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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 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).
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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).

Mentions: Bacterial load values at each milk maturation stage are shown in Figure 1. 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. Median values for colostrum, transition and mature milk were around 106 bacterial cells per ml, with no significant differences between the three time points. Data from other researchers had indicated bacterial densities of 103–104 per ml of breast milk, but they were based on laboratory culture (Heikkilä and Saris, 2003), or on qPCR methods calibrated by culture (Khodayar-Pardo et al., 2014), which account for a limited fraction of total bacteria in human samples. In addition, a significant fraction of microorganisms were found to be adhered to the extracellular matrix of human cells (see Section Bacterial Distribution in Milk Below), which could further prevent the growth on culture media. The molecular approach used in the current manuscript expands these pioneering estimates, allowing now the study of potential relationships between bacterial load and other parameters. Although our molecular-based methods suggest bacterial loads between two and three orders of magnitude higher than those estimated by culture, it has to be taken into account that DNA from non-viable bacteria and extracellular DNA would also be amplified by qPCR, and therefore the real number of viable bacteria would probably be lower.


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

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

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).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4837678&req=5

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).
Mentions: Bacterial load values at each milk maturation stage are shown in Figure 1. 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. Median values for colostrum, transition and mature milk were around 106 bacterial cells per ml, with no significant differences between the three time points. Data from other researchers had indicated bacterial densities of 103–104 per ml of breast milk, but they were based on laboratory culture (Heikkilä and Saris, 2003), or on qPCR methods calibrated by culture (Khodayar-Pardo et al., 2014), which account for a limited fraction of total bacteria in human samples. In addition, a significant fraction of microorganisms were found to be adhered to the extracellular matrix of human cells (see Section Bacterial Distribution in Milk Below), which could further prevent the growth on culture media. The molecular approach used in the current manuscript expands these pioneering estimates, allowing now the study of potential relationships between bacterial load and other parameters. Although our molecular-based methods suggest bacterial loads between two and three orders of magnitude higher than those estimated by culture, it has to be taken into account that DNA from non-viable bacteria and extracellular DNA would also be amplified by qPCR, and therefore the real number of viable bacteria would probably be lower.

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