Relationship between Milk Microbiota, Bacterial Load, Macronutrients, and Human Cells during Lactation

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 10(6) 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.

Keywords: 16S rRNA; bacterial load; breast milk; flow cytometry; human microbiome; lactation; qPCR; somatic cells.

Figures

Figure 1

Bacterial load over lactational stages. (A) Data show the median with ranges (maximum and minimum values for each group) of bacterial load at the three time points. C, colostrum samples (n = 19); T, transition milk samples (n = 20); M, mature milk samples (n = 17). (B) Lines show individual bacterial load for each mother at the three time points (n = 17).

Figure 2

Bacterial taxonomic composition of human breast milk. The bars show the proportion of bacterial genera as inferred by PCR amplification and pyrosequencing of the 16S rRNA gene in healthy mothers (n = 12). Each number in the X axis represent a donor, with first column representing the colostrum sample, second the transition milk and third the mature milk samples. In some cases, data from the three breastfeeding stages could not be obtained due to sample unavailability or sequencing failure. Bacterial genera that were under 1% were grouped in the “Others” category.

Figure 3

Bacterial diversity of human breast milk. (A) Shows the proportion of each bacterial genera in the three lactational-stages, as inferred by PCR amplification and pyrosequencing of the 16S rRNA gene. (B) Shows the rarefaction curves of the three groups, relating the sequencing effort with an estimate of the number of bacterial species, as inferred by the number of OTUs. An OTU is a cluster of 16SrRNA sequences that were >95% identical, a conservative estimate for the boundary between species, established at 97% for full-length 16S rRNA sequences. The inlet Venn's diagram shows the number of bacterial genera shared between and unique to the three sample types, excluding bacterial genera present at <1% proportion. Seven genera are shared at the three breastfeeding stages: Finegoldia, Streptococcus, Corynebacterium, Staphylococcus, Acinetobacter, Peptoniphilus, and Pseudomonas. C, colostrum samples (n = 11); T, transition samples (n = 11); M, mature samples (n = 8).

Figure 4

Relationship between number of somatic cells and bacterial load in milk samples. The graph shows a comparison between the number of bacterial cells per milliliter (estimated by qPCR) and the number of somatic cells per milliliter, estimated with an Integrated Milk Testing Fossomatic 5000 (FOSS) cytometer. (n = 38, R2 = 0.0066). C, colostrum samples (n = 12); T, transition samples (n = 15); M, mature samples (n = 11).

Figure 5

Relationships between bacterial composition and nutritional or cellular content of human breastmilk. The figure shows a heatmap where samples have been clustered according to its compositional profile. Bacterial genera appear color-coded according to their under- (red) or over-representation (blue) in the samples, and its proportion is correlated to the amount of protein content (indicated as “prot” in the figure), fat content (Fat), lactose content (Lact), and non-fatty solid content (NFS), as well as the density of bacterial and human somatic cells. n = 30.

Figure 6

Richness and diversity of milk samples. (A) Shows the richness in the samples as inferred by computation of Chao1 index, compared with bacterial load in cells per ml, as estimated by qPCR. (B) Shows the diversity in the samples as inferred by Shannon index, compared with bacterial load. (n = 30 in both cases).

Figure 7

Bacterial fractions in human breast milk. (A) Proportion of bacteria present in a free-living, “planktonic” state and aggregated to human immune cells in colostrum and mature milk samples. Bacteria from 10 ml of milk were counted and sorted by size and complexity using a Moflo cytometer. *indicates a p < 0.05, Mann–Whitney test. (B) Planktonic bacteria in milk observed by SEM microscopy. (C) Bacteria associated to human immune cells, observed with SEM microscopy. (D) Bacteria associated to human immune cells, observed with fluorescence microscopy. DNA was stained with DAPI fluorophore. Bacteria are indicated with arrows. IC, human immune cell; B, bacteria.