Bifidobacteria are associated with a host of health benefits and are typically dominant in the gut microbiota of healthy, breast-fed infants. A key adaptation, facilitating the establishment of these ...species, is their ability to consume particular sugars, known as human milk oligosaccharides (HMO), which are abundantly found in breastmilk. In the current study, we aimed to characterise the co-operative metabolism of four commercial infant-derived bifidobacteria (Bifidobacterium bifidum R0071, Bifidobacterium breve M-16V, Bifidobacterium infantis R0033, and Bifidobacterium infantis M-63) when grown on HMO. Three different HMO substrates (2'-fucosyllactose alone and oligosaccharides isolated from human milk representing non-secretor and secretor status) were employed. The four-strain combination resulted in increased bifidobacterial numbers (> 21%) in comparison to single strain cultivation. The relative abundance of B. breve increased by > 30% during co-cultivation with the other strains despite demonstrating limited ability to assimilate HMO in mono-culture. HPLC analysis revealed strain-level variations in HMO consumption. Metabolomics confirmed the production of formate, acetate, 1,2-propanediol, and lactate with an overall increase in such metabolites during co-cultivation. These results support the concept of positive co-operation between multiple bifidobacterial strains during HMO utilisation which may result in higher cell numbers and a potentially healthier balance of metabolites.
Bifidobacteria represent one of the dominant microbial groups that are present in the gut of various animals, being particularly prevalent during the suckling stage of life of humans and other ...mammals. However, the overall genome structure of this group of microorganisms remains largely unexplored. Here, we sequenced the genomes of 42 representative (sub)species across the Bifidobacterium genus and used this information to explore the overall genetic picture of this bacterial group. Furthermore, the genomic data described here were used to reconstruct the evolutionary development of the Bifidobacterium genus. This reconstruction suggests that its evolution was substantially influenced by genetic adaptations to obtain access to glycans, thereby representing a common and potent evolutionary force in shaping bifidobacterial genomes.
Abstract
Bifidobacteria have been shown to produce exopolysaccharides (EPS), which are polymeric structures composed of various carbohydrates, commonly containing glucose, galactose, and rhamnose. ...EPS are produced by different bifidobacterial taxa commonly identified in the human gut, such as Bifidobacterium breve and Bifidobacterium longum subsp. longum, and have been suggested to modulate the interaction of bifidobacterial cells with other members of the human gut microbiota as well as with their host. In this study, we evaluated if bifidobacterial EPS production of four selected EPS-producing strains is associated with enhanced resistance to antibiotic treatments through MIC analysis when compared to bacterial cultures that do not produce exopolysaccharides. Our results showed that an increase in EPS production by modifying the growth medium with different carbon sources, i.e. glucose, galactose or lactose and/or by applying stressful conditions, such as bile salts and acidity, is associated with a tolerance enhancement of bifidobacterial cells toward various beta-lactam antibiotics. In addition, after analyzing the production of EPS at the phenotypic level, we explored the genes involved in the production of these structures and evaluated their expression, in presence of various carbon sources, using RNAseq. Overall, this study provides preliminary experimental evidence showing how bifidobacterial EPS modifies the level of susceptibility of these bacteria towards antibiotics.
Potential involvment of bifidobacterial exopolysaccharides in antibiotic resistance.
Summary
Available 3D structures of bacteriophage modules combined with predictive bioinformatic algorithms enabled the identification of adhesion modules in 57 siphophages infecting Streptococcus ...thermophilus (St). We identified several carbohydrate‐binding modules (CBMs) in so‐called evolved distal tail (Dit) and tail‐associated lysozyme (Tal) proteins of St phage baseplates. We examined the open reading frame (ORF) downstream of the Tal‐encoding ORF and uncovered the presence of a putative p2‐like receptor‐binding protein (RBP). A 21 Å resolution electron microscopy structure of the baseplate of cos‐phage STP1 revealed the presence of six elongated electron densities, surrounding the core of the baseplate, that harbour the p2‐like RBPs at their tip. To verify the functionality of these modules, we expressed GFP‐ or mCherry‐coupled Tal and putative RBP CBMs and observed by fluorescence microscopy that both modules bind to their corresponding St host, the putative RBP CBM with higher affinity than the Tal‐associated one. The large number of CBM functional domains in St phages suggests that they play a contributory role in the infection process, a feature that we previously described in lactococcal phages and beyond, possibly representing a universal feature of the siphophage host‐recognition apparatus.
We found that for each streptococcal phage several CBMs are present in the virions and are involved in phage adhesion. Moreover, we uncovered an additional ORF that harbours a predicted Receptor Binding Protein (RBP) at its C‐terminus. To confirm these predictions, we determined by electron microscopy the structure of streptococcal phage STP1 baseplate and assessed the binding capability of the CBM and the RBP‐like domain to their specific host using fluorescent binding assays.
Development of the human gut microbiota commences at birth, with certain bifidobacterial species representing dominant and early colonisers of the newborn gastrointestinal tract. The molecular basis ...of Bifidobacterium colonisation, persistence and presumed communication with the host has remained obscure. We previously identified tight adherence (Tad) pili from Bifidobacterium breve UCC2003 as an essential colonisation factor. Here, we demonstrate that bifidobacterial Tad pili also promote in vivo colonic epithelial proliferation. A significant increase in cell proliferation was detectable 5 days postadministration of B. breve UCC2003. Using advanced functional genomic approaches, bacterial strains either (a) producing the Tad2003 pili or (b) lacking the TadE or TadF pseudopilins were created. Analysis of the ability of these mutant strains to promote epithelial cell proliferation in vivo demonstrated that the pilin subunit, TadE, is the bifidobacterial molecule responsible for this proliferation response. These findings were confirmed in vitro using purified TadE protein. Our data imply that bifidobacterial Tad pili may contribute to the maturation of the naïve gut in early life through the production of a specific scaffold of extracellular protein structures, which stimulate growth of the neonatal mucosa.
Bifidobacteria are among the first colonisers of the newborn gastrointestinal tract, and appreciated to positively impact on host health. Previously, we identified bifidobacterial Tad pili as an essential and conserved colonisation factor. Here, we show that Tad pili, and specifically a pseudopilin subunit, TadE, are involved in host signalling, leading to in vivo colonic epithelial proliferation. These findings could have several therapeutic implications, particularly in the management of preterm babies with suboptimal mucosal maturation.
The gut microbiota composition of elderly hospitalized patients with Clostridium difficile infection (CDI) exposed to previous antibiotic treatment is still poorly investigated. The aim of this study ...was to compare the microbiota composition by means of 16S rRNA microbial profiling among three groups of hospitalized elderly patients (age ≥ 65) under standard diet including 25 CDI-positive (CDI group), 29 CDI-negative exposed to antibiotic treatment (AB+ group) and 30 CDI-negative subjects not on antibiotic treatment (AB- group). The functional properties of the gut microbiomes of CDI-positive vs CDI-negative subjects were also assessed by shotgun metagenomics. A significantly lower microbial diversity was detected in CDI samples, whose microbiomes clustered separately from CDI-negative specimens. CDI was associated with a significant under-representation of gut commensals with putative protective functionalities, including Bacteroides, Alistipes, Lachnospira and Barnesiella, and over-representation of opportunistic pathogens. These findings were confirmed by functional shotgun metagenomics analyses, including an in-depth profiling of the Peptostreptococcaceae family. In CDI-negative patients, antibiotic treatment was associated with significant depletion of few commensals like Alistipes, but not with a reduction in species richness. A better understanding of the correlations between CDI and the microbiota in high-risk elderly subjects may contribute to identify therapeutic targets for CDI.
Ulcerative colitis (UC) is associated with a substantial alteration of specific gut commensals, some of which may be involved in microbiota-mediated protection. In this study, microbiota cataloging ...of UC patients by 16S rRNA microbial profiling revealed a marked reduction of bifidobacteria, in particular the Bifidobacterium bifidum species, thus suggesting that this taxon plays a biological role in the aetiology of UC. We investigated this further through an in vivo trial by testing the effects of oral treatment with B. bifidum PRL2010 in a wild-type murine colitis model. TNBS-treated mice receiving 109 cells of B. bifidum PRL2010 showed a marked reduction of all colitis-associated histological indices as well as maintenance of mucosal integrity as it was shown by the increase in the expression of many tight junction-encoding genes. The protective role of B. bifidum PRL2010, as well as its sortase-dependent pili, appears to be established through the induction of an innate immune response of the host. These results highlight the importance of B. bifidum as a microbial biomarker for UC, revealing its role in protection against experimentally induced colitis.
We present the first metagenomic study focusing on the biodiversity of the bifidobacterial communities of UC patients while also providing a detailed overview of possible UC-associated (bifido)bacterial biomarkers.
The tail tape measure protein (TMP) of tailed bacteriophages (also called phages) dictates the tail length and facilitates DNA transit to the cell cytoplasm during infection. Here, a thorough ...mutational analysis of the TMP from lactococcal phage TP901-1 (TMP
) was undertaken. We generated 56 mutants aimed at defining TMP
domains that are essential for tail assembly and successful infection. Through analysis of the derived mutants, we determined that TP901-1 infectivity requires the N-terminal 154 aa residues, the C-terminal 60 residues and the first predicted hydrophobic region of TMP
as a minimum. Furthermore, the role of TMP
in tail length determination was visualized by electron microscopic imaging of TMP-deletion mutants. The inverse linear correlation between the extent of TMP
-encoding gene deletions and tail length of the corresponding virion provides an estimate of TMP
regions interacting with the connector or involved in initiator complex formation. This study represents the most thorough characterisation of a TMP from a Gram-positive host-infecting phage and provides essential advances to understanding its role in virion assembly, morphology and infection.
Streptococcus thermophilus strain ST64987 was exposed to a member of a recently discovered group of S. thermophilus phages (the 987 phage group), generating phage‐insensitive mutants, which were then ...characterized phenotypically and genomically. Decreased phage adsorption was observed in selected bacteriophage‐insensitive mutants, and was partnered with a sedimenting phenotype and increased cell chain length or aggregation. Whole genome sequencing of several bacteriophage‐insensitive mutants identified mutations located in a gene cluster presumed to be responsible for cell wall polysaccharide production in this strain. Analysis of cell surface‐associated glycans by methylation and NMR spectroscopy revealed a complex branched rhamno‐polysaccharide in both ST64987 and phage‐insensitive mutant BIM3. In addition, a second cell wall‐associated polysaccharide of ST64987, composed of hexasaccharide branched repeating units containing galactose and glucose, was absent in the cell wall of mutant BIM3. Genetic complementation of three phage‐resistant mutants was shown to restore the carbohydrate and phage resistance profiles of the wild‐type strain, establishing the role of this gene cluster in cell wall polysaccharide production and phage adsorption and, thus, infection.
Bacteriophage‐insensitive mutants of S. thermophilus were analyzed phenotypically and genotypically. Mutants were found to be lacking a cell wall polysaccharide which is thus proven to be essential for phage adsorption and, hence, infection.
Members of the Bifidobacteriaceae family represent both dominant microbial groups that colonize the gut of various animals, especially during the suckling stage of their life, while they also occur ...as pathogenic bacteria of the urogenital tract. The pan-genome of the genus Bifidobacterium has been explored in detail in recent years, though genomics of the Bifidobacteriaceae family has not yet received much attention. Here, a comparative genomic analyses of 67 Bifidobacteriaceae (sub) species including all currently recognized genera of this family, i.e., Aeriscardovia, Alloscardovia, Bifidobacterium, Bombiscardovia, Gardnerella, Neoscardovia, Parascardovia, Pseudoscardovia and Scardovia, was performed. Furthermore, in order to include a representative of each of the 67 (currently recognized) (sub) species belonging to the Bifidobacteriaceae family, we sequenced the genomes of an additional 11 species from this family, accomplishing the most extensive comparative genomic analysis performed within this family so far.
Phylogenomics-based analyses revealed the deduced evolutionary pathway followed by each member of the Bifidobacteriaceae family, highlighting Aeriscardovia aeriphila LMG 21773 as the deepest branch in the evolutionary tree of this family. Furthermore, functional analyses based on genome content unveil connections between a given member of the family, its carbohydrate utilization abilities and its corresponding host. In this context, bifidobacterial (sub) species isolated from humans and monkeys possess the highest relative number of acquired glycosyl hydrolase-encoding genes, probably in order to enhance their metabolic ability to utilize different carbon sources consumed by the host.
Within the Bifidobacteriaceae family, genomics of the genus Bifidobacterium has been extensively investigated. In contrast, very little is known about the genomics of members of the other eight genera of this family. In this study, we decoded the genome sequences of each member of the Bifidobacteriaceae family. Thanks to subsequent comparative genomic and phylogenetic analyses, the deduced pan-genome of this family, as well as the predicted evolutionary development of each taxon belonging to this family was assessed.
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