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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 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.
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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.

Mentions: After quality filtering and length trimming, 174,886 16S rRNA sequences were analyzed, with an average number of taxonomically assigned, high-quality sequences of 4353 reads per sample. The taxonomic assignment of the sequences showed that human breast milk composition is dominated by Staphylococcaceae, which account for >62% of the total number of sequences obtained (Figure 2). At the three lactation times, the most common genera was Staphylococcus, followed by Acinetobacter in colostrum, Pseudomonas and Streptococcus in transition milk and also Acinetobacter in mature milk samples (Figure 3A). Milk from the three lactation points showed different patterns of bacterial diversity, but no statistically significant differences were found between timepoints for any bacterial genus. Rarefaction curves after analyzing 35,000 reads per lactation time point indicated 223 OTUs in colostrum samples, 251 in transition and 203 in mature samples when sequences were clustered at 97% sequence identity (the consensus value for determining species boundaries; Figure 3B). The number of OTUs obtained suggests values of several hundred species in human breast milk, with transition samples having higher diversity than colostrum and mature milk, containing up to nine genera that were only found at that stage (Figure 3B). Similar estimates of several hundred bacterial species were also obtained by other studies (Hunt et al., 2011, Cabrera-Rubio et al., 2012a), confirming that human breast milk is highly diverse. However, most diversity in the samples corresponded to a few bacterial genera, which appeared to be dominant. Among them, we found a core of seven genera that were present at the three time points: Finegoldia, Streptococcus, Corynebacterium, Staphylococcus, Acinetobacter, Peptoniphilus, and Pseudomonas. Although determining the bacterial species composition with partial 16S rRNA sequences has to be taken with care, the relatively long sequences obtained by pyrosequencing (average read length 718 bp) allowed us to assign reads to the species taxonomic level with some degree of reliability. This analysis revealed that the most common species within Staphylococci was S. epidermidis, and S. aureus was not detected in these healthy mothers (a full list of species composition can be found in Table 1). It must be underlined that although some bacteria typically associated to human breast milk like Bifidobacterium spp (Collado et al., 2009) were detected at low proportions in our samples, this could be due to low amplification efficiency of “universal” primers in these high G+C content taxa (Sim et al., 2012).


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: After quality filtering and length trimming, 174,886 16S rRNA sequences were analyzed, with an average number of taxonomically assigned, high-quality sequences of 4353 reads per sample. The taxonomic assignment of the sequences showed that human breast milk composition is dominated by Staphylococcaceae, which account for >62% of the total number of sequences obtained (Figure 2). At the three lactation times, the most common genera was Staphylococcus, followed by Acinetobacter in colostrum, Pseudomonas and Streptococcus in transition milk and also Acinetobacter in mature milk samples (Figure 3A). Milk from the three lactation points showed different patterns of bacterial diversity, but no statistically significant differences were found between timepoints for any bacterial genus. Rarefaction curves after analyzing 35,000 reads per lactation time point indicated 223 OTUs in colostrum samples, 251 in transition and 203 in mature samples when sequences were clustered at 97% sequence identity (the consensus value for determining species boundaries; Figure 3B). The number of OTUs obtained suggests values of several hundred species in human breast milk, with transition samples having higher diversity than colostrum and mature milk, containing up to nine genera that were only found at that stage (Figure 3B). Similar estimates of several hundred bacterial species were also obtained by other studies (Hunt et al., 2011, Cabrera-Rubio et al., 2012a), confirming that human breast milk is highly diverse. However, most diversity in the samples corresponded to a few bacterial genera, which appeared to be dominant. Among them, we found a core of seven genera that were present at the three time points: Finegoldia, Streptococcus, Corynebacterium, Staphylococcus, Acinetobacter, Peptoniphilus, and Pseudomonas. Although determining the bacterial species composition with partial 16S rRNA sequences has to be taken with care, the relatively long sequences obtained by pyrosequencing (average read length 718 bp) allowed us to assign reads to the species taxonomic level with some degree of reliability. This analysis revealed that the most common species within Staphylococci was S. epidermidis, and S. aureus was not detected in these healthy mothers (a full list of species composition can be found in Table 1). It must be underlined that although some bacteria typically associated to human breast milk like Bifidobacterium spp (Collado et al., 2009) were detected at low proportions in our samples, this could be due to low amplification efficiency of “universal” primers in these high G+C content taxa (Sim et al., 2012).

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