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Human Milk Bacteria: Seeding the Infant Gut?

2020; Cell Press; Volume: 28; Issue: 2 Linguagem: Inglês

10.1016/j.chom.2020.07.017

1934-6069

Moira K. Differding, Noel T. Mueller,

Pediatric health and respiratory diseases

Human milk harbors its own microbiota, but whether the microbes seed the infant gut and are modified by breastfeeding practices is unresolved. In this issue, Fehr et al., 2020Fehr K. Moossavi S. Sbihi H. Boutin R.C.T. Bode L. Robertson B. Yonemitsu C. Field C.J. Becker A.B. Mandhane P.J. et al.Breastmilk feeding practices are associated with the co-occurrence of bacteria in mothers' milk and the infant gut: the CHILD Cohort Study.Cell Host Microbe. 2020; 28 (this issue): 285-297Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar sequence breastmilk and infant stool samples from mother-infant dyads to investigate the co-occurrence of milk-gut bacteria and the impact of breastfeeding practices. Human milk harbors its own microbiota, but whether the microbes seed the infant gut and are modified by breastfeeding practices is unresolved. In this issue, Fehr et al., 2020Fehr K. Moossavi S. Sbihi H. Boutin R.C.T. Bode L. Robertson B. Yonemitsu C. Field C.J. Becker A.B. Mandhane P.J. et al.Breastmilk feeding practices are associated with the co-occurrence of bacteria in mothers' milk and the infant gut: the CHILD Cohort Study.Cell Host Microbe. 2020; 28 (this issue): 285-297Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar sequence breastmilk and infant stool samples from mother-infant dyads to investigate the co-occurrence of milk-gut bacteria and the impact of breastfeeding practices. Human milk is the optimal nourishment for the newborn because of its essential nutrients and bioactive components, including over 200 structures of oligosaccharides that are only broken down by the infant's gut bacteria (Thomson et al., 2018Thomson P. Medina D.A. Garrido D. Human milk oligosaccharides and infant gut bifidobacteria: Molecular strategies for their utilization.Food Microbiol. 2018; 75: 37-46Crossref PubMed Scopus (112) Google Scholar). As early as the 1920s it was observed that human milk may also contain its own bacteria (Dudgeon and Jewesbury, 1924Dudgeon L.S. Jewesbury R.C. The Bacteriology of Human Milk.J. Hyg. (Lond.). 1924; 23: 64-76Crossref PubMed Scopus (7) Google Scholar), but only since the 21st century advent of next-generation sequencing have we come to appreciate potentially symbiotic milk microbiota. Yet important questions still remain about human milk microbiota, including: what are the core constituents, do they seed the infant gut microbiota, and is this transmission impacted by exclusivity, duration, and mode of milk feeding? In this issue of Cell Host & Microbe, Fehr and colleagues aimed to address these questions using the longitudinal CHILD Cohort Study—a birth cohort of singleton mother-infant dyads enrolled at four Canadian cities and one rural site. The team performed 16S rRNA sequencing focusing on bacterial amplicon sequence variants (ASVs; i.e., unique bacterial taxa) from 877 breastmilk samples collected at 3 months and 702 and 742 infant stool samples at 3 months and 1 year, respectively. The primary exposures of interest in this study included breastmilk exclusivity, duration, and feeding mode, assessed by questionnaires. Microbiome measurements included diversity metrics and composition. Furthermore, the paired study, in which 3-month (n = 266) and 1-year (n = 288) infant stool samples were paired to 3-month maternal breastmilk, allowed investigators to examine the co-occurrence, or "sharing," of bacterial ASVs between human milk and the infant gut. Moreover, the rich covariate data allowed for model adjustment by maternal pre-pregnancy body mass index, gestational weight gain, delivery mode, antibiotics, parity, and human milk oligosaccharide concentration. Infants exclusively breastfed for at least 3 months had low gut microbiota diversity at 3 months that sharply increased by 12 months, whereas non-exclusively breastfed infants already had higher diversity at 3 months. Breastfeeding exclusivity was also associated with overall gut microbiota composition at 3 months, including higher abundance of Bifidobacterium and Veillonella dispar. Although high microbiota diversity is generally considered beneficial for adults, diversity in breastfed infants is generally very low, with a preponderance of Bifidobacterium spp. that may help prevent colonization by opportunistic pathogens (Thomson et al., 2018Thomson P. Medina D.A. Garrido D. Human milk oligosaccharides and infant gut bifidobacteria: Molecular strategies for their utilization.Food Microbiol. 2018; 75: 37-46Crossref PubMed Scopus (112) Google Scholar). High gut microbiota diversity in early infancy (<3 months) has also been linked with higher childhood body mass index (Stanislawski et al., 2018Stanislawski M.A. Dabelea D. Wagner B.D. Iszatt N. Dahl C. Sontag M.K. Knight R. Lozupone C.A. Eggesbø M. Gut microbiota in the first 2 years of life and the association with body mass index at age 12 in a Norwegian birth cohort.MBio. 2018; 9 (e01751-18)Crossref PubMed Scopus (62) Google Scholar), raising the possibility that prematurely high diversity may be on the pathway to later-life development of metabolic diseases. The authors then explored whether the infant gut was seeded by microbes from breastmilk collected at 3 months of age. The overall bacterial composition of breastmilk and infant gut samples differed dramatically from each other, and there was not a universal core milk microbiome across mothers. Yet certain bacteria did co-occur, including Streptococcus (shared in 61% of dyads), Veillonella (36%), Staphylococcus (31%), and Bifidobacterium (27%), and related dyads were more likely to share bacterial ASVs than random milk-gut pairs. Interestingly, feeding mode (direct versus pumped breastmilk), but not breastfeeding exclusivity, impacted milk-gut bacteria co-occurrence, with direct feeding having overall more shared ASVs, including greater abundance of V. dispar, Streptococcus spp., and Bifidobacterium spp. These findings support the hypothesis that breastmilk bacteria is vertically transmitted to the infant gut but that this process may be interrupted by breastmilk pumping and/or storage. Next, to determine whether specific breastmilk bacteria impacted the overall infant gut microbiota composition, the authors conducted analyses examining 13 commonly shared bacteria (i.e., ASVs shared between at least 15 mother-infant dyads). Collectively, these 13 bacteria—particularly Streptococcus and Bifidobacterium—were associated with overall infant gut microbiota composition at 3 months of age, and sharing of these bacteria explained a similar amount of variance as breastfeeding exclusivity, delivery mode, intrapartum antibiotics, and number of siblings. A notable finding was that, although breastmilk feeding mode modified the concordance of these 13 shared milk-gut bacteria, it was not associated with the overall infant gut microbiota composition. Taken together, these findings suggest that breastmilk pumping and storage does not result in a major perturbation to infant gut microbiota development, as do C-section, antibiotics, and non-breastmilk feeding. It should also be noted that the associations of breastmilk exclusivity, duration, and feeding mode with microbiota features may be influenced by residual confounding of socioeconomic or lifestyle factors (e.g., maternal diet) associated with both breastfeeding practices and the microbiome. Overall, this work raises three key questions that merit further discussion: First, how confident are we that human milk bacteria are not contaminants? The non-aseptic breastmilk collection methods used by Fehr et al., 2020Fehr K. Moossavi S. Sbihi H. Boutin R.C.T. Bode L. Robertson B. Yonemitsu C. Field C.J. Becker A.B. Mandhane P.J. et al.Breastmilk feeding practices are associated with the co-occurrence of bacteria in mothers' milk and the infant gut: the CHILD Cohort Study.Cell Host Microbe. 2020; 28 (this issue): 285-297Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar may capture more skin bacteria (e.g., Staphylococcus) than aseptic collection techniques (Sakwinska et al., 2016Sakwinska O. Moine D. Delley M. Combremont S. Rezzonico E. Descombes P. Vinyes-Pares G. Zhang Y. Wang P. Thakkar S.K. Microbiota in breast milk of chinese lactating mothers.PLoS One. 2016; 11: e0160856Crossref PubMed Scopus (69) Google Scholar), which could be viewed as either being incorrectly attributed to human milk or as a more accurate reflection of the actual microbiota transfer from mother to infant at the time of direct breastfeeding (or pumped feeding). Sequencing data from low-biomass samples such as human milk are also vulnerable to laboratory contamination and batch effects, which could introduce bias if not adequately addressed in downstream sequence processing methods. To detect and reduce sequencing artifacts, Fehr et al., 2020Fehr K. Moossavi S. Sbihi H. Boutin R.C.T. Bode L. Robertson B. Yonemitsu C. Field C.J. Becker A.B. Mandhane P.J. et al.Breastmilk feeding practices are associated with the co-occurrence of bacteria in mothers' milk and the infant gut: the CHILD Cohort Study.Cell Host Microbe. 2020; 28 (this issue): 285-297Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar employed rigorous statistical methods that incorporated data from negative controls (extraction and sequencing), positive controls (mock communities with known microbial composition), and duplicated samples. These methods not only increased the internal validity, but also the external comparability of their results. Indeed, the obligate anaerobic Bifidobacterium and V. dispar and the facultative anaerobic Streptococcus they observed in human milk have also been isolated from aseptically collected milk via culturing and PCR-based techniques (Jost et al., 2014Jost T. Lacroix C. Braegger C.P. Rochat F. Chassard C. Vertical mother-neonate transfer of maternal gut bacteria via breastfeeding.Environ. Microbiol. 2014; 16: 2891-2904Crossref PubMed Scopus (345) Google Scholar; Sakwinska et al., 2016Sakwinska O. Moine D. Delley M. Combremont S. Rezzonico E. Descombes P. Vinyes-Pares G. Zhang Y. Wang P. Thakkar S.K. Microbiota in breast milk of chinese lactating mothers.PLoS One. 2016; 11: e0160856Crossref PubMed Scopus (69) Google Scholar). Future studies using 16S rRNA data from low-biomass specimens would do well to follow the decontamination approach used by Fehr and colleagues. Second, where do these bacteria come from? It is intriguing to think that bacteria reach human milk via translocation from the maternal gut, and that this bacterial entero-mammary pathway then seeds the infant gut (Jost et al., 2014Jost T. Lacroix C. Braegger C.P. Rochat F. Chassard C. Vertical mother-neonate transfer of maternal gut bacteria via breastfeeding.Environ. Microbiol. 2014; 16: 2891-2904Crossref PubMed Scopus (345) Google Scholar). This mother-to-newborn transmission would provide opportunities to manipulate the milk-gut bacteria connection through maternal interventions. However, as mentioned by the authors, it is also possible that infant oral bacteria disperse to both maternal breastmilk via retrograde transfer during breastfeeding and to their own gut (Figure 1). Indeed, V. dispar and Streptococcus, higher among women who breastfed directly from the breast, are core constituents of the infant oral microbiota (Williams et al., 2019Williams J.E. Carrothers J.M. Lackey K.A. Beatty N.F. Brooker S.L. Peterson H.K. Steinkamp K.M. York M.A. Shafii B. Price W.J. et al.Strong multivariate relations exist among milk, oral, and fecal microbiomes in mother-infant dyads during the first six months postpartum.J. Nutr. 2019; 149: 902-914Crossref PubMed Scopus (64) Google Scholar). Thus, it is possible that infant oral bacteria may explain at least part of the milk-gut bacteria concordance in mother-infant dyads. Unfortunately, Fehr et al., 2020Fehr K. Moossavi S. Sbihi H. Boutin R.C.T. Bode L. Robertson B. Yonemitsu C. Field C.J. Becker A.B. Mandhane P.J. et al.Breastmilk feeding practices are associated with the co-occurrence of bacteria in mothers' milk and the infant gut: the CHILD Cohort Study.Cell Host Microbe. 2020; 28 (this issue): 285-297Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar could not test the retrograde inoculation hypothesis because they did not collect infant oral samples. As such, investigation is still needed to disentangle infant oral bacteria from human milk bacteria. Third, what functions might these bacteria have? Several of the bacteria "shared" between breastmilk and the infant gut are involved in either lactate production (Bifidobacterium, Streptococcus, Staphylococcus) or utilization (Veillonella). Co-presence of lactate producers and utilizers in the infant gut may facilitate a trophic chain that avoids lactate build up and its detrimental sequelae (Pham et al., 2017Pham V.T. Lacroix C. Braegger C.P. Chassard C. Lactate-utilizing community is associated with gut microbiota dysbiosis in colicky infants.Sci. Rep. 2017; 7: 11176Crossref PubMed Scopus (40) Google Scholar). Bifidobacterium spp. also produce the short-chain fatty acid acetate through saccharolytic fermentation of oligosaccharides (Thomson et al., 2018Thomson P. Medina D.A. Garrido D. Human milk oligosaccharides and infant gut bifidobacteria: Molecular strategies for their utilization.Food Microbiol. 2018; 75: 37-46Crossref PubMed Scopus (112) Google Scholar). Together, acetate and lactate help maintain a low intestinal pH, which not only protects against enteropathogenic infections but also facilitates the transport of acetate into the gut epithelium for use by the host (Thomson et al., 2018Thomson P. Medina D.A. Garrido D. Human milk oligosaccharides and infant gut bifidobacteria: Molecular strategies for their utilization.Food Microbiol. 2018; 75: 37-46Crossref PubMed Scopus (112) Google Scholar). A fascinating line of inquiry that warrants more research is how milk bacteria alters milk composition before reaching the infant gut (Moossavi et al., 2019aMoossavi S. Atakora F. Miliku K. Sepehri S. Robertson B. Duan Q.L. Becker A.B. Mandhane P.J. Turvey S.E. Moraes T.J. et al.Integrated analysis of human milk microbiota with oligosaccharides and fatty acids in the CHILD cohort.Front. Nutr. 2019; 6: 58Crossref PubMed Scopus (52) Google Scholar). Unfortunately, the 16S rRNA sequencing employed by Fehr et al., 2020Fehr K. Moossavi S. Sbihi H. Boutin R.C.T. Bode L. Robertson B. Yonemitsu C. Field C.J. Becker A.B. Mandhane P.J. et al.Breastmilk feeding practices are associated with the co-occurrence of bacteria in mothers' milk and the infant gut: the CHILD Cohort Study.Cell Host Microbe. 2020; 28 (this issue): 285-297Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar is not sufficient to reliably characterize microbial taxa at the species/strain level or to identify their function. Future research using shotgun sequencing, metabolomics, and metatranscriptomics is needed to truly determine whether the differentially shared bacteria are functionally relevant to human milk and the infant gut. In addition, to resolve whether breastmilk bacteria seeds the infant gut and impacts health, one might consider randomizing infants who cannot breastfeed to receive either pasteurized or non-pasteurized human milk. In summary, Fehr and colleagues provided invaluable insights into the extent to which breastmilk bacteria may seed the infant gut and how breastfeeding exclusivity, duration, and feeding mode may alter this process. Building on their prior work showing that breastmilk pumping is associated with milk bacteria composition (Moossavi et al., 2019bMoossavi S. Sepehri S. Robertson B. Bode L. Goruk S. Field C.J. Lix L.M. de Souza R.J. Becker A.B. Mandhane P.J. et al.Composition and variation of the human milk microbiota are influenced by maternal and early-life factors.Cell Host Microbe. 2019; 25: 324-335.e4Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), here they found that pumping modifies the co-occurrence of certain milk bacteria in the infant gut but is not associated with overall infant gut microbiota composition. This suggests that pumping does not negate the benefits of breastmilk. Nonetheless, milk-gut bacteria sharing does appear to occur and to explain a similar percentage of variation in overall infant gut microbiota composition as well-established determinants. Potentially vertically transmissible milk bacteria include Bifidobacterium spp., V. dispar, and Streptococcus spp., but the origin and function of these bacteria remains an open question, as does the role of non-bacterial (e.g., fungi) microbes in this process. In particular, research is needed to understand the contribution of the infant oral bacteria to the observed milk-gut bacteria co-occurrence. Finally, whether the associations herein are ultimately linked to childhood health is yet to be determined, but thanks to Fehr et al., 2020Fehr K. Moossavi S. Sbihi H. Boutin R.C.T. Bode L. Robertson B. Yonemitsu C. Field C.J. Becker A.B. Mandhane P.J. et al.Breastmilk feeding practices are associated with the co-occurrence of bacteria in mothers' milk and the infant gut: the CHILD Cohort Study.Cell Host Microbe. 2020; 28 (this issue): 285-297Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, investigators are now in a better position to address this consequential question.