Impact of expression mode and timing of sample collection, relative to milk ejection, on human milk bacterial DNA profiles

AbstractAnalysis of the human milk microbiome is complicated by the presence of a variable quantity of fat. The fat fraction of human milk is typically discarded prior to analysis. It is assumed that all cells are pelleted out of human milk by high speed centrifugation; however, studies of bovine milk have reported that bacteria may remain trapped within the fat fraction. Here, the bacterial DNA profiles of the fat fraction and cell pellet of human milk (n = 10) were analysed. Human and bacterial DNA was consistently recovered from the fat fraction of human milk (average of 12.4% and 32.7%, respectively). Staphylococcus epidermidis was significantly more abundant in the cell pellet compared to the fat fraction (P = 0.038), and three low-abundance species (< 5% relative abundance) were recovered from one fraction only. However, inclusion of fat reduced the efficiency of DNA extraction by 39%. Culture-based methods were used to quantify the distribution of an exogenously added strain of Staphylococcus aureus in human milk fractions. S. aureus was consistently recovered from the fat fraction (average 28.9%). Bacterial DNA profiles generated from skim milk or cell pellets are not representative of the entire human milk microbiome. These data have critical implications for the design of future work in this field.

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Comparison of Bacterial DNA Profiles in Mid-Trimester Amniotic Fluid Samples From Preterm and Term Deliveries

Frontiers in Microbiology ◽

10.3389/fmicb.2020.00415 ◽

2020 ◽

Vol 11 ◽

Cited By ~ 3

Author(s):

Lisa Stinson ◽

Maria Hallingström ◽

Malin Barman ◽

Felicia Viklund ◽

Jeffrey Keelan ◽

...

Keyword(s):

Amniotic Fluid ◽

Bacterial Dna ◽

Dna Profiles

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The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles

Methods and Protocols ◽

10.3390/mps3020039 ◽

2020 ◽

Vol 3 (2) ◽

pp. 39 ◽

Cited By ~ 1

Author(s):

Anna Ojo-Okunola ◽

Shantelle Claassen-Weitz ◽

Kilaza S. Mwaikono ◽

Sugnet Gardner-Lubbe ◽

Heather J. Zar ◽

...

Keyword(s):

Human Milk ◽

Dna Extraction ◽

Bacterial Population ◽

Illumina Miseq ◽

Molecular Techniques ◽

Rrna Gene ◽

Bacterial Dna ◽

Dna Recovery ◽

Culture Independent ◽

Zymo Research

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp® DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of Enhydrobacter and Acinetobacter. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.

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The Viable Microbiome of Human Milk Differs from the Metataxonomic Profile

Nutrients ◽

10.3390/nu13124445 ◽

2021 ◽

Vol 13 (12) ◽

pp. 4445

Author(s):

Lisa F. Stinson ◽

Michelle L. Trevenen ◽

Donna T. Geddes

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Human Milk ◽

Dna Content ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Bacterial Dna ◽

Cutibacterium Acnes ◽

Rrna Gene Sequencing

Bacteria in human milk contribute to the establishment of the infant gut microbiome. As such, numerous studies have characterized the human milk microbiome using DNA sequencing technologies, particularly 16S rRNA gene sequencing. However, such methods are not able to differentiate between DNA from viable and non-viable bacteria. The extent to which bacterial DNA detected in human milk represents living, biologically active cells is therefore unclear. Here, we characterized both the viable bacterial content and the total bacterial DNA content (derived from viable and non-viable cells) of fresh human milk (n = 10). In order to differentiate the living from the dead, a combination of propidium monoazide (PMA) and full-length 16S rRNA gene sequencing was used. Our results demonstrate that the majority of OTUs recovered from fresh human milk samples (67.3%) reflected DNA from non-viable organisms. PMA-treated samples differed significantly in their bacterial composition compared to untreated samples (PERMANOVA p < 0.0001). Additionally, an OTU mapping to Cutibacterium acnes had a significantly higher relative abundance in PMA-treated (viable) samples. These results demonstrate that the total bacterial DNA content of human milk is not representative of the viable human milk microbiome. Our findings raise questions about the validity of conclusions drawn from previous studies in which viability testing was not used, and have broad implications for the design of future work in this field.

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4052 A TL1 Team Approach to Personalization of Donor Human Milk for Preterm Infants

Journal of Clinical and Translational Science ◽

10.1017/cts.2020.286 ◽

2020 ◽

Vol 4 (s1) ◽

pp. 91-91

Author(s):

Natalie Harrison ◽

Marion M Bendixen ◽

Josef Neu ◽

Leslie A. Parker ◽

Graciela L. Lorca

Keyword(s):

16S Rrna ◽

Human Milk ◽

Preterm Infants ◽

16S Rrna Sequencing ◽

Team Approach ◽

Microbial Count ◽

Sample Collection ◽

Donor Human Milk ◽

Cell Counts ◽

Rrna Sequencing

OBJECTIVES/GOALS: Feeding preterm infants with mother’s own milk (MOM) lowers rates of sepsis, decreases necrotizing enterocolitis, and shortens hospital stay. Our objective is to determine whether a similar microbial diversity to MOM can be obtained when fresh or frozen MOM is inoculated in donor human milk (DHM). METHODS/STUDY POPULATION: Subjects included 12 mothers of infants born 100ml of MOM per day and were excluded if they had taken antibiotics within 3 days of the 1-time pumped MOM sample collection. MOM sample was divided into fresh (processed immediately) and frozen (−20°C) for 24h fractions. MOM was inoculated in DHM [referred to as refaunated milk (RM)] at 10% (RM10) and 30% (RM30) dilutions, then incubated at timepoints: 0h, 2h, 4h at 37°C. At each timepoint, total viable microbial cell counts were performed in differential or selective media along with future 16S rRNA sequencing. RESULTS/ANTICIPATED RESULTS: Microbiota expansion was detected in MOM, RM10 and RM30 over time whether fresh or frozen milk was used as the inoculum. Incubated fresh and frozen MOM had similar bacterial loads when tested on nutrient agar (10^5-10^6 CFU/mL), mannitol salt (10^6 CFU/mL), MacConkey (10^2-10^5 CFU/mL), blood agar (10^6 CFU/mL) and MRS (10^4 CFU/mL) plates. Based on these CFU counts, RM30 incubated for 2h and RM10 at 4h showed similar counts to that of MOM at 0h. DISCUSSION/SIGNIFICANCE OF IMPACT: RM, inoculated with fresh or frozen MOM, obtained a similar microbial count compared to MOM at 0h indicates that fresh or frozen MOM can inoculate DHM. 16s rRNA sequencing is ongoing. Future studies are needed to support an inoculation protocol to be used in clinical practice and human milk banking.

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Gleaning a Human DNA Profile from Trace Swabs Collected from Animal Hairs

Acta veterinaria ◽

10.1515/acve-2016-0016 ◽

2016 ◽

Vol 66 (2) ◽

pp. 187-202 ◽

Cited By ~ 1

Author(s):

Branimira Špoljarić ◽

Maja Popović ◽

Josip Crnjac ◽

Zrinka Žderić Savatović ◽

Martina Ratko ◽

...

Keyword(s):

Dna Extraction ◽

Sample Collection ◽

Dna Profile ◽

Human Dna ◽

Intraspecific Variations ◽

Morphological Differences ◽

Dna Profiles ◽

Collection Methods

Abstract Animal hairs are an apt surface for retention of forensic trace epithelial samples. The aim of this study was threefold: to evaluate different methods of sample collection (moistened and dry swabs) and DNA extraction (Chelex® 100 method, Qiagen EZ1® DNA Investigator Kit), as well as to examine the morphological differences of hair fibres between two species (dog, sheep) and their ultimate impact on sample collection and processing. Our preliminary findings suggest that the use of EZ1® DNA Investigator Kit yields donor DNA profiles of higher quality. The results of different sample collection methods have shown intraspecific variations that require further investigation. The ability of retention and subsequent extraction of trace DNA appears to be similar between the two species, despite significant morphological differences between their coat hairs.

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Measurements of the elasticity of the mammary gland as related to the human milk ejection reflex

Bulletin of Experimental Biology and Medicine ◽

10.1007/bf00811629 ◽

1961 ◽

Vol 52 (1) ◽

pp. 765-767

Author(s):

M. G. Zaks ◽

I. A. Mazhbits

Keyword(s):

Mammary Gland ◽

Human Milk ◽

Milk Ejection ◽

Milk Ejection Reflex

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Human Milk Oligosaccharide Profiles and Associations with Maternal Nutritional Factors: A Scoping Review

Nutrients ◽

10.3390/nu13030965 ◽

2021 ◽

Vol 13 (3) ◽

pp. 965

Author(s):

Caren Biddulph ◽

Mark Holmes ◽

Anna Kuballa ◽

Peter S. W. Davies ◽

Pieter Koorts ◽

...

Keyword(s):

Body Composition ◽

Nutritional Status ◽

Human Milk ◽

Dietary Intake ◽

Milk Sample ◽

Sample Collection ◽

Body Composition Assessment ◽

Nutritional Factors ◽

The Impact ◽

Maternal Dietary Intake

Human milk oligosaccharides (HMOs) are complex unconjugated glycans associated with positive infant health outcomes. This study has examined current knowledge of the effect of maternal diet and nutritional status on the composition of HMOs in breast milk. Using the PRISMA-ScR guidelines, a comprehensive, systematic literature search was conducted using Scopus, Web of Science, Global Health (CABI), and MEDLINE. Titles and abstracts were screened independently by two reviewers against predefined inclusion and exclusion criteria. Fourteen studies met the inclusion criteria and reported on maternal dietary intake (n = 3), maternal body composition indices (n = 9), and dietary supplementation interventions (n = 2). In total, data from 1388 lactating mothers (4011 milk samples) were included. Design methodologies varied substantially across studies, particularly for milk sample collection, HMO analysis, dietary and body composition assessment. Overall, this review has identified potential associations between maternal dietary intake and nutritional status and the HMO composition of human milk, though an abundance and sufficiency of evidence is lacking. Standardised procedures for human milk sample collection and HMO analysis, along with robust and validated nutrition assessment techniques, should be employed to further investigate the impact of maternal nutritional factors on HMO composition.

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Human Milk Metabolic Hormones: Analytical Methods and Current Understanding

International Journal of Molecular Sciences ◽

10.3390/ijms22168708 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8708

Author(s):

Majed A. Suwaydi ◽

Zoya Gridneva ◽

Sharon L. Perrella ◽

Mary E. Wlodek ◽

Ching Tat Lai ◽

...

Keyword(s):

Human Milk ◽

Analytical Methods ◽

Growth And Development ◽

Metabolic Reprogramming ◽

Sample Collection ◽

Infant Outcomes ◽

Metabolic Hormones ◽

Born Infant ◽

Validation Parameters ◽

Hormone Analysis

Human milk (HM) contains a wide array of peptide hormones including leptin and adiponectin, which are involved in the regulation of infant growth and development. These essential hormones might play an important role in the regulation of metabolic reprogramming of the new-born infant. However, HM hormone studies are sparse and heterogeneous in regard to the study design, sample collection, preparation and analysis methods. This review discussed the limitations of HM hormone analysis highlighting the gaps in pre-analytical and analytical stages. The methods used to quantify HM metabolic hormones (leptin, adiponectin, ghrelin, insulin, obestatin, resistin and apelin) can be classified as immunoassay, immunosensor and chromatography. Immunoassay methods (ELISA and RIA) have been predominantly used in the measurement of these HM hormones. The relative validity parameters of HM hormones analysis are often overlooked in publications, despite the complexity and differences of HM matrix when compared to that of plasma and urine. Therefore, appropriate reports of validation parameters of methodology and instrumentation are crucial for accurate measurements and therefore better understanding of the HM metabolic hormones and their influences on infant outcomes.

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