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Abstract Endometriosis is a chronic systemic disease, which results in endometrial-type tissue growing outside the uterus, and affects approximately 10% of reproductive-aged women worldwide. Its aetiology is poorly understood, and there is currently no long-term cure. Development and persistence of the disease depend on several coexisting factors including the vaginal microbiome. However, the role played by this important entity in endometriosis and its systemic involvement is not fully understood. Here, we investigated the vaginal microbiota, the serum and urine glycome, and antibody glycosylation in endometriosis patients. We reveal an endometriosis-specific vaginal microbiota in patients, being distinct from that present in a control group. Endometriosis patients were typified by a loss of the dominant Lactobacillus species, i.e. Lactobacillus iners, increased bacterial diversity and the presence of species such as Anaerococcus senegalensis, Prevotella jejuni, Porphyromonas bennonis and Anaerococcus octavius. The presence of trigalactosylated and triantennary serum glycans and urine core fucosylated mono-antennary glycans from IgG correlated with the vaginal presence of the bacterium A. senegalensis in endometriosis patients. Urine glycans did not differ in endometriosis, but urine IgG identified four novel sulfated glycans differing from serum IgG indicating functional relevance. Our findings contribute to understanding the relationships between the vaginal microbiota and the serum and urine glycome on the one hand, and endometriosis on the other. Further functional studies are warranted.

Bacteriocins are broad or narrow-spectrum antimicrobial compounds that have received significant scientific attention due to their potential to treat infections caused by antibiotic-resistant pathogenic bacteria. The genome of Bifidobacterium pseudocatenulatum MM0196, an antimicrobial-producing, fecal isolate from a healthy pregnant woman, was shown to contain a gene cluster predicted to encode Pseudocin 196, a novel lantibiotic, in addition to proteins involved in its processing, transport and immunity. Following antimicrobial assessment against various indicator strains, protease-sensitive Pseudocin 196 was purified to homogeneity from cell-free supernatant. MALDI TOF mass spectrometry confirmed that the purified antimicrobial compound corresponds to a molecular mass of 2679 Da, which is consistent with that deduced from its genetic origin. Pseudocin 196 is classified as a lantibiotic based on its similarity to lacticin 481, a lanthionine ring-containing lantibiotic produced by Lactococcus lactis. Pseudocin 196, the first reported bacteriocin produced by a B. pseudocatenulatum species of human origin, was shown to inhibit clinically relevant pathogens, such as Clostridium spp. and Streptococcus spp. thereby highlighting the potential application of this strain as a probiotic to treat and prevent bacterial infections.

ABSTRACTMembers 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.

ABSTRACT The vaginal microbiome is a key player in the etiology of spontaneous preterm birth. This study aimed to illustrate maternal environmental factors associated with vaginal microbiota composition and function in pregnancy. Women in healthy pregnancy had vaginal microbial sampling from the posterior vaginal fornix performed at 16 weeks gestation. After shotgun metagenomic sequencing, heatmaps of relative abundance data were generated. Community state type (CST) was assigned, and alpha diversity was calculated. Demography, obstetric history, well-being, exercise, and diet using food frequency questionnaires were collected and compared against microbial parameters. A total of 119 pregnant participants had vaginal metagenomic sequencing performed. Factors with strongest association with beta diversity were dietary lysine (adj-R2 0.113, P = 0.002), valine (adj-R2 0.096, P = 0.004), leucine (adj-R2 0.086, P = 0.003), and phenylalanine (adj-R2 0.085, P = 0.005, Fig. 2D). Previous vaginal delivery and breastfeeding were associated with vaginal beta diversity (adj-R2 0.048, P = 0.003; adj-R2 0.045, P = 0.004), accounting for 8.5% of taxonomy variation on redundancy analysis. Dietary fat, starch, and maltose were positively correlated with alpha diversity (fat +0.002 SD/g, P = 0.025; starch +0.002 SD/g, P = 0.043; maltose +0.440 SD/g, P = 0.013), particularly in secretor-positive women. Functional signature was associated with CST, maternal smoking, and dietary phenylalanine, accounting for 8.9%–11% of the variation in vaginal microbiome functional signature. Dietary amino acids, previous vaginal delivery, and breastfeeding history were associated with vaginal beta diversity. Functional signature of the vaginal microbiome differed with community state type, smoking, dietary phenylalanine, and vitamin K. Increased alpha diversity correlated with dietary fat and starch. These data provide a novel snapshot into the associations between maternal environment, nutrition, and the vaginal microbiome.IMPORTANCEThis secondary analysis of the MicrobeMom randomized controlled trial reveals that dietary amino acids, macronutrients, previous vaginal birth, and breastfeeding have the strongest associations with vaginal taxonomy in early pregnancy. Function of the vaginal niche is associated mainly by species composition, but smoking, vitamin K, and phenylalanine also play a role. These associations provide an intriguing and novel insight into the association between host factors and diet on the vaginal microbiome in pregnancy and highlight the need for further investigation into the complex interactions between the diet, human gut, and vaginal microbiome.

ABSTRACT The human gut microbiota possesses the capacity to synthesize vitamins, especially B group vitamins, which are recognized as indispensable for various biological processes both among members of these bacterial communities and host cells. Accordingly, vitamin production by intestinal commensals has attracted significant interest. Nevertheless, our current understanding of bacterial vitamin synthesis is primarily based on individual genomic and monoculture investigations, therefore not providing an overall view of the biosynthetic potential of complex microbial communities. In the current study, we utilized over 100 bacterial genes known to be involved in the biosynthesis of B group and K vitamins to assess the corresponding vitamin biosynthetic potential of approximately 8,000 human gut microbiomes. Our analyses reveal that host-associated factors, such as age and geographical origin, appear to influence the diversity and abundance of vitamin biosynthetic pathways. Furthermore, we identify gut microbiota members that substantially contribute to these biosynthetic functions at each stage of human life. Interestingly, inference of microbial co-associations and network relationships uncovered the apparent key role played by folate and cobalamin in equilibrium establishment of the infant and adult gut microbial communities, respectively.IMPORTANCEOverall, this study expands our understanding of microbe-mediated vitamin biosynthesis in the human gut and may provide potential novel targets to improve availability of these essential micronutrients in the host.

Background: Recent advances have significantly expanded our understanding of the gut microbiome's influence on host physiology and metabolism. However, the specific role of certain microorganisms in gestational health and fetal development remains underexplored. Objective: This study investigates the impact of Bifidobacterium breve UCC2003 on fetal brain metabolism when colonized in the maternal gut during pregnancy. Methods: Germ-free pregnant mice were colonized with or without B. breve UCC2003 during pregnancy. The metabolic profiles of fetal brains were analyzed, focusing on the presence of key metabolites and the expression of critical metabolic and cellular pathways. Results: Maternal colonization with B. breve resulted in significant metabolic changes in the fetal brain. Specifically, ten metabolites, including citrate, 3-hydroxyisobutyrate, and carnitine, were reduced in the fetal brain. These alterations were accompanied by increased abundance of transporters involved in glucose and branched-chain amino acid uptake. Furthermore, supplementation with this bacterium was associated with elevated expression of critical metabolic pathways such as PI3K-AKT, AMPK, STAT5, and Wnt-β-catenin signaling, including its receptor Frizzled-7. Additionally, there was stabilization of HIF-2 protein and modifications in genes and proteins related to cellular growth, axogenesis, and mitochondrial function. Conclusions: The presence of maternal B. breve during pregnancy plays a crucial role in modulating fetal brain metabolism and growth. These findings suggest that Bifidobacterium could modify fetal brain development, potentially offering new avenues for enhancing gestational health and fetal development through microbiota-targeted interventions.

Bifidobacteria are well known as common and abundant colonizers of the human gut and are able to exert multiple beneficial effects on their host, although the cooperative and competitive relationships that may occur among bifidobacterial strains are still poorly investigated. Therefore, to dissect possible molecular interactions among bifidobacterial species that typically colonize the human gut, three previously identified bifidobacterial prototypes, i.e., B. bifidum PRL2010, B. breve PRL2012, and B. longum PRL2022 were cultivated individually as well as in bi- and tri-association in a human gut-simulating medium. Transcriptomic analyses of these co-associations revealed up-regulation of genes predicted to be involved in the production of extracellular structures including pili (i.e., flp pilus assembly TadE protein gene), exopolysaccharides (i.e., GtrA family protein gene) and teichoic acids (i.e., ABC transporter permease), along with carbohydrate, amino acid and vitamin metabolism-related genes (i.e., exo-alpha-sialidase; beta-galactosidase and pyridoxamine kinase), suggesting that co-cultivation of bifidobacteria induces a response, in individual bifidobacterial strains, aimed at enhancing their proliferation and survival, as well as their ability to cooperate with their host to promote their persistence. Furthermore, exposure of the selected prototypes to human cell line monolayers unveiled the ability of the bifidobacterial tri-association to communicate with their host by increasing the expression of genes involved in adherence to/interaction with intestinal human cells. Lastly, bifidobacterial tri-association promoted the transcriptional upregulation of genes responsible for maintaining the integrity and homeostasis of the intestinal epithelial barrier.

Bifidobacteria are commonly encountered members of the human gut microbiota that possess the enzymatic machinery necessary for the metabolism of certain plant-derived, complex carbohydrates. In the current study we describe differential growth profiles elicited by a panel of 21 newly isolated Bifidobacterium pseudocatenulatum strains on various plant-derived glycans. Using a combination of gene-trait matching and comparative genome analysis, we identified two distinct xylanases responsible for the degradation of xylan. Furthermore, three distinct extracellular α-amylases were shown to be involved in starch degradation by certain strains of B. pseudocatenulatum. Biochemical characterization showed that all three α-amylases can cleave the related substrates amylose, amylopectin, maltodextrin, glycogen and starch. The genes encoding these enzymes are variably found in the species B. pseudocatenulatum, therefore constituting a strain-specific adaptation to the gut environment as these glycans constitute common plant-derived carbohydrates present in the human diet. Overall, our study provides insights into the metabolism of these common dietary carbohydrates by a human-derived bifidobacterial species.

Abstract The distinct conjugation machineries encoded by plasmids pNP40 and pUC11B represent the most prevalent plasmid transfer systems among lactococcal strains. In the current study, we identified genetic determinants that underpin pNP40‐ and pUC11B‐mediated, high‐frequency mobilisation of other, non‐conjugative plasmids. The mobilisation frequencies of the smaller, non‐conjugative plasmids and the minimal sequences required for their mobilisation were determined, owing to the determination of the oriT sequences of both pNP40 and pUC11B, which allowed the identification of similar sequences in some of the non‐conjugative plasmids that were shown to promote their mobilisation. Furthermore, the auxiliary gene mobC, two distinct functional homologues of which are present in several plasmids harboured by the pNP40‐ and pUC11B‐carrying host strains, was observed to confer a high‐frequency mobilisation phenotype. These findings provide mechanistic insights into how lactococcal conjugative plasmids achieve conjugation and promote mobilisation of non‐conjugative plasmids. Ultimately, these insights would be harnessed to optimise conjugation and mobilisation strategies for the rapid and predictable development of robust and technologically improved strains.

ABSTRACTBacteria have evolved diverse defense mechanisms to counter bacteriophage attacks. Genetic programs activated upon infection characterize phage–host molecular interactions and ultimately determine the outcome of the infection. In this study, we applied ribosome profiling to monitor protein synthesis during the early stages of sk1 bacteriophage infection in Lactococcus cremoris. Our analysis revealed major changes in gene expression within 5 minutes of sk1 infection. Notably, we observed a specific and severe downregulation of several pyr operons which encode enzymes required for uridine monophosphate biosynthesis. Consistent with previous findings, this is likely an attempt of the host to starve the phage of nucleotides it requires for propagation. We also observed a gene expression response that we expect to benefit the phage. This included the upregulation of 40 ribosome proteins that likely increased the host’s translational capacity, concurrent with a downregulation of genes that promote translational fidelity (lepA and raiA). In addition to the characterization of host–phage gene expression responses, the obtained ribosome profiling data enabled us to identify two putative recoding events as well as dozens of loci currently annotated as pseudogenes that are actively translated. Furthermore, our study elucidated alterations in the dynamics of the translation process, as indicated by time-dependent changes in the metagene profile, suggesting global shifts in translation rates upon infection. Additionally, we observed consistent modifications in the ribosome profiles of individual genes, which were apparent as early as 2 minutes post-infection. The study emphasizes our ability to capture rapid alterations of gene expression during phage infection through ribosome profiling.IMPORTANCEThe ribosome profiling technology has provided invaluable insights for understanding cellular translation and eukaryotic viral infections. However, its potential for investigating host–phage interactions remains largely untapped. Here, we applied ribosome profiling to Lactococcus cremoris cultures infected with sk1, a major infectious agent in dairy fermentation processes. This revealed a profound downregulation of genes involved in pyrimidine nucleotide synthesis at an early stage of phage infection, suggesting an anti-phage program aimed at restricting nucleotide availability and, consequently, phage propagation. This is consistent with recent findings and contributes to our growing appreciation for the role of nucleotide limitation as an anti-viral strategy. In addition to capturing rapid alterations in gene expression levels, we identified translation occurring outside annotated regions, as well as signatures of non-standard translation mechanisms. The gene profiles revealed specific changes in ribosomal densities upon infection, reflecting alterations in the dynamics of the translation process.

Abstract Background Typically, animal models studying gastrointestinal microbiotas compromised in early life have employed either germ-free animals or mice treated with a cocktail of antibiotics. Such studies intend to mimic scenarios of infants born by caesarean section and/or subjected to antibiotic treatment. However, the antibiotics used in these studies are rarely prescribed to infants. Therefore, an early life model was developed in which the murine gastrointestinal microbiota was severely disrupted by clindamycin treatment. Results In this mouse model, we investigated the extent supplementation with a synbiotic mixture of prebiotics, being scGOS/lcFOS with the human milk oligosaccharide 2’-Fucosyllactose (2’-FL), in combination with or without single strain or mix of “infant type” bifidobacteria, can rescue an antibiotic-compromised microbiota. Shotgun metagenomic sequencing showed that the microbiota was severely disrupted by the clindamycin challenge. No recovery was observed 3 weeks post-challenge in the scGOS/lcFOS/2’FL group, while the group that received the synbiotic treatment of scGOS/lcFOS/2’-FL with Bifidobacterium breve NRBB01 showed partial recovery. Strikingly in the scGOS/lcFOS/2’-FL group receiving the mixture of bifidobacteria resulted in a recovery of the microbiota disruption. Histological analyses showed that the clindamycin-treated animals at the end of the experiment still suffered from mild oedema and villi/colonic crypt irregularities which was ameliorated by the synbiotic intervention. Conclusion Our study demonstrates that supplementation of synbiotic mixture of scGOS/lcFOS/2’-FL in combination with a specific mix of infant-type bifidobacterial strains is able to partially revive an antibiotic-perturbed gastrointestinal microbiota. Video Abstract

Abstract Although compositional variation in the gut microbiome during human development has been extensively investigated, strain-resolved dynamic changes remain to be fully uncovered. In the current study, shotgun metagenomic sequencing data of 12,415 fecal microbiomes from healthy individuals are employed for strain-level tracking of gut microbiota members to elucidate its evolving biodiversity across the human life span. This detailed longitudinal meta-analysis reveals host sex-related persistence of strains belonging to common, maternally-inherited species, such as Bifidobacterium bifidum and Bifidobacterium longum subsp. longum. Comparative genome analyses, coupled with experiments including intimate interaction between microbes and human intestinal cells, show that specific bacterial glycosyl hydrolases related to host-glycan metabolism may contribute to more efficient colonization in females compared to males. These findings point to an intriguing ancient sex-specific host-microbe coevolution driving the selective persistence in women of key microbial taxa that may be vertically passed on to the next generation.

Members of the genus Bifidobacterium are among the first microorganisms colonizing the human gut. Among these species, strains of Bifidobacterium breve are known to be commonly transmitted from mother to her newborn, while this species has also been linked with activities supporting human wellbeing. In the current study, an in silico approach, guided by ecology- and phylogenome-based analyses, was employed to identify a representative strain of B. breve to be exploited as a novel health-promoting candidate. The selected strain, i.e., B. breve PRL2012, was found to well represent the genetic content and functional genomic features of the B. breve taxon. We evaluated the ability of PRL2012 to survive in the gastrointestinal tract and to interact with other human gut commensal microbes. When co-cultivated with various human gut commensals, B. breve PRL2012 revealed an enhancement of its metabolic activity coupled with the activation of cellular defense mechanisms to apparently improve its survivability in a simulated ecosystem resembling the human microbiome.

Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of the putative adhesins of the pNP40 and pUC11B conjugation systems, encoded by traAd and trsAd, respectively. Despite their significant sequence divergence, TraAd and TrsAd represent the most conserved component between the pNP40- and the pUC11B-encoded conjugation systems and share similar peptidoglycan-hydrolase domains. Protein structure prediction using AlphaFold2 highlighted the structural similarities between their predicted domains, as well as the potential homo-dimeric state of both proteins. Expression of the putative surface adhesins resulted in a cell clumping phenotype not only among cells expressing these surface adhesins but also between adhesin-expressing and non-producing cells. Furthermore, mutant derivatives of plasmids pNP40 or pUC11B carrying a mutation in traAd or trsAd, respectively, were shown to act as efficient donors provided the corresponding recipient expresses either traAd or trsAd, thus demonstrating in trans reciprocal complementarity of these proteins in conjugation systems.

Lactococcal conjugative plasmids are poorly characterized compared to those harbored by numerous other Gram-positive bacteria, despite their significance in dairy fermentations and starter culture development. Furthermore, the transcriptional landscape of these lactococcal conjugation systems and their regulation have not been studied in any detail. Lactococcal plasmids pNP40 and pUC11B possess two genetically distinct and prevalent conjugation systems. Here, we describe the detailed transcriptional analysis of the pNP40 and pUC11B conjugation-associated gene clusters, revealing three and five promoters, respectively, for which the corresponding transcriptional start sites were identified. Regulation of several of these promoters, and therefore conjugation, is shown to involve the individual or concerted activities of the corresponding relaxase and transcriptional repressor(s) encoded by each conjugative plasmid. This work highlights how the conjugative potential of these systems may be unlocked, with significant implications for the starter culture and food fermentation industry.

Abstract The human gut microbiota (HGM) is comprised of a very complex network of microorganisms, which interact with the host thereby impacting on host health and well-being. β-glucan has been established as a dietary polysaccharide supporting growth of particular gut-associated bacteria, including members of the genera Bacteroides and Bifidobacterium, the latter considered to represent beneficial or probiotic bacteria. However, the exact mechanism underpinning β-glucan metabolism by gut commensals is not fully understood. We show that mycoprotein represents an excellent source for β-glucan, which is consumed by certain Bacteroides species as primary degraders, such as Bacteroides cellulosilyticus WH2. The latter bacterium employs two extracellular, endo-acting enzymes, belonging to glycoside hydrolase families 30 and 157, to degrade mycoprotein-derived β-glucan, thereby releasing oligosaccharides into the growth medium. These released oligosaccharides can in turn be utilized by other gut microbes, such as Bifidobacterium and Lactiplantibacillus, which thus act as secondary degraders. We used a cross-feeding approach to track how both species are able to grow in co-culture.

Abstract A significant proportion of the infant gut microbiome is considered to be acquired from the mother during and after birth. Thus begins a lifelong and dynamic relationship with microbes that has an enduring impact on host health. Based on a cohort of 135 mother-infant (F = 72, M = 63) dyads (MicrobeMom: ISRCTN53023014), we investigated the phenomenon of microbial strain transfer, with a particular emphasis on the use of a combined metagenomic-culture-based approach to determine the frequency of strain transfer involving members of the genus Bifidobacterium, including species/strains present at low relative abundance. From the isolation and genome sequencing of over 449 bifidobacterial strains, we validate and augment metagenomics-based evidence to reveal strain transfer in almost 50% of dyads. Factors important in strain transfer include vaginal birth, spontaneous rupture of amniotic membranes, and avoidance of intrapartum antibiotics. Importantly, we reveal that several transfer events are uniquely detected employing either cultivation or metagenomic sequencing, highlighting the requirement for a dual approach to obtain an in-depth insight into this transfer process.

Abstract During infancy, gut microbiota development is a crucial process involved in the establishment of microbe–host interactions which may persist throughout adulthood, and which are believed to influence host health. To fully understand the complexities of such interactions, it is essential to assess gut microbiota diversity of newborns and its associated microbial dynamics and relationships pertaining to health and disease. To explore microbial biodiversity during the first 3 years of human life, 10,935 shotgun metagenomic datasets were taxonomically and functionally classified. Microbial species distribution between infants revealed the presence of eight major Infant Community State Types (ICSTs), being dominated by 17 bacterial taxa, whose distribution was shown to correspond to the geographical origin and infant health status. In total, 2390 chromosomal sequences of the predominant taxa were reconstructed from metagenomic data and used in combination with 44,987 publicly available genomes to trace the distribution of microbial Population Subspecies (PS) within the different infant groups, revealing patterns of multistrain coexistence among ICSTs. Finally, implementation of a metagenomic- and metatranscriptomic-based metabolic profiling highlighted different enzymatic expression patterns of the gut microbiota that allowed us to acquire insights into mechanistic aspects of health-gut microbiota interplay in newborns. Comparison between metagenomic and metatranscriptomic data highlights how a complex environment like the human gut must be investigated by employing both sequencing methodologies and possibly supplemented with metabolomics approaches. While metagenomic analyses are very useful for microbial classification aimed at unveiling key players driving microbiota balances, using these data to explain functionalities of the microbiota is not always warranted.

Commercially produced cyanobacteria preparations sold under the name spirulina are widely consumed, due to their traditional use as a nutrient-rich foodstuff and subsequent marketing as a superfood. Despite their popularity, the microbial composition of ponds used to cultivate these bacteria is understudied. A total of 19 pond samples were obtained from small-scale spirulina farms and subjected to metagenome and/or virome sequencing, and the results were analysed. A remarkable level of prokaryotic and viral diversity was found to be present in the ponds, with Limnospira sp. and Arthrospira sp. sometimes being notably scarce. A detailed breakdown of prokaryotic and viral components of 15 samples is presented. Twenty putative Limnospira sp.-infecting bacteriophage contigs were identified, though no correlation between the performance of these cultures and the presence of phages was found. The high diversity of these samples prevented the identification of clear trends in sample performance over time, between ponds or when comparing successful and failed fermentations.

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.

ABSTRACTThe human gut microbiota is a key contributor to host metabolism and physiology, thereby impacting in various ways on host health. This complex microbial community has developed many metabolic strategies to colonize, persist and survive in the gastrointestinal environment. In this regard, intracellular glycogen accumulation has been associated with important physiological functions in several bacterial species, including gut commensals. However, the role of glycogen storage in shaping the composition and functionality of the gut microbiota offers a novel perspective in gut microbiome research. Here, we review what is known about the enzymatic machinery and regulation of glycogen metabolism in selected enteric bacteria, while we also discuss its potential impact on colonization and adaptation to the gastrointestinal tract. Furthermore, we survey the presence of such glycogen biosynthesis pathways in gut metagenomic data to highlight the relevance of this metabolic trait in enhancing survival in the highly competitive and dynamic gut ecosystem.

Abstract In vitro gut cultivation models provide host-uncoupled, fast, and cost-efficient solutions to investigate the effects of intrinsic and extrinsic factors impacting on both composition and functionality of the intestinal microbial ecosystem. However, to ensure the maintenance and survival of gut microbial players and preserve their functions, these systems require close monitoring of several variables, including oxygen concentration, pH, and temperature, as well as the use of a culture medium satisfying the microbial nutritional requirements. In this context, in order to identify the macro- and micro-nutrients necessary for in vitro cultivation of the infant gut microbiota, a meta-analysis based on 1669 publicly available shotgun metagenomic samples corresponding to fecal samples of healthy, full-term infants aged from a few days to three years was performed to define the predominant species characterizing the “infant-like” gut microbial ecosystem. A subsequent comparison of growth performances was made using infant fecal samples that contained the most abundant bacterial taxa of the infant gut microbiota, when cultivated on 18 different culture media. This growth analysis was performed by means of flow cytometry-based bacterial cell enumeration and shallow shotgun sequencing, which allowed the formulation of an optimized growth medium, i.e., Infant Gut Super Medium (IGSM), which maintains and sustains the infant gut microbial biodiversity under in vitro growth conditions. Furthermore, this formulation was used to evaluate the in vitro effect of two drugs commonly used in pediatrics, i.e., acetaminophen and simethicone, on the taxonomic composition of the infant gut microbiota.

BackgroundMicrobial dysbiosis in infancy can influence long-term health outcomes such as childhood obesity. The aim of this study is to explore relationships among maternal well-being during pregnancy, breastfeeding, and the infant gut microbiome.MethodsThis is a secondary analysis of healthy pregnant women from the MicrobeMom study, a double-blind randomized control trial of maternal probiotic supplementation (Bifidobacterium breve 702258) versus placebo antenatally and up to 3 months postpartum. Maternal well-being was assessed using the WHO-5 well-being index at 16 weeks’ and 34 weeks’ gestation. Breastfeeding practices were recorded at discharge from hospital and at 1 month postpartum. Infant stool samples were obtained at 1 month of age. Next generation shotgun sequencing determined infant microbial diversity. Independent sample t-tests and Mann-Whitney U tests informed adjusted regression analysis, which was adjusted for delivery mode, antibiotics during delivery, maternal age and body mass index (BMI), and probiotic vs. control study group.ResultsWomen (n = 118) with at least one measure of well-being were on average 33 years (SD 3.93) of age and 25.09 kg/m2 (SD 3.28) BMI. Exclusive breastfeeding was initiated by 65% (n = 74). Any breastfeeding was continued by 69% (n = 81) after 1 month. In early and late pregnancy, 87% (n = 97/111) and 94% (n = 107/114) had high well-being scores. Well-being was not associated with infant microbial diversity at 1 month. In adjusted analysis, exclusive breastfeeding at discharge from hospital was associated with infant microbial beta diversity (PC2; 0.254, 95% CI 0.006, 0.038). At 1 month postpartum, any breastfeeding was associated with infant microbial alpha diversity (Shannon index; −0.241, 95% CI −0.498, −0.060) and observed species; (−0.325, 95% CI −0.307, −0.060), and infant microbial beta diversity (PC2; 0.319, 95% CI 0.013, 0.045). Exclusive breastfeeding at 1 month postpartum was associated with infant alpha diversity (Shannon index −0.364, 95% CI −0.573, −0.194; Simpson index 0.339, 95% CI 0.027, 0.091), and infant’s number of observed microbial species (−0.271, 95% CI −0.172, −0.037).ConclusionBreastfeeding practices at 1 month postpartum were associated with lower microbial diversity and observed species in infants at 1 month postpartum, which is potentially beneficial to allow greater abundance of Bifidobacterium.Clinical trial registrationISRCTN53023014.

ABSTRACT Multiple millennia of human evolution have shaped the chemical composition of breast milk toward an optimal human body fluid for nutrition and protection and for shaping the early gut microbiota of newborns. This biological fluid is composed of water, lipids, simple and complex carbohydrates, proteins, immunoglobulins, and hormones. Potential interactions between hormones present in mother’s milk and the microbial community of the newborn are a very fascinating yet unexplored topic. In this context, insulin, in addition to being one of the most prevalent hormones in breast milk, is also involved in a metabolic disease that affects many pregnant women, i.e., gestational diabetes mellitus (GDM). Analysis of 3,620 publicly available metagenomic data sets revealed that the bifidobacterial community varies in relation to the different concentrations of this hormone in breast milk of healthy and diabetic mothers. Starting from this assumption, in this study, we explored possible molecular interactions between this hormone and bifidobacterial strains that represent bifidobacterial species commonly occurring in the infant gut using ‘omics’ approaches. Our findings revealed that insulin modulates the bifidobacterial community by apparently improving the persistence of the Bifidobacterium bifidum taxon in the infant gut environment compared to other typical infant-associated bifidobacterial species. IMPORTANCE Breast milk is a key factor in modulating the infant's intestinal microbiota composition. Even though the interaction between human milk sugars and bifidobacteria has been extensively studied, there are other bioactive compounds in human milk that may influence the gut microbiota, such as hormones. In this article, the molecular interaction of the human milk hormone insulin and the bifidobacterial communities colonizing the human gut in the early stages of life has been explored. This molecular cross talk was assessed using an in vitro gut microbiota model and then analyzed by various omics approaches, allowing the identification of genes associated with bacterial cell adaptation/colonization in the human intestine. Our findings provide insights into the manner by which assembly of the early gut microbiota may be regulated by host factors such as hormones carried by human milk.

Abstract 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.

Bacteria are engaged in a constant battle against preying viruses, called bacteriophages (or phages). These remarkable nano-machines pack and store their genomes in a capsid and inject it into the cytoplasm of their bacterial prey following specific adhesion to the host cell surface. Tailed phages possessing dsDNA genomes are the most abundant phages in the bacterial virosphere, particularly those with long, non-contractile tails. All tailed phages possess a nano-device at their tail tip that specifically recognizes and adheres to a suitable host cell surface receptor, being proteinaceous and/or saccharidic. Adhesion devices of tailed phages infecting Gram-positive bacteria are highly diverse and, for the majority, remain poorly understood. Their long, flexible, multi-domain-encompassing tail limits experimental approaches to determine their complete structure. We have previously shown that the recently developed protein structure prediction program AlphaFold2 can overcome this limitation by predicting the structures of phage adhesion devices with confidence. Here, we extend this approach and employ AlphaFold2 to determine the structure of a complete phage, the lactococcal P335 phage TP901-1. Herein we report the structures of its capsid and neck, its extended tail, and the complete adhesion device, the baseplate, which was previously partially determined using X-ray crystallography.

Abstract 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 are extensively exploited for the formulation of probiotic food supplements due to their claimed ability to exert health-beneficial effects upon their host. However, most commercialized probiotics are tested and selected for their safety features rather than for their effective abilities to interact with the host and/or other intestinal microbial players. In this study, we applied an ecological and phylogenomic-driven selection to identify novel B. longum subsp. longum strains with a presumed high fitness in the human gut. Such analyses allowed the identification of a prototype microorganism to investigate the genetic traits encompassed by the autochthonous bifidobacterial human gut communities. B. longum subsp. longum PRL2022 was selected due to its close genomic relationship with the calculated model representative of the adult human-gut associated B. longum subsp. longum taxon. The interactomic features of PRL2022 with the human host as well as with key representative intestinal microbial members were assayed using in vitro models, revealing how this bifidobacterial gut strain is able to establish extensive cross-talk with both the host and other microbial residents of the human intestine.

Background The Human Respiratory Tract (HRT) is colonized by various microbial taxa, known as HRT microbiota, in a manner that is indicative of mutualistic interaction between such microorganisms and their host.Aim To investigate the microbial composition of the HRT and its possible correlation with the different compartments of the respiratory tract.Methods In the current study, we performed an in-depth meta‐analysis of 849 HRT samples from public shotgun metagenomic datasets obtained through several distinct collection methods.Results The statistical robustness provided by this meta-analysis allowed the identification of 13 possible HRT-specific Community State Types (CSTs), which appear to be specific to each anatomical region of the respiratory tract. Furthermore, functional characterization of the metagenomic datasets revealed specific microbial metabolic features correlating with the different compartments of the respiratory tract.Conclusion The meta-analysis here performed suggested that the variable presence of certain bacterial species seems to be linked to a location-related abundance gradient in the HRT and seems to be characterized by a specific microbial metabolic capability.

Successful bacteriophage infection starts with specific recognition and adhesion to the host cell surface. Adhesion devices of siphophages infecting Gram-positive bacteria are very diverse and remain, for the majority, poorly understood. These assemblies often comprise long, flexible, and multi-domain proteins, which limits their structural analyses by experimental approaches such as X-ray crystallography and electron microscopy. However, the protein structure prediction program AlphaFold2 is exquisitely adapted to unveil structural and functional details of such molecular machineries. Here, we present structure predictions of whole adhesion devices of five representative siphophages infecting Streptococcus thermophilus, one of the main lactic acid bacteria used in dairy fermentations. The predictions highlight the mosaic nature of these devices that share functional domains for which active sites and residues could be unambiguously identified. Such AlphaFold2 analyses of phage-encoded host adhesion devices should become a standard method to characterize phage-host interaction machineries and to reliably annotate phage genomes.

Pecorino Siciliano PDO is a semi-hard cheese that is produced in wooden vats using raw sheep’s milk and its associated autochthonous microbial community. In the present study, we evaluated the microbial ecology of the milk, curd and whey from five Pecorino Siciliano PDO-producing farms in Sicily using a combination of metagenomic and microbiological approaches. We present an overview of the species and strain-level diversity of dairy lactococcal and streptococcal isolates using established genotyping tools and compare the lactic acid bacterial populations present in samples from these farms. Whole genome sequences of representative isolates of Lactococcus spp. and Streptococcus thermophilus were elucidated and the genetic diversity of the strains was established through analysis of predicted phage-resistance systems and prophage-associated regions. The analysis revealed farm-specific dairy lactococcal and streptococcal isolates that possess diverse genotypic features including newly described phage-resistance systems.

Members of the genus Bifidobacterium are dominant and symbiotic inhabitants of the mammalian gastrointestinal tract. Being vertically transmitted, bifidobacterial host colonization commences immediately after birth and leads to a phase of host infancy during which bifidobacteria are highly prevalent and abundant to then transit to a reduced, yet stable abundance phase during host adulthood. However, in order to reach and stably colonize their elective niche, i.e. the large intestine, bifidobacteria have to cope with a multitude of oxidative, osmotic and bile salt/acid stress challenges that occur along the gastrointestinal tract (GIT). Concurrently, bifidobacteria not only have to compete with the myriad of other gut commensals for nutrient acquisition, but they also require protection against bacterial viruses. In this context, Next-Generation Sequencing (NGS) techniques, allowing large-scale comparative and functional genome analyses have helped to identify the genetic strategies that bifidobacteria have developed in order to colonize, survive and adopt to the highly competitive mammalian gastrointestinal environment. The current review is aimed at providing a comprehensive overview concerning the molecular strategies on which bifidobacteria rely to stably and successfully colonize the mammalian gut.

Gram-positive bacterial cell walls are characterised by the presence of a thick peptidoglycan layer which provides protection from extracellular stresses, maintains cell integrity and determines cell morphology, while it also serves as a foundation to anchor a number of crucial polymeric structures. For ovococcal species, including streptococci, enterococci and lactococci, such structures are represented by rhamnose-containing cell wall polysaccharides, which at least in some instances appear to serve as a functional replacement for wall teichoic acids. The biochemical composition of several streptococcal, lactococcal and enterococcal rhamnose-containing cell wall polysaccharides have been elucidated, while associated functional genomic analyses have facilitated the proposition of models for individual biosynthetic pathways. Here, we review the genomic loci which encode the enzymatic machinery to produce rhamnose-containing, cell wall-associated polysaccharide (Rha cwps) structures of the afore-mentioned ovococcal bacteria with particular emphasis on gene content, biochemical structure and common biosynthetic steps. Furthermore, we discuss the role played by these saccharidic polymers as receptors for bacteriophages and the important role phages play in driving Rha cwps diversification and evolution.

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.

ABSTRACT It has been widely reported that members of the genus Lactobacillus dominate the vaginal microbiota, which is represented by the most prevalent species Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners. L. crispatus is furthermore considered an important microbial biomarker due to its professed beneficial implications on vaginal health. In order to identify molecular mechanisms responsible for health-promoting activities that are believed to be elicited by L. crispatus, we performed in silico investigations of the intraspecies biodiversity of vaginal microbiomes followed by in vitro experiments involving various L. crispatus strains along with other vaginal Lactobacillus species mentioned above. Specifically, we assessed their antibacterial activities against a variety of pathogenic microorganisms that are associated with vaginal infections. Moreover, coculture experiments of L. crispatus strains showing the most antibacterial activity against different pathogens revealed distinct ecological fitness and competitive properties with regard to other microbial colonizers. Interestingly, we observed that even phylogenetically closely related L. crispatus strains possess unique features in terms of their antimicrobial activities and associated competitive abilities, which suggests that they exert marked competition and evolutionary pressure within their specific environmental niche. IMPORTANCE The human vaginal microbiota includes all microorganisms that colonize the vaginal tract. In this context, a vaginal microbiota dominated by Lactobacillus and specifically by Lactobacillus crispatus is considered a hallmark of health. The role of L. crispatus in maintaining host health is linked to its modulatory activity toward other members of the vaginal ecosystem and toward the host. In this study, in vitro experiments followed by genetic analyses of the mechanisms used by L. crispatus in colonizing the vaginal ecological niche, particularly in the production of different antimicrobial compounds, were evaluated, highlighting some intriguing novel aspects concerning the genetic variability of this species. Our results indicate that this species has adapted to its niche and may still undergo adaptation to enhance its competitiveness for niche colonization.

Abstract Shotgun metagenomic sequencing is a valuable tool for the taxonomic and functional profiling of microbial communities. However, this approach is challenging in samples, such as milk, where a low microbial abundance, combined with high levels of host DNA, result in inefficient and uneconomical sequencing. Here we evaluate approaches to deplete host DNA or enrich microbial DNA prior to sequencing using three commercially available kits. We compared the percentage of microbial reads obtained from each kit after shotgun metagenomic sequencing. Using bovine and human milk samples, we determined that host depletion with the MolYsis complete5 kit significantly improved microbial sequencing depth compared to other approaches tested. Importantly, no biases were introduced. Additionally, the increased microbial sequencing depth allowed for further characterization of the microbiome through the generation of metagenome-assembled genomes (MAGs). Furthermore, with the use of a mock community, we compared three common classifiers and determined that Kraken2 was the optimal classifier for these samples. This evaluation shows that microbiome analysis can be performed on both bovine and human milk samples at a much greater resolution without the need for more expensive deep-sequencing approaches.

Summary In this paper, we reveal and characterize cross‐feeding behaviour between the common gut commensal Bacteroides cellulosilyticus (Baccell) and certain bifidobacterial strains, including Bifidobacterium breve UCC2003, when grown on a medium containing Larch Wood Arabinogalactan (LW‐AG). We furthermore show that cross‐feeding is dependent on the release of β‐1,3‐galacto‐di/trisaccharides (β‐1,3‐GOS), and identified that the bga gene cluster of B. breve UCC2003 allows β‐1,3‐GOS metabolism. The product of bgaB is presumed to be responsible for the import of β‐1,3‐GOS, while the bgaA gene product, a glycoside hydrolase family 2 member, was shown to hydrolyse both β‐1,3‐galactobiose and β‐1,3‐galactotriose into galactose monomers. This study advances our understanding of strain‐specific syntrophic interactions between two glycan degraders in the human gut in the presence of AG‐type dietary polysaccharides.

Abstract Initial establishment of the human gut microbiota is generally believed to occur immediately following birth, involving key gut commensals such as bifidobacteria that are acquired from the mother. The subsequent development of this early gut microbiota is driven and modulated by specific dietary compounds present in human milk that support selective colonization. This represents a very intriguing example of host-microbe co-evolution, where both partners are believed to benefit. In recent years, various publications have focused on dissecting microbial infant gut communities and their interaction with their human host, being a determining factor in host physiology and metabolic activities. Such studies have highlighted a reduction of microbial diversity and/or an aberrant microbiota composition, sometimes referred to as dysbiosis, which may manifest itself during the early stage of life, i.e., in infants, or later stages of life. There are growing experimental data that may explain how the early human gut microbiota affects risk factors related to adult health conditions. This concept has fueled the development of various nutritional strategies, many of which are based on probiotics and/or prebiotics, to shape the infant microbiota. In this review, we will present the current state of the art regarding the infant gut microbiota and the role of key commensal microorganisms like bifidobacteria in the establishment of the first microbial communities in the human gut.

Appropriate development of the intestinal microbiota during infancy is known to be important for human health. In fact, aberrant alterations of the microbial composition during childhood may cause short- and/or long-term negative health effects. Many factors influence the initial assembly and subsequent progression of the gut microbiota of a neonate, such as feeding type, delivery mode, gestational age, maternal metabolic status and antibiotic exposure.In the current study, the composition of the infant gut core-microbiota was explored, revealing particular variations of this core-microbiota during the first three years as influenced by delivery mode and feeding type. A multi-population cohort meta-analysis was performed by selecting 15 publicly available datasets pertaining to taxonomic profiles of 1035 fecal samples of healthy infants, as obtained by means of a 16S rRNA gene-based profiling approach. Interestingly, this multi-population cohort meta-analysis revealed great microbial complexity and specific taxonomic shifts in children older than six months, suggesting a major impact by the introduction of solid foods which prompts progression of infant gut microbiota towards that typical of adults. The taxonomic data sets employed in this multi-population cohort meta-analysis possess the statistical robustness to allow the identification of infant community state types (ICSTs). Our analysis therefore reveals the existence of specific taxonomic patterns that correspond to particular nutritional and developmental stages of early life, and that had previously been obscured by the high variability typical of such infant gut microbiota.

ABSTRACT The field of metagenomics has rapidly expanded to become the go-to method for complex microbial community analyses. However, there is currently no straightforward route from metagenomics to traditional culture-based methods of strain isolation, particularly in (bacterio)phage biology, leading to an investigative bottleneck. Here, we describe a method that exploits specific phage receptor binding protein (RBP)-host cell surface receptor interaction enabling isolation of phage-host combinations from an environmental sample. The method was successfully applied to two complex sample types—a dairy-derived whey sample and an infant fecal sample, enabling retrieval of specific and culturable phage hosts. IMPORTANCE PhRACS aims to bridge the current divide between in silico genetic analyses (i.e., phageomic studies) and traditional culture-based methodology. Through the labeling of specific bacterial hosts with fluorescently tagged recombinant phage receptor binding proteins and the isolation of tagged cells using flow cytometry, PhRACS allows the full potential of phageomic data to be realized in the wet laboratory.

Probiotic bacteria are widely administered as dietary supplements and incorporated as active ingredients in a variety of functional foods due to their purported health-promoting features. Currently available probiotic products may have issues with regards to their formulation, such as insufficient levels of viable probiotic bacteria, complete lack of probiotic strains that are stated to be present in the product, and the presence of microbial contaminants. To avoid the distribution of such unsuitable or misleading products, we propose here a novel approach named Probiotic Identity Card (PIC), involving a combination of shotgun metagenomic sequencing and bacterial cell enumeration by flow cytometry. PIC was tested on 12 commercial probiotic supplements revealing several inconsistencies in the formulation of five such products based on their stated microbial composition and viability.

Bacillus velezensis ML122-2 is an antimicrobial-producing strain isolated from the leaf of Assam tea or Miang [Camellia sinensis var. assamica (J.W.Mast.) Kitam.]. The cell-free supernatant (CFS) of strain ML122-2 exhibits a broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria as well as the mold Penicillium expansum. The genome of B. velezensis ML122-2 was sequenced and in silico analysis identified three potential bacteriocin-associated gene clusters, that is, those involved in the production of mersacidin, amylocyclicin, and LCI. Furthermore, six gene clusters exhibiting homology (75–100% DNA sequence identity) to those associated with the secondary metabolites bacilysin, bacillibactin, surfactin, macrolactin H, bacillaene, and plipastatin were identified. Individual antimicrobial activities produced by B. velezensis ML122-2 were purified and characterized by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis, revealing three antimicrobial peptides with molecular masses corresponding to surfactin, plipastatin, and amylocyclicin. Transcriptional analysis of specific genes associated with mersacidin (mrsA), amylocyclicin (acnA), plipastatin (ppsA), and surfactin (srfAA) production by B. velezensis ML122-2 showed that the first was not transcribed under the conditions tested, while the latter three were consistent with the presence of the associated peptides as determined by mass spectrometry analysis. These findings demonstrate that B. velezensis ML122-2 has the genetic capacity to produce a wide range of antimicrobial activities that may support a specific community structure and highlight the biotechnological properties of Assam tea.

In the current study the ability of four previously characterized bifidobacterial β-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.

Bacteriophages, or phages, are the most abundant biological entities on Earth. They possess molecular nanodevices to package and store their genome, as well as to introduce it into the cytoplasm of their bacterial prey. Successful phage infection commences with specific recognition of, and adhesion to, a suitable host cell surface. Adhesion devices of siphophages infecting Gram-positive bacteria are very diverse and remain, for the majority, poorly understood. These assemblies often comprise long, flexible, and multi-domain proteins, which limit their structural analyses by experimental approaches. The protein structure prediction program AlphaFold2 is exquisitely adapted to unveil structural and functional details of such molecular machineries. Here, we present structure predictions of adhesion devices from siphophages belonging to the P335 group infecting Lactococcus spp., one of the most extensively applied lactic acid bacteria in dairy fermentations. The predictions of representative adhesion devices from types I-IV P335 phages illustrate their very diverse topology. Adhesion devices from types III and IV phages share a common topology with that of Skunavirus p2, with a receptor binding protein anchored to the virion by a distal tail protein loop. This suggests that they exhibit an activation mechanism similar to that of phage p2 prior to host binding.

The microbial communities present within fermented foods are diverse and dynamic, producing a variety of metabolites responsible for the fermentation processes, imparting characteristic organoleptic qualities and health-promoting traits, and maintaining microbiological safety of fermented foods. In this context, it is crucial to study these microbial communities to characterise fermented foods and the production processes involved. High Throughput Sequencing (HTS)-based methods such as metagenomics enable microbial community studies through amplicon and shotgun sequencing approaches. As the field constantly develops, sequencing technologies are becoming more accessible, affordable and accurate with a further shift from short read to long read sequencing being observed. Metagenomics is enjoying wide-spread application in fermented food studies and in recent years is also being employed in concert with synthetic biology techniques to help tackle problems with the large amounts of waste generated in the food sector. This review presents an introduction to current sequencing technologies and the benefits of their application in fermented foods.

Abstract Background Lactobacillus brevis is a member of the lactic acid bacteria (LAB), and strains of L. brevis have been isolated from silage, as well as from fermented cabbage and other fermented foods. However, this bacterium is also commonly associated with bacterial spoilage of beer. Results In the current study, complete genome sequences of six isolated L. brevis strains were determined. Five of these L. brevis strains were isolated from beer (three isolates) or the brewing environment (two isolates), and were characterized as beer-spoilers or non-beer spoilers, respectively, while the sixth isolate had previously been isolated from silage. The genomic features of 19 L. brevis strains, encompassing the six L. brevis strains described in this study and thirteen L. brevis strains for which complete genome sequences were available in public databases, were analyzed with particular attention to evolutionary aspects and adaptation to beer. Conclusions Comparative genomic analysis highlighted evolution of the taxon allowing niche colonization, notably adaptation to the beer environment, with approximately 50 chromosomal genes acquired by L. brevis beer-spoiler strains representing approximately 2% of their total chromosomal genetic content. These genes primarily encode proteins that are putatively involved in oxidation-reduction reactions, transcription regulation or membrane transport, functions that may be crucial to survive the harsh conditions associated with beer. The study emphasized the role of plasmids in beer spoilage with a number of unique genes identified among L. brevis beer-spoiler strains.

Abstract Whole metagenome shotgun (WMGS) sequencing is a method that provides insights into the genomic composition and arrangement of complex microbial consortia. Here, we report how WMGS coupled with a cultivation approach allows the isolation of novel bifidobacteria from animal fecal samples. A combination of in silico analyses based on nucleotide and protein sequences facilitate the identification of genetic material belonging to putative novel species. Consequently, the prediction of metabolic properties by in silico analyses permits the identification of specific substrates that are then employed to isolate these species through a cultivation method.

Some microorganisms may be transferred across the food production chain and, potentially, colonize the human gut. Here, Milani et al. provide strain-level evidence supporting that dairy cattle-associated bacteria can be transferred to the human gut via consumption of Parmesan cheese.

Abstract Bifidobacteria are among the first and most abundant bacterial colonizers of the gastrointestinal tract of (breast-fed) healthy infants. Their success of colonising the infant gut is believed to be, at least partly, due to their ability to metabolize available carbon sources by means of secreted or intracellular glycosyl hydrolases (GHs). Among these, β-galactosidases are particularly relevant as they allow bifidobacteria to grow on β-galactosyl-linked saccharidic substrates, which are present in copious amounts in the milk-based diet of their infant host (e.g. lactose and human milk oligosaccharides). In the present study we employed an in silico analysis to identify GH family 2 and 42 β-galactosidases encoded by typical infant-associated bifidobacteria. Comparative genome analysis followed by characterisation of selected β-galactosidases revealed how these GH2 and GH42 members are distributed among these infant-associated bifidobacteria, while their hydrolytic activity towards growth substrates commonly available in the infant gut were also assessed.