Patient characteristics
In this study 41 preterm born infants were included. The GA ranged between 25 and 30 weeks (median 27.6 weeks IQR 26.0-28.1). Table 1 summarizes the baseline characteristics. Detailed patient characteristics per week are shown in Table 2. Important for understanding this cohort, the mode of delivery was strongly associated with the birth weight Z-score for GA and gender (Fig. 1a). Infants delivered by CS had a higher GA on average than their vaginally born counterparts (median 28.1 IQR 26.7-28.2) vs 26.0 IQR 25.3-26.9 weeks, p=0.002), yet were of lower BW (median 778 IQR 641- 923 vs 835 IQR 800-980 grams, p=0.03). Underlying causes of prematurity for all vaginally delivered infants (13/13) were intra-uterine infection, (prolonged) premature rupture of membranes and/or cervix insufficiency while most infants delivered by C-section were born preterm because of placental insufficiency (22/27) (Table 1). Placental insufficiency was negatively associated with the Z-score for birthweight (p=0.0005, Fig. 1b).
Intestinal microbiome development over time
We analyzed 142 samples (3.5 samples per patient on average, range 2-4), including meconium. An overview of the abundances (% of reads per sample) of the most important bacterial groups (species/genera) in these 4 timepoints is shown in Fig. 2 and exact details of oligotype abundances are given in Supplementary Information file 1. Within meconium samples the Staphylococcus genus, mostly consisting out of S. epidermidis, was frequently the most dominant bacterial group (40% of reads on average) followed by 4 members from the Enterobacteriaceae family; Enterobactercloacae (11%), Escherichia (coli) (10%), Klebsiella oxytoca (5.9%) and Klebsiella pneumoniae (5.7%). In week 2 an increase in the abundance of Staphylococcus was observed in this cohort (50% of reads on average). A decline however of Staphylococcus is observed in weeks 3 and 4 (17% and 11%, respectively) with the Enterobacteriaceae family becoming dominant in most samples (32%, 27%, 52% and 54% on average for weeks 1, 2, 3 and 4, respectively). Bifidobacteria and lactobacilli were groups of relatively minor importance within this cohort.
Mode of delivery and intestinal microbiota development
One of the most striking patterns in the data, alluded to in Fig. 2, is that the mode of delivery has a significant influence on the microbiome composition. This was most evident within the first three weeks after birth (Fig. 3). Abundance of Staphylococcus, a genus of typical skin bacteria, was significantly associated with CS delivery in samples from the first 3 weeks of life (p=0.0008, p=0.0003 p=0.046 and p=0.28 for weeks 1-4, respectively). The combined prevalence the facultative anaerobe E. coli and members of the aero-tolerant anaerobic Bacteroides genus, typical maternal fecal representatives, were significantly associated with vaginal delivery during these first 3 weeks (p=0.021, p=0.0002, p=0.028 and p=1 for weeks 1-4, respectively).
Mode of delivery and infant weight development
In this cohort the mode of delivery was significantly associated with BW and GA (Fig. 1). The association between the infant’s current weight (not corrected for GA) at sampling time and the mode of delivery remains largely unchanged during the first four weeks (p=0.04, p=0.03, p=0.02 and p=0.04 for weeks 1-4, respectively), as no difference was present in weight gain in g/week of infants delivered vaginally and by CS (p=0.9; p=0.8; p=0.6 for weeks 2-4, respectively). Z-scores from BW were strongly correlated with absolute weight in all 4 weeks (Spearman r correlation coefficients of 0.70, 0.67, 0.66 and 0.52, respectively).
Infant weight and intestinal microbiome development over time
While the mode of delivery was the most important determinant for the infants’ initial microbiome (Figs. 2 and 3), the increase of Enterobacteriaceae in weeks 3 and 4 appeared associated with absolute weight at sampling time (Fig. 4). When comparing infants with an above-median weight (³835g) with their lighter counterparts, little difference was observed in the microbiome composition of the meconium (week 1). In week 2, infants with an above median weight (³860g) contained significantly less S. epidermidis (p=0.013). This particular association was however partially indirect, as infants delivered by CS had a lower median BW and were more frequently colonized during delivery by S. epidermidis (Fig. 3a). The weight of infants increased from mean 860g in week 2 to 969g in week 3 to 1095g in week 4, respectively. In week 3 Enterobacteriaceae became more dominant again at the expense of S. epidermidis, as the influence of the mode of delivery declined. In week 4 the correlation coefficient between abundance of Enterobacteriaceae and current weight was significant (r=0.4, p=0.04). More specifically, at a body weight of >1100g nearly all samples were dominated by Enterobacteriaceae. In general, the absolute weight of infants, irrespective of the week of sample collection, appeared to be (independently) associated with the shift from a S. epidermidis dominated gut towards one dominated by Enterobacteriaceae.
Gut microbiome maturation vs infant weight or postconceptional age
A simple linear regression analysis of this dataset shows that weight is significantly correlated with the prevalence of Staphylococcus and Enterobacteriaceae (% Enterobacteriaceae - % Staphylococcus) but that this prevalence is not linked with postconceptional age (Fig. 5a). A Mixed Effect Regression analysis was subsequently performed to take the longitudinal and paired aspect of this sample-set into account (multiple samples per infant over time) and came to the same conclusions (Figs. 5b-d); the abundance of Staphylococcus and Enterobacteriaceae are associated with weight (p=0.01, both) but not with postconceptional age in this dataset.
Intestinal microbiota development and health
While staphylococci were associated with low absolute weight in this cohort, they did not appear detrimental to health as 1) the amount of weight gain during any single week did not correlate with the gut microbiome composition (or any of the individual species) at the start of that week and 2) their previously reported negative association with necrotizing enterocolitis development was similarly found within this cohort in meconium samples (p=0.034). Overall infant mortality in this cohort, in part caused by necrotizing enterocolitis (n=8 in total; 3 caused by NEC), was not aligned with the gut microbiome composition or with BW (p=0.36) but it was negatively associated with GA at birth (p=0.005) and positively but not significantly (p=0.08) with prolonged premature rupture of membranes (PPROM).
Exogenous factors and microbiota development
Exogenous factors such as antibiotics use and/or feeding regime were found to be of ancillary importance in comparison with patterns associated with the mode of delivery or with absolute body weight. Associations of bacterial groups with antibiotics use were either found to be non-significant or disappeared when adjusting the analyses for mode of delivery. The current number of subjects was insufficient to unravel significant associations between microbiome development with antibiotics and feeding regimes.