Reuteri DSM Research Articles

Molecular and Probiotic Characterizations of <i>Lactobacillus reuteri</i> DSM 12246 and Impact of pH on Biomass and Metabolic Profile in Batch-Culture

Lactobacillus reuteri is a powerful probiotic and adjunct functional culture candidate received a lot of scientific attention as it is one of the few endogenous “Lactobacillus” species found in the gastrointestinal tract of vertebrates, including humans, rats, pigs and chicken. The organism has been widely utilized as a probiotic in humans and animals for many years. In the present work L. reuteri DSM 12243; a high reuteri producer strain was molecularly characterized by 16SrRNA and RAPD analyses further, the probiotic properties including acid resistance, bile tolerance, adhesion to epithelial gastric cells, and antibiotic susceptibility were also assessed. Furthermore, the effect of pH on biomass production and metabolic profile of L. reuteri DSM 12246 in batch-culture was studied. The L. reuteri DSM 12246 showed a high similarity with L. reuteri strain I49 KR 36477 (100%) and type strain ATCC 55730 (99% of identity). The strain adhered well to CaCO2 cells and showed to be a highly resistance to acid juice (pH 3.0), with 0.7 log10 cfu/ml reduction in count after 60 min exposition. There is no significant change in the cell count after exposure to bile salts. In batch-cultures, at low pH values both glucose consumption and metabolites were low while the production of lactic acid was noticeable. Maximum biomass was reached at pH 5.5, with growth rate of μ = 0.641/h. The switch in pH values from 3.7 to 6.7 resulted in raising of glucose depletion as well as in the yield of acetate and ethanol. It is concluded that L. reuteri DSM 12246 was deemed as a successful candidate to be used as potential probiotic.

Probiotics in Functional Gastrointestinal Disorders.

Infantile colic, constipation, functional abdominal pain (FAP), and irritable bowel syndrome (IBS) are the most common functional gastrointestinal disorders (FGID). This chapter will review current evidence on the role of probiotics in the treatment of these FGID. The etiology of FGID is considered multifactorial, but the importance of intestinal microbiota in their development has been repeatedly emphasized. As a consequence, the potential role of probiotics in their treatment is being increasingly scrutinized. Presently, the strongest evidence of efficacy is for the use of Lactobacillus reuteri (L reuteri) DSM 17938 at the dose of 108CFU/day for the treatment of infantile colic in breastfed infants. Limited, yet encouraging, evidence exists for Lactobacillus rhamnosus GG (LGG) at the dose of 3×109 CFU and for a multi-strain preparation for the treatment of IBS. In the treatment of FAP, there is some evidence for the use of L reuteri DSM 17938 at the dose of at least 108CFU/day.

Variations in the Functional Properties of Soybean Flour Fermented with Lactic Acid Bacteria (LAB)-Consortium

The functional properties of soybean flour fermented with lactic acid (LAB)-consortium was evaluated. Soybean was processed into flour, fermented spontaneously and with LAB-consortium previously isolated from maize (Lactobacillus plantarum WCFS1+Lactobacillus rhamnosus GG, ATCC 53/03+Lactobacillus nantensis LP33+Lactobacillus fermentum CIP 102980+Lactobacillus reuteri DSM 20016) and sorghum (Pediococcus acidilactici DSM 20284+Lactobacillus fermentum CIP 102980+Lactobacillus brevis ATCC 14869+Lactobacillus nantensis LP33+Lactobacillus plantarum WCFS1) to evaluate their effects on the functional properties of the flour at 12 h intervals using standard techniques. The result shows gradual decrease in bulk density with increasing fermentation period ranging from 0.74 ± 0.03 g/mL to 0.72 ± 0.03 g/mL (natural), from 0.74 ± 0.03 g/mL to 0.70 ± 0.02 g/mL (LAB-consortium from maize) and from 0.74 ± 0.03 g/mL to 0.70 ± 0.02 g/mL (LAB-consortium from sorghum) fermentation. The swelling capacity decreased from 0.77 ± 0.03 g/mL to 0.64 ± 0.01 g/mL, from 0.77 ± 0.03 g/mL to 0.59 ± 0.01 g/mL and from 0.77 ± 0.03 g/mL to 0.61 ± 0.03 g/mL in natural, LAB-consortium from maize and sorghum fermentations respectively. Water holding capacity decreased from 2.4 ± 0.03 mL/g to 1.9 ± 0.03 mL/g, from 2.4 ± 0.03 mL/g to 2.0 ± 0.03 mL/g and from 2.4 ± 0.03 mL/g to 1.9 ± 0.03 mL/g in natural, LAB-consortium from maize and sorghum fermentation respectively. Oil holding capacity increased significantly (p<0.05) with increasing fermentation time, ranging traditional bread and 0.62-0.69 g/mL reported by Igbabul et al. on fermented mahogany beansfrom 8.92 ± 0.02 mL/g to 9.30 ± 0.03 mL/g (natural), 8.92 ± 0.01 mL/g to 9.63 ± 0.03 mL/g (LAB-consortium from maize) and from 8.92 ± 0.03 mL/g to 9.69 ± 0.03 mL/g (LAB-consortium from sorghum) fermentations. The least gelation concentration ranged from 3.0% (unfermented) to 6.0% (other fermentation products). Emulsion capacity (EC) increased from 35.88 ± 3.12% to 44.33 ± 1.33%, from 35.88 ± 3.12% to 46.83 ± 3.18% and from 35.88 ± 3.12 % to 45.99 ± 2.21% in natural, LAB-consortium from maize and sorghum fermentations respectively. This suggests the potentials of LAB-consortia fermentation in improving nutritional and functional properties of soybean flour.

Lactobacillus reuteri DSM 17938: Review of Evidence in Functional Gastrointestinal Disorders

Lactobacillus reuteri DSM 17938 has been one of the most extensively studied probiotic in children and adults with functional gastrointestinal disorders. In order to gather evidence on the efficacy of Lactobacillus reuteri DSM 17938 for treating and preventing some of these disorders. MEDLINE and the Cochrane Library were searched till February 2018 for relevant randomized controlled trials, retrospective studies, and meta-analyses with no language restrictions. The recent probiotic literature strongly suggests that Lactobacillus reuteri may be effective in the prevention and treatment of certain clinical conditions, such as infantile colic, infantile regurgitation, functional constipation, acute gastroenteritis, acute diarrhea, antibiotic-associated diarrhea, and necrotizing enterocolitis. No safety concerns with regard to the use of Lactobacillus reuteri in non-immunocompromised subjects were identified. A recent meta-analysis also supports the safety of using Lactobacillus reuteri even in preterm infants. This review presents the history of Lactobacillus reuteri DSM 17938 and the evidence base for its application in the health and disease.

Proximate Composition and In-vitro Starch/Protein Digestibility of Bambara Groundnut Flour Fermented with Lactic Acid Bacteria (LAB)-Consortium Isolated from Cereals

The effect of lactic acid bacteria (LAB) consortium fermentation on the proximate and in-vitro starch and protein digestibility of bambara groundnut flour was evaluated. Bambara groundnuts were processed into flour and fermented with LAB consortium previously isolated from maize (Lactobacillus plantarum WCFS1+Lactobacillus rhamnosus GG, ATCC53/03+Lactobacillus nantensis LP33+Lactobacillus fermentum CIP102980+Lactobacillus reuteri DSM 20016) and from sorghum (Pediococcus acidilactici DSM 20284+Lactobacillus fermentum CIP102980+Lactobacillus brevis ATCC14869+Lactobacillus nantensis LP33+Lactobacillus plantarum WCFS1). The sample was also fermented naturally for comparison. The results showed significant (p<0.05) increase in the moisture, ash and protein contents of the bambara groundnut flour as fermentation time increased. The lipid and crude fibre contents decreased significantly (p<0.05) with the increasing fermentation time in all the samples. The carbohydrate content decreased gradually from 50.66 ± 0.23% to 44.96 ± 0.10%. The in-vitro starch digestibility increased significantly (p<0.05) with increasing fermentation periods from 42.48 ± 0.01% to 50.36 ± 0.02%. The in-vitro protein digestibility increased significantly (p<0.05) from 68.70 ± 1.73% to 85.24 ± 1.21%. There was gradual increase in the bacteria load of the sample as fermentation time increased. However, fungal growth was only observed in natural fermentation. The occurrence of the isolates showed that the starter organisms were the only organisms isolated from the consortia fermentation set up while the natural fermentation have mixed microbial growth. This study has shown the effectiveness of LAB-consortium fermentation in improving the nutritional quality and the in-vitro starch and protein digestibility of flours from bambara groundnut.

Is It Useful to Administer Probiotics Together With Proton Pump Inhibitors in Children With Gastroesophageal Reflux?

Background/AimsGastroesophageal reflux disease (GERD) is a frequent condition diagnosed in children and treated with proton pump inhibitors (PPI). Long-term PPI administration can alter intestinal bacterial population by suppressing the gastric acid barrier and may cause diarrhea. The aim of this study is to evaluate the prevalence of small intestinal bacterial overgrowth assessed by glucose hydrogen breath test among children that received 12 weeks of PPI with or without probiotics (Lactobacillus reuteri DSM 17938) associated, compared to controls.MethodsGlucose hydrogen breath test was performed before PPI treatment and after 12 weeks of PPI treatment to 128 consecutive children with GERD (1–18 years old) and a control group (120 healthy children). The children with GERD were randomized into 2 groups: placebo group (64 who received PPI and placebo for 12 weeks) and probiotics group (64 who received PPI and probiotics for 12 weeks).ResultsAfter 12 weeks of treatment, dysbiosis was detected among 56.2% of children from placebo group (36/64), compared to 6.2% of children from the probiotics group (4/64, P < 0.001). Bacterial overgrowth was detected in 5% of controls (6/120). Probiotics group had a lower prevalence of dysbiosis, similar to controls (P = 0.740).ConclusionProbiotics administration decreased the rate of dysbiosis among children treated with PPI.

Adenosine A2A Receptor Deletion Blocks the Beneficial Effects of Lactobacillus reuteri in Regulatory T-Deficient Scurfy Mice

The lack of a functional Foxp3 transcription factor and regulatory T (Treg) cells causes lethal, CD4+ T cell-driven autoimmune diseases in scurfy (SF) mice and humans. Recent studies have shown that adenosine A2A receptor activation limits inflammation and tissue damage, thereby playing an anti-inflammatory role. However, the role of the adenosine A2A receptor in the development of disease in SF mice remains unclear. Using a genetic approach, we found that adenosine A2A receptor deletion in SF mice (SF) does not affect early life events, the development of a lymphoproliferative disorder, or hyper-production of pro-inflammatory cytokines seen in the Treg-deficiency state. As shown previously, Lactobacillus reuteri DSM 17938 treatment prolonged survival and reduced multiorgan inflammation in SF mice. In marked contrast, A2A receptor deletion completely blocked these beneficial effects of L. reuteri in SF mice. Altogether, these results suggest that although absence of the adenosine A2A receptor does not affect the development of disease in SF mice, it plays a critical role in the immunomodulation by L. reuteri in Treg-deficiency disease. The adenosine A2A receptor and its activation may have a role in treating other Treg dysfunction-mediated autoimmune diseases.

Efficacy of Lactobacillus reuteri DSM 17938 for infantile colic: Systematic review with network meta-analysis.

Background:5% to 40% of infants cry excessively, usually accompanied by fussiness and excessive of gas. There are no uniform criteria for treatment of infantile colic. Lactobacillus reuteri DSM 17938 has been used with promising results. The objective of this network-meta-analysis (NMA) is to compare the efficacy of L reuteri DSM 17938 with other interventions for infantile colic.Methods:RCTs, published between 1960 and 2015 for the treatment of infantile colic were included. Primary outcome was duration of crying after 21 to 28 days of treatment. Different databases were searched. Information was analyzed using control group as central axis. A random effect model was used. Hedges standard mean difference (SMD) and odds ratio (OR) were calculated. A SUCRA analysis was performed to evaluate superiority for each intervention.Results:32 RCTs were analyzed, including 2242 patients. Studies with L reuteri DSM 17938 versus Ctrl., Diet versus Ctrl. and Acupuncture versus Ctrl. were the most influential studies in the NMA. L reuteri DSM 17938 [WMD −51.3 h (CI95% −72.2 to −30.5 h), P .0001] and dietetic approaches [WMD −37.4 h (CI95% −56.1 to −18.7 h), P .0001] were superior compared to the other treatments.Conclusions:L reuteri DSM 17938 and some dietetic approaches are better to other interventions for treatment of infantile colic.

Impact of Lactobacillus reuteri colonization on gut microbiota, inflammation, and crying time in infant colic

Infant colic is a distressing condition of unknown etiology. An aberrant gastrointestinal microbiota has been associated, and Lactobacillus reuteri supplementation has been shown to reduce crying and/or fussing time (‘crying time’) in some infants with colic. The relationship between L. reuteri gut colonization and crying time has not been examined. We investigated the relationship between L. reuteri colonization and fecal microbiota (microbial diversity and Escherichia coli), intestinal inflammation, and crying time in infants with colic, using a subset of 65 infants from the Baby Biotics trial, which randomized healthy term infants aged <13 weeks with infant colic to receive probiotic L. reuteri DSM 17938 (1 × 108 colony forming units) or placebo daily for 28 days. We observed an overall reduction in median crying time, regardless of L. reuteri colonization status (n = 14 colonized). There were no differences in E. coli colonization rates or densities, microbial diversity or intestinal inflammation by L. reuteri colonization status. We found that L. reuteri density positively correlated with crying time, and E. coli density negatively correlated with microbial diversity. As density of L. reuteri was associated with increased crying time, L. reuteri supplementation may not be an appropriate treatment for all infants with colic.

Functional cream cheese supplemented with Bifidobacterium animalis subsp. lactis DSM 10140 and Lactobacillus reuteri DSM 20016 and prebiotics

The aim of this study was to develop a functional fresh cream cheese with Bifidobacterium animalis subsp. lactis DSM 10140 or Lactobacillus reuteri DSM 20016 and prebiotics (inulin, FOS and lactulose). The research was divided into two steps: in vitro evaluation of the effects of prebiotic compounds; validation at laboratory level with production of functional cream mini-cheeses.Prebiotics showed a protective effect: B. animalis subsp. lactis DSM 10140 cultivability on Petri dishes was positively influenced by lactulose, whereas fructooligosaccharides (FOS) were the prebiotic compounds able to prolong Lb. reuteri DSM 20016 cultivability. At 30 °C, a prolongation of the death time (more than 300 days) was observed, while the controls showed death time values about 100 days. At 45 °C, death time values increased from 32.2 (control) to 33, 35, and 38 days in the samples added with FOS, inulin and lactulose, respectively.Lactulose and FOS were chosen to be added to cream mini-cheeses inoculated with B. animalis subsp. lactis DSM 10140 and Lb. reuteri DSM 20016, respectively; the proposed functional cream cheese resulted in a product with favourable conditions for the viability of both probiotics which maintained cultivable cells above the recommended level during 28 days of storage at 4 °C with good sensory characteristics.

Effect of a Partially Hydrolysed Whey Infant Formula Supplemented with Starch and Lactobacillus reuteri DSM 17938 on Regurgitation and Gastric Motility.

Functional regurgitation (FR) is common in early infancy and represents a major drain on healthcare resources. This double-blind, randomized controlled trial investigated the effects of a formula containing partially hydrolysed, 100% whey protein, starch and Lactobacillus reuteri (DSM 17938) on gastric emptying rate (GErate) and regurgitation frequency in infants with FR. Enrolled infants were randomly allocated to receive either the test formula or a standard starter formula for four weeks. Ultrasound GErate assessment was performed at baseline (week 0) and at week 4; the number of regurgitations, feed volumes and potential adverse events were recorded in a daily diary. Eighty infants aged four weeks to five months were enrolled; 72 (test group = 37; control group = 35) completed the study. Compared to controls, the test group showed greater percentage changes in GErate (12.3% vs. 9.1%, p < 0.01). Mean daily regurgitations decreased from 7.4 (0.8) at week 0 to 2.6 (1.0) at week 4 in the test group and from 7.5 (1.0) to 5.3 (1.0) in controls (between-group difference, p < 0.0001). Compared to a standard formula, a starch-thickened partially hydrolysed whey protein formula supplemented with Lactobacillus reuteri is more effective in decreasing the frequency of regurgitation and improving GErate, and can be of benefit to infants with FR.

Rapid enteric testing to permit targeted antimicrobial therapy, with and without Lactobacillus reuteri probiotics, for paediatric acute diarrhoeal disease in Botswana: A pilot, randomized, factorial, controlled trial.

IntroductionDiarrhoeal disease is the second-leading cause of death in young children. Current guidelines recommend treating children with acute non-bloody diarrhea with oral rehydration solutions and zinc, but not antimicrobials. However, in many resource-limited settings, infections with treatable enteric bacterial and protozoan pathogens are common. Probiotics have shown promise as an adjunct treatment for diarrhoea but have not been studied in sub-Saharan Africa.MethodsWe conducted a pilot, factorial, randomized, placebo-controlled trial of children aged 2–60 months hospitalized in Botswana for acute non-bloody diarrhoea. A rapid test-and-treat intervention, consisting of multiplex PCR testing of rectal swabs taken at enrolment, accompanied by targeted antimicrobial therapy if treatable pathogens were detected, was compared to the reference standard of no stool testing. Additionally, Lactobacillus reuteri DSM 17938 x 60 days was compared to placebo treatment. The main objective of this pilot study was to assess feasibility. The primary clinical outcome was the increase in age-standardized height (HAZ) at 60 days adjusted for baseline HAZ.ResultsSeventy-six patients were enrolled over a seven-month study period. We judged that the recruitment rate, lab processing times, communication protocols, provision of specific antimicrobials, and follow-up rates were acceptable. Compared to the reference arm (no stool testing and placebo treatment), the combination of the rapid test-and-treat strategy plus L. reuteri DSM 17938 was associated with an increase of 0.61 HAZ (95% CI 0.09–1.13) and 93% lower odds of recurrent diarrhoea (OR 0.07, 95%CI 0.01–0.61) at 60 days.DiscussionWe demonstrated that it was feasible to evaluate the study interventions in Botswana. Despite the small sample size, we observed a statistically significant increase in HAZ at 60 days and significantly lower odds of recurrent diarrhoea in children receiving both rapid test-and-treat and L. reuteri. There is sufficient evidence to warrant proceeding with a larger follow-up trial in a similar setting.

In silico analysis, molecular cloning, expression and characterization of l-asparaginase gene from Lactobacillus reuteri DSM 20016.

l-Asparaginase is employed in leukaemic treatment and in processing starchy foods. The in silico analysis of Lactobacillus reuteri DSM 20016 reveals the presence of an l-asparaginase gene with theoretical pI value of 4.99. 3D structure prediction was carried out and one model was selected based on the validation scores of 86.293 for ERRAT, 92.10% for VERIFY 3D and Ramachandran plot. Multiple sequence alignment of the protein sequences of l-asparaginases I and II of Escherichia coli, Erwinia chrysanthemum and Homo sapiens shows their sequence similarity. The ORF LREU_RS09880 from L. reuteri DSM 20016 genome was cloned and expressed in E. coli. The recombinant protein was purified to homogeneity using Ni-NTA chromatography and showed higher substrate specificity for l-asparagine. Kinetic parameters like Km and Vmax of recombinant l-asparaginase were calculated as 0.3332mM, 14.06mM/min, respectively. Temperature and pH profile of recombinant l-asparaginase were analysed and maximum activity was found between 30 and 40°C and at pH 6. The recombinant enzyme was thermally stable up to 24h at 28°C. Recombinant l-asparaginase has a recovery percentage of 92 and 10.5 fold purification. HPLC-MS-MS and SDS-PAGE analysis of the purified protein indicated a molecular weight of 35kDa as a monomer.

Probiotic Supplementation in Preterm: Feeding Intolerance and Hospital Cost.

We hypothesized that giving the probiotic strain Lactobacillus reuteri (L. reuteri) DSM 17938 to preterm, formula-fed infants would prevent an early traumatic intestinal inflammatory insult modulating intestinal cytokine profile and reducing the onset of feeding intolerance. Newborn were randomly allocated during the first 48 h of life to receive either daily probiotic (108 colony forming units (CFUs) of L. reuteri DSM 17938) or placebo for one month. All the newborns underwent to gastric ultrasound for the measurement of gastric emptying time. Fecal samples were collected for the evaluation of fecal cytokines. Clinical data on feeding intolerance and weight gain were collected. The costs of hospital stays were calculated. The results showed that the newborns receiving L. reuteri DSM 17938 had a significant decrease in the number of days needed to reach full enteral feeding (p < 0.01), days of hospital stay (p < 0.01), and days of antibiotic treatment (p < 0.01). Statistically significant differences were observed in pattern of fecal cytokine profiles. The anti-inflammatory cytokine interleukin (IL)-10, was increased in newborns receiving L. reuteri DSM 17938. Pro-inflammatory cytokines: IL-17, IL-8, and tumor necrosis factor (TNF)-alpha levels were increased in newborns given placebo. Differences in the gastric emptying and fasting antral area (FAA) were also observed. Our study demonstrates an effective role for L. reuteri DSM 17938 supplementation in preventing feeding intolerance and improving gut motor and immune function development in bottle-fed stable preterm newborns. Another benefit from the use of probiotics is the reducing cost for the Health Care service.

Efficacy of Lactobacillus Reuteri DSM 17938 for the Treatment of Acute Gastroenteritis in Children: Protocol of a Randomized Controlled Trial.

BackgroundAcute gastroenteritis (AGE) is one of the most common diseases among children. Oral rehydration therapy is the key treatment. However, despite proven efficacy, it remains underused. This is because oral rehydration solution neither reduces the frequency of bowel movements and fluid loss nor shortens the duration of illness. Hence, there is interest in adjunctive treatments. According to the 2014 guidelines developed by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition, the use of the following probiotics may be considered in the management of children with AGE in addition to rehydration therapy: Lactobacillus rhamnosus GG (low quality of evidence; strong recommendation) and Saccharomyces boulardii (low quality of evidence; strong recommendation). Less compelling evidence is available for Lactobacillus reuteri DSM 17938 (very low quality of evidence; weak recommendation).ObjectiveConsidering that evidence on L reuteri remains limited, the goal of the study is to assess the effectiveness of L reuteri DSM 17938 in the treatment of AGE in children. Children vaccinated and not vaccinated against rotavirus will be evaluated separately.MethodsThis will be a double-blind, placebo-controlled, randomized trial. Children between 1 and 60 months of age with AGE, defined as a change in stool consistency to loose or liquid form (according to the Bristol Stool Form scale or Amsterdam Stool Form scale) and/or an increase in the frequency of evacuations (typically ≥3 in 24 h) lasting for no longer than 5 days, will be recruited. A total of 72 children will receive either L reuteri DSM 17938 at a dose of 2×108colony-forming units twice daily or matching placebo for 5 consecutive days. A similar sample size for rotavirus vaccinated and nonvaccinated children is planned. The primary outcome measure is the duration of diarrhea. Two separate studies and reports for rotavirus vaccinated and nonvaccinated children are planned.ResultsThe recruitment started in January 2017 and is planned to be finalized in June 2018 for rotavirus nonvaccinated children. The recruitment of rotavirus-vaccinated children may be slower due to a relatively low coverage rate in Poland. Data analysis and submission to a peer-reviewed journal is expected within 3 months after completion of the study.ConclusionThis study will add to current knowledge on the efficacy of L reuteri DSM 17938 for the management of AGE.Trial registrationClinicalTrials.gov NCT02989350; https://clinicaltrials.gov/ct2/show/NCT02989350 (Archived by WebCite at http://www.webcitation.org/6slOFkyTH)

Metabolism of Foodborne Heterocyclic Aromatic Amines by Lactobacillus reuteri DSM 20016.

The heterocyclic aromatic amine (HAA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is converted into 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1) via a chemical reaction with 3-hydroxypropionaldehyde or acrolein derived from glycerol by reuterin producing gut bacteria. Because it is unknown whether this reaction also applies to other HAAs, seven foodborne HAAs (2-amino-9H-pyrido[2,3-b]indole (AαC), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethyl-3H-imidazo[4,5-f]quinoxaline (MeIQx), 9H-pyrido[3,4-b]indole (norharman), and 1-methyl-9H-pyrido[3,4-b]indole (harman)) were anaerobically incubated with Lactobacillus reuteri DSM 20016 in the presence of glycerol. The extent of conversion, as analyzed by HPLC-DAD/FLD, was dependent on both the studied HAAs and the glucose/glycerol ratio, indicating reuterin to be involved in HAA metabolism. Based on HRMS analyses, PhIP-M1-type metabolites were detected for AαC, Trp-P-1, IQ, MeIQ, MeIQx, harman, and norharman. In the case of AαC, this was confirmed by metabolite isolation (AαC-M8, 2,3,4,10-tetrahydro-1H-indolo[2,3-b][1,8]naphthyridin-2-ol) and one- (1H) and two-dimensional (HSQC, HMBC, COSY, DOSY) NMR spectroscopy. In addition, based on HRMS and/or NMR spectroscopy, a new type of HAA metabolite, resulting from the reaction with two molecules of 3-hydroxypropionaldehyde or acrolein, is hypothesized for AαC, Trp-P-1, IQ, MeIQ, and MeIQx.

Lactobacillus caviae sp. nov., an obligately heterofermentative bacterium isolated from the oral cavity of a guinea pig (Cavia aperea f. porcellus).

A Gram-stain-positive, facultatively anaerobic, and catalase- and oxidase-negative bacterial strain designated MOZM2T, having 98.4 % 16S rRNA gene sequence identity with Lactobacillus reuteri DSM 20016T, was isolated from a swab of the oral cavity of a home-bred guinea pig. Comparative analyses based on the hsp60, pheS and tuf genes confirmed L. reuteri as its closest relative species, with calculated sequence similarities of 92.8, 88.8 and 96.9 %, respectively. DNA-DNA hybridisation revealed a 42 % degree of genetic similarity between the novel strain and L. reuteri DSM 20016T. Strain MOZM2T degrades carbohydrates via the 6-phosphogluconate/phosphoketolase pathway, evidenced by its production of gas from glucose and the end products of hexose catabolism. Comparative analysis of the cellular fatty acid profiles determined significant differences between MOZM2T and L. reuteri DSM 20016T in their proportions of C8 : 0, C14 : 1, C17 : 0, C18 : 2ω6t and C20 : 0 fatty acids. Results of genotypic analyses also demonstrated differences between these two strains. They also differed in DNA G+C content, and some biochemical and physiological characteristics. We therefore believe that the examined bacterial isolate should be considered as a new taxon within the group of obligately heterofermentative lactobacilli. The species name Lactobacillus caviae sp. nov. is proposed, of which the type strain is MOZM2T (=CCM 8609T=DSM 100239T=LMG 28780T).

Efficacy of a standardized extract of Matricariae chamomilla L., Melissa officinalis L. and tyndallized Lactobacillus acidophilus (HA122) in infantile colic: An open randomized controlled trial.

Infant colic (IC) is a prevalent physiological event of infants, which can disrupt the child's home environment. We aimed to investigate the effectiveness of a mixture of Matricariae chamomilla L., Melissa officinalis L. and tyndallized Lactobacillus acidophilus (HA122) compared with Lactobacillus reuteri DSM 17938 and with simethicone for the treatment of IC. A multicenter randomized comparative study was conducted in infants with colic, according to Rome III criteria, who were randomly assigned to receive M.chamomilla L., M.officinalis L. and tyndallized L.acidophilus (HA122) (Colimil® Plus® ; Milte Italia Spa, Milan, Italy) (Group A), L.reuteri DSM 17938 (Group B) and simethicone (Group C). Treatment was given to subjects for 28days. One-hundred and seventy-six patients completed the study. Mean daily crying time at day 28 was significantly lower in group A (-44, 95% CI: -58 to -30, P<.001) and group B (-35, 95% CI: -49 to -20, P<.001) when compared to group C. No significant difference was observed between Group A and Group B (mean difference: -9minutes, 95% CI -23 to +5, P=.205). At day 28, 39 of 57 (68.4%) of infants in Group C responded to the treatment compared with 57 out of 60 patients (95%) of Group A and 51 out of 59 (86.4%) of Group B (P<.001). This study suggests that administration of M.chamomilla L., M.officinalis L. and tyndallized L.acidophilus (HA122) and L.reuteri DSM 17938 are significantly more effective than simethicone in IC. ClinicalTrials.gov: NCT02708238.

Detection and Physicochemical Characterization of Membrane Vesicles (MVs) of Lactobacillus reuteri DSM 17938.

Membrane vesicles (MVs) are bilayer structures which bleb from bacteria, and are important in trafficking biomolecules to other bacteria or host cells. There are few data about MVs produced by the Gram-positive commensal-derived probiotic Lactobacillus reuteri; however, MVs from this species may have potential therapeutic benefit. The aim of this study was to detect and characterize MVs produced from biofilm (bMVs), and planktonic (pMVs) phenotypes of L. reuteri DSM 17938. MVs were analyzed for structure and physicochemical characterization by Scanning Electron Microscope (SEM) and Dynamic Light Scattering (DLS). Their composition was interrogated using various digestive enzyme treatments and subsequent Transmission Electron Microscopy (TEM) analysis. eDNA (extracellular DNA) was detected and quantified using PicoGreen. We found that planktonic and biofilm of L. reuteri cultures generated MVs with a broad size distribution. Our data also showed that eDNA was associated with pMVs and bMVs (eMVsDNA). DNase I treatment demonstrated no modifications of MVs, suggesting that an eDNA-MVs complex protected the eMVsDNA. Proteinase K and Phospholipase C treatments modified the structure of MVs, showing that lipids and proteins are important structural components of L. reuteri MVs. The biological composition and the physicochemical characterization of MVs generated by the probiotic L. reuteri may represent a starting point for future applications in the development of vesicles-based therapeutic systems.

Therapeutic effect of Lactobacillus reuteri DSM 17938 on Treg-deficiency-induced autoimmunity (IPEX syndrome) via the inosine-adenosine 2A receptors

Abstract Regulatory T-cell (Treg) deficiency causes lethal, CD4+T cell-driven autoimmune diseases. Stem cell transplantation is used to treat these diseases, but this procedure is limited by the availability of a suitable donor. The intestinal microbiota drives host immune homeostasis by regulating the development of Treg, Th1 and Th2 cells. It is currently unclear if Treg-deficiency autoimmune disorders can be treated by targeting the enteric microbiota. Our aims are to determine the autoimmunity, gut microbiota, and plasma metabolomics affected by probiotic Lactobacillus reuteri DSM 17938 (LR), and to further identify the mechanism of modulated metabolite(s) in suppressing autoimmunity in Treg-deficient scurfy (SF) mice. We demonstrated that Foxp3+Treg deficiency results in gut microbial dysbiosis and autoimmunity over the lifespan of SF mouse. Remodeling microbiota with LR prolonged survival and reduced multi-organ inflammation in SF mice. LR changed the metabolomics profile disrupted by Treg-deficiency with a major effect of restoring levels of the purine metabolite inosine. Feeding inosine itself prolonged life and inhibited multi-organ inflammation by reducing Th1/Th2 cells and their associated cytokines. Mechanistically, the inhibition of inosine on the differentiation of Th1 and Th2 cells in vitro depended on adenosine A2A receptors. Both A2A receptor specific antagonist or genetically knockout A2A to SF mice reversed the anti-inflammatory effects of both inosine and LR in vivo. In conclusions, A2A receptors mediate a substantial protective effect of inosine and LR. The LR-modulated-microbiota-inosine-A2A axis might represent a potential avenue for combatting autoimmune diseases mediated by Treg dysfunction.