Background
Gastrointestinal microorganisms are a diverse community of bacteria, archaea, fungi, unicellular organisms, and viruses, which inhabit the intestines of all mammals. Studies on humans and other mammals have shown that gut microbes are involved in a series of physiological processes that are critical to host health, including energy homeostasis, metabolism, intestinal epithelial health, immune activity, and neurobehavioral development [
1,
2]. Changes in gastrointestinal microbiota are reported to associate with the occurrences and developments of multisystem diseases, including inflammatory bowel disease, asthma, obesity, metabolic syndrome, cardiovascular disease, immune system disease and neurodevelopmental conditions, such as autism spectrum disorders [
3‐
6]. The interaction between the gut microbes and the host has a profound impact on the development of health and diseases [
7].
Human gut microbiota plays an important role in health and disease from prenatal to childhood. Studies have shown that the microorganisms in the human body at the early stage of birth play an important role in the regulation of human development and maturity [
8]. A study on human microbiota in the first 1000 days (from pregnancy to 2 years old) showed that intestinal microbial succession has an important impact on the growth and development of children [
9]. There are studies on the gut microecology of preterm infants and the influence of feeding methods on the gut microflora development of preterm infants [
10], the influence of delivery mode on the composition of gut microflora [
11], and the gut microflora of children with autism spectrum disorder [
12,
13]. From infant to pre-school age (0–6 years old) is a critical period for the growth and development of children’s various systems, but the development and succession of gut microflora of healthy 0–6-year-old children remains poorly revealed.
The gastrointestinal microorganism is considered as a metabolic “organ” and central regulator of host metabolism which can not only help decompose and absorb of nutrients and energy from food but also produce numerous metabolites and regulate host metabolism [
14]. Recently, with the further research of gastrointestinal microorganism, the short chain fatty acids (SCFAs) and bile acids (BAs) are two of the major metabolites produced by gut microbiota and paly important role in human homeostasis and health [
15,
16]. Dietary fibers are metabolized into SCFAs, mainly acetate, propionate, and butyrate by the fermentation of intestinal microorganisms [
17]. In this process, the host biosynthesis of primary bile acids (PBAs), which then enters the intestine through the liver-gut axis and is uncoupled and transformed into secondary bile acids (SBAs) by intestinal microbiota [
18], and that’s why the bile acid metabolism is also a typical example of host-gut microbial co-metabolism. From the beginning of life, the composition and function of intestinal microflora are dynamic and mature with age. The composition and development of intestinal microflora are affected by diet structure, environment, and other factors. At the same time, it affects host health and disease occurrence and development by regulating the metabolism level of the host.
In order to reveal the composition and function changes of gut flora in preschool children from birth to 6 years old, we analyzed and compared the gut flora composition of 120 newborns born in the First Affiliated Hospital of Kunming Medical University in 2015 and 150 healthy children aged from 6 months to 6 years old who underwent physical examination in the First Affiliated Hospital of Kunming Medical University from 2015 to 2016 in the present study. Meanwhile the levels of BAs and SCFAs in the feces of these participants, which can reflect the influence of gut flora on metabolic level in a certain extent, were also determined.
Discussion
In the past few years, more and more reports have revealed that there is a critical relationship between gut microflora and various chronic disease. The intestinal microecology acted on human health through gut-liver axis, gut-lung axis and gut-brain axis and so on [
21]. Intestinal microbes play an important role in mammalian homeostasis and health [
22], including providing essential nutrients [
23], metabolizing dietary fiber into short chain fatty acids (SCFAs) [
24], and ensuring the normal development of the immune system [
25]. Therefore, intestinal microbiota was considered to be a key factor affecting early life development and lifelong health.
As previous studies reported, the complete colonization of gut microbiota may occurred at prior to birth [
26], and the gut microbiota developed into a relatively stable, adult-like configuration within the first 3 years of life [
27]. The results showed that from birth to 6 m after birth, the formation of intestinal microflora in infants was mainly related to delivery mode, feeding mode, antibiotic exposure and environmental factors; then mainly affected by weaning and solid food intake from 6 m to 18 m; and gradually tended to be a relatively stable and adult-like formation from 18 m to 36 m [
11]. In addition, some studies have demonstrated that the development of pediatric intestinal flora lasts from childhood to adolescence [
28,
29]. Ringel-Kulka T et al. [
29] has reported that the diversity of intestinal microflora (both abundance and richness) in adults is significantly greater than that in 1–4 years old children, and there are differences in multiple phylogenetic groups between children and adults at the genus level.
The intestinal microflora diversity was evaluated by alpha diversity and beta diversity in our research. As we all know, alpha diversity refers to the diversity within a specific environment or ecosystem, mainly used to reflect species richness and evenness and sequencing depth and alpha diversity is mainly through the indexes of Chao1, Observed species, Goods coverage, Shannon and Simpson were used to reflect the richness and uniformity of the species in samples. Beta diversity refers to the species diversity among different environmental communities. Beta diversity and alpha diversity together constitute the overall diversity or the biological heterogeneity of certain environmental communities. The aim of PCoA analysis is to observe the difference between individual samples or groups of samples, the distance between different samples represents the difference of species composition. Our results showed that the Chao 1 index which indicated the abundance of gut flora was lower at neonatal period and obviously increased at 6 m while the Shannon index which indicated the uniformity of gut flora was gradually increased from birth to 6 years old (Fig.
1). After 3 years old, the intestinal microbial community of children has shown a similar composition to that of adults, and is mainly characterized by two major phyla:
Bacteroidetes and
Firmicutes (Fig.
3). These results are consistent with previous reports and suggested that it may be a critical period for the development of intestinal flora in infants around 6 months of birth.
In addition to intestinal microflora, a large number of intestinal microbial metabolites are also one of the components of intestinal microecology. SCFAs (mainly acetate, butyrate, and propionate) produced by intestinal microbial ferment dietary fiber are important functional substances in intestinal microenvironment, which are produced by intestinal microbial ferment dietary fiber [
16,
24]. The diversity of intestinal microflora plays an important role in the production of SCFAs. Most SCFAs are produced in cecum and proximal colon,
Bacteroidetes and
Firmicutes are the two dominant bacteria producing SCFAs [
30]. Acetate, butyrate, and propionate are the most abundant in intestinal SCFAs, and butyric acid can be produced by
Firmicutes and
Bacteroidetes [
31]. As shown in Figs.
3 and
6, the content of SCFAs in feces all increased with the increase of the abundance of
Firmicutes and
Bacteroidetes from 6-month-old. SCFAs are considered to be an endogenous protective substance, which can decrease the gut lumen PH, inhibit the growth of pathogens, and regulate immune cell function through SCFA receptor on cell surface to maintain intestinal microecology stability [
16]. In particular, butyric acid can enhance the intestinal epithelial barrier and modulate the gut immunol function [
32]. As our results shown, the concentrations of three major SCFAs in stools, acetate (AA), propionate (PA), and butyrate (BA), were significantly higher than isobutyrate (IBA) and isovalerate (IVA), and the concentrations of fecal SCFAs kept in a lower lever during the neonatal period (0–28 d after birth), then increased at 6 months old and gradually tended to be stable after 2 years old (Fig.
6). The change of SCFAs was observed to be accompanied with the development of gut flora from birth to 6 years old.
Another important function of human intestinal microorganisms is to decompose primary bile acids (PBAs) through the liver-gut circulation of bile acids, and then convert them into secondary bile acids (SBAs) which are more meaningful for human health [
15]. The microbial metabolism of bile acids increases the diversity of bile acids and the hydrophobicity of bile acid pools, which is conducive to the fecal excretion of bile acids. The mass of SBAs produced by intestinal flora is about 5% of the total bile acids [
33]. Most bile acids (90%) are reabsorbed at the end of the ileum via the apical membrane Na dependent bile salt transporter (ASBT). The PBAs that are not reabsorbed by ASBT enter the colon and are metabolized into SBAs by 7-dehydroxylation under the action of
Firmicutes in the intestinal flora [
15]. In addition,
Firmicutes and
Bacteroidetes are also involved in the microbial transformation of bile acids, which produce oxygen- (or keto-) bile acids under the action of these bacteria containing hydroxysteroid dehydrogenase (HSDHs) [
15,
33]. From the results of this study, the fecal contents of bile acids, especially SBAs, increased from 6-month-old, which accompanied with the increasing of the abundances of
Firmicutes and
Bacteroidetes in intestinal flora after neonate period (seen Figs.
3 and
5). Bile acids have endocrine functions and participate in the regulation of host metabolism, especially fat metabolism [
15]. Therefore, bile acid composition is of great significance to human health. The crucial relationships between microbial bile acid metabolism and inflammatory bowel disease (IBD, i.e., ulcerative colitis and Crohn’s Disease), liver cirrhosis, liver cancer, irritable bowel syndrome, jejunal syndrome, obesity and other diseases have been confirmed [
15,
34]. As seen in Fig.
5, the concentrations of fecal PBAs had no significant change from birth to 6 years old, while the fecal SBAs kept in a lower level during neonatal period and began to increase from 6 months old. Because the production of SBAs was more depended on the composition of gut microbiota, the content of fecal SBAs in infants was also according to the development of intestinal flora. At the same time, the time when SBAs content changed significantly and the change trend with age was consistent with the change of SCFAs content (Figs.
5 and
6). About 6 months after birth was a key time point for the increase of SBAs and SCFAs.
However, this study also has several limitations. First and most important, since this study is not a prospective cohort study, the effects of possible confounding factors such as mode of production (natural delivery, cesarean delivery), feeding mode (breastfeeding, milk powder feeding or mixed feeding), dietary conditions (dietary fiber, dietary culture or food habits) on the development of children’s intestinal flora were not considered, the results of this study may have conclusion deviation. Second, because the participants involved in the study were not the same group of children who were followed up from birth to preschool, but nine groups of participants from different ages, the research results could not directly reflect the dynamic changes of intestinal flora development in the growth process from newborns to preschool children, but only reflected the differences in the composition of intestinal flora of subjects at different ages. In order to get more accurate conclusions, in the follow-up study, the subjects need to be followed up longitudinally to obtain continuous data, and then the dynamic development and changes of intestinal flora in the growth and development of preschool children are confirmed through prospective longitudinal study. As well, in the process of the research, factors such as delivery mode, feeding mode, dietary structure and changes in health status should be included in the scope of research, so as to analyze whether the development and changes of intestinal flora are affected by these factors.
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