Background
Although there is evidence that the prevalence of childhood obesity is stabilising at different levels in different countries[
1], the number of children and adolescents being overweight or obese is still dramatically high[
2,
3]. The major concern is that these children are at high risk of developing severe co-morbidities such as metabolic syndrome, non-alcohol fatty liver disease, type 2 diabetes mellitus and premature cardiovascular diseases[
4,
5]. Moreover, obese children are highly prone to become obese adults, especially when having a high body mass index (BMI) or an obese parent[
6,
7]. In order to combat childhood obesity and related complications, prevention is crucial. At the moment, the most important strategies to manage childhood obesity are therapeutic lifestyle changes, such as changing dietary habits and the physical activity level. However, these are often difficult to achieve. When lifestyle modifications continue to fail, pharmacological interventions and possibly bariatric surgery could be considered.
Nowadays, it is generally accepted that the development of obesity is caused by gene-environment interactions, generating a chronic positive energy balance[
8]. However, physiological and environmental predispositions underlying obesity and associated metabolic disorders are still underexplored. Recent evidence suggests that our gut microbiota is involved in energy regulation as well as inflammation[
9], and should therefore be considered as an environmental factor playing a role in the pathophysiology of obesity[
10,
11]. Although energy intake can affect the gut microbiota composition[
12], it is still unclear whether the gut microbiota play a causal role in the development of obesity in humans.
So far, several studies in humans and mice have shown differences in gut microbiota composition between obese and lean subjects. These differences were mostly detected at the phylum level of mainly Firmicutes and Bacteroidetes[
11‐
14]. Obesity in humans has already been associated with low intestinal concentrations of Bacteroidetes and high concentrations of Firmicutes, although this finding has been contradicted by other studies[
15,
16]. Only few studies have investigated the prevalence of faecal bacterial phyla in obese children and adolescents. One study demonstrated low concentrations of Bacteroidetes and high concentrations of Firmicutes in the distal gut of obese adolescents living in Spain[
17]. Another study in Sweden, did not find significant differences in the concentrations of
Bacteroides fragilis group,
Lactobacillus spp. and
Bifidobacterium spp. between preschool children with excessive body weight and normal-weight children[
18]. By contrast, Vael et al.[
19] demonstrated that a high intestinal concentration of
Bacteroides fragilis group present in early infancy was associated with a higher risk of obesity later in life. In general, limited and contradictory findings with regard to the composition of the gut microbiota in obese children indicate that further in-depth analysis of the role of the intestinal microbiota in childhood obesity is warranted.
The principal aim of this study is to evaluate and compare the presence of certain gut bacterial species in faecal samples of obese and lean children and adolescents. Quantitative culturing was used to identify and determine the concentrations of the following bacterial genera: Bacteroides fragilis group, Bifidobacterium, Clostridium, Staphylococcus and Lactobacillus. In addition to quantitative culturing, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) was used for in-depth analysis of species belonging to the Bacteroides fragilis group. Quantitative real-time polymerase chain reaction (qPCR) was applied to quantify Bacteroides-Prevotella-Porphyromonas spp., Bifidobacterium spp., Clostridium coccoides-Eubacterium rectale group, Clostridium leptum group, Staphylococcus spp. and Lactobacillus spp. The Firmicutes-to-Bacteroidetes ratio was calculated based on the qPCR results. Finally, analysed gut bacterial species were associated with dietary compounds and energy intake, which were assessed by dietary records. Moreover, concentrations of biochemical blood parameters were measured in overweight and obese subjects.
Discussion
At birth, the gut microbiota of an infant is sterile but rapidly assembles over days or months[
20]. Mode of delivery (natural delivery versus caesarean section) and feeding method (breast feeding versus bottle feeding) have an early impact on the development of a child’s gut microbiome[
21]. At the age of four, the gut microbiota is fully mature[
22]. Eventually, each person develops a unique gut microbiota which is stable over time in healthy adults[
23].
In this cross-sectional study, the obese gut microbiota composition was compared with that of a lean one. We focused on two major phyla Bacteroidetes and Firmicutes, next to the
Bacteroides fragilis group,
Bifidobacterium, Clostridium,
Staphylococcus and
Lactobacillus. Different bacterial groups were selected according to the frequency to which they have been described in relevant literature[
17‐
19,
24] and the ease of detection by the techniques used. On the one hand, quantitative plating was used as the ‘gold standard’ technique to isolate and characterise the selected bacterial groups. However, only 10 to 50% of all bacteria associated with the human body can be cultivated successfully[
23,
25]. Subsequently, high-throughput culture-independent techniques, which use DNA sequences encoding the 16S ribosomal RNA subunit, were applied in order to assign an organism to a phylogenetic classification more accurately[
25].
To our knowledge, our study was the first to perform an in-depth analysis of species belonging to the Bacteroides fragilis group by means of MALDI-TOF MS. Overall, our results reveal a high Firmicutes-to-Bacteroidetes ratio in faeces of obese children including alterations at species level.
Selective media have been used successfully to identify and enumerate
Bacteroides fragilis group from human faeces[
26]. For the first time, a further in-depth analysis of species of the
Bacteroides fragilis group revealed reduced relative proportions of
B. vulgatus in obese children and adolescents. One study reported decreased relative proportions of
B. vulgatus in the faeces of type 2 diabetic subjects using species specific PCR-denaturing gradient gel electrophoresis (DGGE)[
27].
B. vulgatus was found to constitute a part of the core gut microbiota in healthy humans and is generally considered to be a beneficial gut commensal[
28]. These findings point towards a possible role for
B. vulgatus in the pathophysiology of Western diseases, such as obesity and diabetes.
Moreover, the qPCR method that was used in this study to detect and quantify Bacteroidetes (
Bacteroides-Prevotella-Porphyromonas spp.)
, Firmicutes (
Clostridium coccoides-Eubacterium rectale group
, Clostridium leptum group
, Staphylococcus spp. and
Lactobacillus spp.), and
Bifidobacterium spp. in human faeces has already been thoroughly evaluated and validated[
29‐
31]. In agreement with the findings of previous studies[
32,
33], we describe higher concentrations of
Lactobacillus spp. in the obese gut microbiota. However, the use of quantitative plating did not permit the detection of a significantly higher concentration of
Lactobacillus spp. in faeces of obese children, which we did see using qPCR. A possible explanation is that
L. gasseri and
L. acidophilus could not be identified in culture due to the presence of vancomycin in the LAMVAB medium[
34]. Nevertheless, both quantitative culturing and qPCR resulted in a similar proportion of
Lactobacillus spp. in the obese gut microbiota. A study conducted by Million et al.[
32], demonstrated that
Lactobacillus reuteri was associated with obesity in adults. By contrast, Santacruz et al.[
33] showed that BMI SDS reduction in obese adolescents led to a concomitant increase in the concentrations of
Lactobacillus spp. These findings thus suggest a possible role of
Lactobacillus at species level in body weight and obesity. Additionally, we showed that the concentration of
Lactobacillus spp. is positively correlated to plasma hs-CRP levels in obese children and adolescents. An increased prevalence of positive Firmicutes to higher levels of plasma hs-CRP was also seen in a study conducted in 51 obese and 28 normal-weight children and adults[
35]. These results seems therefore to suggest a possible role for
Lactobacillus spp. in “low-grade” inflammation, a major pathophysiological process of obesity.
Interestingly, we detected an elevated Firmicutes-to-Bacteroidetes ratio in the gut microbiota of obese children and adolescents. Previous investigators also showed significant associations between this ratio and obesity in mice and humans[
11‐
14]. The results of our study are similar to a study in Spanish children, demonstrating increased concentrations of Firmicutes and decreased concentrations of Bacteroidetes in the obese gut[
17]. Contrary to these findings, other studies described no or even opposite differences in the Firmicutes-to-Bacteroidetes ratio between obese and lean subjects[
15,
16]. Possibly, these variations in study outcome are related to the fact that different methodologies were applied in these studies.
To further elucidate the complex role of gut microbiota in host physiology, a more thorough examination of the influence of diet on gut microbiota is recommended. In order to do so, we analysed the relationship between the presence of certain gut bacterial species with dietary compounds and energy intake. Here, we demonstrate that, independent of the BMI status, children and adolescents with a high energy intake (expressed in kcal/d) possess high faecal concentrations of
Staphylococcus spp. analysed by quantitative culture. Note that the regression coefficient β of energy intake is low in all cases. This is due to the fact that values of energy intake are expressed in kcal/d. Given the range of energy intake (1635.53 to 2669.64 kcal), results in effect on mean concentration of
Staphylococcus spp. of 1.27 to 2.08 are obtained. These results are not negligible and a real significant association has been detected. However, caution must be taken when translating these findings into a biological meaningful interpretation. Hence, more detailed research on this topic is necessary. Nevertheless, the importance of
Staphylococcus spp. in childhood obesity has already been demonstrated by Kalliomaki et al.[
24] who showed that a greater faecal concentration of
Staphylococcus spp. during infancy predicted the development of overweight during childhood. A possible role of
Staphylococcus spp. in energy harvesting during childhood is thus suggested.
One major limitation of the current study is the small sample size and therefore these results should be interpreted with caution. In addition, pregnancy related factors, social status, and the period of being obese prior to inclusion were not taken into account.
Further longitudinal research on the cause-effect relationship between gut microbiota and obesity is highly justified, since different bacterial species could play a significant role in the human energy harvest and weight regulation. Moreover, consideration of lifestyle factors in gut microbiota studies is highly recommended, since changes in dietary pattern and physical activity could influence gut microbiota composition and the development of obesity. Finally, we suggest to focus future research not only on the elucidation of gut microbiota composition in obese subjects, but also on the study of gut metabolites, i.e. “metabolomics”. This suggestion for future research aims at expanding our knowledge on the complex interplay between gut microbiota, energy homeostasis and obesity.
In the future, modification of the gut microbiota composition by the administration of pro-, pre- or synbiotics in early childhood could offer an opportunity to prevent and/or treat obesity[
36]. However, additional research is required.
Competing interests
All authors declare that there are no competing financial interests in relation to the work described.
Authors’ contributions
KD, CV, HG and VVK conceived and designed the study. LB, KVH, IK and CVN collected the data. LB carried out experiments. LB, IK, CVN and NH statistically analysed the data. LB, KVH, NH and VVK interpreted the data. LB, IK and CVN did literature research. LB generated figures and tables. LB wrote the manuscript with help of KVH and VVK. KVH, NH, KD, CV, HG and VVK revised the paper. LB had full access to all of the data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis. All authors had final approval of the submitted and published versions.