Rewilding the Human Gut: Reintroduction of the Species Limosilactobacillus Reuteri

The large intestine is home to trillions of microbes, known as the gut microbiome, which perform essential functions, such as digesting food and fighting disease. The diversity of microbes present in our gut microbiome is influenced by lifestyle factors, such as dietary patterns, medication usage, and sanitation practices. Research shows that the diversity of the human gut microbiome decreases as societies undergo industrialization. For example, fecal samples from rural Papua New Guineans contain an additional 50 microbial species, such as Limosilactobacillus reuteri, not found in people living in the United States. What has caused the disappearance of L. reuteri in industrialized countries is currently unknown. However, diet is a major factor influencing the composition of the gut microbiome. Microbiota-accessible carbohydrates (MACs) are indigestible carbohydrates that are a primary source of energy for gut microbes. North Americans consume far less of these carbohydrates (which are contained in foods such as beans, yams, and artichokes) than rural Papua New Guineans. The overall aim of this controlled feeding study is to determine if a strain of L. reuteri isolated from rural Papua New Guinea can be established in the gut of Canadians when taken as a probiotic alongside a non-industrialized-type diet designed to promote its growth. Furthermore, the study will determine: (i) the physiological and immunological effects of both L. reuteri and the non-industrialized-type diet, and (ii) the effects of both L. reuteri and the non-industrialized-type diet on gut microbiome ecology.

There is now consistent evidence that industrialization has substantially decreased the bacterial diversity of the gut microbiota (Segata, 2015), likely due to a combination of factors such as use of antibiotics, modern clinical practices, sanitation, and changes in dietary habits. However, the only factor for which empirical evidence exists is the low content of Microbiota-Accessible Carbohydrates (MACs) in Western diets, which are indigestible dietary carbohydrates that become available to the microbes colonizing the intestine (Sonnerburg et al., 2015). Previous work has confirmed the overall premise of 'microbiome depletion' by demonstrating higher diversity in the fecal microbiota of individuals from rural tribes in Papua New Guinea, which contain an additional of 50 species completely undetectable in North Americans (Martínez et al., 2015). One species detectable in every Papua New Guinean individual by 16S rRNA sequencing but not in a single US control was Limosilactobacillus reuteri (L. reuteri). Interestingly, this species, which is also used as a probiotic, was regularly detected in humans in studies conducted around 1960, but is very rarely found in contemporary humans, suggesting a recent decline of the L. reuteri population in Westerners (Walter et al., 2011). Most importantly, L. reuteri is a member of the gut microbiota in many vertebrate species and exerts benefits towards host immune functions and development, as demonstrated in a number of highly cited publications (Zelante et al. 2013; Buffington et al. 2016; Lamas et al. 2016; He et al. 2017). It is currently unclear what caused the drop in the L. reuteri population. However, it is likely due to the importance of non-digestible carbohydrates that are present at very low amounts in Western diets, while being abundant in the diet of rural Papua New Guineans, a population that consumes a predominantly plant-based diet. The goal of this study is to demonstrate that a bacterial species dominant in the non-westernized microbiome can be 'reintroduced' in the gut of Canadians fed a non-industrialized type diet designed to promote the growth of gut bacteria. This study will also determine how this 'reintroduction' and the non-industrialized-type diet influences immune function of the host and host-diet-microbiome metabolic interactions, and explore associations between them. It will further explore the effects of the microbial treatment and the diet on gut microbiome ecology. The central hypothesis is that an isolate of L. reuteri, originating from rural Papua New Guinea, can be established in the gut of Canadians fed a diet containing the carbohydrates known to facilitate the growth of this microbe. It is also hypothesized that this 'reintroduction' and consuming the non-industrialized type diet will be associated with immunological and metabolic benefits to the host. To achieve these goals, the following aims are proposed: To conduct a human trial to determine if a Limosilactobacillus reuteri strain isolated from rural Papua New Guinea (PNG) can be established in the gut of healthy Canadians. To determine if colonization can be improved by feeding a diet specifically designed to provide growth substrates for L. reuteri. To determine how both L. reuteri and the non-industrialized-type diet change the human microbiome, metabolome, cardiometabolic surrogate endpoints, and immune biomarkers of inflammation. This study will establish if a 'lost species' of bacteria can be reintroduced into the human gut and may provide mechanistic insight to inform how such dietary modulation may be applied to reduce the risk of chronic diseases. Since the L. reuteri strain isolated from rural Papua New Guineans is functionally different from western strains, evidenced by different growth rates on substrates of MACs, this study will further identify potential probiotic strains that were previously uncharacterized due to their overall absence from the industrialized gut microbiome.

Crossover design for dietary intervention (non-industrialized-type diet compared to participants' usual diet); parallel design for L. reuteri probiotic (L. reuteri PB-W1, L. reuteri DSM-20016T, and placebo).

Participants will receive the non-industrialized-type diet for 3 weeks, followed by a crossover to 3 weeks of consuming their usual diet after a 3-week washout period. Participants will be provided with a one-time dose of L. reuteri PB-W1 strain on day 4 of each diet period. The L. reuteri PB-W1 strain will be provided as a drinkable solution (approximately 2.25x10^10 viable cells will be provided in 50 ml of water).

Participants will receive the non-industrialized-type diet for 3 weeks, followed by a crossover to 3 weeks of consuming their usual diet after a 3-week washout period. Participants will be provided with a one-time dose of L. reuteri DSM20016T strain on day 4 of each diet period. The L. reuteri DSM20016T strain will be provided as a drinkable solution (approximately 2.25x10^10 viable cells will be provided in 50 ml of water).

Participants will receive the non-industrialized-type diet for 3 weeks, followed by a crossover to 3 weeks of consuming their usual diet after a 3-week washout period. Participants will be provided with a one-time dose of a placebo solution on day 4 of each diet period. The placebo solution will be provided as a drinkable solution (2 g maltodextrin dissolved in 50 ml water in food grade conditions).

Participants will consume their usual diet for 3 weeks, followed by a crossover to 3 weeks of consuming the provided non-industrialized-type diet after a 3-week washout period. Participants will be provided with a one-time dose of L. reuteri PB-W1 strain on day 4 of each diet period. The L. reuteri PB-W1 strain will be provided as a drinkable solution (approximately 2.25x10^10 viable cells will be provided in 50 ml of water).

Participants will consume their usual diet for 3 weeks, followed by a crossover to 3 weeks of consuming the provided non-industrialized-type diet after a 3-week washout period. Participants will be provided with a one-time dose of L. reuteri DSM20016T strain on day 4 of each diet period. The L. reuteri DSM20016T strain will be provided as a drinkable solution (approximately 2.25x10^10 viable cells will be provided in 50 ml of water).

Participants will consume their usual diet for 3 weeks, followed by a crossover to 3 weeks of consuming the provided non-industrialized-type diet after a 3-week washout period. Participants will be provided with a one-time dose of a placebo solution on day 4 of each diet period. The placebo solution will be provided as a drinkable solution (2 g maltodextrin dissolved in 50 ml water in food grade conditions).

L. Reuteri PB-W1 strain will be prepared in accordance to the standardized operating procedures to prepare Limosilactobacillus reuteri in food grade conditions

L. Reuteri DSM20016T strain will be prepared in accordance to the standardized operating procedures to prepare Limosilactobacillus reuteri in food grade conditions

Non-industrialized-type diet will be prepared in a metabolic kitchen, with all meals and snacks provided to participants for three weeks.

The primary outcome of this study is to measure L. reuteri (PB-W1 and DSM20016T strains) establishment in the gut of Canadian individuals. This will be measured by selective bacterial culture from fecal samples and quantified through quantitative PCR using species specific primers.

Enhanced persistence of colonization of L. reuteri (PB-W1 & DSM20016T strains) following consumption of non-industrialized-type diet designed to provide growth substrates for L. reuteri

We will measure if the persistence of L. reuteri in the gut of Canadian individuals is enhanced by the consumption of a non-industrialized-type diet specifically designed to provide growth substrates (MACs) for L. reuteri. This will be measured by selective bacterial culture from fecal samples and quantified through quantitative PCR using species specific primers.

Effect of L. reuteri strains and the non-industrialized-type diet on cardiometabolic surrogate endpoints: fasting glucose and lipid panel.

Plasma from blood samples will be analyzed for changes in glucose, triglycerides, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, non-HDL cholesterol, and total cholesterol (mmol/L).

Effect of L. reuteri strains and the non-industrialized-type diet on cardiometabolic surrogate endpoints: fasting insulin levels.

Effect of L. reuteri strains and the non-industrialized-type diet on cardiometabolic surrogate endpoints: homeostatic model assessment of insulin resistance and quantitative insulin sensitivity check index.

Homeostatic model assessment of insulin resistance and quantitative insulin sensitivity check index will be calculated based on fasting glucose and insulin levels.

Effect of L. reuteri strains and the non-industrialized-type diet on cardiometabolic surrogate endpoints: fasting C-reactive protein levels.

Effect of L. reuteri strains and the non-industrialized-type diet on cardiometabolic surrogate endpoints: body weight.

Effect of L. reuteri strains and the non-industrialized-type diet on inflammatory bowel disease surrogate endpoint: fecal calprotectin levels.

Effect of L. reuteri strains and the non-industrialized-type diet on biomarkers of gut barrier function: lipopolysaccharide binding protein.

Plasma from blood samples will be analyzed for changes in lipopolysaccharide binding protein (µg/mL).

Effect of L. reuteri strains and the non-industrialized-type diet on biomarkers of gut barrier function: fecal zonulin levels.

Effect of L reuteri strains and the non-industrialized-type diet on the composition of the fecal microbiome.

o Fecal samples will be analyzed using 16S rRNA-sequencing to measure changes in the fecal microbiome at the phylum, class, order, family, genus, and amplicon sequencing variant levels. Fecal microbiome composition will also be analyzed using whole metagenome sequencing to measure changes at the phylum, class, order, family, genus, species, and species-level genome bin levels.

Effect of L reuteri strains and the non-industrialized-type diet on the function of the fecal microbiome: enzyme levels.

Fecal samples will be analyzed using whole metagenome sequencing to measure changes in enzymes encoded by gut microbiota.

Effect of L reuteri strains and the non-industrialized-type diet on the function of the fecal microbiome: short-chain fatty acid levels.

Short-chain fatty acids (acetate, propionate, butyrate, valerate) and branched-chain fatty acids (isovalerate, isobutyrate) will be measured in fecal samples using gas chromatography mass spectrometry (µmol/g).

Effect of L reuteri strains and the non-industrialized-type diet on the function of the fecal microbiome: pH.

Plasma metabolome will be assessed to determine changes in molecules with known immunological functions such as indole derivatives of tryptophan and bile acids following the provision of the intervention. This will be measured via high performance chemical isotope labeling liquid chromatography mass spectrometry platform.

Host immune responses will be measured in blood samples by quantifying IgA, selected cytokines, and white blood cell phenotyping using flow cytometry. Mononuclear cells will be isolated from whole blood on ficoll gradients and the ability of cells to respond to challenges (peptidoglycan, phytohemagglutinin, and lipopolysaccharide) will be determined ex vivo.

Effect of L. reuteri strains and the non-industrialized-type diet on changes in psychological mood state.

Changes in individual mood state will be measured by the profile of mood states questionnaire (scored between -32 and 200; lower scores indicate more stable mood profiles).

Effect of L. reuteri strains and the non-industrialized-type diet on changes in gastrointestinal symptoms.

Changes in individual gastrointestinal symptoms will be measured by a gastrointestinal symptom questionnaire (scored on a scale of 0-5; higher scores indicating more symptoms).

Inclusion Criteria: Healthy individuals with a body mass index between 20-29.9 kg/m² Have at least one bowel movement per day Willing to consume prepared study foods (breakfast, lunch dinner, snacks) for a period of 3 weeks Men and pre-menopausal, non-pregnant or non-lactating women Non-vegetarian, non-smoking, and alcohol intake ≤8 drinks/week, and willing to consume 8 drinks per week or less during the course of the study. If consuming probiotic containing foods, willing to discontinue eating same, and substitute with non-probiotic containing foods -≤5 h/week of moderate-vigorous exercise. Quantity of L. reuteri in screening fecal sample below 10^4 CFU/g Exclusion Criteria: History of diabetes, acute or chronic GI illnesses, conditions, or history of GI surgical intervention antibiotic treatment in the last 3 months use of dietary supplements (including prebiotics and probiotics, fiber supplements/bars, digestive enzymes/beano)- if consumed, willing to undergo 4 week pre-intervention washout period, and remain free of supplements for duration of study. Exception: multivitamin or vitamin d supplement (1 week washout period) use of antihypertensive, lipid-lowering, anti-diabetic, anti-inflammatory (i.e corticosteroids or chronic NSAID use), or laxative medications known food allergies or intolerances (including dairy allergic or lactose intolerant)

Individual participant data will be shared that underlies the results reported after deidentification (text, tables, figures, and appendices). Data will be made available through a safe and secure publicly available data sharing repository.

Anyone who wishes to access the data may do so to achieve aims in the approved proposal and for individual participant data meta-analysis.

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