined by HPLC. There were no predicted genes responsible for biosynthesis of respiratory lipoquinones, mycolic acids and lipopolysaccharides. Genes associated with synthesis of teichoic and lipoteichoic acids, diaminopimelic acid, polar lipids and polyamines were present. According to the 16S rRNA gene sequence phylogeny, strain DSM 24645(T) formed, together with several uncultured oral clones, a separate branch within the family Lachnospiraceae, with the highest sequence similarity to the type strain of Moryella indoligenes at 94.2 %. Based on distinct phenotypic and genotypic characteristics, we suggest that strain DSM 24645(T) represents a novel species in a new genus, for which the name Stomatobaculum longum gen. nov., sp. nov. is proposed. The type strain of Stomatobaculum longum is DSM 24645(T) ( = HM-480(T); deposited in BEI Resources, an NIH collection managed by the ATCC).

Regulation of Peripheral Inflammation by a Non-Viable, Non-Colonizing Strain of Commensal Bacteria.

The gastrointestinal tract represents one of the largest body surfaces that is exposed to the outside world. It is the only mucosal surface that is required to simultaneously recognize and defend against pathogens, while allowing nutrients containing foreign antigens to be tolerated and absorbed. It differentiates between these foreign substances through a complex system of pattern recognition receptors expressed on the surface of the intestinal epithelial cells as well as the underlying immune cells. These immune cells actively sample and evaluate microbes and other particles that pass through the lumen of the gut. This local sensing system is part of a broader distributed signaling system that is connected to the rest of the body through the enteric nervous system, the immune system, and the metabolic system. While local tissue homeostasis is maintained by commensal bacteria that colonize the gut, colonization itself may not be required for the activation of distributed signaling networks that can result in modulation of peripheral inflammation. Herein, we describe the ability of a gut-restricted strain of commensal bacteria to drive systemic anti-inflammatory effects in a manner that does not rely upon its ability to colonize the gastrointestinal tract or alter the mucosal microbiome. Orally administered EDP1867, a gamma-irradiated strain of Veillonella parvula, rapidly transits through the murine gut without colonization or alteration of the background microbiome flora. In murine models of inflammatory disease including delayed-type hypersensitivity (DTH), atopic dermatitis, psoriasis, and experimental autoimmune encephalomyelitis (EAE), treatment with EDP1867 resulted in significant reduction in inflammation and immunopathology. Ex vivo cytokine analyses revealed that EDP1867 treatment diminished production of pro-inflammatory cytokines involved in inflammatory cascades. Furthermore, blockade of lymphocyte migration to the gut-associated lymphoid tissues impaired the ability of EDP1867 to resolve peripheral inflammation, supporting the hypothesis that circulating immune cells are responsible for promulgating the signals from the gut to peripheral tissues. Finally, we show that adoptively transferred T cells from EDP1867-treated mice inhibit inflammation induced in recipient mice. These results demonstrate that an orally-delivered, non-viable strain of commensal bacteria can mediate potent anti-inflammatory effects in peripheral tissues through transient occupancy of the gastrointestinal tract, and support the development of non-living bacterial strains for therapeutic applications.

Diversity of bacteria and glycosyl hydrolase family 48 genes in cellulolytic consortia enriched from thermophilic biocompost.

The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species.

Aerococcus urinae and Globicatella sanguinis Persist in Polymicrobial Urethral Catheter Biofilms Examined in Longitudinal Profiles at the Proteomic Level.

Aerococcus urinae (Au) and Globicatella sanguinis (Gs) are gram-positive bacteria belonging to the family Aerococcaceae and colonize the human immunocompromised and catheterized urinary tract. We identified both pathogens in polymicrobial urethral catheter biofilms (CBs) with a combination of 16S rDNA sequencing, proteomic analyses, and microbial cultures. Longitudinal sampling of biofilms from serially replaced catheters revealed that each species persisted in the urinary tract of a patient in cohabitation with 1 or more gram-negative uropathogens. The Gs and Au proteomes revealed active glycolytic, heterolactic fermentation, and peptide catabolic energy metabolism pathways in an anaerobic milieu. A few phosphotransferase system (PTS)-based sugar uptake and oligopeptide ABC transport systems were highly expressed, indicating adaptations to the supply of nutrients in urine and from exfoliating squamous epithelial and urothelial cells. Differences in the Au vs Gs metabolisms pertained to citrate lyase and utilization and storage of glycogen (evident only in Gs proteomes) and to the enzyme Xfp that degrades d-xylulose-5'-phosphate and the biosynthetic pathways for 2 protein cofactors, pyridoxal 6'-phosphate and 4'-phosphopantothenate (expressed only in Au proteomes). A predicted ZnuA-like transition metal ion uptake system was identified for Gs while Au expressed 2 LPXTG-anchored surface proteins, one of which had a predicted pilin D adhesion motif. While these proteins may contribute to fitness and virulence in the human host, it cannot be ruled out that Au and Gs fill a niche in polymicrobial biofilms without being the direct cause of injury in urothelial tissues.

A simple cleanup method for the removal of humic substances from soil protein extracts using aluminum coagulation.

Soil proteomics, the large-scale characterization of the entire protein complement in soils, provides a promising approach for deciphering the role of microbial functioning in terrestrial ecosystems. However, the extraction of soil proteins in sufficient quantities and of adequate purity remains a challenging task mainly due to the co-extraction of interfering humic substances. Up to now, the treatment of soil extracts with liquid phenol has been the "gold standard" for reducing humics, while the NoviPure cleanup kit was recently launched as a non-toxic approach. The present study describes an alternative method for delivering high-purity proteins based on humic coagulation with trivalent aluminum ions (Al3+). Various experimental parameters were optimized individually in order to maximize protein yield and diminish co-extracted humics. The optimized method was applied on a set of soil samples with diverse physicochemical characteristics and a comparison with the other two techniques was conducted. The amount of residual humics resulting from Al3+-based method was 26 and 35% higher than that from phenol treatment and NoviPure Kit, respectively, but these differences were of marginal statistical significance. With regard to extracted proteins, the average yields of the three methods were comparable, without showing any statistically significant differences. Overall, humic coagulation with Al3+ offers comparable cleanup performance in terms of protein yield and purity, but it is less toxic and less complex than the phenol-partitioning method, whereas it is far less expensive than the NoviPure Kit. The new technique is expected to facilitate the implementation of proteomic studies in soils.

Actinobaculum massiliense Proteome Profiled in Polymicrobial Urethral Catheter Biofilms.

Actinobaculum massiliense, a Gram-positive anaerobic coccoid rod colonizing the human urinary tract, belongs to the taxonomic class of Actinobacteria. We identified A. massiliense as a cohabitant of urethral catheter biofilms (CB). The CBs also harbored more common uropathogens, such as Proteus mirabilis and Aerococcus urinae, supporting the notion that A. massiliense is adapted to a life style in polymicrobial biofilms. We isolated a clinical strain from a blood agar colony and used 16S rRNA gene sequencing and shotgun proteomics to confirm its identity as A. massiliense. We characterized this species by quantitatively comparing the bacterial proteome derived from in vitro growth with that of four clinical samples. The functional relevance of proteins with emphasis on nutrient import and the response to hostile host conditions, showing evidence of neutrophil infiltration, was analyzed. Two putative subtilisin-like proteases and a heme/oligopeptide transporter were abundant in vivo and are likely important for survival and fitness in the biofilm. Proteins facilitating uptake of xylose/glucuronate and oligopeptides, also highly expressed in vivo, may feed metabolites into mixed acid fermentation and peptidolysis pathways, respectively, to generate energy. A polyketide synthase predicted to generate a secondary metabolite that interacts with either the human host or co-colonizing microbes was also identified. The product of the PKS enzyme may contribute to A. massiliense fitness and persistence in the CBs.

Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C.

We developed a procedure to culture microorganisms below freezing point on solid media (cellulose powder or plastic film) with ethanol as the sole carbon source without using artificial antifreezes. Enrichment from soil and permafrost obtained on such frozen solid media contained mainly fungi, and further purification resulted in isolation of basidiomycetous yeasts of the genera Mrakia and Leucosporidium as well as ascomycetous fungi of the genus Geomyces. Contrary to solid frozen media, the enrichment of liquid nutrient solutions at 0 degrees C or supercooled solutions stabilized by glycerol at -1 to -5 degrees C led to the isolation of bacteria representing the genera Polaromonas, Pseudomonas and Arthrobacter. The growth of fungi on ethanol-microcrystalline cellulose media at -8 degrees C was exponential with generation times of 4.6-34 days, while bacteria displayed a linear or progressively declining curvilinear dynamic. At -17 to -0 degrees C the growth of isolates and entire soil community on 14C-ethanol was continuous and characterized by yields of 0.27-0.52 g cell C (g of C-substrate)(-1), similar to growth above the freezing point. The 'state of maintenance,' implying measurable catabolic activity of non-growing cells, was not confirmed. Below -18 to -35 degrees C, the isolated organisms were able to grow only transiently for 3 weeks after cooling with measurable respiratory and biosynthetic (14CO2 uptake) activity. Then metabolic activity declined to zero, and microorganisms entered a state of reversible dormancy.

Novel facultative anaerobic acidotolerant Telmatospirillum siberiense gen. nov. sp. nov. isolated from mesotrophic fen.

Three facultative anaerobic acidotolerant Gram-negative motile spirilla strains designated 26-4b1, 26-2 and K-1 were isolated from mesotrophic Siberian fen as a component of methanogenic consortia. The isolates were found to grow chemoorganotrophically on several organic acids and glucose under anoxic and low oxygen pressure in the dark, tolerant up to 5kPa of oxygen. At low oxygen supply, faint autotrophic growth on the H(2):CO(2) mixture was also observed. All three isolates were able to fix N(2). Major cellular fatty acids were 18:1 omega7c, 17:0 cyclopropane and 16:0. Phylogenetic analyses of the 16S rRNA gene sequences revealed that they formed a deep branch within the family Rhodospirillaceae of the Alphaproteobacteria with the highest similarity of 90.9-92.5% with members of genera Phaeospirillum and Magnetospirillum. Phylogenetic study of nifH (nitrogenase) and cbbL (RuBisCO) amino acid sequence identities confirmed that the new isolates represent a novel group. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strains 26-4b1, 26-2 and K-1 represent a new species of a novel genus for which the name Telmatospirillum siberiense gen. nov. sp. nov. is proposed.

TM7 detection in human microbiome: Are PCR primers and FISH probes specific enough?

TM7 appears important and omnipresent because it is repeatedly detected by molecular techniques in diverse environments. Here we report that most of primers and FISH probes thought to be TM7-specific do hybridize with multiple species from oral and vaginal cavity. This calls for re-examination of TM7 distribution and abundance.

High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae.

Here we report a summary classification and the features of five anaerobic oral bacteria from the family Peptostreptococcaceae. Bacterial strains were isolated from human subgingival plaque. Strains ACC19a, CM2, CM5, and OBRC8 represent the first known cultivable members of "yet uncultured" human oral taxon 081; strain AS15 belongs to "cultivable" human oral taxon 377. Based on 16S rRNA gene sequence comparisons, strains ACC19a, CM2, CM5, and OBRC8 are distantly related to Eubacterium yurii subs. yurii and Filifactor alocis, with 93.2 - 94.4 % and 85.5 % of sequence identity, respectively. The genomes of strains ACC19a, CM2, CM5, OBRC8 and AS15 are 2,541,543; 2,312,592; 2,594,242; 2,553,276; and 2,654,638 bp long. The genomes are comprised of 2277, 1973, 2325, 2277, and 2308 protein-coding genes and 54, 57, 54, 36, and 28 RNA genes, respectively. Based on the distinct characteristics presented here, we suggest that strains ACC19a, CM2, CM5, and OBRC8 represent a novel genus and species within the family Peptostreptococcaceae, for which we propose the name Peptoanaerobacter stomatis gen. nov., sp. nov. The type strain is strain ACC19a(T) (=HM-483(T); =DSM 28705(T); =ATCC BAA-2665(T)).

Polaromonas hydrogenivorans sp. nov., a psychrotolerant hydrogen-oxidizing bacterium from Alaskan soil.

Psychrotolerant (0-25 degrees C), chemolithotrophic Gram-negative cocci were isolated from Alaskan forest soil. The novel isolate was found to grow autotrophically on H(2) : CO(2) mixtures and to switch to heterotrophic growth on media containing organic substrates. The novel strain utilized a wide range of organic acids, some simple sugars and alcohols. Naphthalene vapour did not support growth. On the basis of 16S rRNA gene sequence similarity, the novel strain is affiliated to the genus Polaromonas, of the class Betaproteobacteria, and is related to Polaromonas naphthalenivorans (99.6 % gene sequence similarity), Polaromonas aquatica (97.4 %) and Polaromonas vacuolata (96.1 %). The membrane phospholipids contained 16 : 1omega7c/16 : 1omega6c, 16 : 0 and 18 : 1omega7c, similar to the fatty acids found for P. naphthalenivorans, P. aquatica and P. vacuolata. On the basis of DNA-DNA hybridization, physiological and biochemical properties, the hydrogen-oxidizing mixotrophic isolate represents a novel species, for which the name Polaromonas hydrogenivorans sp. nov. is proposed. The type strain is DSM 17735(T) (=NRRL B-41369(T)).

Isolation and characterization of oligotrophic acido-tolerant methanogenic consortia from a Sphagnum peat bog.

Two dense and highly enriched (up to 10(9) cells ml(-1), <10% of bacterial satellites) acido-tolerant (pH 4.0-6.5) methanogenic consortia, '26' and 'K', were isolated from the peat beneath a Sphagnum-Eriophorum-Carex community in West Siberia. Both consortia produced methane from CO2:H2 on chemically defined, diluted N-free media containing Ti(III)citrate as reducing agent. The phylogenetic analysis of 16S ribosomal DNA revealed three archaeal and nine bacterial sequence types. Consortium '26' contained single archaea Methanobacterium sp., represented by rods of 1.5-10x0.5-1.0 microm. In consortium 'K', there were two archaeal phylotypes, the respective methanogens were further differentiated morphologically with the fluorescence in situ hybridization technique: one less abundant (<2%) population of the long-curved rods (50-100x0.3-0.4 microm) fell into the order of Methanomicrobiales, while the dominant organism ( approximately 98%), represented by straight rods with abrupt rectangular ends (3-9x0.5 microm), was affiliated with earlier uncultured 'Rice cluster I'. The main bacterial satellite used citrate as a single carbon and energy source; it was similar in both consortia, and after isolation in pure culture, it was identified as a new member of the alpha-subclass of Proteobacteria. The other bacterial satellites were distributed among four taxonomic groups: the delta-subclass of Proteobacteria, the Flavobacterium-Bacteroides-Cytophaga line of descent, the Acidobacterium-Fibrobacter line of descent and the Green non-sulfur bacteria line of descent. At least 11 out of 12 components of acido-tolerant consortia are new to science at the species, genus and order levels; their existence until now was evident only from environmental gene retrievals. The Sphagnum wetlands, attracting attention only recently because of their global environmental role, are shown to be an especially valuable source of novel prokaryotic organisms.