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Numerous studies suggest that infants delivered by cesarean section are at a greater risk of non-communicable diseases than their vaginal counterparts. In particular, epidemiological studies have linked Cesarean delivery with increased rates of asthma, allergies, autoimmune disorders, and obesity. Mode of delivery has also been associated with differences in the infant microbiome. It has been suggested that these differences are attributable to the \"bacterial baptism\" of vaginal birth, which is bypassed in cesarean deliveries, and that the abnormal establishment of the early-life microbiome is the mediator of later-life adverse outcomes observed in cesarean delivered infants. This has led to the increasingly popular practice of \"vaginal seeding\": the iatrogenic transfer of vaginal microbiota to the neonate to promote establishment of a \"normal\" infant microbiome. In this review, we summarize and critically appraise the current evidence for a causal association between Cesarean delivery and neonatal dysbiosis. We suggest that, while Cesarean delivery is certainly associated with alterations in the infant microbiome, the lack of exposure to vaginal microbiota is unlikely to be a major contributing factor. Instead, it is likely that indication for Cesarean delivery, intrapartum antibiotic administration, absence of labor, differences in breastfeeding behaviors, maternal obesity, and gestational age are major drivers of the Cesarean delivery microbial phenotype. We, therefore, call into question the rationale for \"vaginal seeding\" and support calls for the halting of this practice until robust evidence of need, efficacy, and safety is available.

Although there has been limited application in the field to date, human milk omics research continues to gain traction. Human milk lipidomics and metabolomics research is particularly important, given the significance of milk lipids and metabolites for infant health. For researchers conducting compositional milk analyses, it is important to consider the origins of these compounds. The current review aims to provide a summary of the existing evidence on the sources of human milk lipids and small metabolites. Here, we describe five major sources of milk lipids and metabolites: de novo synthesis from mammary cells, production by the milk microbiota, dietary consumption, release from non-mammary tissue, and production by the gut microbiota. We synthesize the literature to provide evidence and understanding of these pathways in the context of mammary gland biology. We recommend future research focus areas to elucidate milk lipid and small metabolite synthesis and transport pathways. Better understanding of the origins of human milk lipids and metabolites is important to improve translation of milk omics research, particularly regarding the modulation of these important milk components to improve infant health outcomes.

Human milk is composed of complex microbial and non-microbial components that shape the infant gut microbiome. Although several maternal and infant factors have been associated with human milk microbiota, no study has investigated this in an Australian population. Therefore, we aimed to investigate associations between human milk bacterial composition of Australian women and maternal factors (body mass index (BMI), mode of delivery, breast pump use, allergy, parity) and infant factors (sex, mode of feeding, pacifier use, and introduction of solids). Full-length 16S rRNA gene sequencing was used to characterise milk bacterial DNA profiles. Milk from mothers with a normal BMI had a higher relative abundance of Streptococcus australis than that of underweight mothers, while milk from overweight mothers had a higher relative abundance of Streptococcus salivarius compared with underweight and obese mothers. Mothers who delivered vaginally had a higher relative abundance of Streptococcus mitis in their milk compared to those who delivered via emergency caesarean section. Milk of mothers who used a breast pump had a higher relative abundance of Staphylococcus epidermidis and Streptococcus parasanguinis . Milk of mothers whose infants used a pacifier had a higher relative abundance of S . australis and Streptococcus gwangjuense . Maternal BMI, mode of delivery, breast pump use, and infant pacifier use are associated with the bacterial composition of human milk in an Australian cohort. The data from this pilot study suggests that both mother and infant can contribute to the human milk microbiome.

Human milk is a complex and variable ecosystem fundamental to the development of newborns. This study aimed to investigate relationships between human milk oligosaccharides (HMO) and human milk bacterial profiles and infant body composition. Human milk samples (n = 60) were collected at two months postpartum. Infant and maternal body composition was measured with bioimpedance spectroscopy. Human milk bacterial profiles were assessed using full-length 16S rRNA gene sequencing and 19 HMOs were quantitated using high-performance liquid chromatography. Relative abundance of human milk bacterial taxa were significantly associated with concentrations of several fucosylated and sialylated HMOs. Individual human milk bacteria and HMO intakes and concentrations were also significantly associated with infant anthropometry, fat-free mass, and adiposity. Furthermore, when data were stratified based on maternal secretor status, some of these relationships differed significantly among infants born to secretor vs non-secretor mothers. In conclusion, in this pilot study the human milk bacterial profile and HMO intakes and concentrations were significantly associated with infant body composition, with associations modified by secretor status. Future research designed to increase the understanding of the mechanisms by which HMO and human milk bacteria modulate infant body composition should include intakes in addition to concentrations.

Analysis of the human milk microbiome is complicated by the presence of a variable quantity of fat. The fat fraction of human milk is typically discarded prior to analysis. It is assumed that all cells are pelleted out of human milk by high speed centrifugation; however, studies of bovine milk have reported that bacteria may remain trapped within the fat fraction. Here, the bacterial DNA profiles of the fat fraction and cell pellet of human milk (n = 10) were analysed. Human and bacterial DNA was consistently recovered from the fat fraction of human milk (average of 12.4% and 32.7%, respectively). Two low-abundance Staphylococcus species (< 0.5% relative abundance) was significantly more abundant in the cell pellet compared to the fat fraction ( P  < 0.04), and three low-abundance species (< 5% relative abundance) were recovered from one fraction only. However, inclusion of fat reduced the efficiency of DNA extraction by 39%. Culture-based methods were used to quantify the distribution of an exogenously added strain of Staphylococcus aureus in human milk fractions. S. aureus was consistently recovered from the fat fraction (average 28.9%). Bacterial DNA profiles generated from skim milk or cell pellets are not representative of the entire human milk microbiome. These data have critical implications for the design of future work in this field.

Short chain fatty acids (SFCAs) are microbial metabolites produced in the gut upon fermentation of dietary fiber. These metabolites interact with the host immune system and can elicit epigenetic effects. There is evidence to suggest that SCFAs may play a role in the developmental programming of immune disorders and obesity, though evidence in humans remains sparse. Here we have quantified human milk (HM) SCFA levels in an international cohort of atopic and non-atopic mothers ( = 109). Our results demonstrate that human milk contains detectable levels of the SCFAs acetate, butyrate, and formate. Samples from atopic mothers had significantly lower concentrations of acetate and butyrate than those of non-atopic mothers. HM SCFA levels in atopic and non-atopic women also varied based on maternal country of residence (Australia, Japan, Norway, South Africa, USA). Reduced exposure to HM SCFA in early life may program atopy or overweight risk in breastfed infants.

The human milk microbiome is dominated by typical oral and skin bacteria, suggesting that bacterial communities from the infant mouth and maternal skin contribute to the development of the human milk microbiome. It is postulated that breastfeeding characteristics, such as breastfeeding frequency and duration, could lead to different levels of exposure to oral and skin bacteria, and subsequently, altered bacterial profiles in human milk. To investigate the associations between breastfeeding characteristics and the human milk microbiome, this study analysed milk samples collected from 56 participants at 3 months postpartum by full-length 16 S rRNA gene sequencing. Breastfeeding characteristics (breastfeeding frequency, total 24 h breastfeeding duration, and 24 h milk removal volume) were recorded by the participants using the 24 h test weighing protocol. The milk microbiome was largely robust to breastfeeding practices, with only one association detected between breastfeeding characteristics and the milk microbiome. Duration of breastfeeding from the sampled breast (per 24 h) was weakly positively associated with the relative abundance of Streptococcus salivarius ( P  = 0.035). No associations with alpha nor beta diversity were detected. In conclusion, variations in breastfeeding characteristics do not have a major impact on the composition or diversity of the human milk microbiome.

Acquisition and development of the oral microbiome are dynamic processes that occur over early life. This study aimed to characterize the temporal development of the oral microbiome of predominantly breastfed infants during the first two years of life. Infant oral samples (n=667) were collected at ten time points from the first week to year two of life from 84 infants. Bacterial DNA profiles were analyzed using full-length 16S rRNA gene sequencing. The oral microbiome was dominated by , , and . Bacterial richness decreased from 1 to 2 months (P = 0.043) and increased from 12 to 24 months (P = 0.038). Shannon diversity increased from 1 week to 1 month and again from 6 to 9 months and 9 to 12 months (all P ≤ 0.04). The composition of the infant oral microbiome was associated with multiple factors, including early pacifier use, intrapartum antibiotic prophylaxis, maternal allergy, pre-pregnancy body mass index, siblings, delivery mode, maternal age, pets at home, and birth season (all P < 0.01). Introduction of solid foods was a significant milestone in oral microbiome development, triggering an increase in bacterial diversity (richness P = 0.0004; Shannon diversity P = 0.0007), a shift in the abundance of seven species, and a change in beta diversity (P = 0.001). These findings underscore the importance of multiple factors, particularly the introduction of solid foods, in shaping the oral microbiome of breastfed infants during early life.

The human microbiome plays a number of critical roles in host physiology. Evidence from longitudinal cohort studies and animal models strongly supports the theory that maldevelopment of the microbiome in early life can programme later-life disease. The early-life microbiome develops in a clear stepwise manner over the first 3 years of life. During this highly dynamic time, insults such as antibiotic use and formula feeding can adversely affect the composition and temporal development of the microbiome. Such experiences predispose infants for the development of chronic health conditions later in life. This review highlights key factors that disrupt the early-life microbiome and highlights major non-communicable diseases which are underpinned by early-life dysbiosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.