Multiple Approaches Detect the Presence of Fungi in Human Breastmilk Samples from Healthy Mothers

Abstract

Human breastmilk contains a variety of bacteria that are transmitted to the infant and have been suggested to contribute to gut microbiota development and immune maturation. However, the characterization of fungal organisms in milk from healthy mothers is currently unknown although their presence has been reported in the infant gut and also in milk from other mammals. Breastmilk samples from healthy lactating mothers (n = 65) within 1 month after birth were analyzed. Fungal presence was assessed by different techniques, including microscopy, growth and identification of cultured isolates, fungal load estimation by qPCR, and fungal composition using 28S rRNA gene high-throughput sequencing. In addition, milk macronutrients and human somatic cells were quantified by spectrophotometry and cytometry. qPCR data showed that 89% of samples had detectable levels of fungal DNA, at an estimated median load of 3,5 × 10⁵ cells/ml, potentially including both viable and non-viable fungi. Using different culture media, 33 strains were isolated and identified, confirming the presence of viable fungal species. Pyrosequencing results showed that the most common genera were Malassezia (44%), followed by Candida (19%) and Saccharomyces (12%). Yeast cells were observed by fluorescence microscopy. Future work should study the origin of these fungi and their potential contribution to infant health.

Figure 1

Fluorescent microscopy images of yeasts detected in breastmilk. Left panels are showing the yeasts stained in green with the EUK516 FISH probe targeting the 18S rRNA gene. Right panels are showing the yeasts stained in blue with calcofluor. (A) Candida parapsilosis isolate FBMI4 (positive control). (B) Yeast from fixed transitional breastmilk sample BMF9. (C) Yeasts from fixed colostrum sample BMF5.

Figure 2

Fungal load in breastmilk over time. The plot shows the median with interquartile ranges of fungal load at three time points in the 89% of samples that showed fungal presence by qPCR. C, colostrum samples (n = 16); T, transitional milk samples (n = 14); M, mature milk samples (n = 28). Detection limit was established at 10³ cells/ml, estimated as the lowest concentration at which 95% of the positive samples are detected.

Figure 3

Fungal taxonomic composition of human breastmilk. Bars show the proportion of fungal genera as inferred by PCR amplification and pyrosequencing of the 28S rRNA gene in healthy mothers (n = 10). Each code in the X axis corresponded to a donor. Fungal genera that were under 1% were grouped in the “Others” category. The majority of the samples presented correspond to mature milk samples, except for BMF5 and BMF8 (colostrum) and BMF9 (transitional milk).

Figure 4

Relationships between fungal taxa relative abundance and nutritional, cellular and bacterial content of human breastmilk. The heatmap shows samples clustered by their compositional profile. Relative abundance of fungal genera is colour-coded according to their negative- (red) or positive- (blue) correlations with the amounts of milk components: fat protein, bacterial load, lactose, fungal load, somatic cells and non-fatty solids (NFS). Significant correlations are represented with an asterisk (*) and are as defined in the results section (n = 10).

Figure 5

Relationships between fungal and bacterial loads with nutritional and cellular content of human breastmilk. Correlations of fungal and bacterial load appear colour-coded according to their negative- (red) or positive- (blue) correlations with fat content, lactose content, protein content, somatic cells density and non-fatty solids content (NFS). Significant correlations are represented with an asterisk (n = 34).