The extracellular proteome of two Bifidobacterium species reveals different adaptation strategies to low iron conditions.

BACKGROUND: Bifidobacteria are among the first anaerobic bacteria colonizing the gut. Bifidobacteria require iron for growth and their iron-sequestration mechanisms are important for their fitness and possibly inhibit enteropathogens. Here we used combined genomic and proteomic analyses to characterize adaptations to low iron conditions of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2, 2 strains isolated from the feces of iron-deficient African infants and selected for their high iron-sequestering ability. RESULTS: Analyses of the genome contents revealed evolutionary adaptation to low iron conditions. A ferric and a ferrous iron operon encoding binding proteins and transporters were found in both strains. Remarkably, the ferric iron operon of B. pseudolongum PV8-2 is not found in other B. pseudolongum strains and likely acquired via horizontal gene transfer. The genome B. kashiwanohense PV20-2 harbors a unique region encoding genes putatively involved in siderophore production. Additionally, the secretomes of the two strains grown under low-iron conditions were analyzed using a combined genomic-proteomic approach. A ferric iron transporter was found in the secretome of B. pseudolongum PV8-2, while ferrous binding proteins were detected in the secretome of B. kashiwanohense PV20-2, suggesting different strategies to take up iron in the strains. In addition, proteins such as elongation factors, a glyceraldehyde-3-phosphate dehydrogenase, and the stress proteins GroEL and DnaK were identified in both secretomes. These proteins have been previously associated with adhesion of lactobacilli to epithelial cells. CONCLUSION: Analyses of the genome and secretome of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2 revealed different adaptations to low iron conditions and identified extracellular proteins for iron transport. The identified extracellular proteins might be involved in competition for iron in the gastrointestinal tract.

High Iron-Sequestrating Bifidobacteria Inhibit Enteropathogen Growth and Adhesion to Intestinal Epithelial Cells In vitro.

The gut microbiota plays an important role in host health, in particular by its barrier effect and competition with exogenous pathogenic bacteria. In the present study, the competition of Bifidobacterium pseudolongum PV8-2 (Bp PV8-2) and Bifidobacterium kashiwanohense PV20-2 (Bk PV20-2), isolated from anemic infant gut microbiota and selected for their high iron sequestration properties, was investigated against Salmonella Typhimurium (S. Typhi) and Escherichia coli O157:H45 (EHEC) by using co-culture tests and assays with intestinal cell lines. Single and co-cultures were carried out anaerobically in chemically semi-defined low iron (1.5 µM Fe) medium (CSDLIM) without and with added ferrous iron (30 µM Fe). Surface properties of the tested strains were measured by bacterial adhesion to solvent xylene, chloroform, ethyl acetate, and to extracellular matrix molecules, mucus II, collagen I, fibrinogen, fibronectin. HT29-MTX mucus-secreting intestinal cell cultures were used to study bifidobacteria competition, inhibition and displacement of the enteropathogens. During co-cultures in CSDLIM we observed strain-dependent inhibition of bifidobacterial strains on enteropathogens, independent of pH, organic acid production and supplemented iron. Bp PV8-2 significantly (P < 0.05) inhibited S. Typhi N15 and EHEC after 24 h compared to single culture growth. In contrast Bk PV20-2 showed less inhibition on S. Typhi N15 than Bp PV8-2, and no inhibition on EHEC. Affinity for intestinal cell surface glycoproteins was strain-specific, with high affinity of Bp PV8-2 for mucin and Bk PV20-2 for fibronectin. Bk PV20-2 showed high adhesion potential (15.6 ± 6.0%) to HT29-MTX cell layer compared to Bp PV8-2 (1.4 ± 0.4%). In competition, inhibition and displacement tests, Bp PV8-2 significantly (P < 0.05) reduced S. Typhi N15 and EHEC adhesion, while Bk PV20-2 was only active on S. Typhi N15 adhesion. To conclude, bifidobacterial strains selected for their high iron binding properties inhibited S. Typhi N15 and EHEC in co-culture experiments and efficiently competed with the enteropathogens on mucus-producing HT29-MTX cell lines. Further studies in complex gut ecosystems should explore host protection effects of Bp PV8-2 and Bk PV20-2 mediated by nutritional immunity mechanism associated with iron-binding.

Bifidobacteria strains isolated from stools of iron deficient infants can efficiently sequester iron.

BACKGROUND: Bifidobacteria is one of the major gut commensal groups found in infants. Their colonization is commonly associated with beneficial effects to the host through mechanisms like niche occupation and nutrient competition against pathogenic bacteria. Iron is an essential element necessary for most microorganisms, including bifidobacteria and efficient competition for this micronutrient is linked to proliferation and persistence. For this research we hypothesized that bifidobacteria in the gut of iron deficient infants can efficiently sequester iron. The aim of the present study was to isolate bifidobacteria in fecal samples of iron deficient Kenyan infants and to characterize siderophore production and iron internalization capacity. RESULTS: Fifty-six bifidobacterial strains were isolated by streaking twenty-eight stool samples from Kenyan infants, in enrichment media. To target strains with high iron sequestration mechanisms, a strong iron chelator 2,2-dipyridyl was supplemented to the agar media. Bifidobacterial isolates were first identified to species level by 16S rRNA sequencing, yielding B. bifidum (19 isolates), B. longum (15), B. breve (11), B. kashiwanohense (7), B. pseudolongum (3) and B. pseudocatenulatum (1). While most isolated bifidobacterial species are commonly encountered in the infantile gut, B. kashiwanohense was not frequently reported in infant feces. Thirty strains from culture collections and 56 isolates were characterized for their siderophore production, tested by the CAS assay. Siderophore activity ranged from 3 to 89% siderophore units, with 35 strains (41%) exhibiting high siderophore activity, and 31 (36%) and 20 (23%) showing intermediate or low activity. The amount of internalized iron of 60 bifidobacteria strains selected for their siderophore activity, was in a broad range from 8 to118 µM Fe. Four strains, B. pseudolongum PV8-2, B. kashiwanohense PV20-2, B. bifidum PV28-2a and B. longum PV5-1 isolated from infant stool samples were selected for both high siderophore activity and iron internalization. CONCLUSIONS: A broad diversity of bifidobacteria were isolated in infant stools using iron limited conditions, with some strains exhibiting high iron sequestration properties. The ability of bifidobacteria to efficiently utilize iron sequestration mechanism such as siderophore production and iron internalization may confer an ecological advantage and be the basis for enhanced competition against enteropathogens.

Complete and Assembled Genome Sequence of Bifidobacterium kashiwanohense PV20-2, Isolated from the Feces of an Anemic Kenyan Infant.

The complete genome sequence of Bifidobacterium kashiwanohense strain PV20-2, an infant feces isolate, was determined using single-molecule real-time sequencing (SMRT). Hierarchical genome assembly resulted in a completely assembled genome of 2,370,978 bp. The B. kashiwanohense PV20-2 genome is the first completely sequenced and assembled genome of the species.

Bifidobacterium pseudolongum Strain PV8-2, Isolated from a Stool Sample of an Anemic Kenyan Infant.

The complete genome sequence of Bifidobacterium pseudolongum PV8-2, isolated from feces of an anemic Kenyan infant, was determined using single-molecule real-time (SMRT) technology. The genome consists of a 2-Mbp chromosome and a 4-kb plasmid.

Novel Polyfermentor intestinal model (PolyFermS) for controlled ecological studies: validation and effect of pH.

In vitro gut fermentation modeling offers a useful platform for ecological studies of the intestinal microbiota. In this study we describe a novel Polyfermentor Intestinal Model (PolyFermS) designed to compare the effects of different treatments on the same complex gut microbiota. The model operated in conditions of the proximal colon is composed of a first reactor containing fecal microbiota immobilized in gel beads, and used to continuously inoculate a set of parallel second-stage reactors. The PolyFermS model was validated with three independent intestinal fermentations conducted for 38 days with immobilized human fecal microbiota obtained from three child donors. The microbial diversity of reactor effluents was compared to donor feces using the HITChip, a high-density phylogenetic microarray targeting small subunit rRNA sequences of over 1100 phylotypes of the human gastrointestinal tract. Furthermore, the metabolic response to a decrease of pH from 5.7 to 5.5, applied to balance the high fermentative activity in inoculum reactors, was studied. We observed a reproducible development of stable intestinal communities representing major taxonomic bacterial groups at ratios similar to these in feces of healthy donors, a high similarity of microbiota composition produced in second-stage reactors within a model, and a high time stability of microbiota composition and metabolic activity over 38 day culture. For all tested models, the pH-drop of 0.2 units in inoculum reactors enhanced butyrate production at the expense of acetate, but was accompanied by a donor-specific reorganization of the reactor community, suggesting a concerted metabolic adaptation and trigger of community-specific lactate or acetate cross-feeding pathways in response to varying pH. Our data showed that the PolyFermS model allows the stable cultivation of complex intestinal microbiota akin to the fecal donor and can be developed for the direct comparison of different experimental conditions in parallel reactors continuously inoculated with the exact same microbiota.