We used a high-throughput phylogenetic microarray to reveal alterations in the gut microbiota composition throughout early childhood. The used microarray has been developed and validated for determining the microbiota diversity and evaluating the relative proportions of genus-like or higher (phylum-like) phylogenetic groups . Moreover, it has been demonstrated that this microarray has a power equal to or higher than deep new generation sequencing  and it has previously been used to compare the microbiota diversity and composition in a variety of studies [33, 42].
Despite the highly significant increase of microbiota diversity with age, the diversity indeces at 18 months of age are still relatively low (~110) when compared to the approximately two-fold higher indexes (150–200) commonly observed in healthy adults . It has been suggested that by the age of 1 to 2 years the microbiota resembles that of an adult [29, 43]. Our results show that microbiota succession continues at least until the age of 18 months and most likely even further, because the bacterial diversity has still not reached the diversity of an adult person. Thus, significant changes can be expected to occur in even after 18 months of age.
Concerning the microbiota composition at 6 months of age, our results are in agreement with earlier studies [5, 29], except that we observed significant colonization by bifidobacteria in most of the children (mean relative abundances 22.9% at 6 months and 12.6% at 18 months of age, respectively) while in the study of Palmer et al.  bifidobacteria were not detected, possibly due to differences in DNA extraction, PCR primers, demographic and geographic origin, dietary patterns of the infants or other confounding factors. Primers used for PCR are often not so optimal for bifidobacteria than for other species and thus, high GC bacteria may perform less well in such PCRs. Further, in our previous studies we have shown that mechanical lysis of faecal bacteria is essential and improves the detection of especially Gram-positive bacteria including bifidobacteria [32, 44]. In the Palmer et al. study , mechanical lysis by bead-beating was not applied, which may have hampered the detection of bifidobacteria. Thus, we consider that the most likely explanation for the different results concerning bifidobacteria in our and Palmer et al.  study is the different DNA extraction methods used.
When comparing healthy and eczematous children we found statistically significant differences in microbiota composition only at 18 months of age. The total microbiota of children with eczema was found to become significantly more diverse than the microbiota of children who remained healthy by 18 months of age. Interestingly, the total microbiota and particularly Firmicutes diversity was higher in the eczema group children, although the difference with the healthy subjects was not statistically significant. Abrahamsson et al. described the infants as having atopic eczema during the first two years of life (diagnostics were done at 6, 12 and 24 months of age), but the age at the onset of symptoms was not clarified . However, it can be concluded from the Abrahamsson et al.  and our study, both taking advantage of the high resolution microbiota assessment techniques, that after 1 year of age the total microbiota diversity in children either developing or having eczema is comparable or even higher than that of healthy children. Secretory IgA has been suggested to play a role in shaping the microbiota composition and diversity. Some early studies showed an association between the low levels of secretory IgA and the risk of developing atopy [45, 46] and could suggest that the low IgA levels permit establishment of a wider variety of bacteria and explain the higher bacterial diversity in children with eczema observed in this study. However, more recent studies have shown a higher concentration of secretory IgA in children with allergic sensitization during the first 2 years of life [47, 48].
Another possible explanation for the increased bacterial diversity in children with eczema is the decreased levels or altered repertoire of antimicrobial peptides secreted into the gut lumen. These peptides, such as alpha- and beta-defensins, have at least two key roles at the mucosal interface: contributing to the host defense against enteric bacterial attachment and homeostatic control of the intestinal bacterial ecosystem [49, 50]. Recently, decreased alpha-defensin levels and increased beta-defensin levels were associated with increased risk of developing atopy . To our knowledge, the levels of faecal antimicrobial peptides in children already having eczema have not been studied. However, a few studies have highlighted the role of alpha-defensins in microbiota composition and intestinal health. For example, genetic mutations resulting in decreased alpha-defensin expression have been associated with the susceptibility and severity of inflammatory bowel disease in humans and decreased alpha-defensins may have an effect on the differences observed in microbiota composition between healthy and diseased subjects . Interestingly, mice deficient in production of active alpha -defensins were shown to have a decrease in Bacteroidetes . The reason for decreased Bacteroidetes levels in children with eczema in this study remains unaccountable, but alpha-defensins provide one possible explanation for our observation. Also other host-dependent factors, such as the amount of mucus secretion and differences in mucus glycosylation (e.g. FUT2 secretor status) may have an influence on the microbiota diversity and composition, as recently reviewed by Maynard et al. . Clearly, the role of intestinal IgA levels, antimicrobial peptides and mucus secretion in shaping the gut microbiota in healthy and eczematous children warrants for further investigation.
Our results emphasize that the microbiota diversity in children with eczema should be further studied by using high-resolution techniques in order to define the favourable course of bacterial succession in early childhood and toddler age and to evaluate possible means to influence it.
It was observed that children with eczema harbour more bacteria belonging to the Clostridium cluster IV and Clostridium cluster XIVa. These bacteria are among the most abundant microbial groups detected in the healthy adult intestine . Thus, prematurely occurring changes towards an adult-type microbiota were observed to take place in children with eczema. It has been suggested that resident bacteria may shape the hosts’ physiology, among others, by modulating the expression of genes involved in intestinal functions, such as postnatal intestinal maturation and the maintenance of mucosal barrier . It may be speculated that an infant-type microbiota supports adequate gut barrier function and tolerance against food allergens in an immature gut. Infant-type microbiota may fortify the normal mucosal barrier function e.g. by affecting the maturation of the gut epithelium and immune functions in an optimal way and decrease the low-grade intestinal inflammation observable in subjects with eczema [53, 56]. Maintenance of adequate mucosal barrier function may also play a role in the level of sensitisation to food-derived compounds [57, 58]. The complex host-microbe interactions in the intestinal epithelium are only recently beginning to be understood [53, 59].
Furthermore, we observed decreased relative abundances of bacteria belonging to Bacteroidetes in children with eczema. Previous studies have reported an association between decreased amounts Bacteroides spp. and the development of atopy and increased risk for atopic sensitization [9, 60, 61]. Bacteria belonging to the Bacteroidetes are among the first groups colonizing the gut [15, 29] and they are typical intestinal habitants in healthy adults . Bacteroides spp. are specialized in the breakdown of complex plant polysaccharides  and their abundance has been associated with increased short-chain fatty acid concentrations in the infant gut after introduction of first solid foods . Furthermore, B. fragilis polysaccharide has been shown in mice model to direct the cellular and physical maturation of the developing immune system via its ability to direct the development of CD4+ T cells, thus inducing the differentiation of Th1 lineage and correction of the Th1/Th2 imbalance . Together with our findings, these results suggest the significance of Bacteroides spp. in the development and maintenance of healthy infant gut and balanced mucosal immunity and necessitate the role of these bacteria to be considered in future studies.
When comparing healthy children with children with eczema we found statistically significant differences in microbiota composition only at 18 months, but not at 6 months of age. Breast-feeding is known as a major factor influencing the microbiota composition in infancy [4, 5]. At 6 months of age, the majority of children included in this study were still nursed and breast-feeding is likely to have had a strong influence on their microbiota composition at that age. Thus, it seems that breast-feeding could have evened up the microbiota differences between the healthy and eczematous children and masked the eczema-associated changes, which came apparent and measurable at 18 months of age after the withdrawal of breast-milk.
While many studies addressed the impact of L. rhamnosus GG on health parameters, the short and long-term effect on the intestinal microbiota has only received limited attention. In the present intervention, the supplementation of L. rhamnosus GG continued until the age of 6 months. Interestingly, no significant effect on the microbiota composition was observed at the age of 6 months, but instead the supplementation of L. rhamnosus GG in early life was observed to a induce long-term effect and small but significant changes between the intervention groups were observed one year later at the age of 18 months. The observation that the C. difficile et rel. group bacteria were lower in the LGG groups as compared to placebo is of particular interest. Previously, Clostridium difficile colonization at the age of 1 month has been associated with a higher risk of a diagnosis of atopic dermatitis at the age of 2 years . The higher Anaerostipes caccae et rel levels in the children that had received the L. rhamnosus GG supplementation is also a potentially beneficial effect, because A. caccae produces butyrate, which is an energy source for epithelial cells of colonic mucosa . Bacteria belonging to the Eubacterium ventriosum et rel group that were higher in the children that received the probiotic supplementation, also have shown to produce butyrate but have been less investigated. In mice, however, it has been shown that E. ventriosum was reduced in colitic mice as compared to non-colitic animals . To our knowledge this is the first high -throughput microbiota analysis study reporting the long-term effects of a probiotic strain on the microbiota composition in early life.