Microbiome mapping in dairy industry reveals new species and genes for probiotic and bioprotective activities.

The resident microbiome in food industries may impact on food quality and safety. In particular, microbes residing on surfaces in dairy industries may actively participate in cheese fermentation and ripening and contribute to the typical flavor and texture. In this work, we carried out an extensive microbiome mapping in 73 cheese-making industries producing different types of cheeses (fresh, medium and long ripened) and located in 4 European countries. We sequenced and analyzed metagenomes from cheese samples, raw materials and environmental swabs collected from both food contact and non-food contact surfaces, as well as operators' hands and aprons. Dairy plants were shown to harbor a very complex microbiome, characterized by high prevalence of genes potentially involved in flavor development, probiotic activities, and resistance to gastro-intestinal transit, suggesting that these microbes may potentially be transferred to the human gut microbiome. More than 6100 high-quality Metagenome Assembled Genomes (MAGs) were reconstructed, including MAGs from several Lactic Acid Bacteria species and putative new species. Although microbial pathogens were not prevalent, we found several MAGs harboring genes related to antibiotic resistance, highlighting that dairy industry surfaces represent a potential hotspot for antimicrobial resistance (AR) spreading along the food chain. Finally, we identified facility-specific strains that can represent clear microbial signatures of different cheesemaking facilities, suggesting an interesting potential of microbiome tracking for the traceability of cheese origin.

Pseudocin 196, a novel lantibiotic produced by Bifidobacterium pseudocatenulatum elicits antimicrobial activity against clinically relevant pathogens.

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.

Novel Dairy Fermentates Have Differential Effects on Key Immune Responses Associated with Viral Immunity and Inflammation in Dendritic Cells.

Fermented foods and ingredients, including furmenties derived from lactic acid bacteria (LAB) in dairy products, can modulate the immune system. Here, we describe the use of reconstituted skimmed milk powder to generate novel fermentates from Lactobacillus helveticus strains SC232, SC234, SC212, and SC210, and from Lacticaseibacillus casei strains SC209 and SC229, and demonstrate, using in vitro assays, that these fermentates can differentially modulate cytokine secretion via bone-marrow-derived dendritic cells (BMDCs) when activated with either the viral ligand loxoribine or an inflammatory stimulus, lipopolysaccharide. Specifically, we demonstrate that SC232 and SC234 increase cytokines IL-6, TNF-α, IL-12p40, IL-23, IL-27, and IL-10 and decrease IL-1ß in primary bone-marrow-derived dendritic cells (BMDCs) stimulated with a viral ligand. In contrast, exposure of these cells to SC212 and SC210 resulted in increased IL-10, IL-1ß, IL-23, and decreased IL-12p40 following activation of the cells with the inflammatory stimulus LPS. Interestingly, SC209 and SC229 had little or no effect on cytokine secretion by BMDCs. Overall, our data demonstrate that these novel fermentates have specific effects and can differentially enhance key immune mechanisms that are critical to viral immune responses, or can suppress responses involved in chronic inflammatory conditions, such as ulcerative colitis (UC), and Crohn's disease (CD).

Ketogenic diet but not free-sugar restriction alters glucose tolerance, lipid metabolism, peripheral tissue phenotype, and gut microbiome: RCT.

Restricted sugar and ketogenic diets can alter energy balance/metabolism, but decreased energy intake may be compensated by reduced expenditure. In healthy adults, randomization to restricting free sugars or overall carbohydrates (ketogenic diet) for 12 weeks reduces fat mass without changing energy expenditure versus control. Free-sugar restriction minimally affects metabolism or gut microbiome but decreases low-density lipoprotein cholesterol (LDL-C). In contrast, a ketogenic diet decreases glucose tolerance, increases skeletal muscle PDK4, and reduces AMPK and GLUT4 levels. By week 4, the ketogenic diet reduces fasting glucose and increases apolipoprotein B, C-reactive protein, and postprandial glycerol concentrations. However, despite sustained ketosis, these effects are no longer apparent by week 12, when gut microbial beta diversity is altered, possibly reflective of longer-term adjustments to the ketogenic diet and/or energy balance. These data demonstrate that restricting free sugars or overall carbohydrates reduces energy intake without altering physical activity, but with divergent effects on glucose tolerance, lipoprotein profiles, and gut microbiome.

Unveiling metabolic pathways of selected plant-derived glycans by Bifidobacterium pseudocatenulatum.

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.

Impact of previous pregnancy and BMI on cellular and serum immune activity from early to late pregnancy.

Immunological adaptions during pregnancy play a crucial role in healthy fetal development. Aberrant immune modifications however contribute to adverse pregnancy outcomes, which may be driven by maternal factors such as previous pregnancies and BMI. This secondary analysis of the MicrobeMom2 RCT investigates the changes to maternal inflammatory biomarkers derived from serum and stimulated peripheral blood mononuclear cells (PBMCs) during pregnancy, and the effects of previous pregnancies (parity) and BMI on maternal immune responses. Changes in immune and metabolic biomarkers from early (11-15 weeks' gestation) to late (28-32 weeks' gestation) pregnancy were compared using paired t-tests. Participants were then split by parity (nulliparous, parous) and BMI (BMI < 25, BMI > = 25), and the relationship between parity and BMI with immune biomarker levels was examined using independent t-tests, paired t-tests, ANCOVA, and linear regression. Equivalent non-parametric tests were used for skewed data. Recruited women (n = 72) were on average 31.17 (SD ± 4.53) years of age and 25.11 (SD ± 3.82) BMI (kg/m2). Of these, 51 (70.8%) had a previous term pregnancy. Throughout gestation, PBMC cytokines displayed contrasting trends to serum, with a dampening of immune responses noted in PBMCs, and enhanced production of cytokines observed in the serum. Significant decreases in PBMC derived TNF-α, IL-10 and IFN-γ were seen from early to late pregnancy. Serum C3, IL-17A, IL-6, TNF-α, CD163, GDF-15 and leptin increased throughout gestation. First pregnancy was associated with higher levels of leptin in late pregnancy, while parous women showed significant decreases in PBMC derived TNF-α, IL10, and IFN-γ with gestation. Differences in levels of C3, IL-17A, TNF-α, GDF-15 and leptin were observed across BMI groups. Overall, serum-derived cytokines exhibit contrasting levels to those derived from stimulated PBMCs. Maternal immune responses undergo significant changes from early to late pregnancy, which are influenced by parity and BMI. These differences aid our understanding as to why first-time mothers are at greater risk of placental disease such as pre-eclampsia and fetal growth restriction.

Metagenomic analysis of the bacterial microbiome, resistome and virulome distinguishes Portuguese Serra da Estrela PDO cheeses from similar non-PDO cheeses: An exploratory approach.

This study aimed to evaluate the microbiome, resistome and virulome of two types of Portuguese cheese using high throughput sequencing (HTS). Culture-dependent chromogenic methods were also used for certain groups/microorganisms. Eight samples of raw ewe's milk cheese were obtained from four producers: two producers with cheeses with a PDO (Protected Designation of Origin) label and the other two producers with cheeses without a PDO label. Agar-based culture methods were used to quantify total mesophiles, Enterobacteriaceae, Escherichia coli, Staphylococcus, Enterococcus and lactic acid bacteria. The presence of Listeria monocytogenes and Salmonella was also investigated. The selected isolates were identified by 16S rRNA gene sequencing and evaluated to determine antibiotic resistance and the presence of virulence genes. The eight cheese samples analyzed broadly complied with EC regulations in terms of the microbiological safety criteria. The HTS results demonstrated that Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus plantarum, Lacticaseibacillus rhamnosus, Enterococcus durans and Lactobacillus coryniformis were the most prevalent bacterial species in cheeses. The composition of the bacterial community varied, not only between PDO and non-PDO cheeses, but also between producers, particularly between the two non-PDO cheeses. Alpha-diversity analyses showed that PDO cheeses had greater bacterial diversity than non-PDO cheeses, demonstrating that the diversity of spontaneously fermented foods is significantly higher in cheeses produced without the addition of food preservatives and dairy ferments. Despite complying with microbiological regulations, both PDO and non-PDO cheeses harbored potential virulence genes as well as antibiotic resistance genes. However, PDO cheeses exhibited fewer of these virulence and antibiotic resistance genes compared to non-PDO cheeses. Therefore, the combination of conventional microbiological methods and the metagenomic approach could contribute to improving the attribution of the PDO label to this type of cheese.

The changing landscape with respect to scientific research and education for second-level students and how they can overlap: the Kefir4All example.

Have you ever deeply considered the intersections between research and education, particularly for second-level students? Traditionally, the convergence of these two realms is most often noted when considering the integration of research findings into educational practices or the involvement of, typically a small number, of students in research activities. While these practices have demonstrated efficacy, the fields of scientific research and education are evolving rapidly, necessitating a reevaluation of how we can optimize their convergence. In our discourse, we delve into these evolving trends, uncover the potential for greater integration, and, ultimately, enhance outcomes using the citizen science initiative Kefir4All as an illustrative example.

GROND: a quality-checked and publicly available database of full-length 16S-ITS-23S rRNA operon sequences.

Sequence comparison of 16S rRNA PCR amplicons is an established approach to taxonomically identify bacterial isolates and profile complex microbial communities. One potential application of recent advances in long-read sequencing technologies is to sequence entire rRNA operons and capture significantly more phylogenetic information compared to sequencing of the 16S rRNA (or regions thereof) alone, with the potential to increase the proportion of amplicons that can be reliably classified to lower taxonomic ranks. Here we describe GROND (Genome-derived Ribosomal Operon Database), a publicly available database of quality-checked 16S-ITS-23S rRNA operons, accompanied by multiple taxonomic classifications. GROND will aid researchers in analysis of their data and act as a standardised database to allow comparison of results between studies.

A secondary model for the effect of pH on the variability in growth fitness of Listeria innocua strains.

Variability in microbial growth is a keystone of modern Quantitative Microbiological Risk Assessment (QMRA). However, there are still significant knowledge gaps on how to model variability, with the most common assumption being that variability is constant. This is implemented by an error term (with constant variance) added on top of the secondary growth model (for the square root of the growth rate). However, this may go against microbial ecology principles, where differences in growth fitness among bacterial strains would be more prominent in the vicinity of the growth limits than at optimal growth conditions. This study coins the term "secondary models for variability", evaluating whether they should be considered in QMRA instead of the constant strain variability hypothesis. For this, 21 strains of Listeria innocua were used as case study, estimating their growth rate by the two-fold dilution method at pH between 5 and 10. Estimates of between-strain variability and experimental uncertainty were obtained for each pH using mixed-effects models, showing the lowest variability at optimal growth conditions, increasing towards the growth limits. Nonetheless, the experimental uncertainty also increased towards the extremes, evidencing the need to analyze both sources of variance independently. A secondary model was thus proposed, relating strain variability and pH conditions. Although the modelling approach certainly has some limitations that would need further experimental validation, it is an important step towards improving the description of variability in QMRA, being the first model of this type in the field.

The gut microbiota in persistent post-operative pain following breast cancer surgery.

Persistent post-surgical pain (PPSP) is defined as pain which continues after a surgical operation in a significant form for at least three months (and is not related to pre-existing painful conditions). PPSP is a common, under-recognised, and important clinical problem which affects millions of patients worldwide. Preventative measures which are currently available include the selection of a minimally invasive surgical technique and an aggressive multimodal perioperative analgesic regimen. More recently, a role for the gut microbiota in pain modulation has become increasingly apparent. This study aims to investigate any relationship between the gut microbiota and PPSP. A prospective observational study of 68 female adult patients undergoing surgery for management of breast cancer was carried out. Stool samples from 45 of these patients were obtained to analyse the composition of the gut microbiota. Measures of pain and state-trait anxiety were also taken to investigate further dimensions in any relationship between the gut microbiota and PPSP. At 12 weeks postoperatively, 21 patients (51.2%) did not have any pain and 20 patients (48.8%) reported feeling pain that persisted at that time. Analysis of the gut microbiota revealed significantly lower alpha diversity (using three measures) in those patients reporting severe pain at the 60 min post-operative and the 12 weeks post-operative timepoints. A cluster of taxa represented by Bifidobacterium longum, and Faecalibacterium prausnitzii was closely associated with those individuals reporting no pain at 12 weeks postoperatively, while Megamonas hypermegale, Bacteroides pectinophilus, Ruminococcus bromii, and Roseburia hominis clustered relatively closely in the group of patients fulfilling the criteria for persistent post-operative pain. We report for the first time specific associations between the gut microbiota composition and the presence or absence of PPSP. This may provide further insights into mechanisms behind the role of the gut microbiota in the development of PPSP and could inform future treatment strategies.

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

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

Kefir4All, a citizen science initiative to raise awareness of the roles that microbes play in food fermentation.

Microorganisms are ubiquitous in nature and are central to human, animal, environmental, and planetary health. They play a particularly important role in the food chain and the production of high-quality, safe, and health-promoting foods, especially fermented foods. This important role is not always apparent to members of the public. Here, we describe Kefir4All, a citizen science project designed to provide the general public with an opportunity to expand their awareness, knowledge, and practical skills relating to microbiology, introduced through the medium of producing fermented food, i.e., milk kefir or water kefir. During the course of Kefir4All, 123 citizen scientists, from second-level school and non-school settings, participated in a study to track changes in the microbial composition of kefirs, by performing and recording details of milk kefir or water kefir fermentations they performed in their homes or schools over the 21-week project. At the start of the study, the citizen scientists were provided with milk or water kefir grains to initiate the fermentations. Both types of kefir grain are semi-solid, gelatinous-like substances, composed of exopolysaccharides and proteins, containing a symbiotic community of bacteria and yeast. The experimental component of the project was complemented by a number of education and outreach events, including career talks and a site visit to our research center (Kefir Day). At the end of the study, a report was provided to each citizen scientist, in which individualized results of their fermenting activities were detailed. A number of approaches were taken to obtain feedback and other insights from the citizen scientists. Evaluations took place before and after the Kefir4All project to gauge the citizen scientist's self-reported awareness, knowledge, and interest in microbiology and fermented foods. Further insights into the level of citizen science participation were gained through assessing the number of samples returned for analysis and the level of participation of the citizen scientists throughout the project. Notably, the survey results revealed a self-reported, increased interest in, and general knowledge of, science among the Kefir4All citizen scientists after undertaking the project and a willingness to take part in further citizen science projects. Ultimately, Kefir4All represents an example of the successful integration of citizen science into existing education and research systems.

The detailed analysis of the microbiome and resistome of artisanal blue-veined cheeses provides evidence on sources and patterns of succession linked with quality and safety traits.

BACKGROUND: Artisanal cheeses usually contain a highly diverse microbial community which can significantly impact their quality and safety. Here, we describe a detailed longitudinal study assessing the impact of ripening in three natural caves on the microbiome and resistome succession across three different producers of Cabrales blue-veined cheese. RESULTS: Both the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome. Lactococcus and the former Lactobacillus genus, among other taxa, showed high abundance in cheeses at initial stages of ripening, either coming from the raw material, starter culture used, and/or the environment of processing plants. Along cheese ripening in caves, these taxa were displaced by other bacteria, such as Tetragenococcus, Corynebacterium, Brevibacterium, Yaniella, and Staphylococcus, predominantly originating from cave environments (mainly food contact surfaces), as demonstrated by source-tracking analysis, strain analysis at read level, and the characterization of 613 metagenome-assembled genomes. The high abundance of Tetragenococcus koreensis and Tetragenococcus halophilus detected in cheese has not been found previously in cheese metagenomes. Furthermore, Tetragenococcus showed a high level of horizontal gene transfer with other members of the cheese microbiome, mainly with Lactococcus and Staphylococcus, involving genes related to carbohydrate metabolism functions. The resistome analysis revealed that raw milk and the associated processing environments are a rich reservoir of antimicrobial resistance determinants, mainly associated with resistance to aminoglycosides, tetracyclines, and ß-lactam antibiotics and harbored by aerobic gram-negative bacteria of high relevance from a safety point of view, such as Escherichia coli, Salmonella enterica, Acinetobacter, and Klebsiella pneumoniae, and that the displacement of most raw milk-associated taxa by cave-associated taxa during ripening gave rise to a significant decrease in the load of ARGs and, therefore, to a safer end product. CONCLUSION: Overall, the cave environments represented an important source of non-starter microorganisms which may play a relevant role in the quality and safety of the end products. Among them, we have identified novel taxa and taxa not previously regarded as being dominant components of the cheese microbiome (Tetragenococcus spp.), providing very valuable information for the authentication of this protected designation of origin artisanal cheese. Video Abstract.

Novel Fermentates Can Enhance Key Immune Responses Associated with Viral Immunity.

Fermented foods have long been known to have immunomodulatory capabilities, and fermentates derived from the lactic acid bacteria of dairy products can modulate the immune system. We have used skimmed milk powder to generate novel fermentates using Lb. helveticus strains SC234 and SC232 and we demonstrate here that these fermentates can enhance key immune mechanisms that are critical to the immune response to viruses. We show that our novel fermentates, SC234 and SC232, can positively impact on cytokine and chemokine secretion, nitric oxide (NO) production, cell surface marker expression, and phagocytosis in macrophage models. We demonstrate that the fermentates SC234 and SC232 increase the secretion of cytokines IL-1ß, IL-6, TNF-α, IL-27, and IL-10; promote an M1 pro-inflammatory phenotype for viral immunity via NO induction; decrease chemokine expression of Monocyte Chemoattractant Protein (MCP); increase cell surface marker expression; and enhance phagocytosis in comparison to their starting material. These data suggest that these novel fermentates have potential as novel functional food ingredients for the treatment, management, and control of viral infection.

Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus.

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Insights into the Adolescent Cystic Fibrosis Airway Microbiome Using Shotgun Metagenomics.

Cystic fibrosis (CF) is an inherited genetic disorder which manifests primarily in airway disease. Recent advances in molecular technologies have unearthed the diverse polymicrobial nature of the CF airway. Numerous studies have characterised the genus-level composition of this airway community using targeted 16S rDNA sequencing. Here, we employed whole-genome shotgun metagenomics to provide a more comprehensive understanding of the early CF airway microbiome. We collected 48 sputum samples from 11 adolescents and children with CF over a 12-month period and performed shotgun metagenomics on the Illumina NextSeq platform. We carried out functional and taxonomic analysis of the lung microbiome at the species and strain levels. Correlations between microbial diversity measures and independent demographic and clinical variables were performed. Shotgun metagenomics detected a greater diversity of bacteria than culture-based methods. A large proportion of the top 25 most-dominant species were anaerobes. Samples dominated by Staphylococcus aureus and Prevotella melaninogenica had significantly higher microbiome diversity, while no CF pathogen was associated with reduced microbial diversity. There was a diverse resistome present in all samples in this study, with 57.8% agreement between shotgun metagenomics and culture-based methods for detection of resistance. Pathogenic sequence types (STs) of S. aureus, Pseudomonas aeruginosa, Haemophilus influenzae and Stenotrophomonas maltophilia were observed to persist in young CF patients, while STs of S. aureus were both persistent and shared between patients. This study provides new insight into the temporal changes in strain level composition of the microbiome and the landscape of the resistome in young people with CF. Shotgun metagenomics could provide a very useful one-stop assay for detecting pathogens, emergence of resistance and conversion to persistent colonisation in early CF disease.

Effects of removing in-feed antibiotics and zinc oxide on the taxonomy and functionality of the microbiota in post weaning pigs.

BACKGROUND: Post weaning diarrhoea (PWD) causes piglet morbidity and mortality at weaning and is a major driver for antimicrobial use worldwide. New regulations in the EU limit the use of in-feed antibiotics (Ab) and therapeutic zinc oxide (ZnO) to prevent PWD. New approaches to control PWD are needed, and understanding the role of the microbiota in this context is key. In this study, shotgun metagenome sequencing was used to describe the taxonomic and functional evolution of the faecal microbiota of the piglet during the first two weeks post weaning within three experimental groups, Ab, ZnO and no medication, on commercial farms using antimicrobials regularly in the post weaning period. RESULTS: Diversity was affected by day post weaning (dpw), treatment used and diarrhoea but not by the farm. Microbiota composition evolved towards the dominance of groups of species such as Prevotella spp. at day 14dpw. ZnO inhibited E. coli overgrowth, promoted higher abundance of the family Bacteroidaceae and decreased Megasphaera spp. Animals treated with Ab exhibited inconsistent taxonomic changes across time points, with an overall increase of Limosilactobacillus reuteri and Megasphaera elsdenii. Samples from non-medicated pigs showed virulence-related functions at 7dpw, and specific ETEC-related virulence factors were detected in all samples presenting diarrhoea. Differential microbiota functions of pigs treated with ZnO were related to sulphur and DNA metabolism, as well as mechanisms of antimicrobial and heavy metal resistance, whereas Ab treated animals exhibited functions related to antimicrobial resistance and virulence. CONCLUSION: Ab and particularly ZnO maintained a stable microbiota composition and functionality during the two weeks post weaning, by limiting E. coli overgrowth, and ultimately preventing microbiota dysbiosis. Future approaches to support piglet health should be able to reproduce this stable gut microbiota transition during the post weaning period, in order to maintain optimal gut physiological and productive conditions.

Seasonal and geographical impact on the Irish raw milk microbiota correlates with chemical composition and climatic variables.

Season and location have previously been shown to be associated with differences in the microbiota of raw milk, especially in milk from pasture-based systems. Here, we further advance research in this area by examining differences in the raw milk microbiota from several locations across Ireland over 12 months, and by investigating microbiota associations with climatic variables and chemical composition. Shotgun metagenomic sequencing was used to investigate the microbiota of raw milk collected from nine locations (n = 241). Concurrent chemical analysis of the protein, fat, lactose, total solids, nonprotein nitrogen contents, and titratable acidity (TA) of the same raw milk were performed. Although the raw milk microbiota was highly diverse, a core microbiota was found, with Pseudomonas_E, Lactococcus, Acinetobacter, and Leuconostoc present in all samples. Microbiota diversity significantly differed by season and location, with differences in seasonality and geography corresponding to 11.8% and 10.5% of the variation in the microbiota. Functional and antibiotic resistance profiles also varied across season and location. The analysis of other metadata revealed additional interactions, such as an association between mean daily air and grass temperatures with the abundance of spoilage taxa like Pseudomonas species. Correlations were identified between pathogenic, mastitis-related species, fat content, and the number of sun hours, suggesting a seasonal effect. Ultimately, this study expands our understanding of the interconnected nature of the microbiota, environment/climate variables, and chemical composition of raw milk and provides evidence of a season- and location-specific microbiota. IMPORTANCE: The microbiota of raw milk is influenced by many factors that encourage or prevent the introduction and growth of both beneficial and undesirable microorganisms. The seasonal and geographical impacts on the microbial communities of raw milk have been previously seen, but the relationships with environmental factors and the chemical composition has yet to be investigated. In this year-long study, we found that while raw milk is highly diverse, a core microbiota was detected for Irish raw milk, with strong evidence of seasonal and geographical influence. We also found associations between groups of microorganisms, environmental factors, and milk composition, which expand current knowledge on the relationships between microbial and chemical composition and the climate. These results provide evidence for the development of a tool to allow for the prediction of raw milk quality and safety.

The gut virome is associated with stress-induced changes in behaviour and immune responses in mice.

The microbiota-gut-brain axis has been shown to play an important role in the stress response, but previous work has focused primarily on the role of the bacteriome. The gut virome constitutes a major portion of the microbiome, with bacteriophages having the potential to remodel bacteriome structure and activity. Here we use a mouse model of chronic social stress, and employ 16S rRNA and whole metagenomic sequencing on faecal pellets to determine how the virome is modulated by and contributes to the effects of stress. We found that chronic stress led to behavioural, immune and bacteriome alterations in mice that were associated with changes in the bacteriophage class Caudoviricetes and unassigned viral taxa. To determine whether these changes were causally related to stress-associated behavioural or physiological outcomes, we conducted a faecal virome transplant from mice before stress and autochthonously transferred it to mice undergoing chronic social stress. The transfer of the faecal virome protected against stress-associated behaviour sequelae and restored stress-induced changes in select circulating immune cell populations, cytokine release, bacteriome alterations and gene expression in the amygdala. These data provide evidence that the virome plays a role in the modulation of the microbiota-gut-brain axis during stress, indicating that these viral populations should be considered when designing future microbiome-directed therapies.