Previous human milk studies have confirmed the existence of a highly diverse bacterial community using culture-independent and targeted culture-dependent techniques. However, culture-enriched molecular profiling of milk microbiota has not been done. Additionally, the impact of storage conditions and milk fractionation on microbiota composition is not understood. In this feasibility study, we optimised and applied culture-enriched molecular profiling to study culturable milk microbiota in eight milk samples collected from mothers of infants admitted to a neonatal intensive care unit. Fresh samples were immediately plated or stored at -80 °C for two weeks (short-term frozen). Long-term samples were stored at -20 °C for more than six months. Samples were cultured using 10 different culture media and incubated both aerobically and anaerobically. We successfully isolated major milk bacteria including Streptococcus, Staphylococcus, and Bifidobacterium from fresh milk samples, but were unable to culture any bacteria from the long-term frozen samples. Short-term freezing shifted the composition of viable milk bacteria from the original composition in fresh samples. Nevertheless, the inter-individual variability of milk microbiota composition was observed even after short-term storage. There was no major difference in the overall milk microbiota composition between milk fractions in this feasibility study. This is among the first studies on culture-enriched molecular profiling of the milk microbiota demonstrating the effect of storage and fractionation on milk microbiota composition.
This study was generously supported by the Trainee Expansion Program from the International Society of Research in Human Milk and Lactation and Family Larsson-Rosenquist Foundation.
We are grateful to the anonymous mothers who provided milk samples, and the NICU personnel at McMaster Children’s Hospital. We would like to thank Prof. Stephanie Atkinson who was instrumental in facilitating access to the fresh milk samples.