Participants

The data consist of Dutch (N = 87) and Finnish (N = 75) children comprising 162 children in total (Table 1). These were selected from large longitudinal cohorts, the Bibo cohort (N = 193) in the Netherlands and the Flora cohort (N = 1223) in Finland, which have been reported previously [30, 31]. The selection criteria of the original studies included birth at term and no serious pregnancy or birth complications. For the current study, we selected vaginally born cases with appropriately stored faecal sample collected at 3 months and information on lifetime antibiotic use and BMI at 5–6 years. The children were measured for weight and height at the age of 5 (Flora) or 6 (Bibo) years.

Table 1 Characteristics of the cohorts (mean ± standard deviation) Full size table

In the Flora study, pregnant mothers, whose infants had increased risk for allergy (at least one parent had a diagnosed allergic disease), were recruited at antenatal clinics and through advertisements in the Helsinki (Finland) suburban area. The Flora study was a probiotic intervention study, and here, we only included infants from the control group. The study was approved by the ethical committee of the Helsinki region hospital district. The Bibo study is a longitudinal study in which mothers and their children were followed from the third trimester of pregnancy on. Pregnant women were recruited through midwife practices in Nijmegen and surrounding areas (the Netherlands).

The median birth weight was 3.4 kg (range 2.3–4.7 kg) among the Finnish infants and 3.6 kg (range 2.7–4.7 kg) among the Dutch infants. In the Finnish cohort, 39 infants were first-born, and in the Dutch cohort, 23 infants were first-born. At the age of 3 months, 12 Finnish infants and 42 Dutch infants were no longer breastfed. The sub-cohorts analysed here did not differ from the total Flora and Bibo cohorts in terms of birth weight, BMI, or duration of breastfeeding. In the total Flora cohort, the mean ± sd birth weight was 3.59 ± 0.49 kg, duration of breastfeeding was 8.42 ± 5.23 months, and BMI at 5 years was 15.86 ± 1.38. In the total Bibo cohort, the mean ± sd birth weight was 3.61 ± 0.47 kg, duration of breastfeeding was 4.19 ± 2.78 months, and BMI at 6 years was 15.64 ± 1.35.

Procedure

A faecal sample was collected from all infants at the age of 3 months, by parents at home, as previously detailed [30, 31]. In addition, information on weight development from birth to 6 months of age was available for a subset of infants in both cohorts (Table 1). Growth during the first 6 months was calculated as change in weight from birth to age 6 months. BMI at 5–6 years was calculated based on the weight and height measured by a paediatrician or a researcher. Lifetime antibiotic use information was available for both cohorts. For the Dutch cohort, data on antibiotic prescriptions were obtained from clinical records from the child’s GP. For the Finnish children, the parents provided information on the number of antibiotic courses the child had received every 3–6 months during the first year, thereafter yearly in questionnaires. The overwhelming majority of the antibiotic courses were given to the children after the collection of the faecal samples; only eight infants received antibiotics before the age of 3 months (Fig. 1). The type of antibiotics used was not available for all infants, and therefore, only the total number of courses was used in the analysis. The level of antibiotic use was higher in the Finnish cohort: the median number of courses was 5 in the Finnish cohort and 2 in the Dutch cohort, corresponding to population-level data from both countries [32].

Fig. 1 Microbiota composition of 3-month-old infants in the Dutch (a) and Finnish (b) cohorts. The 16 most abundant genus-level taxa are shown. Colour codes are from top down on each column. Each column represents an individual child, and the squares below the columns show the BMI of the child at 5–6 years (white = 13, black = 21) and lifetime antibiotic use (white = 0 courses, black = 8 or more courses). Asterisks indicate infants who had received a course of antibiotics before sample collection Full size image

Microbiota analysis

The faecal samples of both cohorts were processed and analysed using a pipeline that has been used in many infant and child microbiota studies [31, 33]. This included DNA extraction using the repeated bead beating method [34] and analysis of the microbiota composition using the HITChip phylogenetic microarray, which contains oligonucleotide probes for hypervariables regions on the 16S rRNA gene [35]. All samples were analysed on two independent microarray experiments, and the data only passed the quality control if the inter-experiment Pearson correlation was >0.97. The signal intensities were normalized using the fRPA method [36] and summarised at different levels of phylogenetic resolution: species, genus, and phylum, except for the Firmicutes, which was further divided to Clostridium clusters and Bacilli. Relative normalized signal intensities were calculated for all samples and all three levels. For the analyses, only bacterial groups with >30% prevalence at a minimum of 0.01% relative abundance were included (Additional file 1).

Statistical analysis

Differences between the countries in bacterial relative abundances were tested using generalised linear models with negative binomial distribution. Associations between bacterial taxa to BMI and early growth were tested using correlations. To adjust for the effects of country, birth weight, breastfeeding duration, and antibiotic use on BMI, we calculated the deviance from the expected BMI, based on a linear model with the aforementioned variables. We then tested for a correlation between the BMI deviance and the log-transformed relative abundances of the bacterial taxa. The models were run using the whole data set and separately for each cohort to ascertain consistency in the associations. In addition, the models were run separately for children with minimal antibiotic exposure (0–1 lifetime courses), and those with several antibiotic courses, to see if the associations were present regardless of antibiotic exposure. Only associations that were significant in the total cohort (p < 0.05) and nearly significant (p < 0.15) in both countries separately were considered robust. All statistical analyses were conducted in R [37] using the package vegan [38]. R script for the analysis is available in Additional file 2.