New insights in gut microbiota establishment in healthy breast fed neonates

Abstract

The establishment of a pioneer gut microbiota is increasingly recognized as a crucial stage in neonatal development influencing health throughout life. While current knowledge is mainly based on either culture or molecular analysis of feces, we opted for a comprehensive approach complementing culture with state-of-the-art molecular methods. The bacterial composition in feces from seven healthy vaginally-delivered, breast-fed neonates was analyzed at days 4-6, 9-14 and 25-30 postnatal, using culture, 16S rRNA gene sequencing of isolates, quantitative PCR and pyrosequencing. Anaerobes outnumbered facultative anaerobes in all seven neonates within the first days of life, owing to high levels of Bifidobacterium and unexpectedly also Bacteroides, which were inversely correlated. Four neonates harbored maternal Bacteroides levels, comprising typical adult species, throughout the neonatal period, while in three only subdominant levels were detected. In contrast, the major adult-type butyrate-producing anaerobic populations, Roseburia and Faecalibacterium, remained undetectable during the neonatal period. The presence of Bacteroidetes as pioneer bacteria in the majority of neonates studied demonstrates that adult-type strict anaerobes may reach adult-like population densities within the first week of life. Consequently the switch from facultative to strict anaerobes may occur earlier than previously assumed in breast-fed neonates, and the establishment of the major butyrate-producing populations may be limited by other factors than the absence of anaerobic conditions. The impact of breast milk components on the timing of establishment of anaerobic pioneer bacteria, as well as opportunistic pathogens should be further studied in regard to priming of the gut-associated immune system and consequences on later health.

Figure 1. Counts of anaerobes and facultative anaerobes (culture) and total bacteria (qPCR) detected in neonatal feces (NF).

Values are expressed as means ± SD at each of the three sampling points, i.e. days 4–6, 9–14 and 25–30 postnatal (n = 7); and compared to means obtained from maternal feces (MF) (n = 7) over the perinatal period.

Figure 2. Gene copy numbers of the major gut-associated bacterial populations detected in neonatal feces (NF) using qPCR.

Values are expressed as means ± SD at each of the three sampling points, i.e. days 4–6, 9–14 and 25–30 postnatal in neonates harboring high (A, n = 4) and low (B, n = 3) Bacteroides population levels, respectively; and comparison to means obtained from maternal feces (MF) (n = 7) over the perinatal period.

Figure 3. Relative 16S rRNA gene abundances of the four major phyla detected in neonatal feces (NF) using pyrosequencing.

Values are expressed as means ± SD at each of the three sampling points, i.e. days 4–6, 9–14 and 25–30 postnatal.

Figure 4. Relative 16S rRNA gene abundances of the 17 most abundant genera detected in neonatal feces (NF) using pyrosequencing.

Values are expressed as means ± SD at each of the three sampling points, i.e. days 4–6, 9–14 and 25–30 postnatal in neonates harboring high (A, n = 4) and low (B, n = 3) Bacteroides population levels, respectively.