Background: Microbial communities in human milk and those in feces from breastfed infants vary within and across populations. However, few researchers have conducted cross-cultural comparisons between populations, and little is known about whether certain “core” taxa occur normally within or between populations and whether variation in milk microbiome is related to variation in infant fecal microbiome. The purpose of this study was to describe microbiomes of milk produced by relatively healthy women living at diverse international sites and compare these to the fecal microbiomes of their relatively healthy infants. Methods: We analyzed milk ( n = 394) and infant feces ( n = 377) collected from mother/infant dyads living in 11 international sites (2 each in Ethiopia, The Gambia, and the US; 1 each in Ghana, Kenya, Peru, Spain, and Sweden). The V1-V3 region of the bacterial 16S rRNA gene was sequenced to characterize and compare microbial communities within and among cohorts. Results: Core genera in feces were Streptococcus, Escherichia/Shigella , and Veillonella , and in milk were Streptococcus and Staphylococcus , although substantial variability existed within and across cohorts. For instance, relative abundance of Lactobacillus was highest in feces from rural Ethiopia and The Gambia, and lowest in feces from Peru, Spain, Sweden, and the US; Rhizobium was relatively more abundant in milk produced by women in rural Ethiopia than all other cohorts. Bacterial diversity also varied among cohorts. For example, Shannon diversity was higher in feces from Kenya than Ghana and US-California, and higher in rural Ethiopian than Ghana, Peru, Spain, Sweden, and US-California. There were limited associations between individual genera in milk and feces, but community-level analyses suggest strong, positive associations between the complex communities in these sample types. Conclusions: Our data provide additional evidence of within- and among-population differences in milk and infant fecal bacterial community membership and diversity and support for a relationship between the bacterial communities in milk and those of the recipient infant's feces. Additional research is needed to understand environmental, behavioral, and genetic factors driving this variation and association, as well as its significance for acute and chronic maternal and infant health.

Human milk provides a very wide range of nutrients and bioactive components, including immune factors, human milk oligosaccharides, and a commensal microbiota. These factors are essential for interconnected processes including immunity programming and the development of a normal infant gastrointestinal microbiome. Newborn immune protection mostly relies on maternal immune factors provided through milk. However, studies dealing with an in-depth profiling of the different immune compounds present in human milk and with the assessment of their natural variation in healthy women from different populations are scarce. In this context, the objective of this work was the detection and quantification of a wide array of immune compounds, including innate immunity factors (IL1β, IL6, IL12, INFγ, TNFα), acquired immunity factors (IL2, IL4, IL10, IL13, IL17), chemokines (IL8, Groα, MCP1, MIP1β), growth factors [IL5, IL7, epidermal growth factor (EGF), granulocyte colony-stimulating factor, granulocyte–macrophage colony-stimulating factor, TGFβ2], and immunoglobulins (IgA, IgG, IgM), in milk produced by healthy women of different ethnicities living in different geographic, dietary, socioeconomic, and environmental settings. Among the analyzed factors, IgA, IgG, IgM, EGF, TGFβ2, IL7, IL8, Groα, and MIP1β were detected in all or most of the samples collected in each population and, therefore, this specific set of compounds might be considered as the “core” soluble immune factors in milk produced by healthy women worldwide. This approach may help define which immune factors are (or are not) common in milk produced by women living in various conditions, and to identify host, lifestyle, and environmental factors that affect the immunological composition of this complex biological fluid.Clinical Trial Registration: , identifier NCT02670278.

Studies conducted in the last years have demonstrated that human milk represents a continuous supply of beneficial bacteria to the infant gut, which contribute to the maturation of the digestive and immune functions in the developing infant. Nevertheless, the origin of bacterial populations in milk is not fully understood yet and they have been proposed to originate from maternal skin, infant's mouth, and (or) endogenously, from the maternal digestive tract through a mechanism involving immune cells. Understanding the composition, functions and assembly of the human milk microbiota has important implications not only for the infant gut microbiota establishment, but also for the mammary health since dysbiosis in the milk bacteria may lead to mastitis. Besides, host, microbial, medical and environmental factors may affect the composition of the human milk microbiome, with implications for the motherinfant health. Application of both culture-dependent and-independent techniques to assess the milk microbiome faces some practical limitations but, together, have allowed providing novel and complementary views on its origin, composition and functioning as summarized in this minireview. In the next future, the application of the ultimate advances in next-generation sequencing and omics approaches, including culturomics, will allow a detailed and comprehensive understanding of the composition and functions of these microbial communities, including their interactions with other milk components, expanding the opportunities to design novel microbiome-based modulation strategies for this ecosystem.

Human milk represents a source of bacteria for the initial establishment of the oral (and gut) microbiomes in the breastfed infant, however, the origin of bacteria in human milk remains largely unknown. While some evidence points towards a possible endogenous enteromammary route, other authors have suggested that bacteria in human milk are contaminants from the skin or the breastfed infant mouth. In this work 16S rRNA sequencing and bacterial culturing and isolation was performed to analyze the microbiota on maternal precolostrum samples, collected from pregnant women before delivery, and on oral samples collected from the corresponding infants. The structure of both ecosystems demonstrated a high proportion of taxa consistently shared among ecosystems, Streptococcus spp. and Staphylococcus spp. being the most abundant. Whole genome sequencing on those isolates that, belonging to the same species, were isolated from both the maternal and infant samples in the same mother-infant pair, evidenced that in 8 out of 10 pairs both isolates were >99.9% identical at nucleotide level. The presence of typical oral bacteria in precolostrum before contact with the newborn indicates that they are not a contamination from the infant, and suggests that at least some oral bacteria reach the infant’s mouth through breastfeeding.

Lactational mastitis is an excellent target to study possible interactions between HMOs, immune factors and milk microbiota due to the infectious and inflammatory nature of this condition. In this work, microbiological, immunological and HMO profiles of milk samples from women with (MW) or without (HW) mastitis were compared. Secretor status in women (based on HMO profile) was not associated to mastitis. DFLNH, LNFP II and LSTb concentrations in milk were higher in samples from HW than from MW among Secretor women. Milk from HW was characterized by a low bacterial load (dominated by Staphylococcus epidermidis and streptococci), high prevalence of IL10 and IL13, and low sialylated HMO concentration. In contrast, high levels of staphylococci, streptococci, IFNγ and IL12 characterized milk from MW. A comparison between subacute (SAM) and acute (AM) mastitis cases revealed differences related to the etiological agent (S. epidermidis in SAM; Staphylococcus aureus in AM), milk immunological profile (high content of IL10 and IL13 in SAM and IL2 in AM) and milk HMOs profile (high content of 3FL in SAM and of LNT, LNnT, and LSTc in AM). These results suggest that microbiological, immunological and HMOs profiles of milk are related to mammary health of women.

Recent work has demonstrated the existence of large inter-individual and inter-population variability in the microbiota of human milk from healthy women living across variable geographical and socio-cultural settings. However, no studies have evaluated the impact that variable sequencing approaches targeting different 16S rRNA variable regions may have on the human milk microbiota profiling results. This hampers our ability to make meaningful comparisons across studies. In this context, the main purpose of the present study was to re-process and re-sequence the microbiome in a large set of human milk samples (n = 412) collected from healthy women living at diverse international sites (Spain, Sweden, Peru, United States, Ethiopia, Gambia, Ghana and Kenya), by targeting a different 16S rRNA variable region and reaching a larger sequencing depth. Despite some differences between the results obtained from both sequencing approaches were notable (especially regarding alpha and beta diversities and Proteobacteria representation), results indicate that both sequencing approaches revealed a relatively consistent microbiota configurations in the studied cohorts. Our data expand upon the milk microbiota results we previously reported from the INSPIRE cohort and provide, for the first time across globally diverse populations, evidence of the impact that different DNA processing and sequencing approaches have on the microbiota profiles obtained for human milk samples. Overall, our results corroborate some similarities regarding the microbial communities previously reported for the INSPIRE cohort, but some differences were also detected. Understanding the impact of different sequencing approaches on human milk microbiota profiles is essential to enable meaningful comparisons across studies.Clinical Trial Registrationwww.clinicaltrials.gov, identifier NCT02670278.

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