Human milk microbiota and its relationship with milk components in health and during lactational mastitis

Resumen

Background: Human milk is nature's ideal food for the nurture and protection of the new-born and growing infant. Recent evidence reported the presence of bacteria in human milk under normal, healthy conditions, which are thought to confer beneficial properties to the infant. However, little is known about the relationship between bacteria and milk macronutrients and human cells, and there is no optimal protocol to estimate bacterial numbers in the samples. Also, the potential presence of fungi in human milk has not been explored to date, despite the fact that fungi has been previously detected in dairy animal's milk and in the neonatal gut. In addition, the aetiology of sub-acute mastitis is not well understood, and information about the composition of the milk microbiota during this process by means of next-generation sequencing and its potential implications in the disease is scarce. This thesis is aimed to improve our understanding of human milk microbiota, its composition and diversity as well as the interactions with other milk components, in health and during sub-acute mastitis. We also explore the potential effect of environmental factors, such as mode of delivery and geographic location, and the lactation stage on milk's microbiota composition. Methods: Next-generation sequencing technologies targeting the bacterial 16S rRNA gene, and the fungal 28S rRNA gene and ITS1 genetic region, in combination with classic microbiological analyses, were used in order to assess the bacterial and fungal composition in milk of healthy mothers, and in mothers suffering sub-acute mastitis. Bacterial and fungal loads in human milk were obtained by qPCR methodology calibrated with flow cytometry. Results: Bacterial composition in human milk has a high inter-individual variability, and also over time, and is predominantly comprised of bacteria from the Staphylococacceae family. A bacterial and fungal "core" were found in the human milk of Spanish donors. Some correlations were observed between bacteria with milk macronutrients and somatic cells, indicating an active relationship between milk microbiota and the environment. Bacterial density appeared to be higher than previously estimated, at a mean of 106 cells/ml, and bacteria were found both in a free-living state and associated to human cells. No correlations were observed between bacterial load with number of somatic cells nor bacterial richness and diversity, indicating that higher bacterial densities under healthy conditions do not trigger an immune response in the mammary gland, nor alter the microbial community. In addition, our results revealed the existence of certain diversity of fungi in human milk that was further confirmed by culture-dependent methods and microscopy. 89% of the Spanish samples analysed had detectable levels of fungal DNA, at a median load of approximately 105 cells/ml. Malassezia, Candida and Saccharomyces prevailed in the samples, and fungi interacted with milk components in different ways. The presence of fungi in milk was further confirmed in samples from distant geographic origins, and it was observed that maternal and delivery factors can impact milk microbial communities. After describing milk microbiota in healthy conditions, we performed an observational, prospective case-control study, where DNA and RNA from human milk microbiota from healthy and sub-acute mastitis-suffering mothers were studied before and after the treatment. Bacterial loads increased during the disease, diversity decreased and alterations in bacterial composition likely reflected a dysbiotic process in the mammary gland. This supports that sub-acute mastitis is a microbial-driven disease.