Quantifying the transport of biologics across intestinal barrier models in real-time by fluorescent imaging.

Unsuccessful clinical translation of orally delivered biological drugs remains a challenge in pharmaceutical development and has been linked to insufficient mechanistic understanding of intestinal drug transport. Live cell imaging could provide such mechanistic insights by directly tracking drug transport across intestinal barriers at subcellular resolution, however traditional intestinal in vitro models are not compatible with the necessary live cell imaging modalities. Here, we employed a novel microfluidic platform to develop an in vitro intestinal epithelial barrier compatible with advanced widefield- and confocal microscopy. We established a quantitative, multiplexed and high-temporal resolution imaging assay for investigating the cellular uptake and cross-barrier transport of biologics while simultaneously monitoring barrier integrity. As a proof-of-principle, we use the generic model to monitor the transport of co-administrated cell penetrating peptide (TAT) and insulin. We show that while TAT displayed a concentration dependent difference in its transport mechanism and efficiency, insulin displayed cellular internalization, but was restricted from transport across the barrier. This illustrates how such a sophisticated imaging based barrier model can facilitate mechanistic studies of drug transport across intestinal barriers and aid in vivo and clinical translation in drug development.

Production of a monolithic fuel cell stack with high power density.

The transportation sector is undergoing a technology shift from internal combustion engines to electric motors powered by secondary Li-based batteries. However, the limited range and long charging times of Li-ion batteries still hinder widespread adoption. This aspect is particularly true in the case of heavy freight and long-range transportation, where solid oxide fuel cells (SOFCs) offer an attractive alternative as they can provide high-efficiency and flexible fuel choices. However, the SOFC technology is mainly used for stationary applications owing to the high operating temperature, low volumetric power density and specific power, and poor robustness towards thermal cycling and mechanical vibrations of conventional ceramic-based cells. Here, we present a metal-based monolithic fuel cell design to overcome these issues. Cost-effective and scalable manufacturing processes are employed for fabrication, and only a single heat treatment is required, as opposed to multiple thermal treatments in conventional SOFC production. The design is optimised through three-dimensional multiphysics modelling, nanoparticle infiltration, and corrosion-mitigating treatments. The monolithic fuel cell stack shows a power density of 5.6 kW/L, thus, demonstrating the potential of SOFC technology for transport applications.

Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut.

Breastfeeding profoundly shapes the infant gut microbiota, which is critical for early life immune development, and the gut microbiota can impact host physiology in various ways, such as through the production of metabolites. However, few breastmilk-dependent microbial metabolites mediating host-microbiota interactions are currently known. Here, we demonstrate that breastmilk-promoted Bifidobacterium species convert aromatic amino acids (tryptophan, phenylalanine and tyrosine) into their respective aromatic lactic acids (indolelactic acid, phenyllactic acid and 4-hydroxyphenyllactic acid) via a previously unrecognized aromatic lactate dehydrogenase (ALDH). The ability of Bifidobacterium species to convert aromatic amino acids to their lactic acid derivatives was confirmed using monocolonized mice. Longitudinal profiling of the faecal microbiota composition and metabolome of Danish infants (n = 25), from birth until 6 months of age, showed that faecal concentrations of aromatic lactic acids are correlated positively with the abundance of human milk oligosaccharide-degrading Bifidobacterium species containing the ALDH, including Bifidobacterium longum, B. breve and B. bifidum. We further demonstrate that faecal concentrations of Bifidobacterium-derived indolelactic acid are associated with the capacity of these samples to activate in vitro the aryl hydrocarbon receptor (AhR), a receptor important for controlling intestinal homoeostasis and immune responses. Finally, we show that indolelactic acid modulates ex vivo immune responses of human CD4+ T cells and monocytes in a dose-dependent manner by acting as an agonist of both the AhR and hydroxycarboxylic acid receptor 3 (HCA3). Our findings reveal that breastmilk-promoted Bifidobacterium species produce aromatic lactic acids in the gut of infants and suggest that these microbial metabolites may impact immune function in early life.

Development of a new generic extraction method for the analysis of pesticides, mycotoxins, and polycyclic aromatic hydrocarbons in representative animal feed and food samples.

Various generic extraction methods have been used to determine pesticide residues, mycotoxins, and polycyclic aromatic hydrocarbons (PAHs) in food and animal feed to ensure consumer safety. However, these methods cannot extract all relevant compounds at an acceptable rate of recovery. This study presents a new extraction method. This new method facilitated the identification of 231 compounds, including 196 pesticides, 11 mycotoxins, and 24 PAHs over a broad range of polarities. These compounds were identified in various sample matrices, including those that are lipid-rich. The processed sample is first extracted with water, acetonitrile, formic acid, and heptane. The addition of ammonium formate results in separation into three phases and enables analysis of the aqueous phase. Solid-phase extraction clean-up procedures were performed as necessary followed by analysis by liquid or gas chromatography and mass spectrometry. Analyte recoveries were typically in the range of 70 - 120% with relative standard deviations below 20%.

Systems-wide effects of short-term feed deprivation in obese mice.

While prolonged fasting induces significant metabolic changes in humans and mice, less is known about systems-wide metabolic changes in response to short-term feed deprivation, which is used in experimental animal studies prior to metabolic challenge tests. We here performed a systems biology-based investigation of connections between gut bacterial composition and function, inflammatory and metabolic parameters in the intestine, liver, visceral adipose tissue, blood and urine in high-fat fed, obese mice that were feed deprived up to 12 h. The systems-wide analysis revealed that feed deprivation linked to enhanced intestinal butyric acid production and expression of the gene encoding the pro-thermogenic uncoupling protein UCP1 in visceral adipose tissue of obese mice. Ucp1 expression was also positively associated with Il33 expression in ileum, colon and adipose tissue as well as with the abundance of colonic Porphyromonadaceae, the latter also correlating to cecal butyric acid levels. Collectively, the data highlighted presence of a multi-tiered system of inter-tissue communication involving intestinal, immune and metabolic functions which is affected by feed deprivation in obese mice, thus pointing to careful use of short-feed deprivation in metabolic studies using obese mice.

Evaluating a tylosin dosage regimen for treatment of Staphylococcus delphini infection in mink (Neovison vison): a pharmacokinetic-pharmacodynamic approach.

Staphylococcus delphini is one of the most common pathogens isolated from mink infections, especially dermatitis. Tylosin (TYL) is used frequently against these infections, although no evidence-based treatment regimen exists. This study aimed to explore the dosage of TYL for infections caused by S. delphini in mink. Two animal experiments with a total of 12 minks were conducted to study the serum pharmacokinetic (PK) characteristics of TYL in mink after 10 mg/kg IV and oral dosing, respectively. The concentration of TYL in serum samples collected before and eight times during 24 h after TYL administration was quantitated with liquid chromatography quadrupole time-of-flight mass spectrometry, and the TYL disposition was analyzed using non-linear mixed effect analysis. The pharmacodynamics (PD) of TYL against S. delphini were studied using semi-mechanistic modeling of in vitro time-kill experiments. PKPD modeling and simulation were done to establish the PKPD index and dosage regimen. The disposition of TYL was described by a two-compartmental model. The area under the free concentration-time curve of TYL over the minimum inhibitory concentration of S. delphini (fAUC/MIC) was determined as PKPD index with breakpoints of 48.9 and 98.7 h for bacteriostatic and bactericidal effect, respectively. The calculated daily oral dose of TYL was 2378 mg/kg, which is 238-fold higher than the currently used TYL oral dosage regimen in mink (10 mg/kg). Accordingly, sufficient TYL concentrations are impossible to achieve in mink plasma, and use of this drug for extra-intestinal infections in this animal species must be discouraged.

Targeted and non-targeted unexpected food contaminants analysis by LC/HRMS: Feasibility study on rice.

A widely applicable analytical LC/HRMS method based on ion source optimization, data treatment optimization on rice matrix was developed. The effects of key parameters of ion source, and their interactions on ESI response were studied on HPLC-QTOF. Compared with center points, 40% and 20% increase of response factors in the positive and negative mode can be achieved by ion source optimization, respectively. Data processing strategies inspired from metabolomics and multi-targeted analysis were compared and developed using case and control rice samples. Highly automated workflow using XCMS achieved highest mass accuracy, highest detection rate of 96% for 5 µg/kg in a non-targeted way. A clear distinction between the control and contaminated samples by PCA and PLS-DA was also achieved by this workflow using XCMS, even for the concentration of 5 µg/kg.

Validating an empiric sulfadiazine-trimethoprim dosage regimen for treatment of Escherichia coli and Staphylococcus delphini infections in mink (Neovison vison).

Antimicrobial agents are used extensively off-label in mink, as almost no agents are registered for this animal species. Pharmacokinetic (PK) and pharmacodynamic (PD) data are required to determine antimicrobial dosages specifically targeting mink bacterial pathogens. The aims of this study were to assess, in a PKPD framework, the empirical dosage regimen for a combination of trimethoprim (TMP) and sulfadiazine (SDZ) in mink, and secondarily to produce data for future setting of clinical breakpoints. TMP and SDZ PK parameters were obtained experimentally in 22 minks following IV or oral administration of TMP/SDZ (30 mg/kg, i.e. 5 mg/kg TMP and 25 mg/kg SDZ). fAUC/MIC with a target value of 24 hr was selected as the PKPD index predictive of TMP/SDZ efficacy. Using a modeling approach, PKPD cutoffs for TMP and SDZ were determined as 0.062 and 16 mg/L, respectively. By incorporating an anticipated potentiation effect of SDZ on TMP against Escherichia coli and Staphylococcus delphini, the PKPD cutoff of TMP was revised to 0.312 mg/L, which is above the tentative epidemiological cutoffs (TECOFF) for these species. The current empirical TMP/SDZ dosage regimen (30 mg/kg, PO, once daily) therefore appears adequate for treatment of wild-type E. coli and S. delphini infections in mink.

Automated micro-solid-phase extraction clean-up of polycyclic aromatic hydrocarbons in food oils for analysis by gas chromatography-orbital ion trap mass spectrometry.

Food can contain unwanted compounds and need to be analyzed for compounds like carcinogenic polycyclic aromatic hydrocarbons to ensure consumer safety. The analytes need to be extracted from the sample matrix and cleaned-up to enable detection. However, established methods for clean-up are labor intensive and have a high expenditure on organic solvents. Here, we show a newly developed micro-solid-phase extraction cartridge method to automate the clean-up for analysis of polycyclic aromatic hydrocarbons in sunflower oil using gas chromatography with quadrupole-orbitrap mass spectrometry with a TriPlus autosampler. This automated micro-solid-phase extraction cartridge method needs only 4 µL of vegetable oil sample and requires only 360 µL acetonitrile for elution, and, therefore, it needs only small amounts of organic solvent. Two different micro-solid-phase extraction cartridge methods were developed, one using two commercially available cartridges with florisil and octadecylsilane/Z-Sep/CarbonX, and the other method using one commercially available cartridge with florisil followed by one self-made cartridge with octadecylsilane/Z-Sep. The latter method showed successful lipid removal and was further validated for 22 of 24 polycyclic aromatic hydrocarbon compounds in sunflower oil at a spiked level of 1090 µg/kg with recoveries ranging from 53 to 118% and relative standard deviation below 22%. This method shows promising short-time clean-up with low expenditure of solvents.

Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders.

BACKGROUND: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed. OBJECTIVES: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats. METHODS: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression. RESULTS: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o-phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two (λ-cyhalothrin, pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions. DISCUSSION: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing. https://doi.org/10.1289/EHP6774.

Grouping of endocrine disrupting chemicals for mixture risk assessment - Evidence from a rat study.

Exposure to mixtures of endocrine disrupting chemicals may contribute to the rising incidence of hormone-related diseases in humans. Real-life mixtures are complex, comprised of chemicals with mixed modes of action, and essential knowledge is often lacking on how to group such chemicals into cumulative assessment groups, which is an essential prerequisite to conduct a chemical mixture risk assessment. We investigated if mixtures of chemicals with diverse endocrine modes of action can cause mixture effects on hormone sensitive endpoints in developing and adult rat offspring after perinatal exposure. Wistar rats were exposed during pregnancy and lactation simultaneously to either bisphenol A and butylparaben (Emix), diethylhexyl phthalate and procymidone (Amix), or a mixture of all four substances (Totalmix). In male offspring, the anogenital distance was significantly reduced and nipple retention increased in animals exposed to Amix and Totalmix, and the mixture effects were well approximated by the dose addition model. The combination of Amix and Emix responded with more marked changes on these and other endocrine-sensitive endpoints than each binary mixture on its own. Sperm counts were reduced by all exposures. These experimental outcomes suggest that the grouping of chemicals for mixture risk assessment should be based on common health outcomes rather than only similar modes or mechanisms of action. Mechanistic-based approaches such as the concept of Adverse Outcome Pathway (AOP) can provide important guidance if both the information on shared target tissues and the information on shared mode/mechanism of action are taken into account.

Standard substances free quantification makes LC/ESI/MS non-targeted screening of pesticides in cereals comparable between labs.

LC/ESI/MS is the technique of choice for qualitative and quantitative food monitoring; however, analysis of a large number of compounds is challenged by the availability of standard substances. The impediment of detection of food contaminants has been overcome by the suspect and non-targeted screening. Still, the results from one laboratory cannot be compared with the results of another laboratory as quantitative results are required for this purpose. Here we show that the results of the suspect and non-targeted screening for pesticides can be made quantitative with the aid of in silico predicted electrospray ionization efficiencies and this allows direct comparison of the results obtained in two different laboratories. For this purpose, six cereal matrices were spiked with 134 pesticides and analysed in two independent labs; a high correlation for the results with the R2 of 0.85.

Microbiological quality of mink feed raw materials and feed production area.

BACKGROUND: The quality of mink feed and raw ingredients affect health and growth. The objectives of this study were to examine the microbiological quality of ready-to-eat mink feed and its raw ingredients, screen the plant part of the feed for mycotoxins, and determine the hygiene of the production environment in the feed processing facilities. The results of the study are important for identification of critical steps in the feed production and for formulation of recommendations for improvements of production processes to obtain better quality feed. Feed and swab samples were taken at three Danish mink feed producers October 2016 and May 2017, respectively. Viable counts, detection of methicillin-resistant Staphylococcus aureus (MRSA), influenza virus and filamentous fungi were performed together with qualitative chemical analyses for bioactive fungal metabolites and mycotoxins. Swab samples were analyzed for total viable counts. RESULTS: Viable counts varied between 7.2 × 102 and 9.3 × 107 cfu/g in raw ingredients and between 107 and 109 cfu/cm2 on different surfaces at the feed production facilities. A pork meat product, pork haemoglobin, pork liver and a poultry mix was found positive for MRSA, while monophasic Salmonella [4,5,12:i:-] was detected in a pork meat product. Neither MRSA nor Salmonella was detected in any ready-to-eat feed. Influenza A virus was not detected in any sample. Filamentous fungi were detected in all analysed samples of ready-to-eat feed while dihydro-demethyl-sterigmatocystin was found in almost 50% of all ready-to-eat feed samples and in 80% of the sugar beet pulp. Fumonisins and other Fusarium toxins were found especially in corn gluten meal and extruded barley and wheat. CONCLUSIONS: Mink feed contained a cocktail of mycotoxins and bacteria, which may not per se cause clinical disease, but may affect organ function and animal performance and well-being.

In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole.

Azoles are effective antifungal agents used in both medicine and agriculture. They typically work by inhibiting cytochrome P450 enzymes, primarily CYP51 of the ergosterol biosynthesis pathway, thus damaging the fungal cell membrane. However, apart from their desired antifungal properties, several azoles also exhibit endocrine disrupting properties in mammals, both in vitro and in vivo. Here, we have tested two currently used agricultural azole fungicides, triticonazole and flusilazole, for their in vitro anti-androgenic activity and potential effects on reproductive parameters. Both fungicides showed strong androgen receptor (AR) antagonism and disruption of steroid biosynthesis in vitro. Following gestational exposure to flusilazole (15 or 45 mg/kg bw/day) or triticonazole (150 or 450 mg/kg bw/day) in time-mated Sprague Dawley rats, triticonazole induced shorter male anogenital distance (AGD). Flusilazole exposure did not affect the AGD, but altered fetal male blood hormone profile, with increased androstenedione and decreased estrone levels. Flusilazole and triticonazole have dissimilar effects on reproductive parameters in vivo, but both show endocrine disrupting activities.

Transfer of amoxicillin to suckling mink (Neovison vison) kits via the milk from dams treated orally or intra-muscularly.

Treatment of mink kits with pre-weaning diarrhea (PWD) can be time-consuming and expensive for the farmer, and the efficacy of the treatment procedure may be questioned. Evidence-based treatment protocols for application on affected animals at farms with outbreaks of PWD are lacking. In Denmark, the dams are sometimes treated with amoxicillin, however, it is unknown if it is passed on to the mink kits via the milk. The aim of the present study was to investigate if amoxicillin is transferred via the milk to the kits after oral (PO) and intramuscular (IM) treatment, respectively, of the dam. Moreover, we estimated the concentrations of amoxicillin continuously in serum from the kits up to 8 h after administration. The concentration of amoxicillin was not affected by the route of administration (P = .64) and serum reached the highest level after 8 h (34 ng/mL, CI95% = [24.3-47.7]). The serum concentrations of amoxicillin in the mink kits achieved within 8 h were judged too low to exert antimicrobial impact on relevant bacterial species.

Whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial.

OBJECTIVE: To investigate whether a whole grain diet alters the gut microbiome and insulin sensitivity, as well as biomarkers of metabolic health and gut functionality. DESIGN: 60 Danish adults at risk of developing metabolic syndrome were included in a randomised cross-over trial with two 8-week dietary intervention periods comprising whole grain diet and refined grain diet, separated by a washout period of ≥6 weeks. The response to the interventions on the gut microbiome composition and insulin sensitivity as well on measures of glucose and lipid metabolism, gut functionality, inflammatory markers, anthropometry and urine metabolomics were assessed. RESULTS: 50 participants completed both periods with a whole grain intake of 179±50 g/day and 13±10 g/day in the whole grain and refined grain period, respectively. Compliance was confirmed by a difference in plasma alkylresorcinols (p<0.0001). Compared with refined grain, whole grain did not significantly alter glucose homeostasis and did not induce major changes in the faecal microbiome. Also, breath hydrogen levels, plasma short-chain fatty acids, intestinal integrity and intestinal transit time were not affected. The whole grain diet did, however, compared with the refined grain diet, decrease body weight (p<0.0001), serum inflammatory markers, interleukin (IL)-6 (p=0.009) and C-reactive protein (p=0.003). The reduction in body weight was consistent with a reduction in energy intake, and IL-6 reduction was associated with the amount of whole grain consumed, in particular with intake of rye. CONCLUSION: Compared with refined grain diet, whole grain diet did not alter insulin sensitivity and gut microbiome but reduced body weight and systemic low-grade inflammation. TRIAL REGISTRATION NUMBER: NCT01731366; Results.

Subacute oral toxicity investigation of selenium nanoparticles and selenite in rats.

Selenium (Se) nanoparticles have been proposed as food supplements. However, the particle formulation may exert unexpected toxicity. The aim was therefore to compare toxicity of low doses of Se nanoparticles and the dissolved, ionized Se species selenite. Female rats were dosed orally for 28 d with either: 0.05, 0.5, or 4 mg Se/kg body weight (bw)/day as 20 nm Se nanoparticles or 0.05 or 0.5 mg Se/kg bw/day as sodium selenite. Male rats were dosed 4 mg Se/kg bw/day as Se nanoparticles. Body weight and clinical appearance were recorded throughout the experiment. At necropsy, blood samples were taken for hematological and clinical chemistry analyses; organ weights were recorded. At the high-dose of Se nanoparticles, overt toxicity occurred and the female animals had to be euthanized prematurely, whereas the male animals were reduced in dose. At all doses of Se nanoparticles and at 0.5 mg Se/kg bw/day as selenite, a lower body weight gain as compared to vehicle occurred. Relative liver weight was increased for both Se formulations at 0.5 mg Se/kg bw/day. Creatinine clearance and urinary pH were affected in some Se dosed groups. There were no effects among dosed groups on brain neurotransmitters or on hematological parameters compared with controls. There were no histological changes in the livers of animals exposed to Se nanoparticles or to selenite. Based on effects on body weight and liver weight, selenium nanoparticles and ionic Se exerted similar toxicity. This suggests that a nanoparticle-specific toxicity of Se did not occur.

A low-gluten diet induces changes in the intestinal microbiome of healthy Danish adults.

Adherence to a low-gluten diet has become increasingly common in parts of the general population. However, the effects of reducing gluten-rich food items including wheat, barley and rye cereals in healthy adults are unclear. Here, we undertook a randomised, controlled, cross-over trial involving 60 middle-aged Danish adults without known disorders with two 8-week interventions comparing a low-gluten diet (2 g gluten per day) and a high-gluten diet (18 g gluten per day), separated by a washout period of at least six weeks with habitual diet (12 g gluten per day). We find that, in comparison with a high-gluten diet, a low-gluten diet induces moderate changes in the intestinal microbiome, reduces fasting and postprandial hydrogen exhalation, and leads to improvements in self-reported bloating. These observations suggest that most of the effects of a low-gluten diet in non-coeliac adults may be driven by qualitative changes in dietary fibres.

Antibiotic treatment of rat dams affects bacterial colonization and causes decreased weight gain in pups.

Intergenerational transmission of bacteria during birth initiates the natural successional development of the intestinal microbiota in mammals. This process can be disrupted by antibiotic exposure, potentially affecting early-life microbiota-dependent metabolic programming. In the present study, we specifically investigate the metabolic consequences of exposing neonate Wistar rats to an antibiotic-perturbed low-diversity microbiota from birth until weaning, without exposing the pups directly to antibiotics. Here, we show that pups born from both amoxicillin and vancomycin-treated dams gain less weight than controls. This was concordant with lower feed intake as well as increased colonic expression of the PYY satiety hormone gene at weaning. The weight difference persists into adulthood even though the initial differences in gut microbiota subsided. Our results demonstrate that early-life exposure to an antibiotic-perturbed low-diversity microbiota is sufficient to cause changes in body weight persisting into adulthood.

Glyphosate has limited short-term effects on commensal bacterial community composition in the gut environment due to sufficient aromatic amino acid levels.

Recently, concerns have been raised that residues of glyphosate-based herbicides may interfere with the homeostasis of the intestinal bacterial community and thereby affect the health of humans or animals. The biochemical pathway for aromatic amino acid synthesis (Shikimate pathway), which is specifically inhibited by glyphosate, is shared by plants and numerous bacterial species. Several in vitro studies have shown that various groups of intestinal bacteria may be differently affected by glyphosate. Here, we present results from an animal exposure trial combining deep 16S rRNA gene sequencing of the bacterial community with liquid chromatography mass spectrometry (LC-MS) based metabolic profiling of aromatic amino acids and their downstream metabolites. We found that glyphosate as well as the commercial formulation Glyfonova®450 PLUS administered at up to fifty times the established European Acceptable Daily Intake (ADI = 0.5 mg/kg body weight) had very limited effects on bacterial community composition in Sprague Dawley rats during a two-week exposure trial. The effect of glyphosate on prototrophic bacterial growth was highly dependent on the availability of aromatic amino acids, suggesting that the observed limited effect on bacterial composition was due to the presence of sufficient amounts of aromatic amino acids in the intestinal environment. A strong correlation was observed between intestinal concentrations of glyphosate and intestinal pH, which may partly be explained by an observed reduction in acetic acid produced by the gut bacteria. We conclude that sufficient intestinal levels of aromatic amino acids provided by the diet alleviates the need for bacterial synthesis of aromatic amino acids and thus prevents an antimicrobial effect of glyphosate in vivo. It is however possible that the situation is different in cases of human malnutrition or in production animals.