Characterization of the Cultivable Microbiota in Fresh and Stored Mature Human Breast Milk.

Besides nutritional components, breast milk contains diverse microbes, which may be involved in colonization of the infant gut. Expressed milk is often stored for few days in the refrigerator. The aim of this study was to determine the abundance, prevalence and diversity of facultative and strict anaerobic bacteria using culture-dependent and -independent methods, and to determine changes in milk microbial and chemical composition during storage. Samples of mature breast milk from 21 women were collected 3-6 months post-partum and were analyzed fresh or after anaerobic storage for 6 days at 4°C. The cultivable bacterial population was analyzed using the most probable number (MPN) method or plate counts and different media. The abundance of major bacterial groups was determined using quantitative PCR and 16S rRNA gene sequencing. Lactose, lactate, short chain fatty acids (SCFA) and human milk oligosaccharides (HMO) were analyzed using chromatography techniques. Highest mean viable cell counts were obtained in yeast casitone fatty acids (YCFA) broth supplied with mucin (log 4.2 ± 1.8 cells/ml) and lactose (log 3.9 ± 1.4 cells/ml), or Columbia broth (log 3.0 ± 0.7 cells/ml). Mean total bacterial counts estimated by qPCR was 5.3 ± 0.6 log cells/ml, with Firmicutes being the most abundant phylum. The most prevalent bacterial groups were Streptococcus spp. (15/19 samples), Enterobacteriaceae (13/19) and Lactobacillus/Lactococcus/Pediococcus group (12/19). While the average total number of bacterial cells did not change significantly during storage, the prevalence of strict anaerobic Bacteroidetes increased threefold, from 3/19 to 9/19, while in 7 samples Clostridium clusters IV or XIVa became detectable after storage. Major HMO were not degraded. Lactate was present in 18/21 samples after storage (2.3-18.0 mM). Butyrate was detected in 15/21 and 18/21 samples before and after storage, respectively, at concentrations ranging from 2.5 to 5.7 mM. We demonstrate enhanced prevalence and/or abundance of viable strict anaerobes from the Bacteroidetes and Clostridiales after 6-day anaerobic storage of human milk. Our data indicate that anaerobic cold storage did not markedly change total viable bacterial load, while HMO profiles were stable. Anaerobic cold storage of human milk for up to 6 days may be suitable for preserving milk quality for potential microbial transfer to the infant gut.

Cutibacterium avidum is phylogenetically diverse with a subpopulation being adapted to the infant gut.

The infant gut harbors a diverse microbial community consisting of several taxa whose persistence depends on adaptation to the ecosystem. In healthy breast-fed infants, the gut microbiota is dominated by Bifidobacterium spp.. Cutibacterium avidum is among the initial colonizers, however, the phylogenetic relationship of infant fecal isolates to isolates from other body sites, and C. avidum carbon utilization related to the infant gut ecosystem have been little investigated. In this study, we investigated the phylogenetic and phenotypic diversity of 28 C. avidum strains, including 16 strains isolated from feces of healthy infants. We investigated the in vitro capacity of C. avidum infant isolates to degrade and consume carbon sources present in the infant gut, and metabolic interactions of C. avidum with infant associated Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum. Isolates of C. avidum showed genetic heterogeneity. C. avidum consumed d- and l-lactate, glycerol, glucose, galactose, N-acetyl-d-glucosamine and maltodextrins. Alpha-galactosidase- and ß-glucuronidase activity were a trait of a group of non-hemolytic strains, which were mostly isolated from infant feces. Beta-glucuronidase activity correlated with the ability to ferment glucuronic acid. Co-cultivation with B. infantis and B. bifidum enhanced C. avidum growth and production of propionate, confirming metabolic cross-feeding. This study highlights the phylogenetic and functional diversity of C. avidum, their role as secondary glycan degraders and propionate producers, and suggests adaptation of a subpopulation to the infant gut.

Colonization of Cutibacterium avidum during infant gut microbiota establishment.

Establishment of the infant gut microbiota affects gut maturation and influences long-term health. Cutibacterium (formerly Propionibacterium) have been identified as early colonizers, but little is known about their function. Using a cultivation-dependent and -independent approach, we determined Cutibacterium prevalence, diversity and functional potential. In feces from a Swiss infant cohort (n = 38), prevalence of Propionibacterium/Cutibacterium decreased from 84% at 2 weeks, to 65% at 4 weeks, 47% at 8 weeks and 41% at 12 weeks of age. Abundance varied among individuals, and persistence depended on the colonization levels at 2 weeks. Cutibacterium isolates (n = 87) were obtained from 10 infants from a smaller cohort (n = 12); restriction fragment length polymorphism clustered isolates in four groups, and all identified as Cutibacterium avidum. Colonization potential and metabolic effects of C. avidum addition were tested in an in vitro continuous intestinal fermentation model mimicking infant proximal colon conditions. Cutibacterium avidum spiked daily at 108 or 109 cells mL-1 colonized, decreased formate and persisted during the washout period. Significant correlations were observed between Propionibacterium/Cutibacterium and lactate-producers and protein-degraders in both reactors and infant feces. Our findings highlight the natural presence of C. avidum and its role as a lactate-consumer and propionate-producer in infants younger than 3 months.