Whole genome sequencing of uropathogenic E. coli from Ireland reveals diverse resistance mechanisms and strong correlation with phenotypic (EUCAST) susceptibility testing.

Urinary tract infections (UTI) caused by uropathogenic Escherichia coli (UPEC) pose a global health concern. Resistance mechanisms, including genetic mutations in antimicrobial target genes, efflux pumps, and drug deactivating enzymes, hinder clinical treatment. These resistance factors often spread through mobile genetic elements. Molecular techniques like whole genome sequencing (WGS), multilocus sequence typing (MLST), and phylotyping help decode bacterial genomes and categorise resistance genes. In this study, we analysed 57 UPEC isolates from different UTI patients following EUCAST guidelines. A selection of 17 representative strains underwent WGS, phylotyping, MLST, and comparative analysis to connect laboratory susceptibility data with predictive genomics based on key resistance genes and chromosomal mutations in antimicrobial targets. Trimethoprim resistance consistently correlated with dfr genes, with six different alleles detected among the isolates. These dfr genes often coexisted with class 1 integrons, with the most common gene cassette combining dfr and aadA. Furthermore, 52.9% of isolates harboured the blaTem-1 gene, rendering resistance to ampicillin and amoxicillin. Ciprofloxacin-resistant strains exhibited mutations in GyrA, GyrB and ParC, plasmid-mediated quinolone resistance genes (qnrb10), and aac(6')-Ib-cr5. Nitrofurantoin resistance in one isolate stemmed from a four amino acid deletion in NfsB. These findings illustrate the varied strategies employed by UPEC to resist antibiotics and the correlation between clinical susceptibility testing and molecular determinants. As molecular testing gains prominence in clinical applications, understanding key resistance determinants becomes crucial for accurate susceptibility testing and guiding effective antimicrobial therapy.

Two extracellular α-arabinofuranosidases are required for cereal-derived arabinoxylan metabolism by Bifidobacterium longum subsp. longum.

Members of the genus Bifidobacterium are commonly found in the human gut and are known to utilize complex carbohydrates that are indigestible by the human host. Members of the Bifidobacterium longum subsp. longum taxon can metabolize various plant-derived carbohydrates common to the human diet. To metabolize such polysaccharides, which include arabinoxylan, bifidobacteria need to encode appropriate carbohydrate-active enzymes in their genome. In the current study, we describe two GH43 family enzymes, denoted here as AxuA and AxuB, which are encoded by B. longum subsp. longum NCIMB 8809 and are shown to be required for cereal-derived arabinoxylan metabolism by this strain. Based on the observed hydrolytic activity of AxuA and AxuB, assessed by employing various synthetic and natural substrates, and based on in silico analyses, it is proposed that both AxuA and AxuB represent extracellular α-L-arabinofuranosidases with distinct substrate preferences. The variable presence of the axuA and axuB genes and other genes previously described to be involved in the metabolism of arabinose-containing glycans can in the majority cases explain the (in)ability of individual B. longum subsp. longum strains to grow on cereal-derived arabinoxylans and arabinan.

A multifaceted investigation of lactococcal strain diversity in undefined mesophilic starter cultures.

The dairy fermentation industry relies on the activity of lactic acid bacteria in robust starter cultures to accomplish milk acidification. Maintenance of the composition of these starter cultures, whether defined or undefined, is essential to ensure consistent and high-quality fermentation end products. To date, limited information exists regarding the microbial composition of undefined starter culture systems. Here, we describe a culture-based analysis combined with a metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, we describe a qPCR-based genotype detection assay, which is capable of discerning nine distinct lactococcal genotypes to characterize these undefined starter cultures, and which can be applied to monitor compositional changes in an undefined starter culture during a fermentation. IMPORTANCE: This study reports on the development of a combined culture-based analysis and metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, a novel qPCR-based genotype detection assay, capable of discerning nine distinct lactococcal genotypes (based on lactococcal cell wall polysaccharide biosynthesis gene clusters), was used to monitor compositional changes in an undefined starter culture following phage attack. These analytical approaches facilitate a multifaceted assessment of starter culture compositional stability during milk fermentation, which has become an important QC aspect due to the increasing demand for consistent and high-quality dairy products.

Transcriptional landscape of the pMP7017 megaplasmid and its impact on the Bifidobacterium breve UCC2003 transcriptome.

The 190 kb megaplasmid pMP7017 of Bifidobacterium breve JCM7017 represents the first conjugative and largest plasmid characterised within this genus to date. In the current study, we adopted an integrated approach combining transcriptomics, whole genome comparative analysis and metagenomic data mining to understand the biology of pMP7017 and related megaplasmids, and to assess the impact of plasmid-carriage on the host strain. The data generated revealed variations within basic features of promoter elements which correlate with a high level of transcription on the plasmid and highlight the transcriptional activity of genes encoding both offensive and defensive adaptations, including a Type IIL restriction-modification system, an anti-restriction system and four Type II toxin-antitoxin systems. Furthermore, a highly transcribed tmRNA, which likely provides translational support to the host strain, was identified, making pMP7017 the first plasmid of the Bifidobacterium genus and the smallest plasmid known to express a tmRNA. Analyses of synteny and variability among pMP7017 and related plasmids indicate substantial diversity in gene organisation and accessory gene cargo highlighting diverse (co-)evolution and potential host-specific rearrangements and adaptations. Systematic analysis of the codon usage profile of transcriptionally active pMP7017-encoded genes suggests that pMP7017 originated from (sub)species of Bifidobacterium longum. Furthermore, mining of metagenomic data suggests the presence of pMP7017-homologues in ~10% of microbiome samples, mostly infants and/or mothers from various geographical locations. Comparative transcriptome analysis of the B. breve UCC2003 chromosome in the presence or absence of pMP7017 revealed differential expression of genes representing 8% of the total gene pool. Genes involved in genetic information processing were exclusively upregulated, while altered expression of genes involved in biofilm production and polysaccharide biosynthesis was also observed.

Identification of novel genera and subcluster classifications for mycobacteriophages.

Aim: To identify novel genera amongst mycobacteriophages (MP) and verify a hypothesised correlation between the taxonomy set by the International Committee on Taxonomy of Viruses (ICTV) and the National Centre for Biotechnology Information (NCBI) with that of the Actinobacteriophage Database, which may help formalise subcluster assignment. Methods: A dataset of 721 MP genomes was analysed using VIRIDIC, a nucleotide alignment-based software that predicts genus assignments. Potentially novel genera were analysed using Gegenees and VICTOR, respectively. These genera were then compared to the subclusters assigned by the Actinobacteriophage Database to verify a hypothesis that one genus can be assigned to one subcluster (i.e., the genus-subcluster hypothesis). Results: Initially, when comparing the current genus classifications of the 721 MP dataset to the Actinobacteriophage database subcluster assignments, 83.3% of subclusters supported the genus-subcluster hypothesis. Following the sequential VIRIDIC, Gegenees and VICTOR analyses, a total of 20 novel genera were identified based on a ≥ 70% and ~ 50% similarity threshold for VIRIDIC and Gegenees, respectively, and a monophyletic nature in the VICTOR output. Interestingly, these criteria also appear to support the creation of 13 novel subclusters, which would increase the support for the genus-subcluster hypothesis to 97.6%. Conclusion: The link between genus and subcluster classifications appears robust, as most subclusters can be assigned a single genus and vice versa. By relating the taxonomic and clustering classification systems, they can be easily kept up to date to best reflect MP diversity, which could aid the rapid selection of related (or diverse) phages for research, therapeutic and diagnostic purposes.

Towards the diversification of lactococcal starter and non-starter species in mesophilic dairy culture systems.

Lactococcus is one of the earliest identified fermentative bacterial genera and among its member species, the dairy-associated Lactococcus lactis and Lactococcus cremoris are undoubtedly the best studied. These two species are believed to have evolved from plant-associated lactococci and through genome decay and acquisition of plasmids, have adapted to the dairy niche. The past decade has witnessed a surge of activity in novel lactococcal species identification from insect, plant and animal sources. Currently, 22 Lactococcus species are described and in this review, we summarise the genome characteristics of and phylogenetic relationships among these species. Furthermore, we explore the role of mobile elements including plasmids and bacteriophages in the diversification of lactococcal species. The pace of identification of novel lactococcal species suggests that the number of lactococcal species is likely to continue to grow. With additional sequence data for the emerging species, it will be possible to perform pathogenicity/virulence risk evaluations and generate extensive insights into the niche adaptation strategies through which they have evolved.

Complete Genome Sequence of Floricoccus penangensis ML061-4 Isolated from Assam Tea Leaf [Camellia sinensis var. assamica (J.W.Mast.) Kitam.].

Floricoccus penangensis is a Gram-positive coccoid organism that is a member of the lactic acid bacteria. F. penangensis ML061-4 was originally isolated from the surface of an Assam tea leaf, and its genome is herein shown to contain gene clusters predicted to be involved in complex carbohydrate metabolism and biosynthesis of secondary metabolites.

Galacto-oligosaccharides as infant prebiotics: production, application, bioactive activities and future perspectives.

Galacto-oligosaccharides (GOS) are non-digestible oligosaccharides characterized by a mix of structures that vary in their degree of polymerization (DP) and glycosidic linkage between the galactose moieties or between galactose and glucose. They have enjoyed extensive scientific scrutiny, and their health-promoting effects are supported by a large number of scientific and clinical studies. A variety of GOS-associated health-promoting effects have been reported, such as growth promotion of beneficial bacteria, in particular bifidobacteria and lactobacilli, inhibition of pathogen adhesion and improvement of gut barrier function. GOS have attracted significant interest from food industries for their versatility as a bioactive ingredient and in particular as a functional component of infant formulations. These oligosaccharides are produced in a kinetically-controlled reaction involving lactose transgalactosylation, being catalyzed by particular ß-galactosidases of bacterial or fungal origin. Despite the well-established technology applied for GOS production, this process may still meet with technological challenges when employed at an industrial scale. The current review will cover relevant scientific literature on the beneficial physiological properties of GOS as a prebiotic for the infant gut microbiota, details of GOS structures, the associated reaction mechanism of ß-galactosidase, and its (large-scale) production.

HMO-primed bifidobacteria exhibit enhanced ability to adhere to intestinal epithelial cells.

The ability of gut commensals to adhere to the intestinal epithelium can play a key role in influencing the composition of the gut microbiota. Bifidobacteria are associated with a multitude of health benefits and are one of the most widely used probiotics for humans. Enhanced bifidobacterial adhesion may increase host-microbe, microbe-nutrient, and/or microbe-microbe interactions, thereby enabling consolidated health benefits to the host. The objective of this study was to determine the ability of human milk oligosaccharides (HMOs) to enhance bifidobacterial intestinal adhesion in vitro. This study assessed the colonisation-promoting effects of HMOs on four commercial infant-associated Bifidobacterium strains (two B. longum subsp. infantis strains, B. breve and B. bifidum). HT29-MTX cells were used as an in vitro intestinal model for bacterial adhesion. Short-term exposure of four commercial infant-associated Bifidobacterium strains to HMOs derived from breastmilk substantially increased the adherence (up to 47%) of these probiotic strains. Interestingly, when strains were incubated with HMOs as a four-strain combination, the number of viable bacteria adhering to intestinal cells increased by >90%. Proteomic analysis of this multi-strain bifidobacterial mixture revealed that the increased adherence resulting from exposure to HMOs was associated with notable increases in the abundance of sortase-dependent pili and glycosyl hydrolases matched to Bifidobacterium bifidum. This study suggests that HMOs may prime infant gut-associated Bifidobacterium for colonisation to intestinal epithelial cells by influencing the expression of various colonization factors.

Host genetic requirements for DNA release of lactococcal phage TP901-1.

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.

Comparative Genomics of Lactiplantibacillus plantarum: Insights Into Probiotic Markers in Strains Isolated From the Human Gastrointestinal Tract and Fermented Foods.

Lactiplantibacillus (Lpb.) plantarum is a versatile species commonly found in a wide variety of ecological niches including dairy products and vegetables, while it may also occur as a natural inhabitant of the human gastrointestinal tract. Although Lpb. plantarum strains have been suggested to exert beneficial properties on their host, the precise mechanisms underlying these microbe-host interactions are still obscure. In this context, the genome-scale in silico analysis of putative probiotic bacteria represents a bottom-up approach to identify probiotic biomarkers, predict desirable functional properties, and identify potentially detrimental antibiotic resistance genes. In this study, we characterized the bacterial genomes of three Lpb. plantarum strains isolated from three distinct environments [strain IMC513 (from the human GIT), C904 (from table olives), and LT52 (from raw-milk cheese)]. A whole-genome sequencing was performed combining Illumina short reads with Oxford Nanopore long reads. The phylogenomic analyses suggested the highest relatedness between IMC513 and C904 strains which were both clade 4 strains, with LT52 positioned within clade 5 within the Lpb. plantarum species. The comparative genome analysis performed across several Lpb. plantarum representatives highlighted the genes involved in the key metabolic pathways as well as those encoding potential probiotic features in these new isolates. In particular, our strains varied significantly in genes encoding exopolysaccharide biosynthesis and in contrast to strains IMC513 and C904, the LT52 strain does not encode a Mannose-binding adhesion protein. The LT52 strain is also deficient in genes encoding complete pentose phosphate and the Embden-Meyerhof pathways. Finally, analyses using the CARD and ResFinder databases revealed that none of the strains encode known antibiotic resistance loci. Ultimately, the results provide better insights into the probiotic potential and safety of these three strains and indicate avenues for further mechanistic studies using these isolates.

BiGAMi: Bi-Objective Genetic Algorithm Fitness Function for Feature Selection on Microbiome Datasets.

The relationship between the host and the microbiome, or the assemblage of microorganisms (including bacteria, archaea, fungi, and viruses), has been proven crucial for its health and disease development. The high dimensionality of microbiome datasets has often been addressed as a major difficulty for data analysis, such as the use of machine-learning (ML) and deep-learning (DL) models. Here, we present BiGAMi, a bi-objective genetic algorithm fitness function for feature selection in microbial datasets to train high-performing phenotype classifiers. The proposed fitness function allowed us to build classifiers that outperformed the baseline performance estimated by the original studies by using as few as 0.04% to 2.32% features of the original dataset. In 35 out of 42 performance comparisons between BiGAMi and other feature selection methods evaluated here (sequential forward selection, SelectKBest, and GARS), BiGAMi achieved its results by selecting 6-93% fewer features. This study showed that the application of a bi-objective GA fitness function against microbiome datasets succeeded in selecting small subsets of bacteria whose contribution to understood diseases and the host state was already experimentally proven. Applying this feature selection approach to novel diseases is expected to quickly reveal the microbes most relevant to a specific condition.

Maximum depth sequencing reveals an ON/OFF replication slippage switch and apparent in vivo selection for bifidobacterial pilus expression.

The human gut microbiome, of which the genus Bifidobacterium is a prevalent and abundant member, is thought to sustain and enhance human health. Several surface-exposed structures, including so-called sortase-dependent pili, represent important bifidobacterial gut colonization factors. Here we show that expression of two sortase-dependent pilus clusters of the prototype Bifidobacterium breve UCC2003 depends on replication slippage at an intragenic G-tract, equivalents of which are present in various members of the Bifidobacterium genus. The nature and extent of this slippage is modulated by the host environment. Involvement of such sortase-dependent pilus clusters in microbe-host interactions, including bacterial attachment to the gut epithelial cells, has been shown previously and is corroborated here for one case. Using a Maximum Depth Sequencing strategy aimed at excluding PCR and sequencing errors introduced by DNA polymerase reagents, specific G-tract sequences in B. breve UCC2003 reveal a range of G-tract lengths whose plasticity within the population is functionally utilized. Interestingly, replication slippage is shown to be modulated under in vivo conditions in a murine model. This in vivo modulation causes an enrichment of a G-tract length which appears to allow biosynthesis of these sortase-dependent pili. This work provides the first example of productive replication slippage influenced by in vivo conditions. It highlights the potential for microdiversity generation in "beneficial" gut commensals.

Insights into Gene Transcriptional Regulation of Kayvirus Bacteriophages Obtained from Therapeutic Mixtures.

Bacteriophages (phages) of the genus Kayvirus of Staphylococcus aureus are promising agents for therapeutic applications. In this study, we isolated Kayvirus phages, SAM1 and SAM2, from the Fersisi commercial phage cocktail (George Eliava Institute, Tbilisi, Georgia), which exhibits high sequence homology with phage K (≥94%, BLASTn). We found that phages SAM1 and SAM2 infected 95% and 86% of 21 MRSA of differing sequence types (MLST, SCCmec type) obtained from the Irish National MRSA collection, respectively. We conducted differential transcriptomic analysis by RNA-Seq on phage SAM1 during host infection, showing differential expression of its genes at different points during host infection. This analysis also allowed the identification of potentially adverse outcomes in the application of these phages to target MRSA as therapy. The interaction of phage SAM1 on the host caused the upregulation of prophage genes. Additionally, phage infection was found to cause the slight upregulation of host genes implicated in virulence factors relating to hemolysins, immune evasion, and adhesion, but also the downregulation of genes associated with enterotoxins. The findings of this study give further insights into the biology of kayviruses and their use as therapeutics.

Selective Isolation of Eggerthella lenta from Human Faeces and Characterisation of the Species Prophage Diversity.

Eggerthella lenta is an anaerobic, high GC, Gram-positive bacillus commonly found in the human digestive tract that belongs to the class Coriobacteriia of the phylum Actinobacteria. This species has been of increasing interest as an important player in the metabolism of xenobiotics and dietary compounds. However, little is known regarding its susceptibility to bacteriophage predation and how this may influence its fitness. Here, we report the isolation of seven novel E. lenta strains using cefotaxime and ceftriaxone as selective agents. We conducted comparative and pangenome analyses of these strains and those publicly available to investigate the diversity of prophages associated with this species. Prophage gene products represent a minimum of 5.8% of the E. lenta pangenome, comprising at least ten distantly related prophage clades that display limited homology to currently known bacteriophages. All clades possess genes implicated in virion structure, lysis, lysogeny and, to a limited extent, DNA replication. Some prophages utilise tyrosine recombinases and diversity generating retroelements to generate phase variation among targeted genes. The prophages have differing levels of sensitivity to the CRISPR/cas systems of their hosts, with spacers from 44 E. lenta isolates found to target only five out of the ten identified prophage clades. Furthermore, using a PCR-based approach targeting the prophage attP site, we were able to determine that several of these elements can excise from the host chromosome, thus supporting the notion that these are active prophages. The findings of this study provide further insights into the diversity of prophages infecting species of the phylum Actinobacteria.

A Proposed Framework to Identify Dispensable and Essential Functions in Bifidobacteria: Case Study of Bifidobacterium breve UCC2003 as a Prototype of Its Genus.

Functional genomics of bacteria commonly aims at establishing genotype-phenotype links in microorganisms of industrial, technological and biomedical relevance. In this regard, random transposon mutagenesis coupled to high-throughput next-generation sequencing approaches, termed transposon-insertion sequencing (TIS), has emerged as a robust, genome-wide alternative to perform functional genome analysis. Though these approaches have been used in a large number of studies involving pathogenic and clinically relevant bacteria, they have received little attention in the fields of commensal and potentially beneficial bacteria, including probiotic microorganisms. In this chapter, we describe the implementation of the TIS method Transposon-Directed Insertion Sequencing to describe the set of essential genes in a representative strain of a genus encompassing several commensal and potentially probiotic bacteria and discuss considerations when applying similar methodological approaches to other Bifidobacterium species/strains of interest.

Diversity of Human-Associated Bifidobacterial Prophage Sequences.

Members of Bifidobacterium play an important role in the development of the immature gut and are associated with positive long-term health outcomes for their human host. It has previously been shown that intestinal bacteriophages are detected within hours of birth, and that induced prophages constitute a significant source of such gut phages. The gut phageome can be vertically transmitted from mother to newborn and is believed to exert considerable selective pressure on target prokaryotic hosts affecting abundance levels, microbiota composition, and host characteristics. The objective of the current study was to investigate prophage-like elements and predicted CRISPR-Cas viral immune systems present in publicly available, human-associated Bifidobacterium genomes. Analysis of 585 fully sequenced bifidobacterial genomes identified 480 prophage-like elements with an occurrence of 0.82 prophages per genome. Interestingly, we also detected the presence of very similar bifidobacterial prophages and corresponding CRISPR spacers across different strains and species, thus providing an initial exploration of the human-associated bifidobacterial phageome. Our analyses show that closely related and likely functional prophages are commonly present across four different species of human-associated Bifidobacterium. Further comparative analysis of the CRISPR-Cas spacer arrays against the predicted prophages provided evidence of historical interactions between prophages and different strains at an intra- and inter-species level. Clear evidence of CRISPR-Cas acquired immunity against infection by bifidobacterial prophages across several bifidobacterial strains and species was obtained. Notably, a spacer representing a putative major capsid head protein was found on different genomes representing multiple strains across B. adolescentis, B. breve, and B. bifidum, suggesting that this gene is a preferred target to provide bifidobacterial phage immunity.

Bifidobacterial ß-Galactosidase-Mediated Production of Galacto-Oligosaccharides: Structural and Preliminary Functional Assessments.

In the current study the ability of four previously characterized bifidobacterial ß-galactosidases (designated here as BgaA, BgaC, BgaD, and BgaE) to produce galacto-oligosaccharides (GOS) was optimized. Of these enzymes, BgaA and BgaE were found to be promising candidates for GOS production (and the corresponding GOS mixtures were called GOS-A and GOS-E, respectively) with a GOS concentration of 19.0 and 40.3% (of the initial lactose), respectively. GOS-A and GOS-E were partially purified and structurally characterized. NMR analysis revealed that the predominant (non-lactose) disaccharide was allo-lactose in both purified GOS preparations. The predominant trisaccharide in GOS-A and GOS-E was shown to be 3'-galactosyllactose, with lower levels of 6'-galactosyllactose and 4'-galactosyllactose. These three oligosaccharides have also been reported to occur in human milk. Purified GOS-A and GOS-E were shown to be able to support bifidobacterial growth similar to a commercially available GOS. In addition, GOS-E and the commercially available GOS were shown to be capable of reducing Escherichia coli adhesion to a C2BBe1 cell line. Both in vitro bifidogenic activity and reduced E. coli adhesion support the prebiotic potential of GOS-E and GOS-A.

Bifidobacterium breve Exopolysaccharide Blocks Dendritic Cell Maturation and Activation of CD4+ T Cells.

Exopolysaccharide (EPS) is a bacterial extracellular carbohydrate moiety which has been associated with immunomodulatory activity and host protective effects of several gut commensal bacteria. Bifidobacterium breve are early colonizers of the human gastrointestinal tract (GIT) but the role of EPS in mediating their effects on the host has not been investigated for many strains. Here, we characterized EPS production by a panel of human B. breve isolates and investigated the effect of EPS status on host immune responses using human and murine cell culture-based assay systems. We report that B. breve EPS production is heterogenous across strains and that immune responses in human THP-1 monocytes are strain-specific, but not EPS status-specific. Using wild type and isogenic EPS deficient mutants of B. breve strains UCC2003 and JCM7017 we show that EPS had strain-specific divergent effects on cytokine responses from murine bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs). The B. breve UCC2003 EPS negative (EPS-) strain increased expression of cytokine genes (Tnfa, Il6, Il12a, and Il23a) relative to untreated BMDCs and BMDCs treated with wild type strain. B. breve UCC2003 and JCM7017 EPS- strains increased expression of dendritic cell (DC) activation and maturation marker genes (Cd80, Cd83, and Cd86) relative to untreated BMDCs. Consistent with this, BMDCs co-cultured with B. breve UCC2003 and JCM7017 EPS- strains engineered to express OVA antigen activated OVA-specific OT-II CD4+ T-cells in a co-culture antigen-presentation assay while EPS proficient strains did not. Collectively, these data indicate that B. breve EPS proficient strains use EPS to prevent maturation of DCs and activation of antigen specific CD4+ T cells responses to B. breve. This study identifies a new immunomodulatory role for B. breve EPS and suggests it may be important for immune evasion of adaptive immunity by B. breve and contribute to host-microbe mutualism.

Infant-Associated Bifidobacterial ß-Galactosidases and Their Ability to Synthesize Galacto-Oligosaccharides.

Galacto-oligosaccharides (GOS) represent non-digestible glycans that are commercially produced by transgalactosylation of lactose, and that are widely used as functional food ingredients in prebiotic formulations, in particular in infant nutrition. GOS consumption has been reported to enhance growth of specific bacteria in the gut, in particular bifidobacteria, thereby supporting a balanced gut microbiota. In a previous study, we assessed the hydrolytic activity and substrate specificity of seventeen predicted ß-galactosidases encoded by various species and strains of infant-associated bifidobacteria. In the current study, we further characterized seven out of these seventeen bifidobacterial ß-galactosidases in terms of their kinetics, enzyme stability and oligomeric state. Accordingly, we established whether these ß-galactosidases are capable of synthesizing GOS via enzymatic transgalactosylation employing lactose as the feed substrate. Our findings show that the seven selected enzymes all possess such transgalactosylation activity, though they appear to differ in their efficiency by which they perform this reaction. From chromatography analysis, it seems that these enzymes generate two distinct GOS mixtures: GOS with a relatively short or long degree of polymerization profile. These findings may be the stepping stone for further studies aimed at synthesizing new GOS variants with novel and/or enhanced prebiotic activities and potential for industrial applications.