GC-MS with Headspace Extraction for Non-Invasive Diagnostics of IBD Dynamics in a Model of DSS-Induced Colitis in Rats.

Inflammatory bowel diseases are extremely common throughout the world. However, in most cases, it is asymptomatic at the initial stage. Therefore, it is important to develop non-invasive diagnostic methods that allow identification of the IBD risks in a timely manner. It is well known that gastrointestinal microbiota secrete volatile compounds (VOCs) and their composition may change in IBD. We propose a non-invasive method to identify the dynamics of IBD development in the acute and remission stage at the level of VOCs in model of dextran sulfate sodium (DSS) with chemically induced colitis measured by headspace GC/MS (HS GC/MS). Methods: VOCs profile was identified using a headspace GC/MS (HS GC/MS). GC/MS data were processed using MetaboAnalyst 5.0 and GraphPad Prism 8.0.1 software. The disease activity index (DAI) and histological method were used to assess intestinal inflammation. The peak of intestinal inflammation activity was reached on day 7, according to the disease activity index. Histological examination data showed changes in the intestine due to different stages of inflammation. As the acute inflammation stage was reached, the metabolomic profile also underwent changes, especially at the short-fatty acids level. A higher relative amounts of acetic acid (p value < 0.025) and lower relative amounts of propanoic acid (p value < 0.0005), butanoic acid (p value < 0.005) and phenol 4-methyl- (p value = 0.053) were observed in DSS7 group on day 7 compared to the control group. In remission stage, disease activity indexes decreased, and the histological picture also improved. But metabolome changes continued despite the withdrawal of the DSS examination. A lower relative amounts of propanoic acid (p value < 0.025), butanoic acid (p value < 0.0005), pentanoic acid (p value < 0.0005), and a significant de-crease of hexanoic acid (p value < 0.0005) relative amounts were observed in the DSS14 group compared to the control group on day 14. A model of DSS-induced colitis in rats was successfully implemented for metabolomic assessment of different stages of inflammation. We demonstrated that the ratios of volatile compounds change in response to DSS before the appearance of standard signs of inflammation, determined by DAI and histological examination. Changes in the volatile metabolome persisted even after visual intestine repair and it confirms the high sensitivity of the microbiota to the damaging effects of DSS. The use of HS GC/MS may be an important addition to existing methods for assessing inflammation at early stages.

Investigating volatile compounds in the Bacteroides secretome.

Microorganisms and their hosts communicate with each other by secreting numerous components. This cross-kingdom cell-to-cell signaling involves proteins and small molecules, such as metabolites. These compounds can be secreted across the membrane via numerous transporters and may also be packaged in outer membrane vesicles (OMVs). Among the secreted components, volatile compounds (VOCs) are of particular interest, including butyrate and propionate, which have proven effects on intestinal, immune, and stem cells. Besides short fatty acids, other groups of volatile compounds can be either freely secreted or contained in OMVs. As vesicles might extend their activity far beyond the gastrointestinal tract, study of their cargo, including VOCs, is even more pertinent. This paper is devoted to the VOCs secretome of the Bacteroides genus. Although these bacteria are highly presented in the intestinal microbiota and are known to influence human physiology, their volatile secretome has been studied relatively poorly. The 16 most well-represented Bacteroides species were cultivated; their OMVs were isolated and characterized by NTA and TEM to determine particle morphology and their concentration. In order to analyze the VOCs secretome, we propose a headspace extraction with GC-MS analysis as a new tool for sample preparation and analysis of volatile compounds in culture media and isolated bacterial OMVs. A wide range of released VOCs, both previously characterized and newly described, have been revealed in media after cultivation. We identified more than 60 components of the volatile metabolome in bacterial media, including fatty acids, amino acids, and phenol derivatives, aldehydes and other components. We found active butyrate and indol producers among the analyzed Bacteroides species. For a number of Bacteroides species, OMVs have been isolated and characterized here for the first time as well as volatile compounds analysis in OMVs. We observed a completely different distribution of VOC in vesicles compared to the bacterial media for all analyzed Bacteroides species, including almost complete absence of fatty acids in vesicles. This article provides a comprehensive analysis of the VOCs secreted by Bacteroides species and explores new perspectives in the study of bacterial secretomes in relation the intercellular communication.

Hominibacterium faecale gen. nov., sp. nov., an anaerobic L-arginine-degrading bacterium isolated from human feces.

Two anaerobic, mesophilic bacteria SF3T and ASD5510 were isolated from human feces in two different countries. Strain SF3T shared 99.9% of 16S rRNA gene sequence similarity with strain ASD5510, and 92.8% similarity with the most similar strain Aminipila butyrica DSM 103574T. Strain SF3T was an anaerobic, Gram-stain-positive bacterium. Cells of strain SF3T were short rods with 0.3-0.4 µm in width × 2.0-2.4 µm in length and occurred mostly in pairs or short chains. Spore formation was not observed. The strain grew optimally at 35 °C (range from 20 to 45 °C), pH 7.5 (pH 6.0-8.5) and without NaCl addition (range from 0 to 20 g l-1 NaCl). Yeast extract was an essential growth factor for strain SF3T, L-arginine and γ-aminobutyrate were utilized as substrates for growth. The major cellular fatty acids were iso-C15:0 and C16:0 DMA. The main polar lipids were aminophospholipid (APL), diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE). The G + C content of the genomic DNA of the strain SF3T was 47.38 mol %. The paired genomic average amino acid identity (AAI) and percentage of conserved proteins (POCP) values showed relatedness of less than 61.0 and 39.4% with type strains of order Eubacteriales. On the basis of phenotypic, phylogenetic and phylogenomic evidence strain SF3T constitutes a novel species in a novel genus, for which the name Hominibacterium faecale gen. nov., sp. nov. is proposed. The type strain is SF3T (= CCAM 730T = JCM 34755T = KCTC 25324T).

Diplocloster agilis gen. nov., sp. nov. and Diplocloster modestus sp. nov., two novel anaerobic fermentative members of Lachnospiraceae isolated from human faeces.

Three novel strains of Gram-stain-negative, obligately anaerobic, spore-forming straight or slightly curved rods with pointed ends occurring singly or in pairs were isolated from the faeces of healthy human children. The strains were characterized by mesophilic fermentative metabolism and production of acetate, ethanol and H2 as the end metabolic products. Strains ASD3451 and ASD5720T were motile, fermented lactose and raffinose, and weakly fermented maltose. Strain ASD4241T was non-motile and did not ferment the carbohydrates listed above but fermented starch. Strains ASD3451 and ASD5720T shared average nucleotide identity higher than 98.5 % with each other, while ASD4241T had only 88.5-89 % identity to them. Based on phylogenetic and chemotaxonomic analyses, we propose Diplocloster agilis gen. nov., sp. nov. (ASD5720T=JCM 34353T=VKM B-3497T) and Diplocloster modestus sp. nov. (ASD4241T=JCM 34351T=VKM B-3498T) within the family Lachnospiraceae.

Hydrogeniiclostidium mannosilyticum gen. nov., sp. nov. isolated from human faeces.

A strain of obligately anaerobic, spore-forming, Gram-positive rods was isolated from child faeces and characterized both phenotypically and genotypically. Phylogenetic analysis based on 16S rRNA gene and whole genome sequencing revealed the strain to represent a member of the family Ruminococcaceae distant from described species and genera. The strain was moderately saccharolytic with mannose as the preferred substrate and produced lactic acid, acetic acid and H2 as the end products. The major cellular long-chain fatty acids were C16 : 0 and C16 : 0 aldehyde. The genomic DNA G+C content was 52.3 mol%. On the basis of chemotaxonomic and genomic properties it was concluded that the strain represents a novel species in a new genus within the family Ruminococcaceae, for which the name Hydrogeniiclostidium mannosilyticum gen. nov., sp. nov. is proposed. The type strain of Hydrogeniiclostidium mannosilyticum is ASD2818T (=VKM B-3268T=JCM 33295T).

Prevotella rara sp. nov., isolated from human faeces.

A strain of obligately anaerobic, Gram-stain-negative rods was isolated from human faeces and characterized both phenotypically and genotypically. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed the strain to represent a member of the genus Prevotella, distant from the species with validly published names, with the closest relationship to Prevotella oryzae. The strain was moderately saccharolytic and proteolytic. The predominant menaquinones were MK-13 and MK-12. The major cellular long-chain fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The genomic DNA G+C content was 45.7 mol%. On the basis of chemotaxonomic and genotypic properties, it was concluded that the strain represent a novel species within the genus Prevotella, for which the name Prevotellarara sp. nov. is proposed. The type strain of Prevotellarara is 109T (=VKM B-2992T=DSM 105141T).

Peptococcus simiae sp. nov., isolated from rhesus macaque faeces and emended description of the genus Peptococcus.

A study of the faecal microbiome in three healthy female rhesus macaques revealed the presence of a novel obligately anaerobic, chemoorganoheterotrophic, non-sporing, coccoid, non-motile, Gram-stain-positive bacterial species. Three strains of this species, designated as M108T, M916-1/1, and M919-2/1, were non-haemolytic, H2S-positive, catalase-positive, bile- and NaCl-sensitive and required peptone for growth. Strains also were asaccharolytic, able to utilize sulfite, thiosulfate and elemental sulfur as electron acceptors, and produced acetic and butyric acids as metabolic end-products. Strain M108T is characterized by the prevalence of C14 : 0, C16 : 0 and C18 : 1ω9cis dimethyl acetal among the cellular fatty acids, and the presence of MK-10 menaquinone. The DNA G+C content was found to be 51 mol%. Phylogenetic analysis of partial 16S rRNA gene sequences of strains M108T, M916-1/1 and M919-2/1 placed these strains into the genus Peptococcus (family Peptococcaceae). On the basis of phenotypic and genotypic properties we conclude that these strains represent a novel bacterial species for which the name Peptococcus simiae sp. nov. is proposed. The type strain is M108T (=DSM 100347T=VKM B-2932T).

Ruthenibacterium lactatiformans gen. nov., sp. nov., an anaerobic, lactate-producing member of the family Ruminococcaceae isolated from human faeces.

Two novel strains of Gram-stain-negative, rod-shaped, obligately anaerobic, non-spore-forming, non-motile bacteria were isolated from the faeces of healthy human subjects. The strains, designated as 585-1T and 668, were characterized by mesophilic fermentative metabolism, production of d-lactic acid, succinic acid and acetic acid as end products of d-glucose fermentation, prevalence of C18 : 1ω9, C18 : 1ω9 aldehyde, C16 : 0 and C16 : 1ω7c fatty acids, presence of glycine, glutamic acid, lysine, alanine and aspartic acid in the petidoglycan peptide moiety and lack of respiratory quinones. Whole genome sequencing revealed the DNA G+C content was 56.4-56.6 mol%. The complete 16S rRNA gene sequences of the two strains shared 91.7/91.6 % similarity with Anaerofilum pentosovorans FaeT, 91.3/91.2 % with Gemmiger formicilis ATCC 27749T and 88.9/88.8 % with Faecalibacterium prausnitzii ATCC 27768T. On the basis of chemotaxonomic and genomic properties it was concluded that the strains represent a novel species in a new genus within the family Ruminococcaceae, for which the name Ruthenibacterium lactatiformans gen. nov., sp. nov. is proposed. The type strain of Ruthenibacterium lactatiformans is 585-1T (=DSM 100348T=VKM B-2901T).

Alistipes inops sp. nov. and Coprobacter secundus sp. nov., isolated from human faeces.

Culture-based study of the faecal microbiome in two adult female subjects revealed the presence of two obligately anaerobic, non-spore-forming, rod-shaped, non-motile, Gram-negative bacterial strains that represent novel species. The first strain, designated 627T, was a fastidious, slow-growing, indole-positive bacterium with a non-fermentative type of metabolism.The strain was characterized by the production of acetic and succinic acids as metabolic end products, the prevalence of iso-C15 : 0 fatty acid and the presence of menaquinones MK-10 and MK-11. The DNA G+C content was found to be 56.6 mol%. The second strain, designated 177T, was capable of fermenting a rich collection of carbohydrate substrates, producing acetic acid as a terminal product. The strain was indole-negative and resistant to bile. The major cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0 (in a 1 : 1 ratio) and the predominant menaquinone was MK-11.The DNA G+C content was 37.8 mol%. A phylogenomic analysis of the draft genomes of strains 627T and 177T placed these bacteria in the genera Alistipes(family Rikenellaceae) and Coprobacter (family Porphyromonadaceae), respectively.On the basis of the phenotypic and genotypic properties of strains 627T and 177T, we conclude that these strains from human faeces represent two novel bacterial species, for which the names Alistipes inops sp. nov. (type strain 627T5DSM 28863T5VKM B-2859T) and Coprobacter secundus sp. nov. (type strain 177T=DSM 28864T=VKM B-2857T) are proposed.

Intraspecies Genomic Diversity and Long-Term Persistence of Bifidobacterium longum.

Members of genus Bifidobacterium are Gram-positive bacteria, representing a large part of the human infant microbiota and moderately common in adults. However, our knowledge about their diversity, intraspecific phylogeny and long-term persistence in humans is still limited. Bifidobacterium longum is generally considered to be the most common and prevalent species in the intestinal microbiota. In this work we studied whole genome sequences of 28 strains of B. longum, including 8 sequences described in this paper. Part of these strains were isolated from healthy children during a long observation period (up to 10 years between isolation from the same patient). The three known subspecies (longum, infantis and suis) could be clearly divided using sequence-based phylogenetic methods, gene content and the average nucleotide identity. The profiles of glycoside hydrolase genes reflected the different ecological specializations of these three subspecies. The high impact of horizontal gene transfer on genomic diversity was observed, which is possibly due to a large number of prophages and rapidly spreading plasmids. The pan-genome characteristics of the subspecies longum corresponded to the open pan-genome model. While the major part of the strain-specific genetic loci represented transposons and phage-derived regions, a large number of cell envelope synthesis genes were also observed within this category, representing high variability of cell surface molecules. We observed the cases of isolation of high genetically similar strains of B. longum from the same patients after long periods of time, however, we didn't succeed in the isolation of genetically identical bacteria: a fact, reflecting the high plasticity of microbiota in children.

Coprobacter fastidiosus gen. nov., sp. nov., a novel member of the family Porphyromonadaceae isolated from infant faeces.

A novel obligately anaerobic, non-spore-forming, rod-shaped, non-motile Gram-reaction-negative bacterium was isolated from infant faeces. The strain, designated NSB1(T), was able to grow on rich media at 30-37 °C, in the presence of up to 2 % (w/v) Oxgall and 2 % (w/v) NaCl. Cells of strain NSB1(T) produced catalase, but not urease and indole. Aesculin was not hydrolysed. The strain was able to utilize d-glucose, lactose, maltose, mannose and raffinose as electron donors. When grown on d-glucose, the main metabolic end products were propionic and acetic acids, with a minor product being succinic acid. The major cellular fatty acids, iso-C15 : 0 and anteiso-C15 : 0, were present at a 1 : 1 molar ratio. The major menaquinone was MK-11. The DNA G+C content was found to be 38.5 mol%. According to 16S rRNA gene sequence analysis strain NSB1(T) is a member of the family Porphyromonadaceae, phylum Bacteroidetes. The closest relatives of the strain were Barnesiella viscericola (88.2 % identity) and Barnesiella intestinihominis (87.4 % identity). On the basis of phenotypic and genotypic properties of strain NSB1(T) we conclude that this strain represent a novel species in a new genus within the family of Porphyromonadaceae for which the name Coprobacter fastidiosus gen. nov., sp. nov. is proposed. The type strain of the species is NSB1(T) ( = DSM 26242(T), = VKM B-2743(T)).

Analysis of a novel 8.9kb cryptic plasmid from Bacteroides uniformis, its long-term stability and spread within human microbiota.

The analysis of plasmid content in dominant Bacteroidales order intestinal strains isolated from the same child at a 5 year interval identified a 8.9 kb plasmid in Bacteroides uniformis BUN24 strain isolated at age 6 and indistinguishably sized plasmids in the isolates of B. uniformis, B. vulgatus, B. intesinalis, and Parabacteroides distasonis at age 11. We sequenced a B. uniformis BUN24 plasmid, designated pBUN24, and using molecular surveys of diverse species we established that this 8944bp molecule (G+C content 43.5%) represents a novel family of small cryptic Bacteroidales plasmids. The replication region of pBUN24 was experimentally localized to a 1707-bp fragment that includes a putative repA gene, coding for a protein of Rep_3 superfamily of replication proteins of theta-type plasmids preceded by a putative iteron-containing origin of replication. The other open reading frames (ORFs) identified in pBUN24 sequence include a putative tad-ata-type toxin-antitoxin and mobA-mobB mobilization modules, as well as seven additional cryptic ORFs. The interaction of Tad and Ada components demonstrated by a pull-down assay and the toxicity of Tad in Escherichia coli host suggests the functionality of the plasmid addiction module. Re-sequencing of plasmids in two Bacteroides strains isolated at the age of 11 showed 100% nucleotide identity to pBUN24. This data supports the notion that this plasmid is transmissible to other Bacteroidales strains in the natural ecosystem. The possible roles of toxin-antitoxin system and other proteins encoded by pBUN24 in providing an apparent ecological advantage to the plasmid-harbouring strains of a bacterial symbiont in the human gut deserve further investigation.

Species composition of Bacteroidales order bacteria in the feces of healthy people of various ages.

A study of species distribution of numerically predominant Bacteroidales order isolates in feces of healthy people aged 1-33 years was accomplished using a combination of amplified ribosomal DNA restriction analysis (ARDRA) and 16S ribosomal DNA (rDNA) sequencing. It was found that the majority of isolates in all age groups belonged to species B. xylanisolvens, B. vulgatus, and B. uniformis. Members of genera Alistipes, Parabacteroides, Odoribacter, Barnesiella, and Prevotella were also detected frequently.

Anti-inflammatory properties of intestinal Bifidobacterium strains isolated from healthy infants.

Certain Bifidobacterium strains have been shown to inhibit inflammatory responses in intestinal epithelial cells. However, the precise mechanisms of these effects, including the chemical nature of the active compounds, remain to be elucidated. Here partial characterization of the anti-inflammatory properties of Bifidobacterium strains isolated from feces of healthy infants is reported. It was found that conditioned media (CM) of all strains studied are capable of attenuating tumor necrosis factor-α (TNF-α) and lipopolysaccharide- (LPS) induced inflammatory responses in the HT-29 cell line. In contrast, neither killed bifidobacterial cells, nor cell-free extracts showed such activities. Further investigations resulted in attribution of this activity to heat-stable, non-lipophilic compound(s) resistant to protease and nuclease treatments and of molecular weight less than 3 kDa. The anti-inflammatory effects were dose- and time-dependent and associated with inhibition of IκB phosphorylation and nuclear factor-κ light chain enhancer of activated B cells (NF-κB)-dependent promoter activation. The combined treatments of cells with CMs and either LPS or TNF-α, but not with CMs alone, resulted in upregulation of transforming growth factor-ß1, IκBζ, and p21(CIP) mRNAs. Our data suggest certain species-specificities of the anti-inflammatory properties of bifidobacteria. This observation should prompt additional validation studies using larger set of strains and employing the tools of comparative genomics.

Characterization of plasmids from human infant Bifidobacterium strains: sequence analysis and construction of E. coli-Bifidobacterium shuttle vectors.

A survey of infant fecal Bifidobacterium isolates for plasmid DNA revealed that a significant portion of the strains, 17.6%, carry small plasmids. The majority of plasmid-harboring strains belonged to the Bifidobacterium longum/infantis group. Most of the plasmids could be assigned into two groups based on their sizes and the restriction profiles. Three plasmids, pB44 (3.6 kb) from B. longum, pB80 (4.9 kb) from Bifidobacterium bifidum, and pB21a (5.2kb) from Bifidobacterium breve were sequenced. While the former two plasmids were found to be highly similar to previously characterized rolling-circle replicating pKJ36 and pKJ56, respectively, the third plasmid, pB21a, does not share significant nucleotide homology with known plasmids. However, it might be placed into the pCIBb1-like group of bifidobacterial rolling-plasmids based on the homology of its Rep protein and the overall molecular organization. Two sets of Escherichia coli-Bifidobacterium shuttle vectors constructed based on pB44 and pB80 replicons were capable of transforming B. bifidum and B. breve strains with efficiency up to 3x10(4)cfu/microg DNA. Additionally, an attempt was made to employ a broad host range conjugation element, RP4, in developing of E. coli-Bifidobacterium gene transfer system.

Application of several molecular techniques to study numerically predominant Bifidobacterium spp. and Bacteroidales order strains in the feces of healthy children.

Bifidobacteria and Bacteroides-like bacteria are strictly anaerobic nonpathogenic members of human intestinal microflora. Here we describe an analysis of the species and subspecies composition of these bacterial populations in healthy children using a combination of culture and molecular methods at two different time points. It was found that B. bifidum and B. longum are the most common dominant taxons in infants aged between 8 and 16 months. The majority of the infants carried several dominant Bifidobacterium strains belonging to different species. Examination of the dominant bifidoflora in some of these children after a 5-year period showed major shifts in both species and strain composition, but the dominant strains remained unchanged in two children. The majority of dominant Bacteroides-like isolates belonged to species B. vulgatus and B. uniformis, but members of genera Alistipes and Barnesiella were common too. In addition, a novel approach to species identification of Bacteroidales order bacteria using amplified ribosomal DNA restriction analysis (ARDRA) is described.

Two families of rep-like genes that probably originated by interspecies recombination are represented in viral, plasmid, bacterial, and parasitic protozoan genomes.

Two families of genes related to, and including, rolling circle replication initiator protein (Rep) genes were defined by sequence similarity and by evidence of intergene family recombination. The Rep genes of circoviruses were the best characterized members of the "RecRep1 family." Other members of the RecRep1 family were Rep-like genes found in the genomes of the Canarypox virus, Entamoeba histolytica, and Giardia duodenalis and in a plasmid, p4M, from the Gram-positive bacterium, Bifidobacterium pseudocatenulatum. The "RecRep2 family" comprised some previously identified Rep-like genes from plasmids of phytoplasmas and similar Rep-like genes from the genomes of Lactobacillus acidophilus, Lactococcus lactis, and Phytoplasma asteris. Both RecRep1 and RecRep2 proteins have a nucleotide-binding domain significantly similar to the helicases (2C proteins) of picorna-like viruses. On the N-terminal side of the nucleotide binding domain, RecRep1 proteins have a domain significantly similar to one found in nanovirus Reps, whereas RecRep2 proteins have a domain significantly similar to one in the Reps of pLS1 plasmids. We speculate that RecRep genes have been transferred from viruses or plasmids to parasitic protozoan and bacterial genomes and that Rep proteins were themselves involved in the original recombination events that generated the ancestral RecRep genes.