Pumping supplies alter the microbiome of pumped human milk: An in-home, randomized, crossover trial

Abstract

Background: The human milk microbiome may contribute to the benefits of breastfeeding by providing bacteria to the infant gastrointestinal tract. Many women pump their milk, but the effect of pumping on the milk microbiome is unknown.

Objectives: Our objective was to determine the effects of pumping supplies on the pumped human milk microbiome.

Methods: This was an in-home, randomized, crossover trial of 2 collection methods. Women (n = 52) pumped twice within 3.5 h, once with their own breast pumps and milk collection supplies (OWN SUPP) and once with a hospital-grade pump and sterile collection supplies (STER SUPP). Pumping order was randomized. The milk microbiome was characterized by aerobic culturing and 16S ribosomal RNA gene sequencing.

Results: Milk collected with OWN SUPP yielded more total aerobic and gram-negative bacteria than milk collected with STER SUPP, reflecting a 6.6 (adjusted OR; 95% CI: 1.7, 25; P = 0.006) higher odds of containing >104 total aerobic CFU/mL and 19 (adjusted OR; 95% CI: 4.1, 88; P < 0.0001) higher odds of yielding culturable gram-negative bacteria. Milk collected with OWN SUPP yielded more Proteobacterias , including higher relative abundances of Acinetobacter and Stenotrophomonas, compared to milk collected with STER SUPP. Results were consistent across pumping-order groups.

Conclusions: We demonstrated that pumping supplies altered the milk microbiome. On average, milk collected with OWN SUPP resulted in elevated levels of culturable total and gram-negative bacteria and proteobacterial DNA compared to milk collected with STER SUPP. More research is needed to assess implications for infant health.

Keywords: Proteobacteria; aerobic bacteria; bacterial counts; breastmilk collection; breastmilk expression/methods; crossover study; gram-negative bacteria; microbiota.

FIGURE 1

Participant flow diagram. Each woman contributed 2 milk samples, one from each collection method. One participant pumped in the opposite order of her randomly assigned assignment. All samples were analyzed by intention-to-treat analysis. HM, human milk; HM + CF, human milk and complementary foods; ITT, intention to-treat; OWN SUPP, personal electric breast pump and milk collection kits; STER SUPP, hospital-grade pump and new, sterile collection kits.

FIGURE 2

Effect of OWN SUPP on cultured bacteria in pumped human milk (n = 104), among all samples (A, B) and according to infant diet and randomly assigned pumping order (C). LS means and SEM from linear mixed-effects regression are shown. *Difference compared with STER SUPP (P ≤ 0.04). All other comparisons were not significant (P ≥ 0.20). HM, human milk; HM + CF, human milk and complementary foods; OWN SUPP, personal electric breast pump and milk collection kits; OWN SUPP First, randomly assigned to use own pumping supplies first; STER SUPP, hospital-grade pump and new, sterile collection kits; STER SUPP First, randomly assigned to use sterile pumping supplies first.

FIGURE 3

Effect of OWN SUPP on the genus-level composition of 16S ribosomal RNA sequences (n = 104), according to infant diet and randomly assigned pumping order (A–C). Asterisks denote differentially abundant genera. Circles scaled by Faith's phylogenetic diversity index. ^†P < 0.05 compared with STER SUPP using mixed-effects regression. OWN SUPP, personal electric breast pump and milk collection kits; PD, phylogenetic diversity; RPO, randomly assigned pumping order; STER SUPP, hospital-grade pump and new, sterile collection kits; Unid., unidentified.

FIGURE 4

Correlations between culturable gram-negative bacteria and 16S microbiome. (A) There was no Spearman correlation between Gram-Neg bacterial counts and percent relative abundances of Proteobacteria 16S in STER SUPP (P = 0.7) but a strong correlation for OWN SUPP (P < 0.001). (B) PCA ordination of Proteobacteria 16S sequences; samples with culturable Gram-Neg bacteria had a different 16S community than samples without culturable Gram-Neg bacteria (permutational ANOVA P < 0.001). (C) The volcano plot shows the log₂ fold change (x-axis) of Proteobacteria asegenera that differed significantly (FDR-adjusted P value y-axis) between samples yielding culturable gram-negative bacteria and those that did not, regardless of pumping supplies used for collection. FDR, false discovery rate; Gram-Neg, gram-negative bacteria; OWN SUPP, personal electric breast pump and milk collection kits; PCA, Principal component analysis; PC1, Principal component 1; PC2, Principal component 2; STER SUPP, hospital-grade pump and new, sterile collection kits.