Lactobacilli in the gastrointestinal tract of dog and wolf

Since the first wolf was taken as a companion animal a lot have happened with the biology of these animals. Is it possible that changes in the gut microbiota of the domestic dog are involved in the increasing numbers of animals with gastrointestinal disorders? Probiotics have been proven to promote the immune system of dogs by increasing concentrations of neutrophils and monocytes, decreasing the fragility of erythrocytes and increasing the serum IgG concentrations. Probiotics have also been shown to improve the fecal consistency, fecal dry matter and defecation frequency of dogs. The aim of this study was to find out if the bacterial counts in the gut of dogs differed from the gut of wolves, with the focus on lactobacilli. The presence of Lactobacillus reuteri and characterization of such strains in wolves and dogs was also analyzed to find a possible probiotic candidate. Fecal specimens from five dogs and intestine content from four wolves were collected and lactobacilli were analyzed and isolated by cultivation on the selective substrate Rogosa. Bacterial enumerations were noted and colonies with morphological differences were collected and isolated before the characterization was done using reuterin assay, rep-PCR and gel electrophoresis. A representative for each rep-PCR type was identified using 16S rRNA gene sequencing. Bacteria identified as L. reuteri was characterized further using methods for testing bile tolerance, detection of the genes ureC and pduC, mucus binding capacity and antibiotic susceptibility. A handful of isolates were also characterized by determining the gene sequence of a cell surface protein specific for L. reuteri. The content of lactobacilli in the fecal samples of dogs ranged from 106 to 108 cfu g-1 feces and in intestine of wolves from 103 to 105 cfu g-1 gut content. Nevertheless, not all of the bacteria grown on the Rogosa agar were lactobacilli, species of Streptococcus were also isolated. Even though the total counts of bacteria were higher in dog, the number of different species of isolated lactobacilli was higher in the wolf. In the samples from dog, two species of Lactobacillus were isolated; L. animalis and L. reuteri while in the wolf samples another five species were isolated. A total of 32 L. reuteri were isolated from the subjects and these could be divided into 10-12 different strains according to the characterization methods. Among the 13 isolated L. reuteri analyzed by the different characterization methods, four were reuterin positive and two were urease positive. Some of the strains bound well to intestinal mucus while others did not bind at all. The bile tolerance also differed between the strains but most of them, except for one strain, survived a concentration of 5% bile. The results from the antibiotic susceptibility test showed a close relation between the strains, with resistance against streptomycin and tetracycline. Some of these strains were isolated from both dog and wolf whereas other strains could only be found in dog or wolf. This was established using the different characterization methods as well as analyzing the gene sequence of a cell surface protein referred to as “protein x”. A new method for isolating L. reuteri were also tested, called immunomagnetic isolation. The results from this method were both positive and negative, this since the beads bound to some strains of L. reuteri but not all. The Dynabeads also bound to lactobacilli in a dog sample but not to any bacteria in a wolf sample.Ända sedan den första vargen domesticerades har en hel del skett när det gäller dessa djurs biologi. Är det möjligt att förändringar i tarmens mikrobiota hos hundar är orsaken bakom den ökande frekvensen av djur med mag- och tarmproblem? Probiotika har bevisats ha positiv effekt på immunförsvaret genom att öka koncentrationen av neutrofiler och monocyter, öka erytrocyters motståndskraft och genom att öka serumkoncentrationen av IgG. Probiotika har även visat sig förbättra konsistensen på hundars träck, torrsubstansen i träcken och antalet tarmtömningar. Syftet med detta arbete var att ta reda på om antalet laktobaciller i tarmen hos hund skiljer sig från antalet hos varg. Förekomsten av Lactobacillus reuteri och karaktäriseringen av sådana stammar i varg och hund analyserades också i ett försök att hitta en probiotisk kandidat. Träckprover från fem hundar och prover med tarminnehåll från fyra vargar samlades in och odlades på medium (Rogosa agar) specifikt anpassat för tillväxt av arter av Lactobacillus. Beräkningar av bakterieantalet antecknades och kolonier med morfologiska skillnader plockades och isolerades innan karaktäriseringen genomfördes med hjälp av reuterin analys, rep-PCR och gelelektrofores. En representant från varje rep-PCR-typ identifierades med hjälp av 16S rRNA gensekvensering. Bakterier identifierade som L. reuteri karaktäriserades ytterligare genom användning av metoder för att undersöka galltolerans, detektionen av generna ureC och pduC, bindning till tarmslem samt känslighet mot antibiotika. Ett fåtal isolat karaktäriserades även genom att bestämma gensekvensen för ett cellyteprotein som är specifikt för L. reuteri. Bakterieinnehållet i träckproven från hund varierande från 106 till 108 cfu g-1 träck och i tarmproven från varg varierade innehållet från 103 till 105 cfu g-1 tarminnehåll. Dock var inte alla bakterier som växt på Rogosa agarplattorna laktobaciller, arter av Streptococcus and Pediococcus kunde även påvisas. Även om det totala antalet bakterier var större i hund än i varg, kunde en större diversitet av bakterier ses i varg. I prover från hund isolerades två olika arter av Lactobacillus, L. animalis och L. reuteri medan det i varg isolerades ytterligare fem arter. Totalt isolerades det 32 L. reuteri från alla prover och dessa isolat kunde sedan bli uppdelade i 10-12 olika stammar med hjälp av de olika karaktäriseringsmetoderna. Av de 13 isolerade L. reuteri som analyserats av de olika karaktäriseringsmetoderna, var fyra reuterin-positiva och två ureas-positiva. Några av stammarna band bra till tarmslem (mucus) medan andra inte band alls. Galltoleransen varierade även mellan de olika stammarna men de flesta, förutom en stam, överlevde en koncentration på 5 % galla. Resultaten från testet för känslighet mot olika antibiotika visade på liknande resultat för samtliga stammar, där resistens mot streptomycin och tetracyklin kunde ses. Några av stammarna isolerades från både hund och varg medan andra bara isolerades från antingen hund eller varg. Detta framgick efter karaktäriseringen med hjälp av de olika metoderna samt genom analys av gensekvensen för ett cellyteprotein kallat ”protein x”. En ny metod som kallas ”immunomagnetic isolation” som skulle användas för att isolera L. reuteri testades också. Resultaten blev både positiva och negativa eftersom kulorna band till några stammar av L. reuteri men inte till alla. Kulorna, eller ”Dynabeadsen”, band också till laktobaciller i prov från hund men inte till några bakterier i prov från varg

Fermentation of barley flour with Lactobacillus reuteri

Fermentation of foodstuffs has beneficial effects on shelf life, taste and texture and possibly also health of the consumer. Products containing microbes with beneficial health effects for the host are defined as probiotics. One probiotic bacterium, Lactobacillus reuteri, has been shown to exert positive effects on a number of diseases and disorders, including a possible protective role against increased intestinal permeability or ‘leaky gut’. Increased intestinal permeability has been linked to e.g. inflammatory bowel disease (IBD), irritable bowel disease (IBS), coeliac disease and infectious diarrhoea. This study examined the effect of different cereal substrates on the growth, general metabolism and production of potential bioactive compounds by L. reuteri, with the aim of establishing a synbiotic formulation effective against increased intestinal permeability. Combinations of six barley varieties and six strains of bacteria were evaluated using standard plate counts, chemical analysis (1H-NMR) and a small intestinal epithelial cell model (IPEC-J2). The overall growth of L. reuteri in barley flour was good and reached higher densities in untreated compared with heat-treated flours. Differences in growth were also seen between bacterial strains and barley varieties. The general metabolism was similar for all strains with a few exceptions, e.g. lower production of succinate from L. reuteri DSM 17938. Two potentially bioactive compounds, γ-aminobutyric acid (GABA) and 3-hydroxypropionic acid (reuterin), were detected in barley fermented by L. reuteri. GABA was present in unfermented flour at similar levels as in the fermented counterpart, indicating no or low production by L. reuteri. Reuterin production was detected as formation of 1,3-propanediol and was strain-specific, being present in fermentation with DSM 17938 and ATCC PTA 6475. Experiments with IPEC-J2 cells revealed an increase in epithelial permeability caused by untreated flour, both fermented and unfermented. Treatment with heat-treated flour had a slight increasing effect on permeability, but recovered over time. Pre-treatments with live bacteria or fermented heat-treated flour before challenge with enterotoxigenic E. coli revealed significantly lower leakage of a molecular probe (FITC-dextran, 4 kDa). However, neither live bacteria nor their metabolites had a protective effect on epithelial permeability, measured as transepithelial electrical resistance (TEER)

Improving the functional properties of Lactobacillus reuteri

Lactobacillus reuteri is one of the most clinically studied probiotic bacteria. It has e.g. been shown to reduce the incidence and alleviate infantile colic and acute diarrhoea in children aged 3-60 months. The mechanisms of action behind the clinical effects are not fully understood, but two possibilities are inhibition of increased intestinal permeability (leaky gut) and alleviation of pain perception through inhibition of one of the key receptors (TRPV1) involved in visceral pain. The most studied strain of L. reuteri is DSM 17938, which has demonstrated good effects in clinical studies. To study whether DSM 17938 and other strains could produce bioactive metabolites with a possible inhibitory effect against leaky gut syndrome, different varieties of barley flour were used as growth substrate. Growth and metabolic activity were analysed for different L. reuteri-barley flour combinations, through an overview of the metabolic profile and more targeted chemical analyses. Catabolites of tryptophan previously shown to be bioactive compounds (e.g. AhR-ligands) were detected. Supernatants from L. reuteri-barley flour combinations with high and low concentrations of these catabolites were evaluated for possible protective effect against increased permeability in an intestinal epithelial cell model. Only a minor protective effect was seen for a few supernatants and no correlations were found between content of AhR-ligands and effect on leakage. In further attempts to improve the clinical effect of L. reuteri, experiments were performed to enhance certain functional properties through the use of experimental evolution. DSM 17938 has been shown to have flaws in certain properties that might contribute to the probiotic efficacy, e.g. freeze-thaw tolerance, bile tolerance and binding to intestinal mucus. Through experimental evolution, these properties were improved for DSM 17938 and, although genetic differences were only seen in two variants compared with the wild type, differences in protein expression were seen for all new variants. Evaluation of the new variants in cell culture models showed that they had similar effects to wild-type DSM 17938 on inhibition of TRPV1, although in vitro tests using intestinal epithelial cells revealed that some of the variants had lost their inhibitory effect against increased permeability. Overall, however, the variants retained most of the properties seen in vitro for wild-type DSM 1793

Pepparmyntans bioaktiva ämnen - hur påverkar de människors hälsa?

Since early civilization, herbal tea has been used as remedies for an abundance of different diseases. Among these herbal remedies, peppermint (Mentha piperita) was used for its effect on bowel related ailments. Even though peppermint was not described until late 17th century, it has been used since 1000 years before Christ by the Egyptians. In present day it is still considered a remedy for stomach pain etcetera, and much of the recent research is concentrated on the positive effects of peppermint on pain and bowel diseases. A large section of the research is also reviewing positive effects of peppermint on cancer, allergy and also on its potential as antibacterial agent. The substances that are known to have a possible effect on these diseases are polyphenols, which acts as antioxidants. Examples of such polyphenols are luteoline and other flavanoids. Luteoline seems to protect against the formation of tumors by inhibiting cell proliferation and inducing apoptosis. It also seems to lower the amount of histamine that is released from mast cells, which reduce the amount of allergic inconveniences. The main volatile components in peppermint, menthol and menthone, also seem to participate to some of the positive properties of peppermint. Menthol and menthone acts as analgesics by influencing the calcium channels in muscle tissue. Even though a lot of experiments have been performed, the number of human studies and clinical studies are limited. Such studies are required to enable a definitive conclusion to be drawn about peppermints health effects. Peppermint seems to have a lot of positive effects, but caution should always be made before conducting any remedies. Herbal remedies can have negative effects if the remedy is prolonged or is carried out in combination with medicine

Extracellular membrane vesicles from Limosilactobacillus reuteri strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5 '-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1 beta and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-gamma and TNF-alpha responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements