Methods and Strategies to Examine the Human Breastmilk Microbiome

LeMay-Nedjelski, Lauren; Copeland, John W.; Wang, Pauline W.; Butcher, James; Unger, Sharon; Stintzi, Alain; O’Connor, Deborah L.

doi:10.1007/978-1-4939-8728-3_5

Cited by 19 publications

(18 citation statements)

References 75 publications

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“…The assessed methodologies included a common commercial kit, QIAamp DNA Stool Mini Kit (with or without the addition of bead beating) 14,17,18,21,23,24,[27][28][29] , DNeasy PowerLyzer PowerSoil 33 , and a high stringency phenol-chloroform method 34,35,49 , which have been used in previously published breast milk microbiome analyses. We are not aware of any published studies in which Sigma-Aldrich Gen-Elute has been used to analyse the breast milk microbiota, however, it has been used in the assessment of other low biomass samples 50 .…”

Section: Discussionmentioning

confidence: 99%

DNA extraction approaches substantially influence the assessment of the human breast milk microbiome

Douglas

Ivey

Papanicolas

et al. 2020

Sci Rep

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In addition to providing nutritional and bioactive factors necessary for infant development, human breast milk contains bacteria that contribute to the establishment of commensal microbiota in the infant. However, the composition of this bacterial community differs considerably between studies. We hypothesised that bacterial DnA extraction methodology from breast milk samples are a substantial contributor to these inter-study differences. We tested this hypothesis by applying five widely employed methodologies to a mock breast milk sample and four individual human breast milk samples. Significant differences in DNA yield and purity were observed between methods (P < 0.05). Microbiota composition, assessed by 16S rRNA gene amplicon sequencing, also differed significantly with extraction methodology (P < 0.05), including in the contribution of contaminant signal. Concerningly, many of the bacterial taxa identified here as contaminants have been reported as components of the breast milk microbiome in other studies. These findings highlight the importance of using stringent, well-validated, DNA extraction methodologies for analysis of the breast milk microbiome, and exercising caution interpreting microbiota data from low-biomass contexts. In addition to nutrients and bioactive components, human breast milk contains bacteria 1 , commonly referred to as the breast milk microbiome. These bacterial populations are thought to contribute to the establishment of commensal bacterial communities in the infant, as well as supporting the maturation of the gut, and development of the immune system 1,2. The potential importance of microbes introduced with breast milk on early-life development has led to increasing efforts to accurately define them. Early culture-and PCR-based methods, predominantly reported the presence of members of the Staphylococcus, Streptococcus, Lactobacillus and Bifidobacterium genera 3-12. More recently, 16S rRNA gene amplicon sequencing has become an increasingly popular approach to characterising the bacterial content of breast milk samples 13-33. By enabling the detection of species that are refractory to standard culture methodologies, as well as providing insight into the relative abundance of constitutive bacteria, 16S rRNA gene sequencing offers substantial analytical advantages. However, the composition of the breast milk microbiome reported in sequencing-based studies varies substantially. For example, while two studies have described a "core" human breast milk microbiome (bacteria that are present in all samples analysed) from healthy mothers, consisting of 7 and 9 taxa respectively, only three of these taxa were present in both (Propionibacterium, Staphylococcus, and Streptococcus) 27,34. Notably, many of the other bacterial taxa often reported as part of the breast milk microbiome, including

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Section: Discussionmentioning

confidence: 99%

DNA extraction approaches substantially influence the assessment of the human breast milk microbiome

Douglas

Ivey

Papanicolas

et al. 2020

Sci Rep

View full text Add to dashboard Cite

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“…In general, low-biomass samples are unstable and their relative abundance depends on subtle changes of sample handling or sequencing methodologies [32,33]. More importantly, low-biomass samples are prone to DNA extraction and library preparation kit contaminants [34].…”

Section: Discussionmentioning

confidence: 99%

Compositional analysis of the bacterial community in colostrum samples from women with gestational diabetes mellitus and obesity

Gámez-Valdez

García-Mazcorro²,

Rincón

et al. 2020

Preprint

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Background. Gestational Diabetes Mellitus (GDM) and obesity are threatening health conditions during pregnancy, as they affect the normal function of multiple systems, including neuro-hormonal networks, and adipose, liver, muscle and placenta. GDM and maternal obesity are main triggers to a vicious cycle of metabolic and cardiovascular diseases perpetuated trans-generationally. One of the first stages of this vicious cycle occurs during early lactation as the infant feeds of “disbalanced” breastmilk microbiota and macromolecule level. Despite the importance of breastmilk microbiota on newborn development, few studies have characterized breastmilk microbiota in association with obesity and GDM. Maternal obesity decreases the diversity of breastmilk microbiota, with increased proportions Staphylococcus compared to Bifidobacterium and Bacteroides. However, the extent to which GDM together with maternal obesity affect breastmilk microbiota is unknown. Here, we applied 16S-rRNA high-throughput sequencing to characterize colostrum microbiota of 43 mothers with exclusive GDM and obesity in order to address the impact of GDM/obesity on breastmilk microbiota.Results. We identified a total of 1,496 amplicon sequence variants (ASVs), being Proteobacteria and Firmicutes the dominant phyla. We found Staphylococcus, Corynebacterium-1, Anaerococcus and Prevotella overrepresented in samples of women with obesity and women with GDM. Population diversity indicators, such as Shannon/Faith phylogenetic index and UniFrac/robust Aitchison distances show distinct microbial composition for GDM (female-newborn subgroup) and obesity (male-newborn subgroup) compared to controls. Finally, Differential abundance analysis showed that Rhodobacteraceae was distinct for GDM and five families (Bdellovibrionaceae, Halomonadaceae, Shewanallaceae, Saccharimonadales and Vibrionaceae) were distinct for obesity groups.Conclusions. Our study indicates that GDM and obesity are related to a higher microbial diversity. Preliminary functional analysis suggests that this could be attributed to overrepresentation of amino acid and carbohydrate metabolism bacterial pathways. However, there is a strong influence of the infant´s gender and the utilization of antibiotics intrapartum. To our knowledge, this study represents the first characterization of the taxonomical changes of colostrum breastmilk in mothers with GDM. We think this study contributes to future design of functional metagenomic studies aiming to understand molecular mechanisms by which breastmilk from mothers with GDM/obesity affect the development and future adult physiology of their suckling infants.

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“…Our results are consistent with previous breastmilk studies [8,11,28] but differ from those published by Meehan et al [29], and Jost et al [30] as they reported primarily enrichment of Firmicutes. Differences in relative abundance can be attributed to the sequencing platform used [31,32] but possibly sample conditions and storage could also interfere. The candidate division WPS-2 was present in 1.8% of total relative abundance in our samples.…”

Section: Discussionmentioning

confidence: 99%

Compositional Analysis Of The Bacterial Community In Colostrum Samples From Women With Gestational Diabetes Mellitus And Obesity

Gámez-Valdez

García-Mazcorro²,

Rincón

et al. 2020

Preprint

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Background. Obesity and Gestational Diabetes Mellitus (GDM) are major concerns during pregnancy as they compromise both mother and newborn health. These pathophysiology leads to variations in gut and breastmilk microbiota, threatening the settlement of neonate’s gut microbiota and the stimulation of immune cells. While several studies have described compositional changes of breast milk, placenta and maternal and newborn intestinal microbiota, it is unknown how breastmilk microbiota is affected under maternal GDM. The molecular mechanisms in which microbial-unbalanced breastmilk affect development of infants is not well understood for the case of obesity and GDM. As a first step, we used 16S- amplicon sequencing to describe the taxonomical bias of breastmilk of 43 women with GDM and women with obesity. We sampled colostrum within the first 24 h taken after birth in order to identify and quantify the first microbial community potentially colonizing infants gut.Results. A total of 1,675 amplicon sequence variants (ASVs) were identified, being Proteobacteria and Firmicutes the dominant phyla. We found Pseudomonas, Gemellales and Enterobacter overrepresented in samples of women with obesity and GDM. We used observed ASV´s, Shannon index and Faith´s phylogenetic diversity scores as alpha diversity indicators and UniFrac and robust Aitchison distances beta diversity indicators. Both metrics show a distinct microbial composition for GDM (female sub-group) and Obesity (male subgroup) compared to controls. Finally, differentially abundance analysis showed that Rhodococcaceae was distinct for GDM and 7 families (Bdellovibrionaceae, Burkholderiaceae, Halomonadaceae, Pseudomonadaceae, Shewanallaceae, TM7-1 and Vibrionaceae) with an absolute effect size greater than 1, of which 5 had a q-value ≤ 0.05 were distinct for obesity groups.Conclusions. To our knowledge, this study represents the first approach to the breastmilk bacteria composition of mothers with GDM. Our work provides the foundation for the design of functional metagnomic studies aiming to understand molecular mechanisms of which breastmilk from individuals with GDM and obesity affect the physiology of the infants.

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Methods and Strategies to Examine the Human Breastmilk Microbiome

Cited by 19 publications

References 75 publications

DNA extraction approaches substantially influence the assessment of the human breast milk microbiome

DNA extraction approaches substantially influence the assessment of the human breast milk microbiome

Compositional analysis of the bacterial community in colostrum samples from women with gestational diabetes mellitus and obesity

Compositional Analysis Of The Bacterial Community In Colostrum Samples From Women With Gestational Diabetes Mellitus And Obesity

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