Diet rich in soluble dietary fibres increases excretion of perfluorooctane sulfonic acid (PFOS) in male Sprague-Dawley rats.

Perfluorooctane sulfonic acid (PFOS) belongs to a large group of anthropogenic compounds with high persistency named per- and polyfluorinated substances (PFAS). Widespread use from industry to household appliances and food-contact materials contributes to PFAS exposure with food as the primary source. Association studies suggest that vegetables and fibre rich diet may reduce PFOS levels in humans, but experimental data remain limited. Here, we investigated PFOS uptake and wash-out after seven days of PFOS (3 mg/kg/day) in two groups of rats (N=12 per group) fed diets either high (HF) or low (LF) in soluble dietary fibres. Two control groups (N=12/group) were fed the same diets without PFOS. Changes in pH and transit time were monitored alongside intestinal and faecal microbiota composition. We quantified systemic and excreted, linear and branched PFOS. Results revealed significantly lower pH and faster intestinal transit in the HF groups. Importantly, HF rats had lower serum PFOS concentrations and higher PFOS concentrations in caecal content and faeces, indicating a more efficient excretion on the fibre rich diet. In both dietary groups, PFOS affected the gut microbiota composition. Our results suggest that a diet rich in soluble dietary fibres accelerates excretion of PFOS and lowers PFOS concentration in serum.

Development of an infant colon simulating in vitro model, I-TIM-2, to study the effects of modulation strategies on the infant gut microbiome composition and function.

The early life stages are critical for the development of the gut microbiome. Variables such as antibiotics exposure, birth-mode via Cesarean section, and formula feeding are associated with disruptions in microbiome development and are related to adverse health effects later in life. Studying the effects of microbiome-modulating strategies in infants is challenged by appropriate ethical constraints. Therefore, we developed I-TIM-2, an infant in vitro colonic model based on the validated, computer-controlled, dynamic model of the colon, TIM-2. The system, consisting of four separate compartments, was inoculated with feces from four healthy, primarily breastfed infants, displaying distinctive microbiome profiles. For each infant's fecal sample, a 96-h experiment was performed, with two compartments receiving an infant diet adapted medium and two compartments additionally receiving five human milk oligosaccharides (HMOs) in physiological concentrations and proportions. Bacterial composition was determined by shotgun metagenomics and qPCR. Concentrations of short-chain fatty acids (SCFAs) and HMOs were determined by LC-MS. Microbial diversity and high amounts of inoculum-derived species were preserved in the model throughout each experiment. Microbiome composition and SCFA concentrations were consistent with published data from infants. HMOs strongly modulated the microbiome composition by stimulating relative proportions of Bifidobacterium. This affected the metabolic output and resulted in an increased production of acetic and formic acid, characteristic of bifidobacterial HMO metabolism. In conclusion, these data demonstrate the development of a valid model to study the dynamics and modulations of the infant gut microbiome and metabolome.IMPORTANCEThe infant gut microbiome is intricately linked to the health of its host. This is partly mediated through the bacterial production of metabolites that interact with the host cells. Human milk shapes the establishment of the infant gut microbiome as it contains human milk sugars that select for primarily bifidobacteria. The establishment can be disrupted by modern interventions such as formula feeding. This can alter the microbiome composition and metabolite production profile, which can affect the host. In this article, we set up an infant in vitro colonic model to study microbiome interactions and functions. In this model, we investigated the effects of human milk sugars and their promotion of bifidobacteria at the expense of other bacteria. The model is an ideal system to assess the effects of various modulating strategies on the infant gut microbiome and its interactions with its host.

Regulation of microbial gene expression: the key to understanding our gut microbiome.

During the past two decades, gut microbiome studies have established the significant impact of the gut microbiota and its metabolites on host health. However, the molecular mechanisms governing the production of microbial metabolites in the gut environment remain insufficiently investigated and thus are poorly understood. Here, we propose that an enhanced understanding of gut microbial gene regulation, which is responsive to dietary components and gut environmental conditions, is needed in the research field and essential for our ability to effectively promote host health and prevent diseases through interventions targeting the gut microbiome.

Potential of using an engineered indole lactic acid producing Escherichia coli Nissle 1917 in a murine model of colitis.

The gut microbiome is a significant factor in the pathophysiology of ulcerative colitis (UC), prompting investigations into the use of probiotic therapies to counter gastrointestinal inflammation. However, while much attention has been given to the therapeutic potential of microbes at the species and strain level, the discovery and application of their metabolic products may offer more precise and controlled solutions in battling disease. In this work, we examined the therapeutic potential of indole lactic acid (ILA) to alleviate inflammation in a murine model of colitis. A previously constructed ILA-producing Escherichia coli Nissle 1917 strain (EcN aldh) and its isogenic non-ILA producing counterpart (EcN) were studied in a murine model of Dextran Sodium Sulfate (DSS) induced colitis. The colitic animals suffered from severe colitic symptoms, with no differentiation between the groups in body weight loss and disease activity index. However, three days after cessation of DSS treatment the EcN aldh-treated mice showed signs of reduced intestinal inflammation, as manifested by lower concentrations of fecal lipocalin-2. Additionally, expression analysis of the inflamed tissue revealed distinct effects of the EcN aldh strain on proteins associated with intestinal health, such as TFF3, occludin and IL-1ß expression. These results show no impact of EcN or EcN aldh on acute DSS-induced colitis, but suggest that in particular EcN aldh may assist recovery from intestinal inflammation.

Advanced microbiome therapeutics as a novel modality for oral delivery of peptides to manage metabolic diseases.

The rising prevalence of metabolic diseases calls for innovative treatments. Peptide-based drugs have transformed the management of conditions such as obesity and type 2 diabetes. Yet, challenges persist in oral delivery of these peptides. This review explores the potential of 'advanced microbiome therapeutics' (AMTs), which involve engineered microbes for delivery of peptides in situ, thereby enhancing their bioavailability. Preclinical work on AMTs has shown promise in treating animal models of metabolic diseases, including obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease. Outstanding challenges toward realizing the potential of AMTs involve improving peptide expression, ensuring predictable colonization control, enhancing stability, and managing safety and biocontainment concerns. Still, AMTs have potential for revolutionizing the treatment of metabolic diseases, potentially offering dynamic and personalized novel therapeutic approaches.

Delivery of E. coli Nissle to the mouse gut by mucoadhesive microcontainers does not improve its competitive ability against strains linked to ulcerative colitis.

For patients with ulcerative colitis (UC), administration of the probiotic E. coli Nissle (EcN) holds promise for alleviation of disease symptoms. The mechanisms are unclear, but it has been hypothesised that a capacity of the probiotic to outcompete potentially detrimental UC-associated E. coli strains plays an important role. However, this could previously not be confirmed in a mouse model of competition between EcN and two UC-associated strains, as reported by Petersen et al. 2011. In the present study, we re-evaluated the idea, hypothesising that delivery of EcN by a micro device dosing system (microcontainers), designed for delivery into the intestinal mucus, could support colonisation and confer a competition advantage compared to classical oral dosing. Six groups of mice were pre-colonised with one of two UC-associated E. coli strains followed by oral delivery of EcN, either in capsules containing microcontainers with freeze-dried EcN powder, capsules containing freeze-dried EcN powder, or as a fresh sucrose suspension. Co-colonisation between the probiotic and the disease-associated strains was observed regardless of dosing method, and no competition advantages linked to microcontainer delivery were identified within this setup. Other approaches are thus needed if the competitive capacity of EcN in the gut should be improved.

Antibiotic induced restructuring of the gut microbiota does not affect oral uptake and accumulation of perfluorooctane sulfonic acid (PFOS) in rats.

Perfluorooctane sulfonic acid (PFOS) is a manmade legacy compound belonging to the group of persistent per- and polyfluorinated substances (PFAS). While many adverse health effects of PFOS have been identified, knowledge about its effect on the intestinal microbiota is scarce. The microbial community inhabiting the gut of mammals plays an important role in health, for instance by affecting the uptake, excretion, and bioavailability of some xenobiotic toxicants. Here, we investigated (i) the effect of vancomycin-mediated microbiota modulation on the uptake of PFOS in adult Sprague-Dawley rats, and (ii) the effects of PFOS exposure on the rat microbiota composition. Four groups of twelve rats were exposed daily for 7 days with either 3 mg/kg PFOS plus 8 mg/kg vancomycin, only PFOS, only vancomycin, or a corn oil control. Vancomycin-induced modulation of the gut microbiota composition did not affect uptake of branched and linear PFOS over a period of 7 days, measured in serum samples. 16S rRNA amplicon sequencing of faecal and intestinal samples revealed that vancomycin treatment lowered microbial alpha-diversity, while PFOS increased the microbial diversity in vancomycin-treated as well as in non-antibiotic treated animals, possibly because an observed decrease in the Enterobacteriaceae abundance allows other microbial species to propagate. Colonic short-chain fatty acids were significantly lower in vancomycin-treated animals but remained unaffected by PFOS. Our results suggest that PFOS exposure may disturb the intestinal microbiota, but that antibiotic-induced modulation of the intestinal ecosystem does not affect systemic uptake of PFOS in rats.

Effects of a wholegrain-rich diet on markers of colonic fermentation and bowel function and their associations with the gut microbiome: a randomised controlled cross-over trial.

Background: Diets rich in whole grains are associated with health benefits. Yet, it remains unclear whether the benefits are mediated by changes in gut function and fermentation. Objective: We explored the effects of whole-grain vs. refined-grain diets on markers of colonic fermentation and bowel function, as well as their associations with the gut microbiome. Methods: Fifty overweight individuals with increased metabolic risk and a high habitual intake of whole grains (~69 g/day) completed a randomised cross-over trial with two 8-week dietary intervention periods comprising a whole-grain diet (≥75 g/day) and a refined-grain diet (<10 g/day), separated by a washout period of ≥6 weeks. A range of markers of colonic fermentation and bowel function were assessed before and after each intervention. Results: The whole-grain diet increased the levels of faecal butyrate (p = 0.015) and caproate (p = 0.013) compared to the refined-grain diet. No changes in other faecal SCFA, BCFA or urinary levels of microbial-derived proteolytic markers between the two interventions were observed. Similarly, faecal pH remained unchanged. Faecal pH did however increase (p = 0.030) after the refined-grain diet compared to the baseline. Stool frequency was lower at the end of the refined-grain period compared to the end of the whole-grain diet (p = 0.001). No difference in faecal water content was observed between the intervention periods, however, faecal water content increased following the whole-grain period compared to the baseline (p = 0.007). Dry stool energy density was unaffected by the dietary interventions. Nevertheless, it explained 4.7% of the gut microbiome variation at the end of the refined-grain diet, while faecal pH and colonic transit time explained 4.3 and 5%, respectively. Several butyrate-producers (e.g., Faecalibacterium, Roseburia, Butyriciococcus) were inversely associated with colonic transit time and/or faecal pH, while the mucin-degraders Akkermansia and Ruminococcaceae showed the opposite association. Conclusion: Compared with the refined-grain diet, the whole-grain diet increased faecal butyrate and caproate concentrations as well as stool frequency, emphasising that differences between whole and refined grains affect both colonic fermentation and bowel habits.

Advancing human gut microbiota research by considering gut transit time.

Accumulating evidence indicates that gut transit time is a key factor in shaping the gut microbiota composition and activity, which are linked to human health. Both population-wide and small-scale studies have identified transit time as a top covariate contributing to the large interindividual variation in the faecal microbiota composition. Despite this, transit time is still rarely being considered in the field of the human gut microbiome. Here, we review the latest research describing how and why whole gut and segmental transit times vary substantially between and within individuals, and how variations in gut transit time impact the gut microbiota composition, diversity and metabolism. Furthermore, we discuss the mechanisms by which the gut microbiota may causally affect gut motility. We argue that by taking into account the interindividual and intraindividual differences in gut transit time, we can advance our understanding of diet-microbiota interactions and disease-related microbiome signatures, since these may often be confounded by transient or persistent alterations in transit time. Altogether, a better understanding of the complex, bidirectional interactions between the gut microbiota and transit time is required to better understand gut microbiome variations in health and disease.

Gut microbiota differs between treatment outcomes early after fecal microbiota transplantation against recurrent Clostridioides difficile infection.

AbstarctIn fecal microbiota transplantation (FMT) against recurrent Clostridioides difficile infection (CDI), clinical outcomes are usually determined after 8 weeks. We hypothesized that the intestinal microbiota changes earlier than this timepoint, and analyzed fecal samples obtained 1 week after treatment from 64 patients diagnosed with recurrent CDI and included in a randomized clinical trial, where the infection was treated with either vancomycin-preceded FMT (N = 24), vancomycin (N = 16) or fidaxomicin (N = 24). In comparison with non-responders, patients with sustained resolution after FMT had increased microbial alpha diversity, enrichment of Ruminococcaceae and Lachnospiraceae, depletion of Enterobacteriaceae, more pronounced donor microbiota engraftment, and resolution of gut microbiota dysbiosis. We found that a constructed index, based on markers for the identified genera Escherichia and Blautia, successfully predicted clinical outcomes at Week 8, which exemplifies a way to utilize clinically feasible methods to predict treatment failure. Microbiota changes were restricted to patients who received FMT rather than antibiotic monotherapy, indicating that FMT confers treatment response in a different way than antibiotics. We suggest that early identification of microbial community structures after FMT is of clinical value to predict response to the treatment.

Stool energy density is positively correlated to intestinal transit time and related to microbial enterotypes.

BACKGROUND: It has been hypothesised that the gut microbiota causally affects obesity via its capacity to extract energy from the diet. Yet, evidence elucidating the role of particular human microbial community structures and determinants of microbiota-dependent energy harvest is lacking. RESULTS: Here, we investigated whether energy extraction from the diet in 85 overweight adults, estimated by dry stool energy density, was associated with intestinal transit time and variations in microbial community diversity and overall structure stratified as enterotypes. We hypothesised that a slower intestinal transit would allow for more energy extraction. However, opposite of what we expected, the stool energy density was positively associated with intestinal transit time. Stratifications into enterotypes showed that individuals with a Bacteroides enterotype (B-type) had significantly lower stool energy density, shorter intestinal transit times, and lower alpha-diversity compared to individuals with a Ruminococcaceae enterotype (R-type). The Prevotella (P-type) individuals appeared in between the B- and R-type. The differences in stool energy density between enterotypes were not explained by differences in habitual diet, intake of dietary fibre or faecal bacterial cell counts. However, the R-type individuals showed higher urinary and faecal levels of microbial-derived proteolytic metabolites compared to the B-type, suggesting increased colonic proteolysis in the R-type individuals. This could imply a less effective colonic energy extraction in the R-type individuals compared to the B-type individuals. Notably, the R-type had significantly lower body weight compared to the B-type. CONCLUSIONS: Our findings suggest that gut microbial energy harvest is diversified among individuals by intestinal transit time and associated gut microbiome ecosystem variations. A better understanding of these associations could support the development of personalised nutrition and improved weight-loss strategies. Video Abstract.

Delivery of streptomycin to the rat colon by use of electrospun nanofibers.

Drug-loaded electrospun nanofibers are potential drug carrier systems that may optimize disease treatment while reducing the impact on commensal microbes. The feasibility of streptomycin-loaded pullulan nanofibers fabricated from a green electrospinning procedure using water as the solvent was assessed. We conducted a rat study including a group treated with streptomycin-loaded nanofibers (STR-F, n = 5), a group treated with similar concentrations of streptomycin in the drinking water (STR-W, n = 5), and a non-treated control group (CTR, n = 5). Streptomycin was successfully loaded into nanofibers and delivered by this vehicle, which minimized the quantity of the drug released in the ileal compartment of the gut. Ingested streptomycin-resistant E. coli colonized of up to 106 CFU/g feces, revealing a selective effect of streptomycin even when given in the low amounts allowed by the nanofiber-based delivery. 16S amplicon sequencing of the indigenous microbiota revealed differential effects in the three groups. An increase of Peptostreptococcaceae in the cecum of STR-F animals may indicate that the fermentation of nanofibers directly or indirectly promoted growth of bacteria within this family. Our results elucidate relevant properties of electrospun nanofibers as a novel vehicle for delivery of antimicrobials to the large intestine.

Cross-generational bacterial strain transfer to an infant after fecal microbiota transplantation to a pregnant patient: a case report.

BACKGROUND: Fecal microbiota transplantation (FMT) effectively prevents the recurrence of Clostridioides difficile infection (CDI). Long-term engraftment of donor-specific microbial consortia may occur in the recipient, but potential further transfer to other sites, including the vertical transmission of donor-specific strains to future generations, has not been investigated. Here, we report, for the first time, the cross-generational transmission of specific bacterial strains from an FMT donor to a pregnant patient with CDI and further to her child, born at term, 26 weeks after the FMT treatment. METHODS: A pregnant woman (gestation week 12 + 5) with CDI was treated with FMT via colonoscopy. She gave vaginal birth at term to a healthy baby. Fecal samples were collected from the feces donor, the mother (before FMT, and 1, 8, 15, 22, 26, and 50 weeks after FMT), and the infant (meconium at birth and 3 and 6 months after birth). Fecal samples were profiled by deep metagenomic sequencing for strain-level analysis. The microbial transfer was monitored using single nucleotide variants in metagenomes and further compared to a collection of metagenomic samples from 651 healthy infants and 58 healthy adults. RESULTS: The single FMT procedure led to an uneventful and sustained clinical resolution in the patient, who experienced no further CDI-related symptoms up to 50 weeks after treatment. The gut microbiota of the patient with CDI differed considerably from the healthy donor and was characterized as low in alpha diversity and enriched for several potential pathogens. The FMT successfully normalized the patient's gut microbiota, likely by donor microbiota transfer and engraftment. Importantly, our analysis revealed that some specific strains were transferred from the donor to the patient and then further to the infant, thus demonstrating cross-generational microbial transfer. CONCLUSIONS: The evidence for cross-generational strain transfer following FMT provides novel insights into the dynamics and engraftment of bacterial strains from healthy donors. The data suggests FMT treatment of pregnant women as a potential strategy to introduce beneficial strains or even bacterial consortia to infants, i.e., neonatal seeding. Video Abstract.

Prevotella abundance and salivary amylase gene copy number predict fat loss in response to wholegrain diets.

Background: Salivary amylase (AMY1) gene copy number (CN) and Prevotella abundance in the gut are involved in carbohydrate digestion in the upper and lower gastrointestinal tract, respectively; and have been suggested as prognostic biomarkers for weight loss among overweight individuals consuming diets rich in fiber and wholegrains. Objective: We hypothesized that Prevotella abundance would be linked to greater loss of body fat after wholegrain consumption among individuals with low AMY1 CN, but not in those with high AMY1 CN. Methods: We reanalyzed data from two independent randomized ad libitum wholegrain interventions (fiber intake ∼33 g/d for 6-8 weeks), to investigate the relationship between baseline Prevotella abundance and body fat loss among healthy, overweight participants stratified into two groups by median AMY1 CN. Individuals with no detected Prevotella spp. were excluded from the main analysis. Results: In both studies, individuals with low AMY1 CN exhibited a positive correlation between baseline Prevotella abundance and fat loss after consuming the wholegrain diet (r > 0.5, P < 0.05), but no correlation among participants with high AMY1 CN (P ≥ 0.6). Following consumption of the refined wheat control diets, there were no associations between baseline Prevotella abundance and changes in body fat in any of the AMY1 groups. Conclusion: These results suggest that Prevotella abundance together with AMY1 CN can help predict fat loss in response to ad libitum wholegrain diets, highlighting the potential of these biomarkers in personalized obesity management.

Local Delivery of Streptomycin in Microcontainers Facilitates Colonization of Streptomycin-Resistant Escherichia coli in the Rat Colon.

Oral antibiotic treatment is often applied in animal studies in order to allow establishment of an introduced antibiotic-resistant bacterium in the gut. Here, we compared the application of streptomycin dosed orally in microcontainers to dosage through drinking water. The selective effect on a resistant bacterial strain, as well as the effects on fecal, luminal, and mucosal microbiota composition, were investigated. Three groups of rats (n = 10 per group) were orally dosed with microcontainers daily for 3 days. One of these groups (STR-M) received streptomycin-loaded microcontainers designed for release in the distal ileum, while the other two groups (controls [CTR] and STR-W) received empty microcontainers. The STR-W group was additionally dosed with streptomycin through the drinking water. A streptomycin-resistant Escherichia coli strain was orally inoculated into all animals. Three days after inoculation, the resistant E. coli was found only in the cecum and colon of animals receiving streptomycin in microcontainers but in all intestinal compartments of animals receiving streptomycin in the drinking water. 16S rRNA amplicon sequencing revealed significant changes in the fecal microbiota of both groups of streptomycin-treated animals. Investigation of the inner colonic mucus layer by confocal laser scanning microscopy and laser capture microdissection revealed no significant effect of streptomycin treatment on the mucus-inhabiting microbiota or on E. coli encroachment into the inner mucus. Streptomycin-loaded microcontainers thus enhanced proliferation of an introduced streptomycin-resistant E. coli in the cecum and colon without affecting the small intestine environment. While improvements of the drug delivery system are needed to facilitate optimal local concentration and release of streptomycin, the application of microcontainers provides new prospects for antibiotic treatment. IMPORTANCE Delivery of antibiotics in microcontainer devices designed for release at specific sites of the gut represents a novel approach which might reduce the amount of antibiotic needed to obtain a local selective effect. We propose that the application of microcontainers may have the potential to open novel opportunities for antibiotic treatment of humans and animals with fewer side effects on nontarget bacterial populations. In the current study, we therefore elucidated the effects of streptomycin, delivered in microcontainers coated with pH-sensitive lids, on the selective effect on a resistant bacterium, as well as on the surrounding intestinal microbiota in rats.

PFOS-induced thyroid hormone system disrupted rats display organ-specific changes in their transcriptomes.

Perfluorooctanesulfonic acid (PFOS) is a persistent anthropogenic chemical that can affect the thyroid hormone system in humans and animals. In adults, thyroid hormones (THs) are regulated by the hypothalamic-pituitary-thyroid (HPT) axis, but also by organs such as the liver and potentially the gut microbiota. PFOS and other xenobiotics can therefore disrupt the TH system at various locations and through different mechanisms. To start addressing this, we exposed adult male rats to 3 mg PFOS/kg/day for 7 days and analysed effects on multiple organs and pathways simultaneously by transcriptomics. This included four primary organs involved in TH regulation, namely hypothalamus, pituitary, thyroid, and liver. To investigate a potential role of the gut microbiota in thyroid hormone regulation, two additional groups of animals were dosed with the antibiotic vancomycin (8 mg/kg/day), either with or without PFOS. PFOS exposure decreased thyroxine (T4) and triiodothyronine (T3) without affecting thyroid stimulating hormone (TSH), resembling a state of hypothyroxinemia. PFOS exposure resulted in 50 differentially expressed genes (DEGs) in the hypothalamus, 68 DEGs in the pituitary, 71 DEGs in the thyroid, and 181 DEGs in the liver. A concomitant compromised gut microbiota did not significantly change effects of PFOS exposure. Organ-specific DEGs did not align with TH regulating genes; however, genes associated with vesicle transport and neuronal signaling were affected in the hypothalamus, and phase I and phase II metabolism in the liver. This suggests that a decrease in systemic TH levels may activate the expression of factors altering trafficking, metabolism and excretion of TH. At the transcriptional level, little evidence suggests that the pituitary or thyroid gland is involved in PFOS-induced TH system disruption.

Amoxicillin does not affect the development of cow's milk allergy in a Brown Norway rat model.

The use of antibiotics as well as changes in the gut microbiota have been linked to development of food allergy in childhood. It remains unknown whether administration of a single clinically relevant antibiotic directly promotes food allergy development when administrated during the sensitisation phase in an experimental animal model. We investigated whether the antibiotic amoxicillin affected gut microbiota composition, development of cow's milk allergy (CMA) and frequencies of allergic effector cells and regulatory T cells in the intestine. Brown Norway rats were given daily oral gavages of amoxicillin for six weeks and whey protein concentrate (WPC) with or without cholera toxin three times per week for the last five weeks. Microbiota composition in faeces and small intestine was analysed by 16S rRNA sequencing. The development of CMA was assessed by WPC-specific IgE in serum, ear swelling response to WPC and body hypothermia following oral gavage of WPC. Allergic effector cells were analysed by histology, and frequencies of regulatory and activated T cells were analysed by flow cytometry. Amoxicillin administration reduced faecal microbiota diversity, reduced the relative abundance of Firmicutes and increased the abundance of Bacteroidetes and Proteobacteria. Despite these effects, amoxicillin did not affect the development of CMA, nor the frequencies of allergic effector cells or regulatory T cells. Thus, amoxicillin does not carry a direct risk for food allergy development when administrated in an experimental model of allergic sensitisation to WPC via the gut. This finding suggests that confounding factors may better explain the epidemiological link between antibiotic use and food allergy.

Human milk oligosaccharides induce acute yet reversible compositional changes in the gut microbiota of conventional mice linked to a reduction of butyrate levels.

Human Milk Oligosaccharides (HMOs) are glycans with prebiotic properties known to drive microbial selection in the infant gut, which in turn influences immune development and future health. Bifidobacteria are specialized in HMO degradation and frequently dominate the gut microbiota of breastfed infants. However, some species of Bacteroidaceae also degrade HMOs, which may prompt selection also of these species in the gut microbiota. To investigate to what extent specific HMOs affect the abundance of naturally occurring Bacteroidaceae species in a complex mammalian gut environment, we conducted a study in 40 female NMRI mice administered three structurally different HMOs, namely 6'sialyllactose (6'SL, n = 8), 3-fucosyllactose (3FL, n = 16), and Lacto-N-Tetraose (LNT, n = 8), through drinking water (5%). Compared to a control group receiving unsupplemented drinking water (n = 8), supplementation with each of the HMOs significantly increased both the absolute and relative abundance of Bacteroidaceae species in faecal samples and affected the overall microbial composition analyzed by 16s rRNA amplicon sequencing. The compositional differences were mainly attributed to an increase in the relative abundance of the genus Phocaeicola (formerly Bacteroides) and a concomitant decrease of the genus Lacrimispora (formerly Clostridium XIVa cluster). During a 1-week washout period performed specifically for the 3FL group, this effect was reversed. Short-chain fatty acid analysis of faecal water revealed a decrease in acetate, butyrate and isobutyrate levels in animals supplemented with 3FL, which may reflect the observed decrease in the Lacrimispora genus. This study highlights HMO-driven Bacteroidaceae selection in the gut environment, which may cause a reduction of butyrate-producing clostridia.

Escherichia coli Promoters with Consistent Expression throughout the Murine Gut.

Advanced microbial therapeutics have great potential as a novel modality to diagnose and treat a wide range of diseases. Yet, to realize this potential, robust parts for regulating gene expression and consequent therapeutic activity in situ are needed. In this study, we characterized the expression level of more than 8000 variants of the Escherichia coli sigma factor 70 (σ70) promoter in a range of different environmental conditions and growth states using fluorescence-activated cell sorting and deep sequencing. Sampled conditions include aerobic and anaerobic culture in the laboratory as well as growth in several locations of the murine gastrointestinal tract. We found that σ70 promoters in E. coli generally maintain consistent expression levels across the murine gut (R2: 0.55-0.85, p value < 1 × 10-5), suggesting a limited environmental influence but a higher variability between in vitro and in vivo expression levels, highlighting the challenges of translating in vitro promoter activity to in vivo applications. Based on these data, we design the Schantzetta library, composed of eight promoters spanning a wide expression range and displaying a high degree of robustness in both laboratory and in vivo conditions (R2 = 0.98, p = 0.000827). This study provides a systematic assessment of the σ70 promoter activity in E. coli as it transits the murine gut leading to the definition of robust expression cassettes that could be a valuable tool for reliable engineering and development of advanced microbial therapeutics.