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Aim: Bifidobacterium longum subsp. infantis uses a glycoside hydrolase (GH) family 42 β-galactosidase (BiBga42A) for hydrolyzing lacto-N-tetraose (LNT), which is the most abundant core structure of human milk oligosaccharides (HMOs). As such, BiBga42A represents one of the pivotal enzymes underpinning the symbiosis between bifidobacteria and breastfed infants. Despite its importance, the structural basis underlying LNT hydrolysis by BiBga42A is not understood. Moreover, no substrate-complexed structures are available to date for GH42 family members.Methods: X-ray crystallography was used to determine the structures of BiBga42A in the apo- and liganded forms. The roles of the amino acid residues that were presumed to be involved in catalysis and substrate recognition were examined by a mutational study, in which kinetic parameters of each mutant were determined using 4-nitrophenyl-β-D-galactoside, lacto-N-biose I, LNT, and lacto-N-neotetraose (LNnT) as substrates. Conservation of those amino acid residues was examined among structure-determined GH42 β-galactosidases.Results: Crystal structures of the wild-type enzyme complexed with glycerol, the E160A/E318A double mutant complexed with galactose (Gal), and the E318S mutant complexed with LNT were determined at 1.7, 1.9, and 2.2 Å resolutions, respectively. The LNT molecule (excluding the Gal moiety at subsite +2) bound to the E318S mutant is recognized by an extensive hydrogen bond network and several hydrophobic interactions. The non-reducing end Gal moiety of LNT adopts a slightly distorted conformation and does not overlap well with the Gal molecule bound to the E160A/E318A mutant. Twelve of the sixteen amino acid residues responsible for LNT recognition and catalysis in BiBga42A are conserved among all homologs including β-1,6-1,3-galactosidase (BlGal42A) from Bifidobacterium animalis subsp. lactis.Conclusion: BlGal42A is active on 3-β-galactobiose similarly to BiBga42A but is inactive on LNT. Interestingly, we found that the entrance of the catalytic pocket of BlGal42A is narrower than that of BiBga42A and seems not easily accessible from the solvent side due to the presence of two bulky amino acid side chains. The specificity difference may reflect the structural difference between the two enzymes. LESS      Full article

Delirium is a clinical syndrome characterized by an acute change in attention, awareness and cognition with fluctuating course, frequently observed in older patients during hospitalization for acute medical illness or after surgery. Its pathogenesis is multifactorial and still not completely understood, but there is general consensus on the fact that it results from the interaction between an underlying predisposition, such as neurodegenerative diseases, and an acute stressor acting as a trigger, such as infection or anesthesia. Alterations in brain insulin sensitivity and metabolic function, increased blood-brain barrier permeability, neurotransmitter imbalances, abnormal microglial activation and neuroinflammation have all been involved in the pathophysiology of delirium. Interestingly, all these mechanisms can be regulated by the gut microbiota, as demonstrated in experimental studies investigating the microbiota-gut-brain axis in dementia. Aging is also associated with profound changes in gut microbiota composition and functions, which can influence several aspects of disease pathophysiology in the host. This review provides an overview of the emerging evidence linking age-related gut microbiota dysbiosis with delirium, opening new perspectives for the microbiota as a possible target of interventions aimed at delirium prevention and treatment. LESS      Full article

Aim: The “gut-joint” axis is suspected to be involved in the pathophysiology of osteoarthritis (OA). The present study aims at investigating the potential of lipoproteins (Lpps) secreted by Bifidobacterium longum to alleviate OA progression in the rat.Methods: Experimental OA was induced in rats harbouring Schaedler Flora maintained in SPF conditions. Two weeks post-injection, 20 rats were randomized to water (n = 10) or 0.3 mg/L Lpps solution (n = 10). Weight and food intake were monitored for 6 weeks. At sacrifice, joints were scored using macroscopic and histological criteria. Serum LPS, Schaedler flora as well as selected intestinal bacteria were analyzed.Results: Lpps intake prevents OA progression. The protected rats showed a significant increase in lactobacilli along the intestine as well as in Mucispirillum schaedleri in the colon and a significant decrease in Parabacteroides goldsteini and Akkermansia in caecum and colon, respectively. There was no significant difference in serum lipopolysaccharide or bacteria translocating in Peyer's patches. Labelled Lpps were not detected in bone marrow of the OA joint. The principal component analysis points out that OA prevention is primarily associated with bacteria involved in the tryptophane degradation pathway and SCFA formation.Conclusion: In rats deprived of bifidobacteria, intake of B.longum Lpps prevented OA development and modulated the intestinal microbiome with a possible impact on the bacterial end-products. The link between Lpps and the gut microbial metabolome warrants further investigation. LESS      Full article

The human gut is home to trillions of microorganisms that influence several aspects of our health. This dense microbial community targets almost all dietary polysaccharides and releases multiple metabolites, some of which have physiological effects on the host. A healthy equilibrium between members of the gut microbiota, its microbial diversity, and their metabolites is required for intestinal health, promoting regulatory or anti-inflammatory immune responses. In contrast, the loss of this equilibrium due to antibiotics, low fiber intake, or other conditions results in alterations in gut microbiota composition, a term known as gut dysbiosis. This dysbiosis can be characterized by a reduction in health-associated microorganisms, such as butyrate-producing bacteria, enrichment of a small number of opportunistic pathogens, or a reduction in microbial diversity. Bifidobacterium species are key species in the gut microbiome, serving as primary degraders and contributing to a balanced gut environment in various ways. Colonization resistance is a fundamental property of gut microbiota for the prevention and control of infections. This community competes strongly with foreign microorganisms, such as gastrointestinal pathogens, antibiotic-resistant bacteria, or even probiotics. Resistance to colonization is based on microbial interactions such as metabolic cross-feeding, competition for nutrients, or antimicrobial-based inhibition. These interactions are mediated by metabolites and metabolic pathways, representing the inner workings of the gut microbiota, and play a protective role through colonization resistance. This review presents a rationale for how microbial interactions provide resistance to colonization and gut dysbiosis, highlighting the protective role of Bifidobacterium species. LESS      Full article

Although research on the role of the gut microbiota (GM) in human health has sharply increased in recent years, what a “healthy” gut microbiota is and how it responds to major stressors is still difficult to establish. In particular, anticancer chemotherapy is known to have a drastic impact on the microbiota structure, potentially hampering its recovery with serious long-term consequences for patients’ health. However, the distinguishing features of gut microbiota recovery and non-recovery processes are not yet known. In this narrative review, we first investigated how gut microbiota layouts are affected by anticancer chemotherapy and identified potential gut microbial recovery signatures. Then, we discussed microbiome-based intervention strategies aimed at promoting resilience, i.e., the rapid and complete recovery of a healthy gut microbial network associated with a better prognosis after such high-impact pharmacological treatments. LESS      Full article

The reconstruction of microbial genome sequences by bioinformatic pipelines and the consequent functional annotation of their genes’ repertoire are fundamental activities aiming at unveiling their biological mechanisms, such as metabolism, virulence factors, and antimicrobial resistances. Here, we describe the development of the MEGAnnotator2 pipeline able to manage all next-generation sequencing methodologies producing short- and long-read DNA sequences. Starting from raw sequencing data, the updated pipeline can manage multiple analyses leading to the assembly of high-quality genome sequences and the functional classification of their genetic repertoire, providing the user with a useful report constituting features and statistics related to the microbial genome. The updated pipeline is fully automated from the installation to the delivery of the output, thus requiring minimal bioinformatics knowledge to be executed. LESS      Full article

Inclusion and investigation of technical controls in microbiome sequencing studies is important for understanding technical biases and errors. Here, we present chkMocks, a general R-based tool that allows researchers to compare the composition of mock communities that are processed along with samples to their theoretical composition. A visual comparison between experimental and theoretical community composition and their correlation is provided for researchers to assess the quality of their sample processing workflows. LESS      Full article

Aim:Clostridia are relevant commensals of the human gut due to their major presence and correlations to the host. In this study, we investigated intestinal Clostridia of 51 healthy subjects and reconstructed their taxonomy and phylogeny. The relatively small number of intestinal Clostridia allowed a systematic whole genome approach based on average amino acid identity (AAI) and core genome with the aim of revising the current classification into genera and determining evolutionary relationships.Methods: 51 healthy subjects’ metagenomes were retrieved from public databases. After the dataset’s validation through comparison with Human Microbiome Project (HMP) samples, the metagenomes were profiled using MetaPhlAn3 to identify the population ascribed to the class Clostridia. Intestinal Clostridia genomes were retrieved and subjected to AAI analysis and core genome identification. Phylogeny investigation was conducted with RAxML and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithms, and SplitsTree for split decomposition.Results: 225 out of 406 bacterial taxonomic units were ascribed to Bacillota [Firmicutes], among which 124 were assigned to the class Clostridia. 77 out of the 124 taxonomic units were referred to a species, altogether covering 87.7% of Clostridia abundance. According to the lowest AAI genus boundary set at 55%, 15 putative genera encompassing more than one species (G1 to G15) were identified, while 19 species did not cluster with any other one and each appeared to belong to a diverse genus. Phylogenetic investigations highlighted that most of the species clustered into three main evolutive clades.Conclusion: This study shed light on the species of Clostridia colonizing the gut of healthy adults and pinpointed several gaps in knowledge regarding the taxonomy and the phylogeny of Clostridia. LESS      Full article

Aim: Dietary plant fibers affect gut microbiota composition; however, the underlying microbial degradation pathways are not fully understood. We previously discovered 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase), a glycoside hydrolase family 39 enzyme involved in the assimilation of side chains of arabinogalactan protein (AGP), from Bifidobacterium longum subsp. longum (B. longum) JCM7052. Although GAfase homologs are not highly prevalent in the Bifidobacterium genus, several Bifidobacterium strains possess the homologs. To explore the differences in substrate specificity among the homologs, a homolog of B. longum GAfase in Bifidobacterium pseudocatenulatum MCC10289 (MCC10289_0425) was characterized.Methods: Gum arabic, larch, wheat AGP, and sugar beet arabinan were used to determine the substrate specificity of the MCC10289_0425 protein. An amino acid replacement was introduced into GAfase to identify a critical residue that governs the differentiation of substrate specificity. The growth of several Bifidobacterium strains on β-L-arabinopyranosyl disaccharide and larch AGP was examined.Results: MCC10289_0425 was identified to be an unprecedented 3-O-β-L-arabinopyranosyl-α-L-arabinofuranosidase (AAfase) with low GAfase activity. A single amino acid replacement (Asn¹¹⁹ to Tyr) at the catalytic site converted GAfase into AAfase. AAfase releases sugar source from AGP, thereby allowing B. pseudocatenulatum growth.Conclusion: Bifidobacteria have evolved several homologous enzymes with overlapping but distinct substrate specificities depending on the species. They have acquired different fitness abilities to respond to diverse plant polysaccharide structures. LESS      Full article

Background: Development and maturation of the immune system begin in utero and continue throughout the neonatal period. Both the maternal and neonatal gut microbiome influence immune development, but the relative importance of the prenatal and postnatal periods is unclear.Methods: In the present study, we characterized immune cell populations in mice in which the timing of microbiome colonization was strictly controlled using gnotobiotic methodology.Results: Compared to conventional (CONV) mice, germ-free (GF) mice conventionalized at birth (EC mice) showed few differences in immune cell populations in adulthood, explaining only 2.36% of the variation in immune phenotypes. In contrast, delaying conventionalization to the fourth week of life (DC mice) affected seven splenic immune cell populations in adulthood, including dendritic cells and regulatory T cells (Tregs), explaining 29.01% of the variation in immune phenotypes. Early life treatment of DC mice with Limosilactobacillus reuteri restored splenic dendritic cells and Tregs to levels observed in EC mice, and there were strain-specific effects on splenic CD4+ T cells, CD8+ T cells, and CD11c+ F4/80+ mononuclear phagocytes.Conclusion: This work demonstrates that the early postnatal period, compared to the prenatal period, is relatively more important for microbial signals to influence immune development in mice. Our findings further show that targeted microbial treatments in early life can redress adverse effects on immune development caused by the delayed acquisition of the neonatal gut microbiome. LESS      Full article

Aim: This study is mainly devoted to determining the ability of ∆FN3.1 protein fragments of Bifidobacterium (B.) longum subsp. longum GT15, namely two FN3 domains (2D FN3) and a C-terminal domain (CD FN3), to bind to tumor necrosis factor-alpha (TNF-α).Methods: Fragments of the fn3 gene encoding the 2D FN3 and CD FN3 were cloned in Escherichia (E.) coli. In order to assess the binding specificity between 2D FN3 and CD FN3 to TNFα, we employed the previously developed sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. The trRosetta software was used to build 3D models of the ∆FN3.1, 2D FN3, and CD FN3 proteins. The detection of polymorphism in the amino acid sequences of the studied proteins and the analysis of human gut-derived bacterial proteins carrying FN3 domains were performed in silico.Results: We experimentally showed that neither 2D FN3 nor CD FN3 alone can bind to TNFα. Prediction of the 3D structures of ΔFN3.1, 2D FN3, and CD FN3 suggested that only ΔFN3.1 can form a pocket allowing binding with TNFα to occur. Polymorphism analysis of amino acid sequences of ΔFN3.1 proteins in B. longum strains uncovered substitutions that can alter the conformation of the spatial structure of the ΔFN3.1 protein. We also analyzed human gut-derived bacterial proteins harboring FN3 domains which allowed us to differentiate between those containing motifs of cytokine receptors (MCRs) in their FN3 domains and those lacking them.Conclusion: Only the complete ∆FN3.1 protein can selectively bind to TNFα. Analysis of 3D models of the 2D FN3, CD FN3, and ΔFN3.1 proteins showed that only the ΔFN3.1 protein is potentially capable of forming a pocket allowing TNFα binding to occur. Only FN3 domains containing MCRs exhibited sequence homology with FN3 domains of human proteins. LESS      Full article

Aim: The human gut Bifidobacterium community has been studied in detail in infants and following dietary interventions in adults. However, the variability of the distribution of Bifidobacterium species and intra-species functions have been little studied, particularly beyond infancy. Here, we explore the ecology of Bifidobacterium communities in a large public dataset of human gut metagenomes, mostly corresponding to adults.Methods: We selected 9.515 unique gut metagenomes from curatedMetagenomicData. Samples were partitioned by applying Dirichlet’s multinomial mixture to Bifidobacterium species. A functional analysis was performed on > 2.000 human-associated Bifidobacterium metagenome-assembled genomes (MAGs) paired with participant gut microbiome and health features.Results: We identified several Bifidobacterium-based partitions in the human gut microbiome differing in terms of the presence and abundance of Bifidobacterium species. The partitions enriched in both B. longum and B. adolescentis were associated with gut microbiome diversity and a higher abundance of butyrate producers and were more prevalent in healthy individuals. B. bifidum MAGs harboring a set of genes potentially related to phages were more prevalent in partitions associated with a lower gut microbiome diversity and were genetically more closely related.Conclusion: This study expands our knowledge of the ecology and variability of the Bifidobacterium community, particularly in adults, and its specific association with the gut microbiota and health. Its findings may guide the rational selection of Bifidobacterium strains for gut microbiome complementation according to the individual’s endogenous Bifidobacterium community. Our results also suggest that gut microbiome stratification for particular genera may be relevant for studies of variations of species and associations with the gut microbiome and health. LESS      Full article

Human breast milk (HBM) is the main source of nutrition for neonates across the critical early-life developmental period. The highest demand for energy is due to rapid neurophysiological expansion post-delivery, which is largely met by human milk lipids (HMLs). These HMLs also play a prebiotic role and potentially promote the growth of certain commensal bacteria, which, via HML digestion, supports the additional transfer of energy to the infant. In tandem, HMLs can also exert bactericidal effects against a variety of opportunistic pathogens, which contributes to overall colonisation resistance. Such interactions are pivotal for sustaining homeostatic relationships between microorganisms and their hosts. However, the underlying molecular mechanisms governing these interactions remain poorly understood. This review will explore the current research landscape with respect to HMLs, including compositional considerations and impact on the early life gut microbiota. Recent papers in this field will also be discussed, including a final perspective on current knowledge gaps and potential next research steps for these important but understudied breast milk components. LESS      Full article

The Human Microbiome Project (HMP) has raised great expectations claiming the far-reaching influence of the microbiome on human health and disease ranging from obesity and malnutrition to effects going well beyond the gut. So far, with the notable exception of fecal microbiota transplantation in Clostridioides difficile infection, practical application of microbiome intervention has only achieved modest clinical effects. It is argued here that we need criteria for the link between microbiome and disease modelled on the links between pathogens and infectious disease in Koch’s postulates. The most important question is whether the microbiome change is a cause of the given disease or a consequence of a pathology leading to disease where the microbiome change is only a parallel event without a causal connection to the disease – in philosophical parlance, an epiphenomenon. Also discussed here is whether human virome research is a necessary complement to the microbiome project with a high potential for practical applications. LESS      Full article

Akkermansia muciniphila is a promising next-generation beneficial microbe due to its natural presence in the mucus layer of the gut, its symbiotic ability to degrade mucus, and its capacity to improve the intestinal barrier function. A. muciniphila is able to counteract weight gain and immuno-metabolic disturbances in several animal models. Many of these disorders, including obesity and auto-immune diseases, have been associated with decreased gut barrier function and consequent increased inflammation. Since A. muciniphila was found to normalize these changes and strengthen the gut barrier function, it is hypothesized that other beneficial effects of A. muciniphila might be caused by this restoration. In search for A. muciniphila’s mode of action in enhancing the gut barrier function and promoting health, we reasoned that secreted components or cell envelope components of A. muciniphila are interesting candidates as they can potentially reach and interact with the epithelial barrier. In this review, we focus on the potential mechanisms through which A. muciniphila can exert its beneficial effects on the host by the production of extracellular and secreted proteins, metabolites and cell envelope components. These products have been studied in isolation for their structure, signaling capacity, and in some cases, also for their effects in preclinical models. This includes the protein known as Amuc_1100, which we here rename as pilus-associated signaling (PAS) protein , the P9 protein encoded by Amuc_1631, the short-chain fatty acids acetate and propionate, and cell envelope components, such as phosphatidylethanolamine and peptidoglycan. LESS      Full article

Aim: There is growing evidence that physical activity modulates gut microbiota composition through complex interactions between diet and microbial species. On the other hand, next-generation sequencing techniques include shotgun metagenomics and 16S amplicon sequencing. These methodologies allow a comprehensive characterisation of microbial communities of athletes from different disciplines as well as non-professional players and sedentary adults exposed to training. This systematic review summarises recent applications of next-generation sequencing to characterise the athletic gut microbiome.Methods: A systematic review of microbiome research was performed to determine the association of microbiota composition profiles with sports performance.Results: Bibliographic analysis revealed the importance of a novel research trend aiming at deciphering the associations between individual microbial species and sports performance. In addition, literature review highlighted the role of butyrate-producing bacteria such as Anaerostipes hadrus, Clostridium bolteae, Faecalibacterium prausnitzii, Roseburia hominis and unidentified species belonging to Clostridiales, Lachnospiraceae and Subdoligranulum species in gut health and sports performance across several disciplines. Interestingly, metabolic activities of Prevotella copri and Veillonella atypica involved in branched amino acid and lactate metabolism may contribute to reducing muscular fatigue. Other microbial metabolic pathways of interest involved in carbohydrate metabolism showed increased proportions in athletes´ metagenomes.Conclusion: Future research will aim at developing personalised nutrition interventions to modulate key species associated with certain components of exercise. LESS      Full article

Aims: C16 monounsaturated fatty acid (C16:1) show antibacterial activity against Staphylococcus aureus, a pathogen associated with various diseases such as atopic dermatitis and bacteremia, while the compound does not exhibit antibacterial activity against Staphylococcus epidermidis, an epidermal commensal that inhibits the growth of S. aureus. In this study, we aimed to find bifidobacterial strains with the ability to produce C16:1 and to find a practical manner to utilize C16:1-producing strains in industry.Methods: Various Bifidobacterium strains were screened for their content of C16:1. The chemical identity of C16:1 produced by a selected strain was analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Medium components that affect the C16:1 content of the selected strain were investigated. Antibacterial activity against staphylococci was compared between the authentic C16:1 isomers and total fatty acids (TFA) extracted from the selected strain.Results: B. adolescentis 12451, B. adolescentis 12-111, B. boum JCM 1211, and Bifidobacterium sp. JCM 7042 showed high C16:1 content among the tested strains. TFA extracted from Bifidobacterium sp. JCM 7042 contained C16:1 at 2.3% as the fatty acid constituent (2.4 mg/L of broth). Through GC-MS and LC-MS analyses, the C16:1 synthesized by Bifidobacterium sp. JCM 7042 was identified as 7-cis-hexadecenoic acid (7-cis-C16:1). The authentic 7-cis-C16:1 showed strong and selective antibacterial activity against S. aureus, similar to 6-cis-C16:1, with a minimum inhibitory concentration (MIC) of < 10 µg/mL. Components that increase C16:1 productivity were not found in the MRS and TOS media; however, Tween 80 was shown to considerably reduce the C16:1 ratio in TFA. Antibacterial activity against S. aureus was observed when the TFA extracted from Bifidobacterium sp. JCM 7042 contained high level of 7-cis-C16:1 (6.1% in TFA) but not when it contained low level of 7-cis-C16:1 (0.1% in TFA).Conclusion: The fatty acid, 7-cis-C16:1, which can selectively inhibit the S. aureus growth, is accumulated in TFA of several bifidobacteria. The TFA extracted from cultured cells of Bifidobacterium sp. JCM 7042 demonstrated antibacterial activity. From a practical viewpoint, our findings are important for developing an efficient method to produce novel skin care cosmetics, functional dairy foods, and other commodities. LESS      Full article

Despite multiple advances in medicine, the management of urinary tract infections (UTIs) in women has remained stalled for decades. To prevent the development of symptomatic recurrences, low-dose antibiotics are the mainstay, while alternative approaches have been attempted with limited success. The use of probiotics was first considered forty years ago, and while some promising studies have been published, additional evidence in larger patient groups is needed to recommend specific strains as a primary preventive regimen. Overall, the role of beneficial microbes in reducing the risk of UTI and other urological diseases, such as urolithiasis, remains a target for researchers. The aim of this perspective is to offer a viewpoint on the status of this approach and recommendations for how to develop novel probiotic therapies. LESS      Full article

The human gut microbiota (HGM) plays a pivotal role in health and disease. Consequently, nutritional and medical research focusing on HGM modulation strategies as a means of improving host health is steadily increasing. In vitro HGM fermentation models offer a valid complement to human and animal studies when it comes to the mechanistic exploration of novel modulation approaches and their direct effects on HGM composition and activity, while excluding interfering host effects. However, in vitro cultivation of HGM can be challenging due to its high oxygen sensitivity and the difficulties of accurately modeling the physio-chemical complexity of the gut environment. Despite the increased use of in vitro HGM models, there is no consensus about appropriate model selection and operation, sometimes leading to major deficiencies in study design and result interpretation. In this review paper, we aim to analyze crucial aspects of the application, setup and operation, data validation and result interpretation of in vitro HGM models. When carefully designed and implemented, in vitro HGM modeling is a powerful strategy for isolating and investigating biotic and abiotic factors in the HGM, as well as evaluating their effects in a controlled environment akin to the gut. Furthermore, complementary approaches combining different in vitro and in vivo models can strengthen the design and interpretation of human studies. LESS      Full article

Recent years have seen the development of high-accuracy and high-throughput genetic manipulation techniques, which have greatly improved our understanding of genetically tractable microbes. However, challenges remain in establishing genetic manipulation techniques in novel organisms, owing largely to exogenous DNA defence mechanisms, lack of selectable markers, lack of efficient methods to introduce exogenous DNA and an inability of genetic vectors to replicate in their new host. In this review, we describe some of the techniques that are available for genetic manipulation of novel microorganisms. While many reviews exist that focus on the final step in genetic manipulation, the editing of recipient DNA, we particularly focus on the first step in this process, the transfer of exogenous DNA into a strain of interest. Examples illustrating the use of these techniques are provided for a selection of human gut bacteria in which genetic tractability has been established, such as Bifidobacterium, Bacteroides and Roseburia. Ultimately, this review aims to provide an information source for researchers interested in developing genetic manipulation techniques for novel bacterial strains, particularly those of the human gut microbiota. LESS      Full article

The human gut microbiota is believed to be responsible for multiple health-impacting host effects. The influence of gut microorganisms on the human host begins immediately after birth, having long-lasting health effects, while the gut microbiota itself continues to develop throughout the host’s entire life. The purported health-associated effects of the gut microbiota have fueled extensive and ongoing research efforts. Nonetheless, the precise mode of action of functionalities exerted by microbial colonizers of the infant intestine is still largely unknown. The current perspective intends to illustrate major future investigative directions concerning the human gut microbiota with a specific focus on infant-associated gut microbes. LESS      Full article

The gut-brain axis is gaining momentum as an interdisciplinary field addressing how intestinal microbes influence the central nervous system (CNS). Studies using powerful tools, including germ-free, antibiotic-fed, and fecal microbiota transplanted mice, demonstrate how gut microbiota perturbations alter the fate of neurodevelopment. Probiotics are also becoming more recognized as potentially effective therapeutic agents in alleviating symptoms of neurological disorders. While gut microbes may directly communicate with the CNS through their effector molecules, including metabolites, their influence on neuroimmune populations, including newly discovered brain-resident T cells, underscore the host immunity as a potent mediator of the gut-brain axis. In this review, we examine the unique immune populations within the brain, the effects of the gut microbiota on the CNS, and the efficacy of specific probiotic strains to propose the novel concept of the gut-immune-brain axis. LESS      Full article

The perinatal period sets the basis for the later physiological and immune homeostasis of the individual, with the intestinal microbiota being an important contributor to driving this homeostasis development. Therefore, the initial establishment and later development of the microbiota during early life may play a key role in later health. This early establishment of the intestinal microbiota is known to be affected by several factors, with gestational age, delivery mode, and feeding habits being extensively studied ones. Other factors are not so well understood, although knowledge has been accumulating in the last years. Among them, a factor of great relevance is the effect of perinatal exposure to antibiotics. Administration of intrapartum antimicrobial prophylaxis (IAP) to women during the delivery process represents the most common form of exposure to antibiotics during the perinatal period, present in around 30% of deliveries. During the last decade, evidence has accumulated demonstrating that IAP alters intestinal microbiota development in neonates. Moreover, recent evidence indicates that this practice may also be altering the infant intestinal resistome by increasing the levels of some antibiotic resistance genes. This evidence, as reviewed in this manuscript, suggests the interest in promoting the rational use of IAP. This practice has significantly reduced the risk of neonatal infections, but now the accumulating knowledge suggests the need for strategies to minimize its impact on the neonatal microbiota establishment. LESS      Full article

Background: The ability of probiotic strains to provide health benefits to the host partially hinges on the survival of gastrointestinal passage and temporary colonization of the digestive tract. This study aims to investigate the colonization profile of individual probiotic strains comprising the commercial product VSL#3^® and determine their impact on the host intestinal microbiota.Methods: Using a cefoperazone-treated mouse model of antibiotic treatment, we investigated the impact of oral gavage with ~10⁸ CFU commercial VSL#3^® product on the intestinal microbiota using 16S-based amplicon sequencing over 7 days. Results: Results showed that probiotic strains in the formulation were detected in treated murine fecal samples, with early colonization by Streptococcus thermophilus and Lactiplantibacillus plantarum subsp. plantarum, and late colonization by Lacticaseibacillus paracasei subsp. paracasei, Bifidobacterium breve and Bifidobacterium animalis subsp. lactis. Overall, VSL#3^® consumption is associated with increased alpha diversity in the cecal microbial community, which is important in the context of antibiotic consumption. Probiotic supplementation resulted in an expansion of Proteobacteria, Bacteroidetes, and Actinobacteria, especially Bifidobacteriaceae and Lachnospiraceae, which are associated with Clostridioides difficile resistance in the murine gut.Conclusion: This study illustrates the need for determining the ability of probiotics to colonize the host and impact the gut microbiota, and suggests that multiple doses may be warranted for extended transient colonization. In addition, follow-up studies should determine whether VSL#3^® can provide resistance against C. difficile colonization and disease in a mouse model. LESS      Full article

Aim: 16S rRNA gene-based microbiota analyses (16S metagenomes) using next-generation sequencing (NGS) technologies are widely used to examine the microbial community composition in environmental samples. However, the sequencing capacity of NGS is sometimes insufficient to cover the whole microbial community, especially when analyzing soil and fecal microbiotas. This limitation may have hampered the detection of minority species that potentially affect microbiota formation and structure.Methods: We developed a simple method, termed 16S metagenome-DRIP (Deeper Resolution using an Inhibitory Primer), that not only enhances minority species detection but also increases the accuracy of their abundance estimation. The method relies on the inhibition of normal amplicon formation of the 16S rRNA gene of a target major (abundant) species during the first PCR step. The addition of a biotinylated primer that is complementary to the variable sequence of the V3-V4 region of the target species inhibits a normal amplification process to form an aberrant short amplicon. The fragment is then captured by streptavidin beads for removal from the reaction mixture, and the resulting mixture is utilized for the second PCR with barcode-tag primers. Thus, this method only requires two additional experimental procedures to the conventional 16S metagenome analysis. A proof-of-concept experiment was first conducted using a mock sample consisting of the genomes of 14 bacterial species. Then, the method was applied to infant fecal samples using a Bifidobacterium-specific inhibitory primer (n = 11).Results: As a result, the reads assigned to the family Bifidobacteriaceae decreased on average from 16,657 to 1718 per sample without affecting the total read counts (36,073 and 34,778 per sample for the conventional and DRIP methods, respectively). Furthermore, the minority species detection rate increased with neither affecting Bray-Curtis dissimilarity calculated by omitting the target Bifidobacterium species (median: 0.049) nor changing the relative abundances of the non-target species. While 115 amplicon sequence variants (ASVs) were unique to the conventional method, 208 ASVs were uniquely detected for the DRIP method. Moreover, the abundance estimation for minority species became more accurate, as revealed thorough comparison with the results of quantitative PCR analysis.Conclusion: The 16S metagenome-DRIP method serves as a useful technique to grasp a deeper and more accurate microbiota composition when combined with conventional 16S metagenome analysis methods. LESS      Full article

Human milk (HM) is the gold standard for infant nutrition during the first months of life. Beyond its nutritional components, its complex bioactive composition includes microorganisms, their metabolites, and oligosaccharides, which also contribute to gut colonization and immune system maturation. There is growing evidence of the beneficial effects of bacteria present in HM. However, current research presents limited data on the presence and functions of other organisms. The potential biological impacts on maternal and infant health outcomes, the factors contributing to milk microbes’ variations, and the potential functions in the infant’s gut remain unclear. This review provides a global overview of milk microbiota, what the actual knowledge is, and what the gaps and challenges are for the next years. LESS      Full article

A microbiome consists of microbes and their genomes, encompassing bacteria, viruses, fungi, protozoa, archaea, and eukaryotes. These elements interact dynamically in the specific environment in which they reside and evolve. In the past decade, studies of various microbiomes have been prevalent in the scientific literature, accounting for the shift from culture-dependent to culture-independent identification of microbes using new high-throughput sequencing technologies that decipher their composition and sometimes provide insights into their functions. Despite tremendous advances in understanding the gut microbiome, relatively little attention has been devoted to the vaginal environment, notably regarding the ubiquity and diversity of glycans which denote the significant role they play in the maintenance of homeostasis. Hopefully, emerging technologies will aid in the determination of what is a healthy vaginal microbiome, and provide insights into the roles of Lactobacillus, glycans and microbiome-related drivers of health and disease. LESS      Full article

Alterations in the intestinal microbiota are associated with various human diseases of the digestive system, including obesity and its associated metabolic diseases, inflammatory bowel diseases (IBD), and colorectal cancer (CRC). All three diseases are characterized by modifications of the richness, composition, and metabolic functions of the human intestinal microbiota. Despite being multi-factorial diseases, studies in germ-free animal models have unarguably identified the intestinal microbiota as a causal driver of disease pathogenesis. However, for an increased mechanistic understanding of microbial signatures in human diseases, models require detailed refinement to closely mimic the human microbiota and reflect the complexity and range of dysbiosis observed in patients. The transplantation of human fecal microbiota into animal models represents a powerful tool for studying the causal and functional role of the dysbiotic human microbiome in a pathological context. While human microbiota-associated models were initially employed to study obesity, an increasing number of studies have applied this approach in the context of IBD and CRC over the past decade. In this review, we discuss different approaches that allow the functional validation of the bacterial contribution to human diseases, with emphasis on obesity and its associated metabolic diseases, IBD, and CRC. We discuss the utility of simple models, such as in vitro fermentation systems of the human microbiota and ex vivo intestinal organoids, as well as more complex whole organism models. Our focus here lies on human microbiota-associated mouse models in the context of all three diseases, as well as highlighting the advantages and limitations of this approach. LESS      Full article

For decades, (poly)phenols have been linked to cardiometabolic health, but population heterogeneity limits their apparent efficacy and the development of tailored, practical protocols in dietary interventions. This heterogeneity is likely determined by the existence of different metabotypes, sub-populations of individuals metabolizing some classes of (poly)phenols differently. The gut microbiota plays a major role in this process. The impact of microbiota-related phenolic metabotypes on cardiometabolic health is becoming evident, although the picture is still incomplete, and data are absent for some classes of (poly)phenols. The lack of a complete understanding of the main microbial actors involved in the process complicates the picture. Elucidation of the mechanisms behind phenolic metabotypes requires novel experimental designs that can dissect the inter-individual variability. This paper, in addition to providing an overview on the current state-of-the-art, proposes wider metabotyping approaches as a means of paving the way towards effective personalized nutrition with dietary (poly)phenols. LESS      Full article

Genome sequencing has fundamentally changed our ability to decipher and understand the genetic blueprint of life and how it changes over time in response to environmental and evolutionary pressures. The pace of sequencing is still increasing in response to advances in technologies, paving the way from sequenced genes to genomes to metagenomes to metagenome-assembled genomes (MAGs). Our ability to interrogate increasingly complex microbial communities through metagenomes and MAGs is opening up a tantalizing future where we may be able to delve deeper into the mechanisms and genetic responses emerging over time. In the near future, we will be able to detect MAG assembly variations within strains originating from diverging sub-populations, and one of the emerging challenges will be to capture these variations in a biologically relevant way. Here, we present a brief overview of sequencing technologies and the current state of metagenome assemblies to suggest the need to develop new data formats that can capture the genetic variations within strains and communities, which previously remained invisible due to sequencing technology limitations. LESS      Full article

The infant gut microbiota is the set of microorganisms colonizing the baby’s intestine. This complex ecosystem appears to be related to various physiological conditions of the host and it has also been shown to act as one of the most crucial determinants of infant’s health. Furthermore, the mother’s endocrine system, through its hormones, can have an effect on the composition of the newborn’s gut microbiota. In this perspective, we summarize the recent state of the art on the intricate relationships involving the intestinal microbiota and the endocrine system of mother/baby to underline the need to study the molecular mechanisms that appear to be involved. LESS      Full article

Intricate interactions among commensal bacteria, dietary substrates and immune responses are central to defining microbiome community composition, which plays a key role in preventing enteric pathogen infection, a dynamic phenomenon referred to as colonisation resistance. However, the impact of diet on sculpting microbiota membership, and ultimately colonisation resistance has been overlooked. Furthermore, pathogens have evolved strategies to evade colonisation resistance and outcompete commensal microbiota by using unique nutrient utilisation pathways, by exploiting microbial metabolites as nutrient sources or by environmental cues to induce virulence gene expression. In this review, we will discuss the interplay between diet, microbiota and their associated metabolites, and how these can contribute to or preclude pathogen survival. LESS      Full article

Elements of the human gut microbiota metabolise many host- and diet-derived, non-digestible carbohydrates (NDCs). Intestinal fermentation of NDCs salvages energy and resources for the host and generates beneficial metabolites, such as short chain fatty acids, which contribute to host health. The development of functional NDCs that support the growth and/or metabolic activity of specific beneficial gut bacteria, is desirable, but dependent on an in-depth understanding of the pathways of carbohydrate fermentation. The purpose of this review is to provide an appraisal of what is known about the roles of, and interactions between, Bacteroides and Bifidobacterium as key members involved in NDC utilisation. Bacteroides is considered an important primary degrader of complex NDCs, thereby generating oligosaccharides, which in turn can be fermented by secondary degraders. In this review, we will therefore focus on Bacteroides as an NDC-degrading specialist and Bifidobacterium as an important and purported probiotic representative of secondary degraders. We will describe cross-feeding interactions between members of these two genera. We note that there are limited studies exploring the interactions between Bacteroides and Bifidobacterium, specifically concerning β-glucan and arabinoxylan metabolism. This review therefore summarises the roles of these organisms in the breakdown of dietary fibre and the molecular mechanisms and interactions involved. Finally, it also highlights the need for further research into the phenomenon of cross-feeding between these organisms for an improved understanding of these cross-feeding mechanisms to guide the rational development of prebiotics to support host health or to prevent or combat disease associated with microbial dysbiosis. LESS      Full article

Irritable bowel syndrome (IBS) affects approximately one tenth of the general population and is characterized by abdominal pain associated with abnormalities in bowel habits. Visceral hypersensitivity, abnormal intestinal motor function, mucosal immune activation, and increased intestinal permeability concur to its pathophysiology. Psychological factors can influence symptom perception at the central nervous system level. In addition, recent evidence suggests that dysbiosis may be a key pathophysiological factor in patients with IBS. Increasing understanding of the pathophysiological mechanisms translates into new and more effective therapeutic approaches. Indeed, in line with this evidence, IBS therapies nowadays include agents able to modulate gut microbiota function and composition, such as diet, prebiotics, probiotics, and antibiotics. In addition, in the last decade, an increasing interest in fecal microbiota transplantation has been paid. An in-depth understanding of the intestinal microenvironment through accurate faucal microbiota and metabolite analysis may provide valuable insights into the pathophysiology of IBS, finally shaping new tailored IBS therapies. LESS      Full article

The human gut microbiome harbors a diverse range of microbes that play a fundamental role in the health and well-being of their host. The early-life microbiome has a major influence on human development and long-term health. Perinatal factors such as maternal nutrition, antibiotic use, gestational age and mode of delivery influence the initial colonization, development, and function of the neonatal gut microbiome. The perturbed early-life gut microbiome predisposes infants to diseases in early and later life. Understanding how perinatal factors guide and shape the composition of the early-life microbiome is essential to improving infant health. The following review provides a synopsis of perinatal factors with the most decisive influences on initial microbial colonization of the infant gut. LESS      Full article

Background: The World Health Organization defines probiotics as “live microorganisms, which when administered in adequate amounts confer a health benefit on the host”. In this framework, probiotic strains should be regarded as safe for human and animal consumption, i.e., they should possess the GRAS (generally recognized as safe) status, notified by the local authorities. Consistently, strains of selected Bifidobacterium species are extensively used as probiotic agents to prevent and ameliorate a broad spectrum of human and/or animal gastrointestinal disorders. Even though probiotic properties are often genus- or species-associated, strain-level differences in the genetic features conferring individual probiotic properties to commercialized bifidobacterial strains have not been investigated in detail. Methods: In this study, we built a genomic database named Integrated Probiotic DataBase (IPDB), whose first iteration consists of common bifidobacterial strains used in probiotic products for which public genome sequences were available, such as members of B. longum subsp. longum, B. longum subsp. infantis, B. bifidum, B. breve, and B. animalis subsp. lactis taxa. Furthermore, the IPDB was exploited to perform comparative genome analyses focused on genetic factors conferring structural, functional, and chemical features predicted to be involved in microbe-host and microbe-microbe interactions.Results and conclusion: Our analyses revealed strain-level genetic differences, underlining the importance of inspecting the strain-specific and outcome-specific efficacy of probiotics. In this context, IPDB represents a valuable resource for obtaining genetic information of well-established bifidobacterial probiotic strains. LESS      Full article

Next-generation sequencing technologies allow accomplishing massive DNA sequencing, uncovering the microbial composition of many different ecological niches. However, the various strategies developed to profile microbiomes make it challenging to retrieve a reliable classification that is able to compare metagenomic data of different studies. Many limitations have been overcome thanks to shotgun sequencing, allowing a reliable taxonomic classification of microbial communities at the species level. Since numerous bioinformatic tools and databases have been implemented, the sequencing methodology is only the first of many choices to make for classifying metagenomic data. Here, we discuss the importance of choosing a reliable methodology to achieve consistent information in uncovering microbiomes. LESS      Full article

Aim: The role of intestinal fungi in human health and disease is becoming more evident. The mycobiota composition and diversity of preterm infants is affected by interactions with bacteria and clinical variables. In this study, we aimed to characterize the composition and the diversity of the preterm infant mycobiota and the effect of clinical variables on it in the first six postnatal weeks.Methods: Preterm infants (n = 50) and full-term infants (n = 6) admitted to Isala Women and Children’s hospital (Zwolle, The Netherlands) who were born during 24-36 or 37-40 weeks of gestation, respectively, were included in this study. Feces were collected during the first six postnatal weeks (n = 109) and their mycobiota composition and diversity were characterized by ITS2 amplicon sequencing.Results: Composition analyses identified fungi and other eukaryotic kingdoms, of which Viridiplantae was most abundant. Of the fungal kingdom, Ascomycota and Basidiomycota were the first and second most prominent phyla in early life of all infants. Candida was the most abundant genus in the first six weeks of life and increased with gestational and postnatal age. Fungal phylogenetic diversity remained stable in the first six postnatal weeks. The individuality and the mode of delivery were identified as significant predictors for the variation in the mycobiota composition. Vaginally delivered infants were enriched in Candida spp., whereas infants delivered through emergency C-section were characterized by Malassezia spp.Conclusion: These results indicate that fungi and other eukaryotic kingdoms are detected in the intestine of preterm and full-term infants in the first six postnatal weeks. Similar to the microbiota, colonization of the preterm intestine with fungi is determined by clinical variables including individuality and mode of delivery. LESS      Full article

There is emerging concern regarding the unintentional and often unrecognized antimicrobial properties of “non-antimicrobial” pesticides. This includes insecticides, herbicides, and fungicides commonly used in agriculture that are known to produce broad ranging, off-target effects on beneficial wildlife, even at seemingly non-toxic low dose exposures. Notably, these obscure adverse interactions may be related to host-associated microbiome damage occurring from antimicrobial effects, rather than the presumed toxic effects of pesticides on host tissue. Here, we critically review the literature on this topic as it pertains to the rhizosphere microbiome of crop plants and gut microbiome of pollinator insects (namely managed populations of the western honey bee, Apis mellifera), since both are frequent recipients of chronic pesticide exposure. Clear linkages between pesticide mode of action and host-specific microbiome functionalities are identified in relation to potential antimicrobial risks. For example, inherent differences in nitrogen metabolism of plant- and insect-associated microbiomes may dictate whether neonicotinoid-based insecticides ultimately exert antimicrobial activities or not. Several other context-dependent scenarios are discussed. In addition to direct effects (e.g., microbicidal action of the parent compound or breakdown metabolites), pesticides may indirectly alter the trajectory of host-microbiome coevolution in honey bees via modulation of social behaviours and the insect gut-brain axis - conceivably with consequences on plant-pollinator symbiosis as well. In summary, current evidence suggests: (1) immediate action is needed by regulatory authorities in amending safety assessments for “non-antimicrobial” pesticides; and (2) that the development of host-free microbiome model systems could be useful for rapidly screening pesticides against functionally distinct microbial catalogues of interest. LESS      Full article

Reports on fermented, animal-sourced foods made by Inuit around the circumpolar North have lacked consideration for their unique microbiota and the geo-socio-cultural contexts in which they are made, often resulting in reinforced negative stereotypes. Deficit-based approaches to studying Inuit fermented foods overlook the fact that they have long been considered healthy and integral to Inuit diets. Inuit have deep knowledge on the harvesting, preparation, sharing, and consumption of fermented foods that research efforts must learn from and acknowledge. Our preliminary research into Inuit animal-sourced fermented foods expands current knowledge about the microorganisms needed to make them, and points to a potential to understand how these and other fermented foods impact the human gut microbiome. We provide recommendations for microbiological research on Inuit fermented foods that centers Inuit knowledge within the specific geographic, social, and cultural contexts in which these foods are made. LESS      Full article

The application of in vitro synthetic microbial communities is an excellent approach to model the ecological interactions between microbes in the human gastrointestinal tract. Although DNA-based studies have provided a wealth of information, they do not consider the ecological properties of the human gut microbiota. Ecological interactions between gut microbes of interest can be studied by applying synthetic communities. This review describes the considerations that should be taken into account when constructing a synthetic community by discussing example research questions that can be answered by using a synthetic microbial community, the choice of microbial species, the growth conditions, possible reactor setups, and the parameters to analyze. LESS      Full article

Food fermentation relies on the activity of robust starter cultures, which are commonly comprised of lactic acid bacteria such as Lactococcus and Streptococcus thermophilus. While bacteriophage infection represents a persistent threat that may cause slowed or failed fermentations, their beneficial role in fermentations is also being appreciated. In order to develop robust starter cultures, it is important to understand how phages interact with and modulate the compositional landscape of these complex microbial communities. Both culture-dependent and -independent methods have been instrumental in defining individual phage-host interactions of many lactic acid bacteria (LAB). This knowledge needs to be integrated and expanded to obtain a full understanding of the overall complexity of such interactions pertinent to fermented foods through a combination of culturomics, metagenomics, and phageomics. With such knowledge, it is believed that factory-specific detection and monitoring systems may be developed to ensure robust and reliable fermentation practices. In this review, we explore/discuss phage-host interactions of LAB, the role of both virulent and temperate phages on the microbial composition, and the current knowledge of phageomes of fermented foods. LESS      Full article

The establishment and development of the human gut microbiota constitutes a dynamic and non-random process, which involves positive and negative interactions between key microbial taxa and their host. Remarkably, these early life microbiota-host communications include key events with long-term health consequences. Bifidobacteria arguably represent the most emblematic microbial taxon of the infant gut microbiota. In this context, the interactions among bifidobacteria, their human host, and other members of the human gut microbiota are far from completely understood, despite the crucial role they play in the development and maintenance of human physiology and immune system. Here, we highlight the ecological as well as genetic and functional features of bifidobacteria residing in the human gut using genomic and ecology-based information. LESS      Full article