Intestinal Immunomodulation and Shifts on the Gut Microbiota of BALB/c Mice Promoted by Two Bifidobacterium and Lactobacillus Strains Isolated from Human Samples

Bifidobacterium animalis subsp. lactis IPLA 20020 and Lactobacillus gasseri IPLA 20212, two strains isolated from human samples, were evaluated for safety and influence over the intestinal microbiota and cytokine production by the intestinal tissue of adult BALB/c mice. Mice were divided into four groups receiving during 8 days PBS or a suspension of each strain, prepared fresh or lyophilized (bifidobacteria), at an amount of 4x108 viable cells/day. This dose could be comparable to the probiotic intake of a human adult who consumed about 100-200 mL of functional fermented milk per day, considering the usual level of probiotics in commercial products. No microbial translocation to liver or alterations in food intake, weight, and behavior were observed in treated mice. Intestinal content of secretory immunoglobulin A (s-IgA) was not affected, discarding any adverse effect on the mucosa-associated immunity. The profile of intestinal proinflammatory/regulatory cytokines after intervention evidenced that the microbial strain administered and its cellular state (fresh or lyophilized) as well as the host tissue analyzed (small or large intestine) influenced the immune response and suggests a moderate shift towards a T helper 1 profile (Th1) in the large intestine after the administration of both strains. Changes on relative levels of some intestinal microbial groups were evidenced after intervention. It is noteworthy that butyrate was positively associated with a balanced pro-Th1 immune response. Therefore, B. animalis subsp. lactis IPLA20020 and L. gasseri IPLA 20212 could be considered potential probiotic candidates to be included in functional foods for balancing the intestinal immune response.Fil: Nogacka, Alicja M.. Instituto de Investigación Sanitaria del Principado de Asturias; España. Consejo Superior de Investigaciones Científicas. Instituto Superior de Productos Lácteos de Asturias; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Oddi, Sofia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Salazar, Nuria. Instituto de Investigación Sanitaria del Principado de Asturias; España. Consejo Superior de Investigaciones Científicas. Instituto Superior de Productos Lácteos de Asturias; EspañaFil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Gueimonde, Miguel. Instituto de Investigación Sanitaria del Principado de Asturias; España. Consejo Superior de Investigaciones Científicas. Instituto Superior de Productos Lácteos de Asturias; EspañaFil: Vinderola, Celso Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: De Los Reyes Gavilán, Clara G.. Consejo Superior de Investigaciones Científicas. Instituto Superior de Productos Lácteos de Asturias; España. Instituto de Investigación Sanitaria del Principado de Asturias; Españ

Influence of 2′-fucosyllactose on the microbiota composition and metabolic activity of fecal cultures from breastfed and formula-fed infants at two months of age

Although breast milk is considered the gold standard of nutrition for infant feeding, some circumstances may make breastfeeding difficult. Several commercial milk preparations include syn-thetic human milk oligosaccharides (HMOs) in their composition. However, the effect of HMOs on the establishment of the intestinal microbiota remains incompletely understood. Independent batch fermentations were performed with feces from six full-term infant donors of two months of age (three breastfed and three formula-fed, exclusively) in the presence of 2′fucosyllactose (2′FL), one of the most abundant HMOs in human milk. Microbiota composition was analyzed by 16S rRNA gene sequencing at baseline and at 24 h of incubation. The 2′FL consumption, gas accumulation, and levels of different metabolites were determined by chromatography. Microbiota profiles at baseline were clearly influenced by the mode of feeding and by the intrinsic ability of microbiotas to degrade 2′FL. The 2′FL degradation rate clustered fecal cultures into slow and fast degraders, regardless of feeding type, this being a determinant factor influencing the evolution of the microbiota during incubation, although the low number of donors precludes drawing sound conclusions. More studies are needed to decipher the extent to which the early intervention with HMOs could influence the microbiota as a function of its ability to utilize 2′FL.This research was funded by a contract of Lallemand Health Solutions Inc. with IPLA-CSIC and by the Intramural CSIC Research project PIE201970E061. S.A. was the recipient of a postdoctoral Juan de la Cierva Contract (Ministry of Science, Innovation and Universities, Ref. IJCI-2017-32156) and NS has a postdoctoral contract awarded by the Biosanitary Research Foundation in Asturias (FINBA, Spain)

Impact of Extreme Obesity and Diet-Induced Weight Loss on the Fecal Metabolome and Gut Microbiota

Scope: A limited number of human studies have characterized fecal microbiota and metabolome in extreme obesity and after diet-induced weight loss. Methods and results: Fecal samples from normal-weight and extremely obese adults and from obese participants before and after moderate diet-induced weight loss are evaluated for their interaction with the intestinal adenocarcinoma cell line HT29 using an impedance-based in vitro model, which reveals variations in the interaction between the gut microbiota and host linked to obesity status. Microbiota composition, short chain fatty acids, and other intestinal metabolites are further analyzed to assess the interplay among diet, gut microbiota, and host in extreme obesity. Microbiota profiles are distinct between normal-weight and obese participants and are accompanied by fecal signatures in the metabolism of biliary compounds and catecholamines. Moderate diet-induced weight loss promotes shifts in the gut microbiota, and the primary fecal metabolomics features are associated with diet and the gut–liver and gut–brain axes. Conclusions: Analyses of the fecal microbiota and metabolome enable assessment of the impact of diet on gut microbiota composition and activity, supporting the potential use of certain fecal metabolites or members of the gut microbiota as biomarkers for the efficacy of weight loss in extreme obesity

In vitro Selection of Probiotics for Microbiota Modulation in Normal-Weight and Severely Obese Individuals: Focus on Gas Production and Interaction With Intestinal Epithelial Cells

The intestinal microbiota plays important roles in the maintenance of health. Strategies aiming at its modulation, such as probiotics, have received a deal of attention. Several strains have been studied in different in vitro models; however, the correlation of results obtained with the in vivo data has been limited. This questions the usefulness of such in vitro selection models, traditionally relying on over-simplified tests, not considering the influence of the accompanying microbiota or focusing on microbiota composition without considering functional traits. Here we assess the potential of six Bifidobacterium, Lactobacillus and Lacticaseibacillus strains in an in vitro model to determine their impact on the microbiota not just in terms of composition but also of functionality. Moreover, we compared the responses obtained in two different population groups: normal-weight and severely obese subjects. Fecal cultures were conducted to evaluate the impact of the strains on specific intestinal microbial groups, on the production of short-chain fatty acids, and on two functional responses: the production of gas and the interaction with human intestinal epithelial cells. The response to the different probiotics differed between both human groups. The addition of the probiotic strains did not induce major changes on the microbiota composition, with significant increases detected almost exclusively for the species added. Higher levels of gas production were observed in cultures from normal-weight subjects than in the obese population, with some strains being able to significantly reduce gas production in the latter group. Moreover, in obese subjects all the Bifidobacterium strains tested and Lacticaseibacillus rhamnosus GG were able to modify the response of the intestinal cells, restoring values similar to those obtained with the microbiotas of normal-weight subjects. Our results underline the need for the screening and selection of probiotics in a target-population specific manner by using appropriate in vitro models before enrolling in clinical intervention trials.The present work was mainly financed by the project AGL2013-43770-R (receiving funds from the Ministry of Economy and Competitiveness—MINECO and the European Union FEDER funds), and partly by projects AGL2017-83653-R (AEI/FEDER, UE), and RTI2018-098288-B-I00 (MCIU/AEI/FEDER, UE) and by a contract with the company Takanashi Milk Products (Yokohama, Japan). AN was the recipient of a predoctoral contract granted by MINECO (reference BES-2014-068796). SA is currently financed by a postdoctoral Juan de la Cierva contract (Ministry of Science, Innovation and Universities, Spain, ref. IJCI-2017-32156) and NS is currently the recipient of a postdoctoral contract awarded by the Fundación para la Investigación y la Innovación Biosanitaria del Principado de Asturias (FINBA)

Early-life development of the bifidobacterial community in the infant gut

The establishment of the gut microbiota poses implications for short and long-term health. Bifidobacterium is an important taxon in early life, being one of the most abundant genera in the infant intestinal microbiota and carrying out key functions for maintaining host-homeostasis. Recent metagenomic studies have shown that different factors, such as gestational age, delivery mode, or feeding habits, affect the gut microbiota establishment at high phylogenetic levels. However, their impact on the specific bifidobacterial populations is not yet well understood. Here we studied the impact of these factors on the different Bifidobacterium species and subspecies at both the quantitative and qualitative levels. Fecal samples were taken from 85 neonates at 2, 10, 30, 90 days of life, and the relative proportions of the different bifidobacterial populations were assessed by 16S rRNA–23S rRNA internal transcribed spacer (ITS) region sequencing. Absolute levels of the main species were determined by q-PCR. Our results showed that the bifidobacterial population establishment is affected by gestational age, delivery mode, and infant feeding, as it is evidenced by qualitative and quantitative changes. These data underline the need for understanding the impact of perinatal factors on the gut microbiota also at low taxonomic levels, especially in the case of relevant microbial populations such as Bifidobacterium. The data obtained provide indications for the selection of the species best suited for the development of bifidobacteria-based products for different groups of neonates and will help to develop rational strategies for favoring a healthy early microbiota development when this process is challenged.This work was funded by the EU Joint Programming Initiative—A Healthy Diet for a Healthy Life (JPI HDHL, http://www.healthydietforhealthylife.eu/, Project EarlyMicroHealth) and the Project AGL2017-83653R funded by the Spanish “Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and FEDER”. S.A. was the recipient of a Juan de la Cierva Postdoctoral Contract (Ministry of Science, Innovation, and Universities, Ref. IJCI-2017-32156) and an EU Horizon2020 research grant under the Marie Sklodowska-Curie program (n. 749255)

Impact of intrapartum antimicrobial prophylaxis upon the intestinal microbiota and the prevalence of antibiotic resistance genes in vaginally delivered full-term neonates

Background: Disturbances in the early establishment of the intestinal microbiota may produce important implications for the infant's health and for the risk of disease later on. Different perinatal conditions may be affecting the development of the gut microbiota. Some of them, such as delivery mode or feeding habits, have been extensively assessed whereas others remain to be studied, being critical to identify their impact on the microbiota and, if any, to minimize it. Antibiotics are among the drugs most frequently used in early life, the use of intrapartum antimicrobial prophylaxis (IAP), present in over 30% of deliveries, being the most frequent source of exposure. However, our knowledge on the effects of IAP on the microbiota establishment is still limited. The aim of the present work was to evaluate the impact of IAP investigating a cohort of 40 full-term vaginally delivered infants born after an uncomplicated pregnancy, 18 of which were born from mothers receiving IAP. Results: Fecal samples were collected at 2, 10, 30, and 90 days of age. We analyzed the composition of the fecal microbiota during the first 3 months of life by 16S rRNA gene sequencing and quantified fecal short chain fatty acids by gas chromatography. The presence of genes for resistance to antibiotics was determined by PCR in the samples from 1-month-old infants. Our results showed an altered pattern of intestinal microbiota establishment in IAP infants during the first weeks of life, with lower relative proportions of Actinobacteria and Bacteroidetes and increased of Preoteobacteria and Firmicutes. A delay in the increase on the levels of acetate was observed in IAP infants. The analyses of specific antibiotic resistance genes showed a higher occurrence of some beta-lactamase coding genes in infants whose mothers received IAP. Conclusions: Our results indicate an effect of IAP on the establishing early microbiota during the first months of life, which represent a key moment for the development of the microbiota-induced host homeostasis. Understanding the impact of IAP in the gut microbiota development is essential for developing treatments to minimize it, favoring a proper gut microbiota development in IAP-exposed neonates

In Vitro Probiotic Modulation of the Intestinal Microbiota and 2′Fucosyllactose Consumption in Fecal Cultures from Infants at Two Months of Age

2-fucosyllactose (2 FL) is one of the most abundant oligosaccharides in human milk, with benefits on neonatal health. Previous results point to the inability of the fecal microbiota from some infants to ferment 2 FL. We evaluated a probiotic formulation, including the strains Lactobacillus helveticus Rosell®-52 (R0052), Bifidobacterium longum subsp. infantis Rosell®-33 (R0033), and Bifidobacterium bifidum Rosell®-71 (R0071), individually or in an 80:10:10 combination on the microbiota and 2 FL degradation. Independent batch fermentations were performed with feces from six full-term infant donors of two months of age (three breastfed and three formula-fed) with added probiotic formulation or the constituent strains in the presence of 2 FL. Microbiota composition was analyzed by 16S rRNA gene sequencing. Gas accumulation, pH decrease and 2 FL consumption, and levels of different metabolites were determined by chromatography. B. bifidum R0071 was the sole microorganism promoting a partial increase of 2 FL degradation during fermentation in fecal cultures of 2 FL slow-degrading donors. However, major changes in microbiota composition and metabolic activity occurred with L. helveticus R0052 or the probiotic formulation in cultures of slow degraders. Further studies are needed to decipher the role of the host intestinal microbiota in the efficacy of these strains.This research was funded by a contract of Lallemand Health Solutions Inc. with IPLA-CSIC (Spanish National Research Council contract number 201266) and by the Intramural Spanish National Research Council project PIE201970E061. S.A. was the recipient of a postdoctoral Juan de la Cierva contract (Ministry of Science and Innovation, Ref. IJCI-2017-32156), and N.S. has a postdoctoral contract awarded by the Biosanitary Research Foundation in Asturias (FINBA, Spain)

In vitro evaluation of different prebiotics on the modulation of gut microbiota composition and function in morbid obese and normal-weight subjects

The gut microbiota remains relatively stable during adulthood; however, certain intrinsic and environmental factors can lead to microbiota dysbiosis. Its restoration towards a healthy condition using best-suited prebiotics requires previous development of in vitro models for evaluating their functionality. Herein, we carried out fecal cultures with microbiota from healthy normal-weight and morbid obese adults. Cultures were supplemented with different inulin-type fructans (1-kestose, Actilight, P95, Synergy1 and Inulin) and a galactooligosaccharide. Their impact on the gut microbiota was assessed by monitoring gas production and evaluating changes in the microbiota composition (qPCR and 16S rRNA gene profiling) and metabolic activity (gas chromatography). Additionally, the effect on the bifidobacterial species was assessed (ITS-sequencing). Moreover, the functionality of the microbiota before and after prebiotic-modulation was determined in an in vitro model of interaction with an intestinal cell line. In general, 1-kestose was the compound showing the largest effects. The modulation with prebiotics led to significant increases in the Bacteroides group and Faecalibacterium in obese subjects, whereas in normal-weight individuals, substantial rises in Bifidobacterium and Faecalibacterium were appreciated. Notably, the results obtained showed differences in the responses among the tested compounds but also among the studied human populations, indicating the need for developing population-specific products

Modulación in vitro de la microbiota intestinal de niños lactantes y del consumo de 2’-fucosillactosa con cepas de bacterias lácticas probióticas

Resumen del trabajo presentado a la 15ª Reunión de la Red Española de Bacterias Lácticas: Bacterias Lácticas en Alimentación y Salud, celebrada en Valencia (España), los días 26 y 27 de mayo de 2022.El establecimiento de la microbiota neonatal en las primeras etapas de la vida está modulado por varios tipos de factores entre los que destaca especialmente el tipo de alimentación (leche maternaLM o leche de fórmula-LF). La 2´-fucosillactosa (2´FL) es uno de los oligosacáridos más abundantes de la leche materna (“human milk oligosaccharides”-HMO), con actividad prebiótica. Los niños que no son amamantados con LM, lo ingieren en LF suplementadas, en combinación con cepas probióticas en muchas ocasiones. Sin embargo, el efecto tanto de este HMO como de la combinación con cepas probióticas a nivel de la microbiota intestinal y sus metabolitos no ha sido estudiado en detalle. Por tanto, es de gran interés la evaluación del efecto de la 2’FL y cepas probióticas destinadas a la alimentación de lactantes sobre la microbiota intestinal infantil (MI)

La profilaxis antimicrobiana intraparto afecta el desarrollo de la microbiota intestinal en el recién-nacido

Trabajo presentado en el VIII Workshop de la Sociedad Española de Probióticos y Prebióticos (SEPyP), celebrado en Santiago de Compostela (España) el 23 y 24 de febrero de 2017Peer reviewe