Early development of the gut ecosystem is crucial for lifelong health. While infant gut bacterial communities have been studied extensively, the infant gut virome remains under-explored. To study the development of the infant gut virome over time and the factors that shape it, we longitudinally assess the composition of gut viruses and their bacterial hosts in 30 women during and after pregnancy and in their 32 infants during their first year of life. Using shotgun metagenomic sequencing applied to dsDNA extracted from Virus-Like Particles (VLPs) and bacteria, we generate 205 VLP metaviromes and 322 total metagenomes. With this data, we show that while the maternal gut virome composition remains stable during late pregnancy and after birth, the infant gut virome is dynamic in the first year of life. Notably, infant gut viromes contain a higher abundance of active temperate phages compared to maternal gut viromes, which decreases over the first year of life. Moreover, we show that the feeding mode and place of delivery influence the gut virome composition of infants. Lastly, we provide evidence of co-transmission of viral and bacterial strains from mothers to infants, demonstrating that infants acquire some of their virome from their mother’s gut.

During pregnancy, the semi-allogeneic nature of the foetus requires maternal immune adaption and acquisition of tolerance at the foetal-maternal interface. Macrophages with regulatory properties and regulatory T (Treg) cells are central in promoting foetal tolerance and are enriched in the decidua (the uterine endometrium during pregnancy). Although tissue-resident decidual stromal cells (DSC) have been implicated in regulatory functions, it is not known if they are able to induce the regulatory phenotype of macrophages and T-cells. In this study we report that maternally derived DSC are able to induce homeostatic M2 macrophages and Treg cells. CD14+ monocytes and CD4+ T-cells from healthy non-pregnant women were cultured in the presence or absence of conditioned medium (CM) from DSC isolated from 1st trimester and term placentas. DSC-CM alone was able to promote the survival of macrophages and to induce a regulatory CD14brightCD163+CD209+CD86dim phenotype, typical for decidual macrophages and similar to that induced by M-CSF. Interestingly, DSC-CM was also able to overrule the pro-inflammatory effects of GM-CSF by upregulating CD14, CD163 and CD209. Protein-profiling showed that M-CSF was secreted by DSC, and blocking of M-CSF partially reversed the M2 phenotype and reduced viability. DSC-CM also expanded CD25brightFoxp3+ Treg cells, an expansion that was abolished by a SMAD3-inhibitor, indicating the contribution of TGF-β signaling. In conclusion, our findings collectively emphasize the role of tissue-resident stromal cells in shaping the tolerogenic environment at the foetal-maternal interface.

Lactobacillus reuteri DSM 17938 supplementation reduces morbidities in very low birth weight infants (<1500 g), while the effect on extremely low birth weight infants (ELBW, <1000 g) is still questioned. In a randomised placebo-controlled trial (ClinicalTrials.gov ID NCT01603368), head growth, but not feeding tolerance or morbidities, improved in L. reuteri-supplemented preterm ELBW infants. Here, we investigate colonisation with the probiotic strain in preterm ELBW infants who received L. reuteri DSM 17938 or a placebo from birth to postmenstrual week (PMW) 36. Quantitative PCR was used on 582 faecal DNA samples collected from 132 ELBW infants at one, two, three, and four weeks, at PMW 36, and at two years of age. Human milk oligosaccharides were measured in 31 milk samples at two weeks postpartum. At least 86% of the ELBW infants in the L. reuteri group were colonised with the probiotic strain during the neonatal period, despite low gestational age, high antibiotic pressure, and independent of infant feeding mode. Higher concentrations of lacto-N-tetraose, sialyl-lacto-N-neotetraose c, and 6′-sialyllactose in mother’s milk weakly correlated with lower L. reuteri abundance. Within the L. reuteri group, higher L. reuteri abundance weakly correlated with a shorter time to reach full enteral feeding. Female sex and L. reuteri colonisation improved head growth from birth to four weeks of age. In conclusion, L. reuteri DSM 17938 supplementation leads to successful colonisation in ELBW infants.

Human milk microbiome studies are currently hindered by low milk bacterial/human cell ratios and often rely on 16S rRNA gene sequencing, which limits downstream analyses. Here, we aimed to find a method to study milk bacteria and assess bacterial sharing between maternal and infant microbiota. We tested four DNA isolation methods, two bacterial enrichment methods and three sequencing methods on mock communities, milk samples and negative controls. Of the four DNA isolation kits, the DNeasy PowerSoil Pro (PS) and MagMAX Total Nucleic Acid Isolation (MX) kits provided consistent 16S rRNA gene sequencing results with low contamination. Neither enrichment method substantially decreased the human metagenomic sequencing read-depth. Long-read 16S-ITS-23S rRNA gene sequencing biased the mock community composition but provided consistent results for milk samples, with little contamination. In contrast to 16S rRNA gene sequencing, 16S-ITS-23S rRNA gene sequencing of milk, infant oral, infant faecal and maternal faecal DNA from 14 mother-infant pairs provided sufficient resolution to detect significantly more frequent sharing of bacteria between related pairs compared to unrelated pairs. In conclusion, PS or MX kit-DNA isolation followed by 16S rRNA gene sequencing reliably characterises human milk microbiota, and 16S-ITS-23S rRNA gene sequencing enables studies of bacterial transmission in low-biomass samples.

Human milk is important for infant development, but few large studies have comprehensively investigated milk composition. Here, we characterized human milk oligosaccharides (HMOs) and milk microbiota, their shaping factors, and their links to infant gut microbiota in the longitudinal Dutch Lifelines NEXT cohort. We measured 24 HMOs in 1,542 milk samples from 524 mothers at 0.5-6 months postpartum, profiled microbiota in milk and maternal and infant feces, genotyped mothers, and recorded 174 environmental, maternal, and infant characteristics. HMO concentrations were associated with maternal genetic loci (FUT2, FUT3/FUT6, ABO, and ST3GAL6), lactation stage, and subclinical mastitis. The human milk microbiota varied during lactation and with different feeding practices. Both HMOs and milk microbiota remained stable across multiple pregnancies in the same individual. Some milk bacteria were present in infant feces, but the milk and infant fecal microbiota diverged as the infant aged. Furthermore, individual HMOs were associated with infant fecal microbiota characteristics.