Microbiota Analysis of Donor Human Milk Collected Over One Year in Italy

Abstract Background The beneficial effects of breast milk on infant health have been well documented from nutritional, physiological, and developmental perspectives. Human milk contains a complex microbiota that plays a key role in initiating infant gut colonization shortly after birth, supplying beneficial bacteria that aid immune maturation and protect against pathogens. Mother's own milk is the optimal source of nutrition for preterm infants; however, since many mothers experience difficulty producing enough, pasteurized donor milk is often used as an alternative. The aim of this study was to profile the milk microbiota of donor human milk collected from mothers over a one-year period. Methods Over the course of one year, 27 mothers collected their breast milk and donated it to the Human Milk Bank in Lombardy (Italy), resulting in a total of 84 samples. All samples were processed by 16S rRNA gene metabarcoding sequencing after Holder pasteurization (62.5 °C for 30 minutes). Relative abundances at the genus level were determined using QIIME 2 with a reference database. Results The relative abundance of the phyla Firmicutes and Proteobacteria was inversely correlated, with Firmicutes predominating in 52% and Proteobacteria in 48% of the samples. At the genus level, 61 taxa were identified, with the most common being Pseudomonas (in 89% of samples), Acinetobacter (83%), Streptococcus (80%), and Staphylococcus (75%). The genus Bacillus was identified in 26% of the samples, among these, B. cereus, a pathogen commonly associated with contamination of donor human milk, was isolated in 36% (n = 8) of the cases. Conclusions In addition to identifying the core commensal bacteria of the milk microbiota, it is crucial to investigate those that vary between individuals, as they may include pathogenic genera. This is particularly relevant, as the milk is intended for preterm infant feeding. Key messages • The high diversity of the donor human milk microbiota highlights its influence by individual factors. • Human milk microbiota knowledge is essential as it shapes the composition and function of the infant gut microbiome.