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      Microbial Communities in Human Milk Relate to Measures of Maternal Weight

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          Abstract

          The process of breastfeeding exposes infants to bioactive substances including a diversity of bacteria from breast milk as well as maternal skin. Knowledge of the character of and variation in these microbial communities, as well as the factors that influence them, is limited. We aimed to identify profiles of breastfeeding-associated microbial communities and their association with maternal and infant factors. Bilateral milk samples were collected from women in the New Hampshire Birth Cohort Study at approximately 6 weeks postpartum without sterilization of the skin in order to capture the infant-relevant exposure. We sequenced the V4–V5 hypervariable region of the bacterial 16S rRNA gene in 155 human milk samples. We used unsupervised clustering (partitioning around medoids) to identify microbial profiles in milk samples, and multinomial logistic regression to test their relation with maternal and infant variables. Associations between alpha diversity and maternal and infant factors were tested with linear models. Four breastfeeding microbiome types (BMTs) were identified, which differed in alpha diversity and in Streptococcus, Staphylococcus, Acinetobacter, and Pseudomonas abundances. Higher maternal pre-pregnancy BMI was associated with increased odds of belonging to BMT1 [OR (95% CI) = 1.13 (1.02, 1.24)] or BMT3 [OR (95% CI) = 1.12 (1.01, 1.25)] compared to BMT2. Independently, increased gestational weight gain was related to reduced odds of membership in BMT1 [OR (95% CI) = 0.66 (0.44, 1.00) per 10 pounds]. Alpha diversity was positively associated with gestational weight gain and negatively associated with postpartum sample collection week. There were no statistically significant associations of breastfeeding microbiota with delivery mode. Our results indicate that the breastfeeding microbiome partitions into four profiles and that its composition and diversity is associated with measures of maternal weight.

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          Most cited references39

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          An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system.

          The mammalian gastrointestinal tract harbors a complex ecosystem consisting of countless bacteria in homeostasis with the host immune system. Shaped by evolution, this partnership has potential for symbiotic benefit. However, the identities of bacterial molecules mediating symbiosis remain undefined. Here we show that, during colonization of animals with the ubiquitous gut microorganism Bacteroides fragilis, a bacterial polysaccharide (PSA) directs the cellular and physical maturation of the developing immune system. Comparison with germ-free animals reveals that the immunomodulatory activities of PSA during B. fragilis colonization include correcting systemic T cell deficiencies and T(H)1/T(H)2 imbalances and directing lymphoid organogenesis. A PSA mutant of B. fragilis does not restore these immunologic functions. PSA presented by intestinal dendritic cells activates CD4+ T cells and elicits appropriate cytokine production. These findings provide a molecular basis for host-bacterial symbiosis and reveal the archetypal molecule of commensal bacteria that mediates development of the host immune system.
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            Exosomes with immune modulatory features are present in human breast milk.

            Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant's immune system. Exosomes are nanovesicles (30-100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant's immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-gamma production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3(+)CD4(+)CD25(+) T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.
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              Composition and Variation of the Human Milk Microbiota Are Influenced by Maternal and Early-Life Factors

              Breastmilk contains a complex community of bacteria that may help seed the infant gut microbiota. The composition and determinants of milk microbiota are poorly understood. Among 393 mother-infant dyads from the CHILD cohort, we found that milk microbiota at 3-4 months postpartum was dominated by inversely correlated Proteobacteria and Firmicutes, and exhibited discrete compositional patterns. Milk microbiota composition and diversity were associated with maternal factors (BMI, parity, and mode of delivery), breastfeeding practices, and other milk components in a sex-specific manner. Causal modeling identified mode of breastfeeding as a key determinant of milk microbiota composition. Specifically, providing pumped breastmilk was consistently associated with multiple microbiota parameters including enrichment of potential pathogens and depletion of bifidobacteria. Further, these data support the retrograde inoculation hypothesis, whereby the infant oral cavity impacts the milk microbiota. Collectively, these results identify features and determinants of human milk microbiota composition, with potential implications for infant health and development.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                20 December 2019
                2019
                : 10
                : 2886
                Affiliations
                [1] 1Department of Epidemiology, Geisel School of Medicine at Dartmouth , Hanover, NH, United States
                [2] 2Institute for Molecular Medicine Finland, University of Helsinki , Helsinki, Finland
                [3] 3Division of Neonatology, Department of Pediatrics, Children’s Hospital at Dartmouth , Lebanon, NH, United States
                [4] 4Department of Community and Family Medicine, Geisel School of Medicine at Dartmouth , Hanover, NH, United States
                [5] 5Josephine Bay Paul Center, Marine Biological Laboratory, Woods Hole , MA, United States
                [6] 6Department of Biomedical Data Science, Geisel School of Medicine at Dartmouth , Hanover, NH, United States
                [7] 7Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth , Hanover, NH, United States
                Author notes

                Edited by: Maria Carmen Collado, Institute of Agrochemistry and Food Technology (IATA), Spain

                Reviewed by: Carlos Gómez-Gallego, University of Eastern Finland, Finland; Hein Min Tun, The University of Hong Kong, Hong Kong

                *Correspondence: Brock C. Christensen, Brock.C.Christensen@ 123456dartmouth.edu

                This article was submitted to Microbial Symbioses, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2019.02886
                6933483
                31921063
                5fda3e31-de3d-47bd-8d11-5481b9e7a8bd
                Copyright © 2019 Lundgren, Madan, Karagas, Morrison, Hoen and Christensen.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 17 September 2019
                : 29 November 2019
                Page count
                Figures: 4, Tables: 4, Equations: 0, References: 80, Pages: 15, Words: 0
                Funding
                Funded by: National Institute of General Medical Sciences 10.13039/100000057
                Funded by: National Institute of Environmental Health Sciences 10.13039/100000066
                Funded by: National Institute of Environmental Health Sciences 10.13039/100000066
                Funded by: U.S. National Library of Medicine 10.13039/100000092
                Funded by: U.S. National Library of Medicine 10.13039/100000092
                Funded by: National Cancer Institute 10.13039/100000054
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                human milk,microbiome,bmi,gestational weight gain,16s rrna gene sequencing
                Microbiology & Virology
                human milk, microbiome, bmi, gestational weight gain, 16s rrna gene sequencing

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