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Background: Maternal stress is associated with negative early-life development and (mental)health outcomes. There is recent evidence that maternal stress in the postpartum period impacts the nutrient composition of human milk (HM). However, it is currently not known whether maternal stress is associated with changes in the HM microbiome during the critical early postpartum period., Methods: In this prospective observational cohort study, lactating women were recruited into a high-stress (HS, n = 23) and control (CTL, n = 69) group. The HS group included mothers with infants hospitalized for at least two days. Maternal stress was assessed using validated questionnaires and cortisol concentrations in hair, saliva and HM. HM was collected at days 10 and 24 and its microbiome was analyzed using 16 s rRNA sequencing. HM bacterial composition was compared between study groups and their correlation with maternal stress levels, maternal characteristics and infant outcomes was determined., Results: HM microbiome β-diversity differed significantly between study groups, with HS mothers displaying decreased abundance of Streptococcus, Gemella, and Veillonella, and increased levels of Staphylococcus, Corynebacterium and Acinetobacter compared to the control group. While the strongest correlation of β-diversity was with stress, HM microbiome β-diversity also correlated significantly with maternal education level and infant sex. No correlation between HM microbiome composition and HM cortisol concentrations was found., Conclusions: This study demonstrates stress-associated alterations in the early HM microbiome that could potentially contribute to early gut colonization and subsequent (mental)health outcomes. Future research is needed to elucidate the physiological significance of these changes for infant development and health., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Diarrhea claims >500,000 lives annually among children under five years of age in low- and middle-income countries. Mortality due to acute diarrhea (<7 days' duration) is decreasing, but prolonged (7-13 days) and persistent (≥14 days of duration) diarrhea remains a massive challenge. Here, we use a case-control study to decipher if fecal gut microbiota compositional differences between Ethiopian children with acute (n=554) or prolonged/persistent (n=95) diarrhea and frequency-matched non-diarrheal controls (n=663) are linked to diarrheal etiology. We show that diarrhea cases are associated with lower bacterial diversity and enriched in Escherichia spp., Campylobacter spp., and Streptococcus spp. Further, diarrhea cases are depleted in gut commensals such as Prevotella copri, Faecalibacterium prausnitzii, and Dialister succinatiphilus, with depletion being most pronounced in prolonged/persistent cases, suggesting that prolonged duration of diarrhea is accompanied by depletion of gut commensals and that re-establishing these via e.g., microbiota-directed food supplements offer a potential treatment strategy., (© 2024. The Author(s).)

The viral fraction of human and experimental animal fecal matter is increasingly attracting research interest due to its newfound influence on the gut microbiome and host health. During the past decade, high-throughput sequencing techniques have seen massive improvements, and in recent years, bioinformatics pipelines for virome analysis have also vastly improved with respect to both user-friendliness and output quality. Yet, the shape and quality of such data are highly dependent on how the viruses are isolated and their genomes extracted and processed to build sequencing libraries.Here we describe a simple protocol for virus isolation from fecal samples suitable for further propagation/characterization or sequencing efforts. It is based on two filtration steps: one for removing large particles such as bacteria and one for removing free DNA and up-concentrating phages and other viruses in the solution. The method is highly scalable, adaptable to a long range of sample types including low-input samples, and has a quantifiable output suitable for both plaquing and sequencing., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Apis mellifera evolved mainly in African, Asian, and European continents over thousands of years, leading to the selection of a considerable number of honey bees subspecies that have adapted to various environments such as hot semi-desert zones and cold temperate zones. With the evolution of honey bee subspecies, it is possible that environmental conditions, food sources, and microbial communities typical of the colonized areas have shaped the honey bee gut microbiota., Methods: In this study the microbiota of two distinct lineages (mitochondrial haplotypes) of bees Apis mellifera ruttneri (lineage A) and Apis mellifera ligustica and carnica (both lineage C) were compared. Honey bee guts were collected in a dry period in the respective breeding areas (the island of Malta and the regions of Emilia-Romagna and South Tyrol in Italy). Microbial DNA from the honey bee gut was extracted and amplified for the V3-V4 regions of the 16S rRNA gene for bacteria and for ITS2 for fungi., Results: The analyses carried out show that the Maltese lineage A honey bees have a distinctive microbiota when compared to Italian lineage C honey bees, with the most abundant genera being Bartonellaceae and Lactobacillaceae, respectively. Lactobacillaceae in Maltese Lineage A honey bees consist mainly of Apilactobacillus instead of Lactobacillus and Bombilactobacillus in the lineage C. Lineage A honey bee gut microbiota also harbors higher proportions of Arsenophonus , Bombella , Commensalibacter , and Pseudomonas when compared to lineage C., Discussion: The environment seems to be the main driver in the acquisition of these marked differences in the gut microbiota. However, the influence of other factors such as host genetics, seasonality or geography may still play a significant role in the microbiome shaping, in synergy with the environmental aspects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gaggìa, Jakobsen, Alberoni, Baffoni, Cutajar, Mifsud, Nielsen and Di Gioia.)

Probiotics are intended to improve gastrointestinal health when consumed. However, the probiotics marketed today only colonize the densely populated gut to a limited extent. Bacteriophages comprise the majority of viruses in the human gut virome and there are strong indications that they play important roles in shaping the gut microbiome. Here, we investigate the use of fecal virome transplantation (FVT, sterile filtrated feces) as a mean to alter the gut microbiome composition to lead the way for persistent colonization of two types of probiotics: Lacticaseibacillus rhamnosus GG (LGG) representing a well-established probiotic and Akkermansia muciniphila (AKM) representing a putative next-generation probiotic. Male and female C57BL/6NTac mice were cohoused in pairs from 4 weeks of age and received the following treatment by oral gavage at week 5 and 6: AKM+FVT, LGG+FVT, probiotic sham (Pro-sham)+FVT, LGG+Saline, AKM+Saline, and control (Pro-sham+Saline). The FVT donor material originated from mice with high relative abundance of A. muciniphila . All animals were terminated at age 9 weeks. The FVT treatment did not increase the relative abundance of the administered LGG or AKM in the recipient mice. Instead FVT significantly ( p  < 0.05) increased the abundance of naturally occurring A. muciniphila compared to the control. This highlights the potential of propagating the existing commensal "probiotics" that have already permanently colonized the gut. Being co-housed male and female, a fraction of the female mice became pregnant. Unexpectedly, the FVT treated mice were found to have a significantly ( p  < 0.05) higher fertility rate independent of probiotic administration. These preliminary observations urge for follow-up studies investigating interactions between the gut microbiome and fertility.

Background: Gut microbiota dysbiosis is associated with the development of non-alcoholic steatohepatitis (NASH) through modulation of gut barrier, inflammation, lipid metabolism, bile acid signaling and short-chain fatty acid production. The aim of this study was to describe the impact of a choline-deficient amino acid defined high fat diet (CDAHFD) on the gut microbiota in a male Göttingen Minipig model and on selected pathways implicated in the development of NASH., Results: Eight weeks of CDAHFD resulted in a significantly altered colon microbiota mainly driven by the bacterial families Lachnospiraceae and Enterobacteriaceae, being decreased and increased in relative abundance, respectively. Metabolomics analysis revealed that CDAHFD decreased colon content of short-chain fatty acid and increased colonic pH. In addition, serum levels of the microbially produced metabolite imidazole propionate were significantly elevated as a consequence of CDAHFD feeding. Hepatic gene expression analysis showed upregulation of mechanistic target of rapamycin (mTOR) and Ras Homolog, MTORC1 binding in addition to downregulation of insulin receptor substrate 1, insulin receptor substrate 2 and the glucagon receptor in CDAHFD fed minipigs. Further, the consequences of CDAHFD feeding were associated with increased levels of circulating cholesterol, bile acids, and glucagon but not total amino acids., Conclusions: Our results indicate imidazole propionate as a new potentially relevant factor in relation to NASH and discuss the possible implication of gut microbiota dysbiosis in the development of NASH. In addition, the study emphasizes the need for considering the gut microbiota and its products when developing translational animal models for NASH., (© 2022. The Author(s).)

Introduction: Human milk provides all macronutrients for growth, bioactive compounds, micro-organisms and immunological components, which potentially interacts with and primes infant growth and, development, immune responses and the gut microbiota of the new-born. Infants with an overweight mother are more likely to become overweight later in life and overweight has been related to the gut microbiome. Therefore, it is important to investigate the mother-milk-infant triad as a biological system and if the maternal weight status influences the human milk composition, infant metabolism and gut microbiome., Methods and Analysis: This study aims to include 200 mother-infant dyads stratified into one of three body mass index (BMI) categories based on mother's prepregnancy BMI. Multiomics analyses include metabolomics, proteomics, glycomics and microbiomics methods, aiming to characterise human milk from the mothers and further relate the composition to infant gut microbiota and its metabolic impact in the infant. Infant gut microbiota is analysed using 16S sequencing of faeces samples. Nuclear magnetic resonance and mass spectrometry are used for the remaining omics analysis. We investigate whether maternal pre-pregnancy BMI results in a distinct human milk composition that potentially affects the initial priming of the infant's gut environment and metabolism early in life., Ethics and Dissemination: The Central Denmark Region Committees on Health Research Ethics has approved the protocol (J-nr. 1-10-72-296-18). All participants have before inclusion signed informed consent and deputy informed consent in accordance with the Declaration of Helsinki II. Results will be disseminated to health professionals including paediatricians, research community, nutritional policymakers, industry and finally the public. The scientific community will be informed via peer-reviewed publications and presentations at scientific conferences, the industry will be invited for meetings, and the public will be informed via reports in science magazines and the general press. Data cleared for personal data, will be deposited at public data repositories., Trial Registration Number: Danish regional committee of the Central Jutland Region, journal number: 1-10-72-296-18, version 6.Danish Data Protection Agency, journal number: 2016-051-000001, 1304., Clinicaltrials: gov, identifier: NCT05111990., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In this study, we examined the effect of a bacteriophage cocktail (tentatively designated as the Foodborne Outbreak Pill (FOP)) on the levels of Listeria monocytogenes in simulated small intestine, large intestine, and Caco-2 model systems. We found that FOP survival during simulated passage of the upper gastrointestinal was dependent on stomach pH, and that FOP robustly inhibited L. monocytogenes levels with effectiveness comparable to antibiotic treatment (ampicillin) under simulated ilium and colon conditions. The FOP did not inhibit the commensal bacteria, whereas ampicillin treatment led to dysbiosis-like conditions. The FOP was also more effective than an antibiotic in protecting Caco-2 cells from adhesion and invasion by L. monocytogenes (5-log reduction vs. 1-log reduction) while not triggering an inflammatory response. Our data suggested that the FOP may provide a robust protection against L. monocytogenes should the bacterium enter the human gastrointestinal tract (e.g., by consumption of contaminated food), without deleterious impact on the commensal bacteria.

The laboratory mouse strain C57BL/6 is widely used as an animal model for various applications. It is becoming increasingly clear that the bacterial enteric community highly influences the phenotype. Eukaryotic viruses represent a sparsely investigated member of the enteric microbiome that might also affect the phenotype. We here investigated the presence of enteric eukaryotic DNA viruses (EDVs) in specific pathogen-free (SPF) C57BL/6N mice purchased from three vendors upon arrival and after being fed a low-fat diet (LFD) or high-fat diet (HFD). We detected genetic fragments of EDVs belonging to the viral families of Herpes-, Mimi-, Baculo- and Phycodnaviridae represented by two genera; Chlorovirus and Prasinovirus. The EDVs were detected in the mice upon arrival and persisted for 13 weeks. However, these signals of EDVs were only detected at notable levels in mice fed LFD from 2 out of 3 vendors, which suggested that the enteric composition of these EDVs were affected by both vendor (p < 0.003) and different dietary regimes (p < 0.013). This highlights the need of additional studies assessing the potential function of these EDVs that may influence the mouse phenotype and the reproducibility of animal studies using this C57BL/6N substrain., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bacterial vaginosis (BV) is characterized by a reduction in Lactobacillus (L.) spp. abundance and increased abundance of facultative anaerobes, such as Gardnerella spp. BV aetiology is not fully understood; however, bacteriophages could play a pivotal role in the perturbation of the vaginal bacterial community. We investigated the vaginal viral community, including bacteriophages and the association to the bacterial community and BV-status. Vaginal samples from 48 patients undergoing IVF treatment for non-female factor infertility were subjected to metagenomic sequencing of purified virus-like particles. The vaginal viral community was characterized and correlated with the BV-status by Nugent score, bacterial community, structure, and the presence of key vaginal bacterial species. The majority of identified vaginal viruses belonged to the class of double-stranded DNA bacteriophages, with eukaryotic viruses constituting 4% of the total reads. Clear links between the viral community composition and BV ( q = 0.006, R = 0.26) as well as the presence of L. crispatus ( q = 0.001, R = 0.43), L. iners , Gardnerella spp., and Atopobium vaginae were found ( q < 0.002, R > 0.15). The eukaryotic viral community also correlated with BV-status ( q = 0.018, R = 0.20). In conclusion, the vaginal virome was clearly linked with bacterial community structure and BV-status.

Gut microbiome (GM) composition and function are linked to human health and disease, and routes for manipulating the GM have become an area of intense research. Due to its high treatment efficacy, the use of fecal microbiota transplantation (FMT) is generally accepted as a promising experimental treatment for patients suffering from GM imbalances (dysbiosis), e.g. caused by recurrent Clostridioides difficile infections (rCDI). Mounting evidence suggests that bacteriophages (phages) play a key role in successful FMT treatment by restoring the dysbiotic bacterial GM. As a refinement to FMT, removing the bacterial component of donor feces by sterile filtration, also referred to as fecal virome transplantation (FVT), decreases the risk of invasive infections caused by bacteria. However, eukaryotic viruses and prophage-encoded virulence factors remain a safety issue. Recent in vivo studies show how cascading effects are initiated when phage communities are transferred to the gut by e.g. FVT, which leads to changes in the GM composition, host metabolome, and improve host health such as alleviating symptoms of obesity and type-2-diabetes (T2D). In this review, we discuss the promises and limitations of FVT along with the perspectives of using FVT to treat various diseases associated with GM dysbiosis., (© FEMS 2020.)

Nicaraguan cocoa bean fermentations of several single local cocoa varieties originating from the same region (North Highlands of Nicaragua, San Jose de Bocay/El Cuá) were compared to fermentations of blended cocoa varietals from other producing regions of the country (Waslala and Nueva Guinea) making use of High Throughput Sequencing techniques, metabolite target analysis and sensory evaluation of cocoa liquor samples. A succession of the important cocoa-related yeasts Hanseniaspora uvarum/opuntiae, Saccharomyces cerevisiae and/or Pichia kudriavzevii was seen for single varietals and Nueva Guinea fermentations, while Kazachstania humilis dominated the mid and end phase of the Waslala cocoa fermentations. Tatumella species (mainly Tatumella terrea and Tatumella punctata) predominated the bacterial community at the onset of all fermentations followed by unusually late (generally 2 days into the fermentations) appearance of Lactobacillus fermentum relative to fermentations in other parts of the World. Acetobacter spp. were the main acetic acid bacteria during all fermentations, but also Gluconobacter spp. were involved in some single-variety fermentations. All fermentations proved complete as determined by metabolite analysis with bean sucrose being fully depleted and pulp sugars exhausted after 48-72 h of fermentation. From an organoleptic point of view, all Nicaraguan cocoas of this study reflected fine fruity (citrus or berry-like) flavours with distinct herbal or caramel notes. Floral notes were associated with the cases where P. kudriavzevii was involved in the later stages of fermentation. Intense citrus/fruity character was related to high pulp and bean citrate concentrations. Off-notes were found in some over-fermented batches where Bacillus spp. was detected. No relation between cut-test results and organoleptic appreciation was seen., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Kombucha is usually obtained from the fermentation of black or green tea by a consortium of acetic acid bacteria and yeasts. In this study, kombucha was prepared from the same starter consortium using green and black teas as well as, for the first time, an infusion of rooibos leaves ( Aspalathus linearis ). Microbial diversity was analysed during fermentation both in the biofilm and in the corresponding kombuchas, using culture-dependent and -independent methods. Polyphenols, flavonoids, ethanol, and acids were quantified and anti-oxidant activities were monitored. All of the Kombuchas showed similarity in bacterial composition, with the dominance of Komagataeibacter spp. Beta diversity showed that the yeast community was significantly different among all tea substrates, between 7 and 14 days of fermentation and between biofilm and kombucha, indicating the influence of the substrate on the fermenting microbiota. Kombucha from rooibos has a low ethanol concentration (1.1 mg/mL), and a glucuronic acid amount that was comparable to black tea. Although antioxidant activity was higher in black and green kombucha compared to rooibos, the latter showed an important effect on the recovery of oxidative damage on fibroblast cell lines against oxidative stress. These results make rooibos leaves interesting for the preparation of a fermented beverage with health benefits.