Abstract
Human breast milk is considered the optimal nutrition for infants, providing essential nutrients and a broad range of bioactive compounds, as well as its own microbiota. However, the interaction among those components and the biological role of milk microorganisms is still uncovered. Thus, our aim was to identify the relationships between milk microbiota composition, bacterial load, macronutrients, and human cells during lactation. Bacterial load was estimated in milk samples from a total of 21 healthy mothers through lactation time by bacteria-specific qPCR targeted to the single-copy gene fusA. Milk microbiome composition and diversity was estimated by 16S-pyrosequencing and the structure of these bacteria in the fluid was studied by flow cytometry, qPCR, and microscopy. Fat, protein, lactose, and dry extract of milk as well as the number of somatic cells were also analyzed. We observed that milk bacterial communities were generally complex, and showed individual-specific profiles. Milk microbiota was dominated by Staphylococcus, Pseudomonas, Streptococcus, and Acinetobacter. Staphylococcus aureus was not detected in any of these samples from healthy mothers. There was high variability in composition and number of bacteria per milliliter among mothers and in some cases even within mothers at different time points. The median bacterial load was 106 bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods. Furthermore, milk bacteria were present in a free-living, “planktonic” state, but also in equal proportion associated to human immune cells. There was no correlation between bacterial load and the amount of immune cells in milk, strengthening the idea that milk bacteria are not sensed as an infection by the immune system.
Highlights
Human milk is a complex fluid adapted to satisfy the nutritional requirements of the infant, and protective compounds which help to create the right microenvironment for gut development and maturation of the immune system (Petherick, 2010; Walker, 2010)
After analyzing 56 milk samples by qPCR, results showed large individual differences in bacterial load over time between samples from the different mothers and in some cases even within individuals at different time points, indicating that human milk samples are highly variable in microbial content
Our estimates of bacterial load provided by molecular methods indicate that a lactating infant feeding 800 ml of breastmilk per day could ingest 107–108 bacterial cells daily, about 100 times higher than previous estimates based on laboratory culture methodologies
Summary
Human milk is a complex fluid adapted to satisfy the nutritional requirements of the infant, and protective compounds which help to create the right microenvironment for gut development and maturation of the immune system (Petherick, 2010; Walker, 2010). Bacterial Load in Breast Milk factors and other components may have health implications. They could be involved in the digestion of nutrients, facilitating the digestion process, the most likely role for these microorganisms is immune modulation (Fernández et al, 2013) Culture-dependent methods have long confirmed the presence of viable bacteria in aseptically collected samples (Heikkilä and Saris, 2003). Partial contamination from skin microbes occurs, the presence of strictly anaerobic species such as Bifidobacterium, Clostridium, and some Bacteroides spp., which are absent in the skin microbiota, supports that breast milk hosts a unique microbiome (Hunt et al, 2011; Cabrera-Rubio et al, 2012a; Jost et al, 2013). Accumulating evidence suggeststhat milk microbiota is influenced by perinatal factors such as mode of delivery, lactation time, gestational age, maternal health, or geographical locations (Khodayar-Pardo et al, 2014; Cabrera-Rubio et al, 2016)
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