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

Boix-Amorós A, Collado MC, Mira A - Front Microbiol (2016)

Bottom Line: 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.The median bacterial load was 10(6) bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Health and Genomics, Center for Advanced Research in Public Health, FISABIO FoundationValencia, Spain; Department of Biotechnology, Institute of Agrochemistry and Food Technology, Spanish National Research CouncilValencia, Spain.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Bacterial loads in planktonic and human cell-associated fractions of nine samples of colostrum and nine samples of mature milk were calculated, showing that the microorganisms were present in both fractions, although aggregated bacteria appeared to be more abundant in colostrum (65.75%), and planktonic bacteria were found to be more abundant in mature samples (63.92%; Figure 7A). Mann-Whitney statistical tests showed significant differences (p < 0.05) between the two time points (but not within the same time point) for both free and human cell-associated bacteria. The high proportion of bacteria associated with human immune cells was confirmed by fluorescence and Scanning Electron microscopy (Figures 7B–D). Bacteria in the aggregated fraction seemed to be adhered to the membrane of human cells (identified as immune cells according to their shape and size) but not intracellular. We confirmed the presence of live bacteria moving inside the extracellular matrix of immune cells (Supplementary Video). Bacterial cells in this extracellular matrix have also been observed in blood samples from pregnant mothers by other researchers (Donnet-Hughes et al., 2010). An “entero-mammary pathway” has been proposed to explain the translocation of bacteria to the mammary gland through blood and/or lymph stream through its association to human immune cells (Martín et al., 2004). If this translocation process is confirmed, the milk cell-bacterial association described here could be a consequence of such a relationship. An alternative explanation would be that bacteria originated from skin and the oral cavity of the lactating child invades the mammary gland and binds to immune cells without eliciting a response (Hagi et al., 2013). Future studies should determine the kind of immune cells involved in the observed bacterial adhesion and the nature of the bacteria-human recognition (Langa, 2006; Perez et al., 2007), including the identification of which microorganisms are free and which ones are human cell-associated.


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

Boix-Amorós A, Collado MC, Mira A - Front Microbiol (2016)

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.
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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.
Mentions: Bacterial loads in planktonic and human cell-associated fractions of nine samples of colostrum and nine samples of mature milk were calculated, showing that the microorganisms were present in both fractions, although aggregated bacteria appeared to be more abundant in colostrum (65.75%), and planktonic bacteria were found to be more abundant in mature samples (63.92%; Figure 7A). Mann-Whitney statistical tests showed significant differences (p < 0.05) between the two time points (but not within the same time point) for both free and human cell-associated bacteria. The high proportion of bacteria associated with human immune cells was confirmed by fluorescence and Scanning Electron microscopy (Figures 7B–D). Bacteria in the aggregated fraction seemed to be adhered to the membrane of human cells (identified as immune cells according to their shape and size) but not intracellular. We confirmed the presence of live bacteria moving inside the extracellular matrix of immune cells (Supplementary Video). Bacterial cells in this extracellular matrix have also been observed in blood samples from pregnant mothers by other researchers (Donnet-Hughes et al., 2010). An “entero-mammary pathway” has been proposed to explain the translocation of bacteria to the mammary gland through blood and/or lymph stream through its association to human immune cells (Martín et al., 2004). If this translocation process is confirmed, the milk cell-bacterial association described here could be a consequence of such a relationship. An alternative explanation would be that bacteria originated from skin and the oral cavity of the lactating child invades the mammary gland and binds to immune cells without eliciting a response (Hagi et al., 2013). Future studies should determine the kind of immune cells involved in the observed bacterial adhesion and the nature of the bacteria-human recognition (Langa, 2006; Perez et al., 2007), including the identification of which microorganisms are free and which ones are human cell-associated.

Bottom Line: 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.The median bacterial load was 10(6) bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Health and Genomics, Center for Advanced Research in Public Health, FISABIO FoundationValencia, Spain; Department of Biotechnology, Institute of Agrochemistry and Food Technology, Spanish National Research CouncilValencia, Spain.

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.

No MeSH data available.


Related in: MedlinePlus