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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., Ministerio de Economía y Competitividad (AGL2013-4190-P), Ministerio de Ciencia e Innovación - RTI2018-095021-J-I00, National Science Foundation (award 36 #1344288), Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria), Fac. de Veterinaria, TRUE, pub

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., Ministerio de Economía y Competitividad (AGL2013-4190-P), Ministerio de Ciencia e Innovación - RTI2018-095021-J-I00, National Science Foundation (award 36 #1344288), Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria), Fac. de Veterinaria, TRUE, pub

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 Registration: www.clinicaltrials.gov, identifier NCT02670278.

[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 inf

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 mother-infant 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.

[Introduction] The infant oral microbiome might impact the infant gut microbiome establishment providing the first commensals that will reach the infant gut (1). Besides, human milk represents a source of bacteria for the initial establishment of the oral (and gut) microbiomes in the breastfed infant (2,3) although little is known on the origin of the human milk microbiome and its impact on the infant oral microbiome. [Objectives] To investigate the maternal precolostrum and infant oral microbiomes to identify bacterial species (strains) shared in the maternal-infant dyad. [Methods] 16S rRNA sequencing microbiome profiling was performed on maternal precolostrum and infant saliva collected from 19 mother-infant pairs. Bacteria were cultured, isolated and identified by MALDI-TOF. RAPD-PCr and full genome sequencing was performed on those isolates that, belonging to the same species, were isolated from both the maternal and infant samples in the same mother-infant pair. [Results] Maternal precolostrum and infant salivary microbiomes significantly differed, although certain taxa were consistently shared, Streptococcus and Staphylococcus spp. being the most abundant. In 13 pairs, at least one isolate from the same species was recovered from both sample types. From those, isolates obtained from precolostrum and infant saliva in 8 of the pairs could not be distinguised by RAPD-PCR profiling. 8 out of 10 fully sequenced isolate pairs were > 99.9% identical at nucleotide level, thus suggesting the sharing of bacteria at strain level between precolostrum and infant oral microbiomes. [Conclusions] The presence of oral bacteria in precolostrum, before contact with the newborn, indicates that they are not a contamination from the infant gut and suggests that at least some oral bacteria colonize the infant's oral cavity through transmission through breastmilk.

[Background] The infant oral microbiome exerts an important role in the establishment of the infant gut microbiome since it contains the first commensals that will reach and stably colonize the infant gut, but it also influences the dental health of the adult. In this context, human milk represents a source of bacteria for the initial establishment of the oral (and gut) microbiomes in the breastfed infant although little is known on the specific mechanisms that condition the early establishment and development of the infant oral microbiomes. [Objectives] To investigate the maternal colostrum and infant oral microbiomes to identify bacterial species (strains) shared in the maternal-infant dyad. [Methods] Maternal pre-colostrum and infant saliva were collected from 19 mother-infant pairs and their microbiomes were analyzed through 16S rRNA metagenomics sequencing. Bacteria were also cultured, isolated and identified by MALDI-TOF. RAPD-PCR was performed on those isolates that, belonging to the same species, were shared in the maternal and infant samples of the same pair. [Conclusions] Maternal pre-colostrum and infant salivary microbiomes were significantly different from each other, although certain groups were consistently shared between both sample types. Streptococcus and Staphylococcus spp were the most abundant groups in all samples. In 13 pairs, at least one isolate from the same species was recovered from the colostrum and the salivary sample. From those, the isolates obtained from colostrum and infant saliva in 8 of the pairs could not be distinguished by RAPD-PCR profiling, thus suggesting the sharing of bacteria at strain level between milk and infant oral microbiomes.

[EN] 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