Insufficient Evidence of a Breastmilk Microbiota at Six-Weeks Postpartum: A Pilot Study

Abstract

Breastmilk is thought to influence the infant gut by supplying prebiotics in the form of human milk oligosaccharides and potentially seeding the gut with breastmilk microbes. However, the presence of a breastmilk microbiota and origins of these microbes are still debated. As a pilot study, we assessed the microbes present in expressed breastmilk at six-weeks postpartum using shotgun metagenomic sequencing in a heterogenous cohort of women who delivered by vaginal (n = 8) and caesarean delivery (n = 8). In addition, we estimated the microbial load of breastmilk at six-weeks post-partum with quantitative PCR targeting the 16S rRNA gene. Breastmilk at six-weeks postpartum had a low microbial mass, comparable with PCR no-template and extraction controls. Microbes identified through metagenomic sequencing were largely consistent with skin and oral microbes, with four samples returning no identifiable bacterial sequences. Our results do not provide convincing evidence for the existence of a breastmilk microbiota at six-weeks postpartum. It is more likely that microbes present in breastmilk are sourced by ejection from the infant's mouth and from surrounding skin, as well as contamination during sampling and processing.

Keywords: breastmilk; contamination; infant; microbiome; retrograde inoculation.

Figure 1

Comparison of total DNA extracted in a single elution of different extraction kits of both whole and skim milk. QIABlood is the QIAamp DNA blood mini kit, QIAMini is the QIAamp DNA mini kit, and QIAFaecal of QIAamp DNA PowerFaecal Pro DNA kit. Extraction controls were all undetectable at less than <0.5 ng/mL.

Figure 2

(A) Comparison of extraction kits from whole and skim milk. Icon represents median, and error bar represents range of triplicate technical replicates. (B) Cycle threshold (C_T) vs. Log(No. of Bacteria) for testing of extraction efficiency from bacteria in PBS (Standard curve) vs. in breastmilk (Spiked breastmilk). For all but breastmilk (orange inverted triangle) icon represents median of technical triplicates; however, range was too small to display. Median only displayed for test breastmilk sample for ease of viewing. (C) Cycle threshold (C_T) vs. Log(No. of Bacteria) for quantification of bacterial load of QFC breastmilk samples. Icon and error bars represent median and range for standard curve. Orange icons are the median values of the QFC samples (n = 16) with Log(No. of Bacteria) calculated from C_T values.

Figure 3

(A) Comparison of number of species detected (minus those present in negative control) in sequences from each facility (Median ± IQR). (B) Non-significant relationship between Log(Bacteria/mL) and No. of Species detected (minus those present in negative control). (C) Comparison of Log(Bacteria/mL) of samples sent to each of Facility A and Facility B. (D) Comparison of Log(no. of reads) produced for each sample following quality control. ns p > 0.05, ** p < 0.01.