Artisanal raw milk cheeses harbour complex microbial communities that drive cheese making and shape sensory quality. Previous work on Idiazabal cheese identified rennet as a major microbial source, although all reservoirs contributed to varying degrees. However, their impact in terms of enzyme-encoding genes related to technological quality of cheese remained unexplored. Building on that, this study draws on metagenome-assembled genomes (MAGs) from cheeses and dairy environments to comprehensively identify enzyme-encoding genes involved in key biochemical processes. In cheese MAGs (Lacticaseibacillus paracasei), protease-encoding genes were dominated by ATP-dependent metalloproteases (M41), carbohydrate-active enzyme-encoding genes (CAZymes) by glycoside hydrolases (GH) and glycosyltransferases (GT), while esterase, lipase, and related-enzyme-encoding genes were restricted to sparse 'GDXG', type-B and esterase D families. Dairy environments emerged as major reservoirs of enzyme-encoding genes, with notable differences among sample types (p <= 0.001). The richest sources of protease-encoding genes were grass (610 genes), linked primarily to Pantoea agglomerans, and rennet (318), mainly related to Basfia sp. and Moraxella sp., dominated by metalloproteases (M23, M38) and serine proteases (S15). The largest reservoirs of CAZyme-encoding genes were food contact surfaces (1550), associated mainly with Salinisphaera sp. and Dietzia sp., and rennet (1505), related to, e.g., Bacteroides pyogenes, Alloprevotella sp., and Lentilactobacillus buchneri. Food contact surfaces were also the richest source of esterase, lipase and related-enzyme-encoding genes (1209), mainly linked to Dietzia sp., Corynebacterium sp., and Brevibacterium aurantiacum. Similarly, aroma-related enzyme-encoding genes (e.g., oppA, pepA, GH13, esterase D) were consistently detected in environmental matrices. These results provide novel insights into dairy microbiomes as functional reservoirs of aroma precursors, revealing their relevance for artisanal PDO cheese production and future biotechnological applications.

BACKGROUND:Broncho-Alveolar Lavage (BAL) is the reference standard for airway surveillance in clinical management of cystic fibrosis (CF), but is invasive and requires general anaesthesia in children. Non-invasive alternatives can lack specificity (Oropharyngeal swabs; OPS), or evaluation in paediatric CF (Middle meatus sampling; MMS). We sought to determine if MMS via nasal-swabs performed better than OPS at representing the microbiological attributes of BAL. METHODS:In a stable preschool CF cohort attending a single specialist centre, we evaluated the microbiological yield of BAL, MMS, and OPS sampling using both standard clinical culturing, and shotgun metagenomic sequencing (Illumina NextSeq 500). RESULTS:Matched BAL, MMS, and OPS from 30 preschool children provided 88 samples. While both culture and metagenomic surveillance performed well at detecting S. pneumoniae in BAL, MMS performed better at detecting S. aureus, M. catarrhalis and Escherichia coli, while OPS performed better at detecting H. Influenzae. Metagenomics revealed a significantly more diverse microbiome in OPS than BAL or MMS. While agreement on pathogen profiles varied widely between metagenomics and culture methods, MMS more accurately represented BAL, particularly for Streptococcus, M. catarrhalis, and Escherichia. CONCLUSIONS:MMS and OPS cultures performed well as proxies for BAL in relation to certain pathogens. Metagenomics detected pathogens in many samples that were unobserved in culture, and showed the oropharynx microbiome to be much more diverse. Lung and nares microbiomes were more similar in composition and diversity. Our data suggest that nasal sampling of the middle meatus may be a more accurate surrogate for lower airway samples.

IntroductionCheddar cheese is a nutritionally dense food matrix containing nutrients and bioactives with the potential to influence gut microbial characteristics. Food matrices influence nutrient absorption and digestibility, therefore the dairy matrix may affect gut microbial responses to dairy food intake. This research aims to identify gut microbial responses to Cheddar cheese consumption, considering aspects of the dairy matrix.MethodsSecondary analysis was conducted on a subset (n = 69) of participants’ data collected during a 6-week parallel 3-armed intervention study. Interventions involved daily consumption of one of the following: (A) 120 g unmelted Cheddar cheese; (B) 120 g melted Cheddar cheese; (C) butter (49 g), calcium caseinate powder (30 g), and Ca supplement (500 mg). Demographics, anthropometry, dietary intake and fecal samples were collected at baseline (V1) and post-intervention (V2). Fecal samples underwent 16S rRNA gene sequencing, followed by bioinformatic processing and statistical analysis.ResultsAt V1, 52% were female, mean age was 58.2 ± 5.4 years, with no significant differences between groups or timepoints. Following sequencing, 12,098 unique bacterial taxa in total were identified. Under a False Discovery Rate (FDR) cutoff of 0.1, Dorea (W = 0.568, FDR = 0.079) and Erysipelotrichaceae UCG-003 (W = 0.887, FDR = 0.097) were significantly increased from V1 to V2 in the unmelted cheese group. At V2, Bacteroides was differentially more abundant in the unmelted cheese group, relative to the melted group (W = 0.587, FDR = 0.034). Bacterial alpha diversity (Shannon, Simpson) significantly increased in the unmelted cheese group only from V1 to V2 (p < 0.05). Beta diversity analysis showed a significant group effect considering both timepoints (F = 1.505, p < 0.01). Considering V2 only, Principal Coordinate Analysis showed the unmelted group clustered more closely relative to the other groups, although the effect was not significant.DiscussionUnmelted Cheddar cheese modulated the gut microbiome by increasing alpha diversity and abundance of several fermenting bacteria. Overall community structure also became more similar following consumption of unmelted cheese, relative to the other groups. Heating cheese and altering its physical structure disrupts the dairy matrix, potentially influencing downstream gut-nutrient interactions and subsequent gut microbial response.

Blueberries, widely recognized as a "superfood," are rich in bioactive compounds whose functionality may be further enhanced through fermentation. This study investigated the nutritional and functional potential of blueberry juice fermented with Leuconostoc mesenteroides VUCC R-037 (or LM2), a native isolate, and Lactiplantibacillus plantarum DSM 25710 (or LP09), an industrial probiotic strain. Fermentation kinetics, microbial viability, and metabolic activity were monitored by microcalorimetry and selective plating, while compositional and functional changes in the fermented blueberry juice (FBJ) (such as total phenolics, antioxidant capability, putative cytotoxicity) were assessed through HPLC, chemical assays (FRAP), and in vitro cellular models (MTT and cytochrome c assay). Strain-specific contributions shaped the final product with LM2 promoting stronger acidification and acetic acid production, while LP09, favoured lactic acid accumulation and increased ferric reducing antioxidant capacity. Additionally, LM2 enhanced radical scavenging activity with co-cultures revealing complementary effects. FBJ supported probiotic viability, showed no cytotoxicity in CaCo-2 intestinal epithelial cells, preserved epithelial barrier integrity, and mitigated oxidative stress. Assays for impact of the FBJ on the gut microbiota was carried out in the micro-MatrixTM ex vivo distal colon model, where it selectively reduced Clostridium perfringens and Escherichia spp., suggesting a role in beneficial gut microbiota modulation. Overall, fermentation with native and probiotic lactic acid bacteria improved the biochemical, functional, and cytoprotective profile of blueberry juice, highlighting its promise as a novel functional beverage. While further clinical validation is forthcoming, these results strengthen the case for fruit-based fermented products as innovative vehicles to deliver both health-promoting microbes and bioactive compounds to consumers.

Milk and water kefir are fermented beverages prepared by inoculating milk or sugar-rich solutions with kefir grains - containing complex microbial consortia of bacteria and yeast. The increasing global popularity of kefir has driven interest in their microbiomes. Beyond their culinary significance, kefir serves as a model system for studying microbial community assembly, ecological filtering, and evolutionary dynamics. Kefir’s suitability as a model is enhanced by its simplicity (including short fermentation times), adaptability to experimental manipulation, and scalability for both controlled laboratory studies and citizen science initiatives. In this study, we employed genome-resolved metagenomics to investigate milk and water kefir microbiomes, produced over 21 weeks from an initial common source milk or water kefir grain by citizen scientists, using diverse substrates and conditions. Early fermentation (weeks 1–9) was marked by rapid compositional, functional and volatile shifts, leading to reproducible transitions between community states, with values not changing considerably thereafter. At the strain level, we identified multiple coexisting strains within dominant species, with initial fluctuations. While rare environmental species occasionally appeared, they were selectively filtered and did not persist. The sustained coexistence of key species and strains, alongside the emergence of reproducible community types, highlights the resilience and stability of kefir microbiomes across both short- and long-term timeframes. Our findings reinforce kefir’s value as a model for investigating microbial interactions, environmental microbial acquisition, and strain-level dynamics. The co-occurrence patterns observed among specific strains and species provide insight into microbial assembly and persistence, with broader relevance to complex ecosystems such as the gut microbiome. By elucidating patterns of compositional and functional change and assessing how selective pressures shape microbial communities, this study underscores kefir’s utility in microbial ecology and evolution.

Background and aims: Disorders of gut-brain interactions (DGBIs), including irritable bowel syndrome (IBS), functional constipation (FC) and functional diarrhoea (FD), have a multifactorial aetiology, with colonic microbiota alterations likely contributing. To investigate how these changes relate to DGBIs, the faecal microbial taxonomic composition and gene abundance in DGBI subjects was characterised and integrated with gastrointestinal and non-gastrointestinal symptoms. Methods: Microbial DNA was extracted and analysed by shotgun sequencing. 239 faecal samples (IBS-constipation/FC, n = 60; IBS-diarrhoea/FD, n = 66; controls, n = 113) were used for integrative analysis with Data Integration Analysis for Biomarker discovery using Latent cOmponents (DIABLO). Results: High-level compositional patterns were similar between FD and IBS-diarrhoea but differed between FC and IBS-constipation, compared to controls. All DGBI subtypes showed altered relative abundance of hydrogen-metabolising taxa (Enterobacteriaceae, Lachnospiraceae, Bilophila, Desulphovibrio, Methanobrevibacter), compared to controls. Relative gene abundance associated to micronutrient homeostasis discriminated IBS-diarrhoea, IBS-constipation and FC, but not FD, from controls. Increased tyrosine metabolism relative gene abundance discriminated FC and IBS-diarrhoea from controls. FC was further distinguished from controls and other DGBIs by increased abundance of facultative anaerobes (Salmonella, Shigella, Escherichia) and genes related to aromatic amine catabolism, secretion systems, and virulence. In constipation- and diarrhoea-predominant DGBIs, Firmicutes negatively correlated with microbial “secondary metabolism” and “phages, prophages, transposable elements, plasmids”, while “aromatic compound metabolism” positively correlated with constipation severity and diarrhoea symptoms (abdominal pain). Conclusion: Distinctive microbial changes suggested FC as a distinct condition from IBS-constipation. Despite taxonomic similarities between FD and IBS-diarrhoea, microbial gene abundance discriminated IBS-diarrhoea but not FD from controls. Integrative analysis revealed potential microbial-symptom relationships in DGBIs.

Rapid advancements in long-read sequencing have facilitated species-level microbial profiling through full-length 16S rRNA sequencing (~ 1500 bp), and more notably, by the newer 16S-ITS-23S ribosomal RNA operon (RRN) sequencing (~ 4500 bp). RRN sequencing is emerging as a superior method for species resolution, exceeding the capabilities of short-read and full-length 16S rRNA sequencing. However, being in its early stages of development, RRN sequencing has several underexplored or understudied elements, highlighting the need for a critical and thorough examination of its methodologies. Key areas that require detailed analysis include understanding how primer pairs, sequencing platforms, and classifiers and databases affect the accuracy of species resolution achieved through RRN sequencing. Our study addresses these gaps by evaluating the effect of primer pairs using four RRN primer combinations, and that of sequencing platforms by employing PacBio and Oxford Nanopore Technologies (ONT) systems. Furthermore, two classification methods (Minimap2 and OTU clustering), in combination with four RRN reference databases (MIrROR, rrnDB, and two versions of GROND) were compared to identify consistent and accurate classification methods with RRN sequencing. Here we demonstrate that RRN primer pair choice and sequencing platform do not substantially bias taxonomic profiles for most of the tested mock communities, while classification methods significantly impact the accuracy of species-level assignments. Of the classification methods tested, Minimap2 classifier in combination with the GROND database most consistently provided accurate species-level classification across the communities tested, irrespective of sequencing platform.

Colostrum quality is influenced by multiple factors, including its microbial load, which is determined by the cleanliness of collection and storage conditions. Additionally, the dam's diet and immune status play a crucial role in shaping colostrum quality by affecting IgG concentrations. Whereas many factors contribute to colostrum quality, this study will specifically use IgG content as the primary measure of quality. It is well established that feeding low-quality colostrum negatively affects calf health and growth, whereas feeding good-quality colostrum leads to better outcomes. However, it remains unclear if feeding colostrum above the recommended quality threshold offers additional benefits for calf health and growth. This study compared the effects of adequate- versus high-quality colostrum on dairy heifer calf growth, health, and the development of the fecal microbiome during the first 15 wk of life. We also measured the levels of apparent efficacy of absorption of IgG in both groups. Colostrum quality was initially determined and measured before feeding using a Brix refractometer (only feeding a minimum of 21% Brix); 93 heifer calves were assigned to treatment based on this data. Subsequently, laboratory analyses were conducted using radial immunodiffusion assays to measure the exact IgG levels in the colostrum; 72 calves were selected for continuation in the experiment based on the highest (n = 36) and lowest (n = 36) IgG levels in the colostrum. For these 72 calves, laboratory analyses were conducted using radial immunodiffusion assays to measure the exact IgG levels in the colostrum at the point of feeding and in the calf serum at 24 h after feeding. To ensure a fair comparison, the groups were balanced for calf birthweight, breed, and dam parity. Serum IgG at 24 h, weight, and health data were analyzed in SAS using linear mixed models and logistic regression. Alpha and β diversity were analyzed using R with ANOVA, permutational multivariate analysis of variance (PERMANOVA), and Benjamini-Hochberg P-value adjustments. Calves fed colostrum with a high IgG content (123.0 mg/mL IgG) exhibited higher rates of passive transfer compared with those fed adequate-quality colostrum (85.2 mg/mL IgG). Both groups had passive transfer rates >23 mg/mL IgG. Health outcomes were similar between the 2 groups, and ADG during the preweaning period was comparable, with calves gaining an average of 0.62 kg/d. Measures of α and β diversity in the fecal microbiome showed similar development in both groups. Apparent absorption efficacy was lower in calves fed high-quality colostrum (24.9%) compared with those fed adequate-quality colostrum (29.3%). The findings of this study support current recommendations for colostrum quality and suggest that calves may have a limited capacity to absorb higher concentrations of IgG. Whereas feeding higher-quality colostrum did not lead to significant improvements in growth, health, or microbiome diversity, it demonstrated that adequate-quality colostrum can be equally effective when combined with best practice management. Further research is needed to better understand the relationships between immunoglobulin absorption efficiency, calf health, microbiome development, and growth performance.

BACKGROUND:Mycobacterium avium complex causes chronic and difficult-to-treat infection in vulnerable patient groups, and incidence is increasing worldwide. Whole genome sequencing has the potential to reveal new information about how M. avium persists over time in the human lung. METHODS:We analysed the genomes of 287 isolates of M. avium that were sampled longitudinally from 56 patients. Our dataset included 50 newly sequenced genomes from a cohort of 20 patients from Ireland who were sampled for up to 10 years, and we compared these to 237 published genomes from 2 pre-existing cohorts from Europe to evaluate strains from Ireland in a wider context. Additionally, we performed a combined analysis across the 3 cohorts to examine the changes that occurred over the course of infection. RESULTS:We identified 2 instances where strains from Ireland clustered with strains from Europe within a 13-SNP threshold, supporting previous observations that dominant circulating clones of M. avium are present internationally. Across the 3 cohorts, we found that the communities of M. avium evolved over time within individual hosts, and we report that acquisition of new strains is frequent. Importantly, our findings suggest that M. avium may adapt to the conditions that it faces in the host, with evidence of positive selection of 13 distinct mycobacterial genes. Notably, multiple virulence-associated genes were under selection, including genes that could confer resistance to antibiotics and host defence mechanisms. CONCLUSIONS:Whole genome sequencing provides novel insights into within-host evolution of M. avium and highlights potentially important mycobacterial strategies to enhance persistence that may provide new targets for therapeutic investigation.

Faecal microbiome and serum metabolome have been studied in human medicine to provide a better understanding of metabolic derangements including diabetes; however, equivalent studies in equine medicine are limited. This was a case–control study conducted to identify differences in faecal microbiota composition and concurrent serum metabolite patterns between metabolically normal Connemara ponies and those with Equine Metabolic Syndrome (EMS). Thirty privately owned Connemara ponies (15 EMS and 15 controls) were included in the study. EMS was diagnosed by oral sugar test (OST). Blood samples were collected before and after an oral sugar challenge. One concurrent faecal sample was collected from each pony. Sequencing of the V3-V4 region of 16S rRNA gene was used to identify the microbial communities in faecal samples and assess the differences in microbial profiles between groups. Serum metabolites were analyzed using liquid chromatography–high-resolution mass spectrometry (LC–MS). Finally, multi-omics analysis was conducted by integration of microbiota-metabolome datasets to determine potential associations between metabolites and microbiota in EMS. The faecal microbiota community composition was significantly different between EMS and control groups (p = 0.04 and r2 = 4.3

Background/Objectives: Antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional antibiotics in livestock, offering a sustainable strategy for controlling bacterial pathogens in food production systems. In addition to their direct antimicrobial effects, AMPs play a key role in modulating host-associated microbiomes, influencing both microbial composition and function. Advances in metagenomic sequencing and bioinformatic tools now enable comprehensive exploration of AMP diversity and activity within complex microbial ecosystems. Methods: In this study, we employed Illumina-based next-generation sequencing (NGS) to analyze intestinal contents from six gut sections of broiler chickens obtained from a Spanish slaughterhouse. Results: Through de novo assembly and bioinformatic annotation, we identified biosynthetic gene clusters (BGCs) encoding ribosomally synthesized and post-translationally modified peptides (RiPPs), other specialized bioactive secondary metabolites, antimicrobial resistance genes (ARGs), virulence factor genes (VFGs), and a diverse microbial community. Among all gut sections, the cecum exhibited the highest genetic richness, characterized by a high diversity of RiPP-like clusters and antimicrobial resistance determinants. Conclusions: These findings highlight the poultry gut, particularly the cecum, as a significant reservoir of antimicrobial peptides (AMPs) with potential implications in antibiotic-free poultry production and enhanced food safety.

Aims: Gestational diabetes mellitus (GDM) is the most common complication of pregnancy and is known to be associated with an increased risk of postpartum metabolic disease. Based on the important role that the intestinal microbiota plays in blood glucose regulation and insulin sensitivity, supplementation of probiotic and postbiotic strains could improve glucose metabolism and tolerance in GDM. Main methods: 56 4-week-old female C57BL/6J-mice were divided into 4 groups (n = 14 animals/group): control (CNT), high-fat/high-sucrose (HFS), pA1c (R) alive (pA1c (R)) and heat-inactivated pA1c (R) (pA1c (R) HI). Serum biochemical parameters were analyzed, gene expression analyses were conducted, and fecal microbiota composition was evaluated by shot-gun sequencing. Key findings: pA1c (R)- and pA1c (R) HI-supplemented groups presented reduced fasting blood glucose levels and reduced insulin resistance during gestation and exhibited lower visceral adiposity and increased muscle tissue, together with an improvement in intrahepatic TGs content and ALT levels. Liver gene expression analyses demonstrated that pA1c (R) and pA1c (R) HI activities were mediated by modulation of the insulin receptor, but also by an overexpression of beta-oxidation genes, and downregulation of fatty acid biosynthesis genes. Shot-gun metagenomics demonstrated that Pediococcus acidilactici was detected in the feces of all the pA1c (R) and pA1c (R) HI-group after the supplementation period (75 % of the microbial profile was Pediococcus acidilactici) in only nine weeks of supplementation, and modulated gut microbiota composition. Significance: These results may be considered as future perspectives for the development of preventive, even therapeutic options for GDM based on hyperglycemia reduction, blood glucose regulation, hepatic steatosis attenuation and insulin resistance alleviation.

Metabolic health improvements in response to exercise and energy restriction may be mediated by the gut microbiome, yet causal evidence in humans remains limited. We used a 3-week exercise and energy restriction intervention to examine changes to the gut microbiome in otherwise healthy sedentary men and postmenopausal women with overweight/obesity. Intervention participants (n = 18) reduced habitual energy intake by 5000 kcal/week and expended 2000 kcal/week in addition to habitual physical activity through treadmill walking at 70% V̇O2Peak. Control participants (n = 12) maintained their usual lifestyle. Participants underwent dual-energy X-ray absorptiometry (DEXA), and samples of faeces, fasted venous blood, subcutaneous adipose tissue and skeletal muscle were collected. Faecal DNA was sequenced and profiled using shotgun metagenomics, Kraken2/Bracken and Human Microbiome Project Unified Metabolic Analysis Network 2 (HUMAnN2). The intervention significantly reduced body mass (mean Δ ± SD: -2.6 ± 1.5 kg), fat mass (-1.5 ± 1.3 kg), fasted insulin (-23.5 ± 38.1 pmol/l), leptin (-10.6 ± 7.3 ng/ml) and total cholesterol (-0.70 ± 0.42 mmol/l) concentrations, and also improved insulin sensitivity (HOMA2%S (homeostatic model of assessment)). Despite these significant metabolic changes the gut microbiome was unchanged in terms of α and β diversity and relative abundance. Thus, despite clinically meaningful improvements in body composition and metabolic health, we found no evidence for changes to the gut microbiome. In conclusion early metabolic changes with weight loss in humans are unlikely to be mediated by changes to the gut microbiome. KEY POINTS: Changes to the gut microbiome could contribute to metabolic improvements associated with weight loss in humans, but there have been limited attempts to address this question using robust randomised controlled trials (RCTs). We used a parallel-group RCT to examine whether a 3-week combined energy intake restriction and vigorous-intensity exercise intervention in people with overweight and obesity was temporally associated with changes to gut microbiome taxonomic composition and functional potential, short-chain fatty acid concentrations and expression of genes related to host-microbiome interactions in skeletal muscle and subcutaneous adipose tissue. We found that the human gut microbiome remains unchanged in the face of an intensive energy intake restriction and vigorous exercise intervention that significantly improved body composition and metabolic health in people with overweight/obesity. These findings indicate that early metabolic changes with weight loss in humans are unlikely to be mediated by changes to the gut microbiome.

Feeding waste milk, a common practice in dairy farming, exposes calves to subtherapeutic levels of antimicrobials, potentially contributing to antibiotic resistance-a growing concern globally. Many dairy farmers, including those in Ireland, continue this practice, feeding waste milk from antibiotic-treated cows to calves. Although previous studies have linked waste milk feeding to changes in calf growth and health during the preweaning period, its effects postweaning remain unclear. This study examined how the duration of antimicrobial exposure at levels equivalent to those found in waste milk influences health and growth outcomes of dairy heifer calves both before and after weaning. It also assessed the prevalence of extended-spectrum β-lactamase (ESBL)-producing antimicrobial-resistant Escherichia coli in feces and changes in the fecal microbiota over time. To mimic waste milk, as derived from a cow treated with an intramammary suspension of antibiotics, a simulated waste milk (SWM) was prepared by adding amoxicillin (1.68 mg/L) and neomycin (2.28 mg/L) to a conventional milk replacer (MR). The study employed a randomized block design with 87 dairy heifer calves assigned to 1 of 3 treatments: (1) long-term antibiotic (LTA), with calves fed SWM until weaning at 12 wk; (2) short-term antibiotic (STA), with SWM fed from 3 to 5 wk; and (3) control (CONT), with calves fed antibiotic-free MR. Calves were weighed weekly, and health scores, including fecal scores (tail and hindquarters cleanliness as diarrhea indicator), were recorded twice per week. Fecal and blood samples were collected to analyze microbiome changes and the shedding of antimicrobial resistance. Blood samples were taken to measure systemic inflammation, using serum amyloid A as a biomarker. Results indicated that SWM feeding did not affect average daily gains before or after weaning. However, higher fecal scores were observed in the LTA group during weaning and after weaning in the STA group. Antibiotic-resistant isolates were present in all groups, with the highest prevalence in LTA. Fecal microbiota analysis revealed treatment-specific microbial community variations, with an increase of Enterococcus faecium genes resistant to macrolide, aminoglycoside, and tetracycline antibiotics in LTA and STA compared with CONT. In summary, SWM feeding did not significantly affect growth or overall health, but it was associated with increased fecal shedding of resistant bacteria and some changes in the microbiota, indicating potential long-term implications for antimicrobial resistance in dairy herds.

In utero exposure to an increased level of maternal inflammation or a disrupted maternal gut microbiome during pregnancy have been linked to several neurodevelopmental disorders in the offspring. Despite the strong links between these two adverse events, few studies looked at the interaction between the maternal gut microbiome and maternal immune activation (MIA) on the neurodevelopmental outcomes in the offspring. Here, we aim to determine if maternal gut microbiome disruption exacerbated the impact of systemic inflammation on brain development, offspring behaviour, and long-term microbiome changes. A low dose of intraperitoneal lipopolysaccharide (LPS) was administered to pregnant Sprague Dawley rats from gestational day 12–18. Concurrently, an antibiotic cocktail (ampicillin, neomycin, vancomycin) was given in the drinking water to disturb the maternal microbiome. Embryos at gestational day 18 were found to have a reduced body size and weight, along with reduced placental weight following exposure to LPS, with some effects also seen with antibiotic exposure. Offspring exposed to LPS in utero were found to have increased anxiety-like behaviours and repetitive-behaviours. No behavioural changes were noted from antibiotic exposure. The expression of 5HT1a receptors in the prefrontal cortex was found to be reduced following LPS exposure. The offspring microbiome varied between the groups, with prenatal antibiotic exposure playing a role in reducing α-diversity and species richness in the periadolescent period. This study highlights the impact of prenatal exposures on different aspects of neurodevelopment. However, additional research is warranted to explore the role of the maternal immune system and microbiome on the offspring development, while also testing the potential therapeutic agents such as probiotics.

Background:Alterations in gut-brain axis communication pathways and the gut microbiota ecosystem caused by early life stress have been extensively described as critical players in the pathophysiology of stress-induced disorders. However, the extent to which stress-induced gut microbiota alterations manifest in early life and contribute to the sex-specific susceptibility to distinct gut-brain phenotypes in adulthood has yet to be defined. Methods:Male and female Sprague-Dawley rat offspring underwent maternal separation (3h/day from postnatal day 2-12). Faecal samples were collected before weaning for gut microbiota 16S rRNA sequencing and metabolomic analysis. Visceral pain sensitivity and negative valence behaviours were assessed in adulthood using colorectal distension and the forced swim test respectively. Behavioural data were processed in a two-step cluster analysis to identify groupings within the dataset. Multi-omics analysis was carried out to investigate if the microbial signatures following early life stress were already defined according to the membership of the adult behavioural phenotypes. Results:Maternal separation resulted in increased visceral hypersensitivity while showing a trend for a sex-dependent increase in negative valence behaviour in adulthood. The cluster analysis revealed four clusters within the dataset representing distinct pathophysiological domains reminiscent of the behavioural consequences of early-life stress: 1. resilient, 2. pain, 3. immobile and 4. comorbid. The early life gut microbiota of each of these clusters show distinct alterations in terms of diversity, genus level differential abundance, and functional modules. Multi-omic integrations points towards a role for different metabolic pathways underlying each cluster-specific phenotype. Conclusion:Our study is the first to identify distinct phenotypes defined by susceptibility or resilience to gut-brain dysfunction induced by early life stress. The gut microbiota in early life shows sex-dependent alterations in each cluster that precede specific behavioural phenotypes in adulthood. Future research is warranted to determine the causal relationship between early-life stress-induced changes in the gut microbiota and to understand the trajectory leading to the manifestation of different behavioural phenotypes in adulthood.

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.

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.

IntroductionPost-weaning diarrhoea (PWD) is a multifactorial disease that affects piglets after weaning, contributing to productive and economic losses. Its control includes the use of in-feed prophylactic antibiotics and therapeutic zinc oxide (ZnO), treatments that, since 2022, are no longer permitted in the European Union due to spread of antimicrobial resistance genes and pollution of soil with heavy metals. A dysbiosis in the microbiota has been suggested as a potential risk factor of PWD onset. Understanding pig’s microbiota development around weaning and its changes in response to ZnO and antibiotics is crucial to develop feasible alternatives to prophylactic and metaphylactic antimicrobial use.MethodsThis study used shotgun metagenomic sequencing to investigate the environmental and faecal microbiota on 10 farms using (Treated) or not using (ZnO-free) in-feed antibiotics and ZnO during the first 14 days post-weaning (dpw). Environmental samples from clean pens were collected at weaning day (0dpw), and faecal samples at 0, 7 and 14dpw. Diarrhoeic faecal samples were collected at 7dpw when available.ResultsThe analysis of data revealed that the faecal microbiota composition and its functionality was impacted by the sampling time point (microbiota maturation after weaning) but not by the farm environment. Treatment with antibiotics and ZnO showed no effects on diversity indices while the analyses of microbiota taxonomic and functional profiles revealed increased abundance of taxa and metabolic functions associated with Phascolarctobacterium succinatutens or different species of Prevotella spp. on the Treated farms, and with Megasphaera elsdenii and Escherichia coli on the ZnO-free farms. The analysis of diarrhoea samples revealed that the treatment favoured the microbiota transition or maturation from 0dpw to 14dpw in Treated farms, resembling the composition of healthy animals, when compared to diarrhoea from ZnO-free farms, which were linked in composition to 0dpw samples.DiscussionThe results provide a comprehensive overview of the beneficial effects of ZnO and antibiotics in PWD in the microbiota transition after weaning, preventing the overgrowth of pathogens such as pathogenic E. coli and revealing the key aspects in microbiota maturation that antibiotics or ZnO alternatives should fulfil.