Metabolomic Alterations of Volatile Organic Compounds and Bile Acids as Biomarkers of Microbial Shifts in a Murine Model of Short Bowel Syndrome.

Pediatric short bowel syndrome (SBS) is a rare condition characterized by a massive loss of the small intestine, leading to the inability to meet nutritional requirements without the use of parenteral or enteral supplementation. SBS causes profound alterations in the intestinal microbiome and metabolome. The aim of this study was a detailed assessment of the intestinal microbiome and metabolome in a murine model of SBS. We performed a 60% proximal small bowel resection versus a sham operation in C57BL/6 mice. Four weeks postoperatively, the microbial communities of different intestinal segments (jejunum, ileum, colon) and stool were assessed by 16S rRNA gene sequencing. Bile acids in serum and stool and volatile organic compounds (VOCs) in the fecal headspace were assessed using LC-MS and GC-MS techniques. The α-diversity of the different intestinal segments did not significantly differ between the two groups. ß-diversity significantly differed between sham and SBS mice. While in the jejunum, Faecalibaculum was significantly increased in SBS animals, a significant reduction in Lactobacillus and Sporosarcina was detected in the ileum of SBS mice. In the colon of SBS mice, a significant decrease in Ruminococcaceae and a significant increase in Proteobacteria such as Faecalibaculum and Escherichia-Shigella were found. Serum levels of deoxycholic, taurocholic and taurochenodeoxycholic acids were significantly higher in the SBS group. Of the 29 VOCs tested, hexane, isoflurane and pentane were significantly higher in the SBS group, and pyrrole was significantly lower. We were able to show that SBS causes shifts in the murine intestinal microbiome and metabolome including serum BAs and fecal VOCs.

Probiotic OMNi-BiOTiC® 10 AAD Reduces Cyclophosphamide-Induced Inflammation and Adipose Tissue Wasting in Mice.

Cancer therapy is often associated with severe side effects such as drug induced weight loss, also known as chemotherapy-induced cachexia. The aim of this study was to investigate the effects of a multispecies probiotic (OMNi-BiOTiC® 10 AAD) in a chemotherapy mouse model. A total of 24 male BALB/c mice were gavage-fed with the probiotic formulation or water, once a day for 3 weeks. In the third week, the mice received intraperitoneal cyclophosphamide. At euthanasia, the organs were dissected, and serum was sampled for cytokine analysis. Tight junction components, myosin light chain kinase, mucins, and apoptosis markers were detected in the ileum and colon using histological analyses and qRT-PCR. Lipolysis was analyzed by enzymatic activity assay, Western blotting analyses, and qRT-PCR in WAT. The fecal microbiome was measured with 16S-rRNA gene sequencing from stool samples, and fecal volatile organic compounds analysis was performed using gas chromatography/mass spectrometry. The probiotic-fed mice exhibited significantly less body weight loss and adipose tissue wasting associated with a reduced CGI58 mediated lipolysis. They showed significantly fewer pro-inflammatory cytokines and lower gut permeability compared to animals fed without the probiotic. The colons of the probiotic-fed animals showed lower inflammation scores and less goblet cell loss. qRT-PCR revealed no differences in regards to tight junction components, mucins, or apoptosis markers. No differences in microbiome alpha diversity, but differences in beta diversity, were observed between the treatment groups. Taxonomic analysis showed that the probiotic group had a lower relative abundance of Odoribacter and Ruminococcus-UCG014 and a higher abundance of Desulfovibrio. VOC analysis yielded no significant differences. The results of this study indicate that oral administration of the multispecies probiotic OMNi-BiOTiC® 10 AAD could mitigate cyclophosphamide-induced chemotherapy side effects.

Insights into the Composition of a Co-Culture of 10 Probiotic Strains (OMNi BiOTiC® AAD10) and Effects of Its Postbiotic Culture Supernatant.

BACKGROUND: We aimed to gain insights in a co-culture of 10 bacteria and their postbiotic supernatant. METHODS: Abundances and gene expression were monitored by shotgun analysis. The supernatant was characterized by liquid chromatography mass spectroscopy (LC-MS) and gas chromatography mass spectroscopy (GC-MS). Supernatant was harvested after 48 h (S48) and 196 h (S196). Susceptibility testing included nine bacteria and C. albicans. Bagg albino (BALBc) mice were fed with supernatant or culture medium. Fecal samples were obtained for 16S analysis. RESULTS: A time-dependent decrease of the relative abundances and gene expression of L. salivarius, L. paracasei, E. faecium and B. longum/lactis and an increase of L. plantarum were observed. Substances in LC-MS were predominantly allocated to groups amino acids/peptides/metabolites and nucleotides/metabolites, relating to gene expression. Fumaric, panthotenic, 9,3-methyl-2-oxovaleric, malic and aspartic acid, cytidine monophosphate, orotidine, phosphoserine, creatine, tryptophan correlated to culture time. Supernatant had no effect against anaerobic bacteria. S48 was reactive against S. epidermidis, L. monocytogenes, P. aeruginosae, E. faecium and C. albicans. S196 against S. epidermidis and Str. agalactiae. In vivo S48/S196 had no effect on alpha/beta diversity. Linear discriminant analysis effect size (LEfSe) and analysis of composition of microbiomes (ANCOM) revealed an increase of Anaeroplasma and Faecalibacterium prausnitzii. CONCLUSIONS: The postbiotic supernatant had positive antibacterial and antifungal effects in vitro and promoted the growth of distinct bacteria in vivo.

Production, Storage Stability, and Susceptibility Testing of Reuterin and Its Impact on the Murine Fecal Microbiome and Volatile Organic Compound Profile.

Background: Probiotics are generally considered as safe, but infections may rarely occur in vulnerable patients. Alternatives to live microorganisms to manage dysbiosis may be of interest in these patients. Reuterin is a complex component system exhibiting broad spectrum antimicrobial activity and a possible candidate substance in these cases. Methods: Reuterin supernatant was cultured from Lentilactobacillus diolivorans in a bioreactor in a two-step process. Storage stability at -20°C and effect of repeated freeze-thaw cycles were assessed by high performance liquid chromatography (HPLC). Antimicrobial activity was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus (S.) aureus, Staphylococcus epidermidis, Streptococcus (S.) agalactiae, Propionibacterium acnes, and Pseudomonas aeruginosae. Male BALBc mice were gavage fed with reuterin supernatant (n = 10) or culture medium (n = 10). Fecal volatile organic compounds (VOC) were assessed by gas chromatography mass spectroscopy; the microbiome was examined by 16S rRNA gene sequencing. Results: The supernatant contained 13.4 g/L reuterin (3-hydroxypropionaldehyde; 3-HPA). 3-HPA content remained stable at -20°C for 35 days followed by a slow decrease of its concentration. Repeated freezing/thawing caused a slow 3-HPA decrease. Antimicrobial activity was encountered against S. aureus, S. epidermidis, and S. agalactiae. Microbiome analysis showed no differences in alpha and beta diversity markers. Linear discriminant effect size (LEfSe) analysis identified Lachnospiraceae_bacterium_COE1 and Ruminoclostridium_5_uncultured_Clostridiales_ bacterium (in the reuterin medium group) and Desulfovibrio_uncultured_ bacterium, Candidatus Arthromitus, Ruminococcae_NK4A214_group, and Eubacterium_xylanophilum_group (in the reuterin group) as markers for group differentiation. VOC analysis showed a significant decrease of heptane and increase of 3-methylbutanal in the reuterin group. Conclusion: The supernatant produced in this study contained acceptable amounts of 3-HPA remaining stable for 35 days at -20°C and exhibiting an antimicrobial effect against S. aureus, S. agalactiae, and S. epidermidis. Under in vivo conditions, the reuterin supernatant caused alterations of the fecal microbiome. In the fecal, VOC analysis decreased heptane and increased 3-methylbutanal were encountered. These findings suggest the high potential of the reuterin system to influence the intestinal microbiome in health and disease, which needs to be examined in detail in future projects.

(S)-Reutericyclin: Susceptibility Testing and In Vivo Effect on Murine Fecal Microbiome and Volatile Organic Compounds.

We aimed to assess the in vitro antimicrobial activity and the in vivo effect on the murine fecal microbiome and volatile organic compound (VOC) profile of (S)-reutericyclin. The antimicrobial activity of (S)-reutericyclin was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Staphylococcus (S.) epidermidis, Streptococcus agalactiae, Pseudomonas aeruginosa and Propionibacterium acnes. Reutericyclin or water were gavage fed to male BALBc mice for 7 weeks. Thereafter stool samples underwent 16S based microbiome analysis and VOC analysis by gas chromatography mass spectrometry (GC-MS). (S)-reutericyclin inhibited growth of S. epidermidis only. Oral (S)-reutericyclin treatment caused a trend towards reduced alpha diversity. Beta diversity was significantly influenced by reutericyclin. Linear discriminant analysis Effect Size (LEfSe) analysis showed an increase of Streptococcus and Muribaculum as well as a decrease of butyrate producing Ruminoclostridium, Roseburia and Eubacterium in the reutericyclin group. VOC analysis revealed significant increases of pentane and heptane and decreases of 2,3-butanedione and 2-heptanone in reutericyclin animals. The antimicrobial activity of (S)-reutericyclin differs from reports of (R)-reutericyclin with inhibitory effects on a multitude of Gram-positive bacteria reported in the literature. In vivo (S)-reutericyclin treatment led to a microbiome shift towards dysbiosis and distinct alterations of the fecal VOC profile.

Characterization of the Luminal and Mucosa-Associated Microbiome along the Gastrointestinal Tract: Results from Surgically Treated Preterm Infants and a Murine Model.

Environmental factors, including nutritional habits or birth mode, are known key determinants for intestinal microbial composition. Investigations of the intestinal microbiome in different species in a multiplicity of studies during recent decades have revealed differential microbial patterns and quantities along the gastrointestinal (GI) tract. Characterization of the microbial pattern in various aspects is a prerequisite for nutritional interventions. In this 16S rRNA amplicon-based approach, we present a characterization of the mucosa-associated microbiome in comparison with the luminal community of four infants at the time of the closure of ileostomies and perform a systematic characterization of the corresponding luminal and mucosal microbiome from jejunal, ileal and colonic regions, as well as collected feces in mice. The most dominant taxa in infant-derived samples altered due to individual differences, and in the mucosa, Enterococcus, Clostridiumsensustricto1, Veillonella, Streptococcus and Staphylococcus were the most abundant. Two less abundant taxa differed significantly between the mucosa and lumen. In murine samples, relative abundances differed significantly, mainly between the intestinal regions. Significant differences between mouse mucosa- and lumen-derived samples could be found in the observed species with a trend to lower estimated diversity in mucosa-derived samples, as well as in the relative abundance of individual taxa. In this study, we examined the difference between the mucosal and luminal bacterial colonization of the gastrointestinal tract in a small sample cohort of preterm infants. Individual differences were characterized and statistical significance was reached in two taxa (Cupriavidus, Ralstonia). The corresponding study on the different murine intestinal regions along the GI tract showed differences all over the intestinal region.

Volatile Organic Compounds, Bacterial Airway Microbiome, Spirometry and Exercise Performance of Patients after Surgical Repair of Congenital Diaphragmatic Hernia.

The aim of this study was to analyze the exhaled volatile organic compounds (VOCs) profile, airway microbiome, lung function and exercise performance in congenital diaphragmatic hernia (CDH) patients compared to healthy age and sex-matched controls. A total of nine patients (median age 9 years, range 6-13 years) treated for CDH were included. Exhaled VOCs were measured by GC-MS. Airway microbiome was determined from deep induced sputum by 16S rRNA gene sequencing. Patients underwent conventional spirometry and exhausting bicycle spiroergometry. The exhaled VOC profile showed significantly higher levels of cyclohexane and significantly lower levels of acetone and 2-methylbutane in CDH patients. Microbiome analysis revealed no significant differences for alpha-diversity, beta-diversity and LefSe analysis. CDH patients had significantly lower relative abundances of Pasteurellales and Pasteurellaceae. CDH patients exhibited a significantly reduced Tiffeneau Index. Spiroergometry showed no significant differences. This is the first study to report the VOCs profile and airway microbiome in patients with CDH. Elevations of cyclohexane observed in the CDH group have also been reported in cases of lung cancer and pneumonia. CDH patients had no signs of impaired physical performance capacity, fueling controversial reports in the literature.

Examination of intestinal ultrastructure, bowel wall apoptosis and tight junctions in the early phase of sepsis.

Gut hyperpermeability can be caused by either apoptosis of the intestinal epithelium or altered status, permeability or porosity of tight junctions. This project aims to elucidate these mechanisms in the early phase of sepsis. Eighteen male wild type mice were randomized to two groups. All mice received one single gavage of fluorescein isothiocyanate (FITC) dextran 30 min before intervention. One group (n = 10) underwent cecal ligation and puncture to induce sepsis. The other group (n = 8) was sham operated. Septic animals exhibited significantly increased permeability for FITC 8 h post-operatively. Significantly increased serum interleukin-6, tumor-necrosis-factor-alpha and interleukin-1-beta confirmed sepsis. Septic animals showed significant bowel wall inflammation of ileum and colon samples. PCR revealed significantly increased expression of claudin-2 and decreased expressions of claudin-4, tight-junction-protein-1 and occludin-1 resembling increased permeability of tight junctions. However, these alterations could not be confirmed at the protein level. Light microscopy revealed significant dilatation of intercellular spaces at the basal sections of intestinal epithelial cells (IEC) in septic animals confirmed by increased intercellular spaces at the level of tight junctions and adherens junctions in electron microscopy (TEM). In small angle X-ray scattering no increase in number or size of nanopores could be shown in the bowel wall. HOECHST staining and PCR of ileum samples for apoptosis markers proofed no relevant differences in intestinal epithelial cell apoptosis between the groups. Intestinal hyperpermeability in septic animals was most likely caused by alterations of the intercellular contacts and not by apoptosis or increased size/number of nanopores of intestinal epithelial cells in this murine model of early sepsis.

The Effects of Prebiotic Supplementation with OMNi-LOGiC® FIBRE on Fecal Microbiome, Fecal Volatile Organic Compounds, and Gut Permeability in Murine Neuroblastoma-Induced Tumor-Associated Cachexia.

Malignant diseases can cause tumor-associated cachexia (TAC). Supplementation with prebiotic non-digestible carbohydrates exerts positive metabolic effects in experimental oncologic diseases. The aim of this project was to assess the effect of prebiotic supplementation with OMNi-LOGiC® FIBRE on intestinal microbiome, bacterial metabolism, gut permeability, and inflammation in a murine model of neuroblastoma (NB)-associated TAC. For this study, 2,000,000 NB cells (MHH-NB11) were implanted into athymic mice followed by daily supplementation with water or 200 mg prebiotic oligosaccharide (POS) OMNi-LOGiC® FIBRE (NB-Aqua, n = 12; NB-POS, n = 12). Three animals of each tumor group did not develop NB. The median time of tumor growth (first visibility to euthanasia) was 37 days (IQR 12.5 days) in the NB-Aqua group and 37 days (IQR 36.5 days) in the NB-POS group (p = 0.791). At euthanasia, fecal microbiome and volatile organic compounds (VOCs), gut permeability (fluorescein isothiocyanate-dextran (FITC-dextran), and gut barrier markers were measured. Values were compared to sham animals following injection of culture medium and gavage of either water or OMNi-LOGiC® FIBRE (SH-Aqua, n = 10; SH-POS, n = 10). Alpha diversity did not differ significantly between the groups. Principal coordinate analysis (PCoA) revealed clustering differences between Aqua and POS animals. Both NB and POS supplementation led to taxonomic alterations of the fecal microbiome. Of 49 VOCs, 22 showed significant differences between the groups. NB animals had significantly higher gut permeability than Aqua animals; POS did not ameliorate these changes. The pore and leak pathways of tight junctions did not differ between groups. In conclusion, our results suggest that NB-induced TAC causes increased gut permeability coupled with compositional changes in the fecal microbiome and VOC profile. Prebiotic supplementation with OMNi-LOGiC® FIBRE seemed to induce modifications of the fecal microbiome and VOC profile but did not improve gut permeability.

The effects of neuroblastoma and chemotherapy on metabolism, fecal microbiome, volatile organic compounds, and gut barrier function in a murine model.

BACKGROUND: Following transplantation of human neuroblastoma (NB) cells into athymic mice, we investigated the effects of tumor growth and cyclophosphamide (CTX) treatment on systemic metabolism, gut inflammation and permeability, fecal microbiome and volatile organic compounds (VOCs). METHODS: NB cells (MHH-NB11) were implanted into athymic mice (n=20); 20 healthy mice served as controls (sham).  CTX was given to 20 animals (10 NB and 10 sham) after 8 and 9 weeks. Metabolic changes were measured. Ileum samples were obtained for RT-PCR (claudins 2 and 4, occludin, tight junction protein 1) and apoptosis rate determination. Fecal microbiome and VOCs were analyzed. Values were compared to sham animals. RESULTS: NB caused reduction of adipose tissue, increases of IL-6 and TNF-α, and decreases of TGF-ß1 and -ß2. Serum FITC-dextrane levels were increased in NB and improved under CTX. Claudin 4 expression was higher in NB versus NB + CTX and sham animals. NB caused increased apoptosis of epithelial cells. NB but also CTX led to a reduction in the abundance of Lactobacillus. NB led to alterations of the fecal VOC profile. CONCLUSIONS: NB caused a catabolic pro-inflammatory state, increased gut permeability, altered fecal VOCs and reductions of Lactobacillus. Further investigations are required to determine if modifications of the intestinal microbiome may reverse some of the observed effects.

Separated by sand, fused by dropping water: habitat barriers and fluctuating water levels steer the evolution of rock-dwelling cichlid populations in Lake Tanganyika.

The conditions of phenotypic and genetic population differentiation allow inferences about the evolution, preservation and loss of biological diversity. In Lake Tanganyika, water level fluctuations are assumed to have had a major impact on the evolution of stenotopic littoral species, though this hypothesis has not been specifically examined so far. The present study investigates whether subtly differentiated colour patterns of adjacent Tropheus moorii populations are maintained in isolation or in the face of continuous gene flow, and whether the presumed influence of water level fluctuations on lacustrine cichlids can be demonstrated in the small-scale population structure of the strictly stenotopic, littoral Tropheus. Distinct population differentiation was found even across short geographic distances and minor habitat barriers. Population splitting chronology and demographic histories comply with our expectation of old and rather stable populations on steeper sloping shore, and more recently established populations in a shallower region. Moreover, population expansions seem to coincide with lake level rises in the wake of Late Pleistocene megadroughts ~100 KYA. The imprint of hydrologic events on current population structure in the absence of ongoing gene flow suggests that phenotypic differentiation among proximate Tropheus populations evolves and persists in genetic isolation. Sporadic gene flow is effected by lake level fluctuations following climate changes and controlled by the persistence of habitat barriers during lake level changes. Since similar demographic patterns were previously reported for Lake Malawi cichlids, our data furthermore strengthen the hypothesis that major climatic events synchronized facets of cichlid evolution across the East African Great Lakes.

Monogamy in the maternally mouthbrooding Lake Tanganyika cichlid fish Tropheus moorii.

Supported by evidence for assortative mating and polygynandry, sexual selection through mate choice was suggested as the main force driving the evolution of colour diversity of haplochromine cichlids in Lakes Malawi and Victoria. The phylogenetically closely related tribe Tropheini of Lake Tanganyika includes the genus Tropheus, which comprises over 100 colour variants currently classified into six morphologically similar, polyphyletic species. To assess the potential for sexual selection in this sexually monochromatic maternal mouthbrooder, we used microsatellite-based paternity inference to investigate the mating system of Tropheus moorii. In contrast to haplochromines in Lake Malawi, multiple paternity is rare or even absent in broods of T. moorii. Eighteen of the 19 analysed families were consistent with genetic monogamy, while either a mutation or more than one sire explained the genotype of one offspring in another brood. We discuss the differences in breeding behaviour between T. moorii and the Lake Malawi haplochromines, and evaluate additional factors or alternatives to sexual selection as promoters of colour diversification. A preliminary survey of other Tropheini species suggested that multiple paternity is infrequent in the entire tribe.