Andrei N. Shkoporov

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‐β1, IκBζ, and p21CIP 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.

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.

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.

Draft Genome Sequences of Two Pairs of Human Intestinal Bifidobacterium longum subsp. longum Strains, 44B and 1-6B and 35B and 2-2B, Consecutively Isolated from Two Children after a 5-Year Time Period

ABSTRACT We report the genome sequences of four isolates of a human gut symbiont, Bifidobacterium longum. Strains 44B and 35B were isolated from two 1-year-old infants, while 1-6B and 2-2B were isolated from the same children 5 years later. The sequences permit investigations of factors enabling long-term colonization of bifidobacteria.

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)).

Complete Genome Sequence of an Enterotoxigenic Bacteroides fragilis Clinical Isolate

ABSTRACT Here we present the complete genome sequence of Bacteroides fragilis isolate BOB25. It is an enterotoxigenic isolate that was obtained from a stool sample of a patient with dysbiosis.

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.

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).