BioMed Research International

BioMed Research International / 2020 / Article

Research Article | Open Access

Volume 2020 |Article ID 6587136 | https://doi.org/10.1155/2020/6587136

Saeam Shin, Ky Young Cho, "Altered Gut Microbiota and Shift in Bacteroidetes between Young Obese and Normal-Weight Korean Children: A Cross-Sectional Observational Study", BioMed Research International, vol. 2020, Article ID 6587136, 19 pages, 2020. https://doi.org/10.1155/2020/6587136

Altered Gut Microbiota and Shift in Bacteroidetes between Young Obese and Normal-Weight Korean Children: A Cross-Sectional Observational Study

Academic Editor: Flavia Prodam
Received20 Jan 2020
Revised26 Jun 2020
Accepted03 Jul 2020
Published18 Aug 2020

Abstract

Emerging data suggest that the gut microbiome is related to the pathophysiology of obesity. This study is aimed at characterizing the gut microbiota composition between obese and normal-weight Korean children aged 5-13. We collected fecal samples from 22 obese and 24 normal-weight children and performed 16S rRNA gene sequencing using the Illumina MiSeq platform. The relative abundance of the phylum Bacteroidetes was lower in the obese group than in the normal-weight group and showed a significant negative correlation with BMI -score. Linear discriminative analysis (LDA) coupled with effect size measurement (LEfSe) analysis also revealed that the Bacteroidetes population drove the divergence between the groups. There was no difference in alpha diversity, but beta diversity was significantly different between the normal-weight and obese groups. The gut microbial community was linked to BMI -score; blood biomarkers associated with inflammation and metabolic syndrome; and dietary intakes of niacin, sodium, vitamin B6, and fat. The gut microbiota of the obese group showed more clustering of genera than that of the normal-weight group. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis revealed that the functions related to carbohydrate and lipid metabolism in the microbiota were more enriched in the normal-weight group than in the obese group. Our data may contribute to the understanding of the gut microbial structure of young Korean children in relation to obesity. These findings suggest that Bacteroidetes may be a potential therapeutic target in pediatric obesity.

1. Introduction

Childhood obesity is a major public health concern worldwide [1]. Obese children have a high risk of developing adult obesity and obesity-related comorbidities, including type 2 diabetes mellitus, cardiovascular disease, and psychological problems [2, 3]. Recently, accumulating evidence suggests that the human gut microbiota is associated with many chronic diseases, including obesity. Previous studies in adults have demonstrated that obese individuals have different microbial compositions than lean individuals [4, 5]. The gut microbiota is closely related to energy harvest and metabolism in humans [6, 7]. Therefore, microbiota-targeted strategies have attracted much attention in the context of obesity treatment [8, 9].

Studies on the obesity-related microbiota in children are still scarce. Gut microbial composition is known to vary with age, ethnicity, and diet [6, 10]. Although previous studies have suggested that the gut microbiota of infants is converted into adult-like composition in the first 1-3 years [11], some evidence indicates that the microbiota continues to change until adolescence [12]. Therefore, valuable information on the obesity-related microbiota could be obtained from different ethnic and age groups.

The objective of this study was to characterize the composition of the gut microbiota among obese and normal-weight Korean children. Amplicons of the 16S rRNA gene were sequenced using Illumina MiSeq to analyze the composition of the gut microbiota. The dietary and lifestyle patterns of the participants and the levels of blood biochemical markers related to inflammation and metabolic disease were measured and examined in relation to the gut microbiota composition. The correlation structure of the microbiota was shown using network analysis, and predictive functional differences between groups were identified by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis.

2. Materials and Methods

2.1. Subjects, Questionnaire, and Anthropometric Measurements

Forty-six children who were 5-13 years of age were enrolled in the study at Hallym University Kangnam Sacred Heart Hospital from December 2017 to March 2018 (NCT03388411). This study protocol was approved by the Institutional Review Board of the Hallym University Kangnam Sacred Heart Hospital (2017-09-015). Written informed consent was obtained from all subjects of the study and their parents, in accordance with the Declaration of Helsinki. Based on the 2017 Korean growth chart [13], subjects with a were classified as the obese group, and subjects with were classified as the normal-weight group. For a month prior to the stool sampling, none of the subjects had taken antibiotics/probiotics/steroids or had diarrhea. None of the subjects had acute infections or chronic disease. Subjects completed questionnaires on lifestyle, bowel habits, and dietary intake and submitted them at the hospital visit. Anthropometric measurements, including height, weight, waist circumference, midarm circumference, hip circumference, thigh circumference, and blood pressure, were performed by professionally trained personnel [14, 15]. Body composition analysis including skeletal muscle mass and total body fat content was measured using the inBody 770 analyzer (Biospace Co. Ltd., Seoul, Korea).

2.2. Dietary Assessments

The children and their parents received specific training from a dietitian to describe in a proper way all the foods and the quantities consumed, including the name/brand of the consumed food, recipes of dishes, method of preparation or cooking, and portion sizes. After training with the dietitian, the participants filled out everything the subjects ate and drank for 3 days: 2 weekdays and 1 weekend day. Using data from the dietary records, nutrient intakes were calculated by a dietitian using the Computer-Aided Analysis Program 4.0 for professionals (CAN-pro 4.0, Korean Nutrition Society, Seoul, Korea).

2.3. Blood Sampling and Biochemical Analysis

After a 12-hour overnight fast, blood samples were taken from the subjects. The levels of glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), insulin, total cholesterol, triglycerides, high-density lipoprotein- (HDL-) cholesterol, low-density lipoprotein- (LDL-) cholesterol, high-sensitivity C-reactive protein (hs-CRP), uric acid, iron, and unsaturated iron binding capacity (UIBC) were measured using a Hitachi 7600 autoanalyzer (Hitachi, Tokyo, Japan). Total iron binding capacity (TIBC) was calculated as the sum of the serum iron and UIBC levels. Transferrin saturation (Tf%) was calculated as . Concentrations of ferritin, insulin, and 25-OH vitamin D were determined using an ADVIA Centaur XP (Siemens Healthcare Diagnostics, Deerfield, IL, USA). The complete blood count was analyzed by an ADVIA 2120i (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). The neutrophil-to-lymphocyte ratio (NLR) was calculated as the ratio of the neutrophil count to the lymphocyte count. The hemoglobin A1c (HbA1c) level was determined using a D-100 system (Bio-Rad Laboratories, Hercules, CA, USA). Insulin resistance and beta-cell function were evaluated by the homeostasis model assessment methods (HOMA-IR and HOMA-%B, respectively). HOMA-IR was calculated as , and HOMA-%B was calculated as .

2.4. Stool Sampling, Bacterial DNA Extraction, Illumina MiSeq Sequencing, and Bioinformatics

The stool samples were collected in sterile containers and immediately frozen at -80°C until DNA extraction. DNA was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Using 2 μL of the extracted DNA, polymerase chain reaction (PCR) amplifications were performed with the primers targeting the V3 to V4 regions of the 16S rRNA gene [16]. The products were then amplified by the second PCR with index primers. Equal concentrations of amplicons were pooled together and purified using an AMPure bead kit (Agencourt Bioscience, Beverly, MA, USA). The product size and quality were assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Sequencing was carried out at ChunLab, Inc. (Seoul, Korea) using the Illumina MiSeq platform (Illumina, San Diego, CA, USA). The raw reads were checked for quality, and low-quality reads () were filtered by a Trimmomatic tool (version 0.32). Then, the paired-end sequences were merged using a PANDASeq Paired-end Assembler [17]. Chimeric sequences were removed using the UCHIME algorithm [18]. The taxonomic classification of each read was performed based on the EzBioCloud database (http://eztaxon-e.ezbiocloud.net) [19]. Sequences that corresponded to the reference sequence with greater than 97% similarity in EzBioCloud were considered to be identified at the species level. To compare the operational taxonomic units (OTUs) between samples, we determined shared OTUs through the EzBioCloud program (Chunlab Inc.). The functional potential of the microbiota was inferred using PICRUSt metagenomics prediction [20] and was categorized into levels 1-3 based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [21].

2.5. Statistical Analysis

All statistical analyses were carried out using R software (version 3.5.2, http://www.r-project.org/). Based on the Shapiro-Wilk normality test, data are presented as the means and standard deviations (for continuous variables with normal distribution) or medians and interquartile ranges (for continuous variables with skewed distribution). Categorical variables are expressed as frequencies and percentages. In analyzing the characteristics, nutrient intake, and blood biochemical profiles of the participants, the -test or the Kruskal-Wallis Rank Sum test was used according to the results of the normality test. For comparison of the gut microbiota between groups, the Mann-Whitney test was used for continuous variables, and chi-square was used for categorical variables. Correlations between continuous variables were calculated using the Pearson correlation test. All analyses were performed after normalizing for the copy number of the bacterial 16S rRNA gene. The ACE index, the number of observed OTUs, the Chao1 richness estimate, and the Jackknife estimate were used to compare gut microbiota richness between samples. The within sample (alpha) diversity was compared using the Simpson diversity index, the Shannon index, and phylogenetic diversity. Cluster analysis was performed with nonmetric multidimensional scaling (NMDS), after computing the Bray-Curtis dissimilarity between each pair of individuals. The Fast UniFrac analysis was used to calculate the (beta) diversity between groups and was visualized with a principal coordinate analysis (PCoA). Differences in beta diversity between the normal-weight and obese groups were tested with nonparametric analysis of variance based on 999 permutations (permutational multivariate analysis of variance (PERMANOVA)). The differently abundant bacterial taxa between the normal-weight and obese groups were identified using the linear discriminant analysis (LDA) coupled with effect size measurement (LEfSe) method [22]. To analyze the ability of specific taxa to predict obesity, we calculated the area under the receiver operating characteristic curve (AUC of ROC). Multivariate analysis for relationships between gut microbial community composition, BMI -score, and blood biochemical markers was performed using canonical correspondence analysis (CCA). Using the R package qgraph [23], we performed a network analysis to identify the difference in the correlation network of the gut microbiota between the normal-weight and obese groups. To obtain the relative robustness, the sample coverage threshold for the identified genera was set at ≥0.5. The resulting values were adjusted for multiple testing with the false discovery rate (FDR) method [24]. values ≤ 0.05 were considered statistically significant.

3. Results

3.1. Participant Characteristics, Lifestyle Questionnaire, and Blood Biochemical Marker

A total of forty-six children were enrolled in this study (obese group: ; normal-weight group: ). The characteristics of the participants are summarized in Table 1. The sex distribution showed no difference between the normal-weight and obese groups (; Table 1). The individuals in the normal-weight group were slightly younger than those in the obese group (; Table 1). All anthropometric measurements, including height (), weight (), BMI (), BMI -score (), waist circumference (), midarm circumference (), hip circumference (), and thigh circumference (), showed significantly higher values in the obese group than in the normal-weight group (Table 1). Systolic/diastolic blood pressure (/), skeletal muscle mass (), and total body fat content () also showed significantly higher values in the obese group than in the normal-weight group (Table 1). The percentage of children born by cesarean section was significantly higher in the obese group (; Table 1). Questionnaires on lifestyle patterns differed between the normal-weight and obese groups. The percentage of children who did not exercise at all was 36.4% in the obese group and 0% in the normal-weight group (; Table 1). The percentage of children who exercised for 30 minutes or more per day was significantly higher in the normal-weight group than in the obese group (; Table 1). The percentage of children who watched television or used electronic devices (video games, smart phones, or computers) more than two hours a day was significantly higher in the obese group than in the normal-weight group (; Table 1). The percentage of children who used electronic devices near bedtime tended to be higher in the obese group than in the normal-weight group (; Table 1).


Normal-weight ()Obese ()

Sex (male, %)18 (75.0%)15 (68.2%)0.853
Age (years)0.048
Anthropometric measurements
 Weight (kg)27.3 (24.9; 30.1)46.4 (42.0; 52.9)<0.001
 Weight (-score)<0.001
 Height (cm)0.002
 Height (-score)0.035
 BMI (kg/m2)16.6 (15.5; 17.6)24.4 (22.9; 25.7)<0.001
 BMI (-score)0.6 (0.4; 0.9)2.5 (2.1; 2.8)<0.001
 Systolic blood pressure98.5 (90.0; 111.5)110.0 (100.0; 120.0)0.002
 Diastolic blood pressure60.0 (60.0; 63.5)70.0 (60.0; 70.0)<0.001
 Waist circumference (cm)58.5 (55.5; 61.8)77.8 (74.0; 81.5)<0.001
 Waist-to-height ratio<0.001
 Midarm circumference (cm)<0.001
 Hip circumference (cm)<0.001
 Thigh circumference (cm)39.0 (35.6; 41.1)46.0 (44.5; 51.5)<0.001
 Total body fat content (%)22.1 (19.6; 26.4)39.2 (33.8; 40.2)<0.001
 Skeletal muscle mass (kg)10.2 (9.3; 11.3)16.4 (13.9; 19.1)<0.001
Delivery type0.012
 Vaginal : cesarean18 (75.0%) : 6 (25.0%)7 (33.3%) : 14 (66.7%)
Lifestyle pattern
 Study time after school0.094
  ≤1 hour7 (29.2%)1 (5.0%)
  >1 hour17 (70.8%)19 (95.0%)
 Exercise during the day0.004
  Yes24 (100.0%)14 (63.6%)
  No0 (0.0%)8 (36.4%)
 Exercise time during the day0.025
  ≤30 min2 (8.3%)9 (40.9%)
  >30 min22 (91.7%)13 (59.1%)
 Exercise time during the day0.219
  ≤1 hour11 (45.8%)15 (68.2%)
  >1 hour13 (54.2%)7 (31.8%)
 Time spent watching TV or using electronic devices0.017
  ≤2 hours20 (83.3%)10 (45.5%)
  >2 hours4 (16.7%)12 (54.5%)
 Use electronic device for more than an hour before sleeping0.072
  Yes4 (16.7%)10 (45.5%)
  No20 (83.3%)12 (54.5%)

Data are expressed as the , median (interquartile range), or a number (%). Significant values are shown in bold. Abbreviations: AST—aspartate aminotransferase.

Analysis of energy and nutrient intakes from three-day dietary records showed significant differences in total energy (), protein (), fat (), carbohydrate (8), cholesterol (), total fatty acid (), polyunsaturated fatty acid (), trace mineral (phosphorus, iron, sodium, potassium, and zinc ()), and vitamin (thiamine, niacin, vitamin B6, and vitamin E ()) intakes between the normal-weight and obese groups (Table 2).


Normal-weight ()Obese ()

Energy (kCal)<0.001
Protein (g)<0.001
Fat (g)0.001
Carbohydrates (g)0.028
Ca (mg)0.801
P (mg)0.011
Iron (mg)0.002
Na (mg)<0.001
K (mg)0.039
Zinc (mg)0.001
Folic acid (μg)0.767
β-Carotene (μg)2430.8 (1691.5; 3646.6)3350.5 (1979.1; 5071.1)0.260
Retinol (μg)100.1 (69.1; 199.6)157.5 (97.3; 305.2)0.104
Thiamine (mg)0.002
Riboflavin (mg)0.296
Niacin (mg)<0.001
Vitamin B6 (mg)1.0 (0.9; 1.4)1.6 (1.3; 2.0)0.001
Vitamin C (mg)95.4 (40.3; 226.9)87.6 (52.2; 123.5)0.532
Vitamin D (μg)2.6 (1.5; 6.0)3.1 (2.0; 4.9)0.891
Vitamin E (mg)0.004
Fiber (g)15.7 (11.8; 19.2)16.9 (16.0; 18.8)0.191
Cholesterol (mg)0.002
Total fatty acid (mg)0.010
Saturated fatty acid (mg)9.7 (6.6; 12.1)12.1 (6.0; 19.9)0.094
Polyunsaturated fatty acid (mg)0.001

Data are expressed as the , median (interquartile range), or a number (%). Significant values are shown in bold.

Among the measured blood biochemical markers, glucose (), ALT (), triglycerides (), LDL-cholesterol (), hs-CRP (), uric acid (), ferritin (), insulin (), HOMA-IR (), HOMA-%B (), mean platelet volume (MPV) (), white blood cell (WBC) count (), neutrophil percentage (), and NLR () were significantly higher in the obese group than in the normal-weight group (Table 3). On the other hand, HDL-cholesterol (), 25-OH vitamin D (), and lymphocyte percentage () were significantly lower in the obese group than in the normal-weight group (Table 3).


Normal weight ()Obese ()

Glucose (mg/dL)0.027
AST (IU/L)29.0 (25.0; 33.0)25.0 (22.0; 26.0)0.027
ALT (IU/L)13.5 (13.0; 19.0)19.5 (16.0; 29.0)0.018
Total cholesterol (mg/dL)0.165
Triglycerides (mg/dL)47.5 (43.0; 67.0)81.0 (61.0; 132.0)0.003
HDL-cholesterol (mg/dL)0.025
LDL-cholesterol (mg/dL)0.021
hs-CRP (mg/L)0.3 (0.2; 0.4)2.1 (0.7; 3.1)<0.001
Uric acid (mg/L)0.032
Iron (μg/dL)0.353
TIBC (μg/dL)0.115
Transferrin saturation (%)0.212
25-OH vitamin D (ng/mL)0.002
Ferritin (ng/mL)<0.001
Insulin (μU/mL)4.9 (3.8; 6.8)13.8 (9.6; 18.6)<0.001
HOMA-IR1.2 (0.9; 1.8)3.5 (2.4; 4.4)<0.001
HOMA-%B51.5 (43.0; 66.4)122.4 (98.8; 172.9)<0.001
Hemoglobin (g/dL)0.955
HbA1c (%)5.2 (5.1; 5.5)5.3 (5.2; 5.5)0.188
Platelet count (×103/μL)0.567
MPV (fL)7.2 (6.7; 7.3)7.4 (7.1; 8.0)0.017
PDW (%)43.0 (41.2; 51.2)48.0 (44.2; 50.4)0.115
WBC count (×103/μL)0.039
Neutrophil count0.003
Neutrophil (%)0.015
Lymphocyte count (×103/μL)0.911
Lymphocyte (%)0.047
Monocyte count (×103/μL)0.519
Eosinophil count (×103/μL)0.853
Basophil count (×103/μL)0.0 (0.0; 0.0)0.0 (0.0; 0.0)0.474
Neutrophil-to-lymphocyte ratio0.8 (0.7; 1.2)1.2 (1.0; 1.7)0.024

Data are expressed as the , median (interquartile range), or a number (%). Significant values are shown in bold. Abbreviations: AST—aspartate aminotransferase; ALT—alanine aminotransferase; HDL—high-density lipoprotein; LDL—low-density lipoprotein; hs-CRP—high-sensitivity C-reactive protein; TIBC—total iron binding capacity; MPV—mean platelet volume; PDW—platelet distribution width; WBC—white blood cell.
3.2. Gut Microbial Composition and Diversity

After filtering low-quality, nontarget, and chimeric amplicons, 16S rRNA gene sequencing resulted in a total of 3.9 million high-quality reads from 46 fecal samples. The median sequencing read was 78,155 (quartiles: 69,929; 85,731). We obtained a median value of 412.5 OTUs per sample (316; 529.3) after excluding low-abundance OTUs (<1% of total).

At the phylum level, the predominant bacterial taxa were Firmicutes and Bacteroidetes, followed by Actinobacteria and Proteobacteria in both groups (Figures 1(a) and 1(b)). The relative abundance of the phylum Bacteroidetes was significantly decreased in obese children (obese group: median 36.6 (0.3; 52.9); normal-weight group: 45.2 (10.5; 69.1)) (; Figure 1(c)). The relative abundance of Bacteroidetes was significantly negatively correlated with BMI-score (Figure 1(d)). The ROC analysis of Bacteroidetes for predicting obesity showed good performance (AUC: 0.7443; 95% confidence interval (CI): 0.603-0.8856), unlike the analysis of Firmicutes (AUC: 0.5606; 95% CI: 0.3839-0.7373) (Figure 1(e)). Multivariate regression analysis showed that the log-transformed BMI -score and the log-transformed relative abundance of major taxa belonging to the phylum Bacteroidetes were negatively associated after adjusting for age, sex, and delivery type (, ; Figure 1(f)). In the stepwise logistic regression model, the odds ratio for risk of obesity associated with Bacteroidetes at the phylum level was 0.87 after adjusting for age, sex, and delivery type (95% CI: 0.80-0.95; ) (Table 4).


LevelTaxaAdjusted odds ratio95% CI

PhylumBacteroidetes0.870.80-0.950.003
ClassBacteroidia0.870.80-0.950.003
OrderBacteroidales0.870.80-0.950.003
FamilyBacteroidaceae0.900.84-0.970.004
GenusBacteroides0.900.84-0.970.004

Adjusted for age, sex, and delivery type. Abbreviation: CI—confidence interval.

There was no significant difference in the relative abundance of Firmicutes (obese: 53.1 (45.1; 73.3); normal-weight: 45.7 (24.7; 76.6)), Actinobacteria (obese: 1.28 (0.56; 10.55); normal-weight: 0.89 (0.31; 2.99)), or Proteobacteria (obese: 8.5 (1.41; 15.0); normal-weight: 5.34 (1.72; 11.12)) (, , and , respectively; Supplementary Figures S1A, S1B, and S1C). The Firmicutes-to-Bacteroidetes (F : B) ratio was significantly elevated in obese children (obese: 1.5 (0.9; 18.4); normal-weight: 1.1 (0.4; 2.9)) (; Supplementary Figure S1D). Among family-level taxa, the relative abundance of Lachnospiraceae was significantly higher in the obese group than in the normal-weight group (obese: 13.49 (10.97; 18.24); normal-weight: 9.9 (7.52; 12.94)) (; Supplementary Figure S2A). Among taxa at the species level, the abundance of Bacteroides ovatus was significantly lower in the obese group than in the normal-weight group (obese: 0.07 (0.02; 1.04); normal-weight: 1.04 (0.67; 2.8)) (; Figure S2B). There was no difference in the abundance of the genus Akkermansia between the groups (obese: 0.01 (0; 0.05); normal-weight: 0.14 (0; 1.27)) (; Supplementary Figure S2C).

We analyzed the correlation between BMI -score and the relative abundance of bacterial taxa (Table 5). The relative abundance of the phylum Bacteroidetes was negatively correlated with BMI -score (Pearson’s correlation coefficient , ; Figure 1(d)). Furthermore, BMI -score was negatively correlated with the abundance of Bacteroidia at the class level; Bacteroidales at the order level; and Bacteroidaceae, Devosia_f, Leptotrichiaceae, Odoribacteraceae, Porphyromonadaceae, Rikenellaceae, and Staphylococcaceae at the family level (Table 5). The relative abundance of the family Lachnospiraceae was positively correlated with BMI -score (; Table 5). The relative abundance of the phylum Firmicutes was not significantly correlated with BMI -score (, ).


LevelTaxa

PhylumBacteroidetes-0.3290.026
ClassBacteroidia-0.3290.026
OrderBacteroidales-0.3290.026
FamilyBacteroidaceae-0.3540.016
Devosia_f-0.4050.005
Lachnospiraceae0.2910.05
Leptotrichiaceae-0.3140.034
Odoribacteraceae-0.3170.032
Porphyromonadaceae-0.3020.041
Rikenellaceae-0.3770.01
Staphylococcaceae-0.3110.035
AB559589_g0.3040.04
Acetatifactor0.3530.016
Acetitomaculum0.3510.017
Acidaminococcus0.2980.044
Alistipes-0.3770.01
Anaerobium0.4550.001
Anaerofilum-0.4680.001
Anaerotruncus-0.380.009
Bacillus-0.2940.047
Bacteroides-0.3540.016
Brevundimonas-0.3430.019
Catenibacterium0.3150.033
Christensenellaceae_uc-0.3240.028
Coprobacter-0.2920.049
Desulfovibrio_g3-0.3950.007
Devosia-0.4050.005
Dielma-0.3550.015
EF404788_g-0.3640.013
Eubacterium_g21-0.3130.034
FJ881296_g0.3630.013
FN436026_g-0.3080.037
GL872355_g0.2940.048
Holdemania-0.3250.027
Hydrogenoanaerobacterium-0.3530.016
JPZU_g-0.2950.047
KE159600_g-0.3470.018
Rikenellaceae_uc-0.3940.007
Senegalimassilia0.3550.016
Staphylococcus-0.3110.035
SpeciesAB506430_s-0.3150.033
ACWW_s-0.4880.001
AF371599_s0.3840.009
AM500802_g_uc-0.3130.034
AY986255_s-0.3050.039
Allisonella histaminiformans0.3140.034
Anaerobium_uc0.3890.008
Anaerotruncus colihominis-0.3720.011
Atopostipes suicloacalis-0.330.025
BCAB_s-0.3040.04
Bacteroides finegoldii-0.3770.01
Bacteroides oleiciplenus-0.3220.029
Bacteroides ovatus-0.3220.029
Bacteroides uniformis-0.3450.019
Butyricicoccus_uc0.3230.029
Caproiciproducens_uc-0.340.021
Catenibacterium mitsuokai0.3150.033
Clostridium_g12_uc0.3480.018
Clostridium_g6_uc0.3170.032
Corynebacterium pseudodiphtheriticum group0.3280.026
DQ805799_s-0.3410.02
DQ807741_s0.3520.017
DQ905770_s-0.3060.038
Desulfovibrio acrylicus group-0.3950.007
Dielma fastidiosa-0.3550.015
EF025278_g_uc0.370.011
EF400498_s-0.3460.018
EF401207_s0.3380.022
EF402071_s-0.3940.007
EF404788_s-0.3440.019
EF404944_s-0.3080.038
EF405506_s-0.3220.029
EF406456_s0.3630.013
EF640143_s0.3480.018
FJ368968_s-0.3630.013
FJ371693_s-0.3310.024
FJ505998_s-0.3610.014
FJ681675_s-0.3660.012
FJ825526_s0.3140.034
FJ880315_s0.3120.035
FN436026_s-0.3080.037
Fusobacterium hwasookii0.3040.04
Fusobacterium nucleatum group0.3050.039
Gordonibacter pamelaeae-0.3110.035
HM123979_g_uc0.3380.022
HM124219_s0.3310.025
HQ716480_s-0.3210.03
HQ789817_s-0.3710.011
HQ810970_s-0.340.021
JPZU_g_uc-0.3020.042
JRNC_s0.3050.039
KE159600_s-0.3470.018
Klebsiella oxytoca group0.3530.016
LARM_s-0.3030.041
Lachnoanaerobaculum orale group-0.3550.015
Megasphaera_uc0.3370.022
PAC000196_s-0.3890.008
PAC000740_s0.3230.029
PAC000748_s-0.3680.012
Parabacteroides_uc-0.3190.031
Pseudogracilibacillus_uc0.3220.029
Romboutsia sedimentorum0.3870.008
Roseburia_uc0.3860.008
Senegalimassilia anaerobia0.3550.016

Pearson’s correlation coefficient. Taxa with values > 0.05 were omitted.

Gut microbiota richness measures showed no obvious difference between samples from normal-weight and obese children (ACE, ; the number of observed OTUs, ; the Chao1 richness estimate, ; and the Jackknife estimate, ). In addition, several alpha diversity estimates, including the Simpson diversity index (), the Shannon index (), and phylogenetic diversity (), were not significantly different between normal-weight and obese children. NMDS analysis revealed separation and clustering of the obese group from the normal-weight group along the NMDS1 axis, while naïve tended to cluster along NMDS2 (Figure 1(g)). We used the Fast UniFrac analysis to measure beta diversity. The PCoA plot of the microbiota from all individuals in the normal-weight and obese groups is shown in Figure 1(h). The beta diversity showed a statistically significant difference between normal-weight and obese children at the genus level (, PERMANOVA on Fast UniFrac distances; Figure 1(h)).

3.3. Taxonomic Differences in the Microbiota between Obese and Normal-Weight Children

To identify the specific microbial profile distinguishing obese and normal-weight children, a metagenomics biomarker discovery approach using the LEfSe method was applied to assess the effect size of each differently abundant taxon (Figure 2). The LDA effect size values are shown in Figure 2(f). Using the LEfSe method, we found that Bacteroidetes at the phylum level; Bacteroidia at the class level; Bacteroidales at the order level; Bacteroidaceae, Porphyromonadaceae, and Rikenellaceae at the family level; and Bacteroides, EF404788_g, Desulfovibrio_g3, Anaerofilum, Alistipes, Bacteroidaceae_uc, Hydrogenoanaerobacterium, EF402988_g, Oscillibacter, and Citrobacter at the genus level were significantly enriched in the normal-weight group (; Figure 2). This population is dominated by bacteria belonging to the Bacteroidetes phyla in the normal-weight group. In addition, Actinomyces, Romboutsia, Weissella, and GL872355_g at the genus level were significantly enriched in the obese group (; Figure 2).

3.4. Relationship between Gut Microbial Community Composition, BMI -Score, Blood Biochemical Markers, and Dietary Intake

We generated a correlogram to visualize the degree of association between BMI -score, major phyla, blood biochemical markers, and dietary intake of energy and nutrients (Figure 3(a)). Variables with highly significant differences between the two groups ( value ≤ 0.1) were selected from the univariate analysis. Variables with a correlation coefficient () of 0.8 or greater were regarded as the same variables, and then one representative variable was selected from the same variables. The BMI -score showed a significant positive correlation with inflammatory markers, including HOMA-IR; neutrophil count; and serum levels of triglycerides, hs-CRP, and ferritin, and with increased dietary intake of calories, fat, niacin, vitamin B6, P, Na, and zinc (Figure 3(a)). On the other hand, serum vitamin D levels and the proportion of Bacteroidetes were negatively correlated with BMI -score (Figure 3(a)). The Actinobacteria population showed negative correlations with the Bacteroidetes and Proteobacteria populations and positive correlations with blood markers including hs-CRP and neutrophil count and with dietary intake of calories and fat (Figure 3(a)). The correlogram showed that Bacteroidetes and Firmicutes exhibited different orientations in regard to correlation with most of the variables, including blood biomarkers and dietary intakes (Figure 3(a)). The Bacteroidetes population showed a negative correlation with the Firmicutes population and a positive correlation with the Proteobacteria population (Figure 3(a)). Moreover, the proportion of Bacteroidetes was negatively correlated with inflammatory markers, including hs-CRP, ferritin, HOMA-IR, and neutrophil count, and with dietary intake of calories, fat, niacin, vitamin B6, P, Na, and zinc (Figure 3(a)). On the other hand, the Firmicutes population showed a negative correlation with the proportion of Proteobacteria and positive correlations with the inflammatory markers hs-CRP and neutrophil count and with dietary intake of calories, fat, niacin, vitamin B6, and Na (Figure 3(a)).

We performed CCA to visualize the relationship between gut microbial community composition, BMI -scores, and blood biochemical markers (Figure 3(b)) or dietary intake (Figure 3(c)). Variables were selected from the same standards with the correlogram. The distance between two points shows the significance of the correlation. The distance between Bacteroidetes and the microbial community in the normal-weight group is shorter than that between Firmicutes and the microbial community in the normal-weight group, suggesting that Bacteroidetes has a strong correlation with the normal-weight group (Figures 3(b) and 3(c)). The length of the blue line is proportional to the degree of importance. As shown in Figure 3(b), BMI -score, HOMA-IR, hs-CRP, ferritin, and neutrophil count are more important in the microbial community of the obese group, whereas vitamin D is an important factor in the microbial community of the normal-weight group. Figure 3(c) shows that fat, Na, and niacin among dietary components, in addition to BMI -score, are more important in the microbial community of the obese group, whereas vitamin B6 intake is an important factor in the microbial community of the normal-weight group. Firmicutes was negatively correlated with serum vitamin D levels and dietary intake of vitamin B6 (Figures 3(b) and 3(c)). Bacteroidetes was negatively correlated with BMI -score, serum ferritin level, and fat intake (Figures 3(b) and 3(c)). Actinobacteria showed a negative correlation with HOMA-IR and dietary intake of Zn, P, niacin, and Na (Figures 3(b) and 3(c)). Proteobacteria was negatively correlated with neutrophil count and hs-CRP (Figures 3(b) and 3(c)).

3.5. Correlation Network

We performed a correlation network analysis to investigate whether obesity was associated with alterations in the overall correlation structure of the gut microbiota (Figure 4 and Supplementary Table S1). Constructed networks revealed that samples from the normal-weight group had fewer edges, a lower mean degree, and a longer mean distance than those from the obese group, which indicates that there were fewer significant correlations and less clustering of genera (Supplementary Table S2). The betweenness centrality was higher in the obese group, which indicates that only a few genes were highly correlated in a network (Supplementary Table S2). Bacteroidetes showed higher positive intraphylum correlations in the normal-weight group, and Firmicutes showed higher positive intraphylum correlations in the obese group (Supplementary Table S2).

3.6. PICRUSt

To investigate the differences in microbial functions between the normal-weight and obese groups, we assessed the microbial community functional potential using PICRUSt analysis. The distribution of tier 1 KEGG functional categories was similar between the normal-weight and obese groups (Figure 5(a)). The largest number of genes (approximately 48%) corresponded to a function that encoded proteins involved in “metabolism” among tier 1 KEGG categories. Then, we examined which metabolic pathways in the tier 2 and tier 3 KEGG categories showed statistically significant differences between the normal-weight and obese groups (Supplementary Table S3). In the tier 2 KEGG categories, the microbiota of the normal-weight group was enriched in the functional abundance of “metabolism of terpenoids and polyketides” (), “lipid metabolism” (), “carbohydrate metabolism” (), and “biosynthesis of other secondary metabolites” () (Figure 5(b)). In the tier 3 KEGG categories, several pathways were enriched in the gut microbiota of the normal-weight group: “biotin metabolism” (), “glycosaminoglycan degradation” (), “glycosphingolipid biosynthesis-ganglio series” (), “glycosphingolipid biosynthesis-globo and isoglobo series” (), “inositol phosphate metabolism” (), “other glycan degradation” (), “phenylpropanoid biosynthesis” (), “phosphonate and phosphinate metabolism” (), “sphingolipid metabolism” (), “steroid hormone biosynthesis” (), and “various types of n-glycan biosynthesis” () (Figure 5(c)). In contrast, several functional pathways were enriched in the microbiota of the obese group: “cysteine and methionine metabolism” (); “peptidoglycan biosynthesis” (); “phenylalanine, tyrosine, and tryptophan biosynthesis” (); “photosynthesis” (); and “seleno-compound metabolism” () (Figure 5(c)).

4. Discussion

The present study showed differences in gut microbial composition between young normal-weight and obese Korean children aged 5-13 years. Obese children showed a significant reduction in Bacteroidetes, an elevated F : B ratio, and significantly different beta diversity compared with the same parameters among normal-weight children, as described by previous studies [4, 25, 26]. The Bacteroidetes population was also detected by LEfSe with a high LDA score, suggesting that it is the key phylotype responsible for the differences between the normal-weight and obese groups. The relative abundance of Firmicutes, however, revealed no significant difference between the groups. The results of our study suggest the importance of Bacteroidetes in pediatric obesity. Recent evidence has indicated that Bacteroidetes is a potentially modifiable therapeutic target because it is more largely influenced by environmental factors rather than host genetics [4, 27, 28]. In the future, prospective intervention studies will be needed to explore the impact of the specific species or strains belonging to the Bacteroidetes phylum on pediatric obesity modulation. Family Lachnospiraceae was significantly correlated with BMI -score. This result is consistent with a previous experimental result showing that the colonization of bacteria belonging to Lachnospiraceae induces the development of diabetes in germ-free ob/ob mice [29]. From these findings, it could be assumed that Lachnospiraceae is involved in the development of metabolic dysfunction in children. Akkermansia is a mucin-degrading bacterium, and its abundance has been reported to be negatively correlated with obesity in previous studies with adults [30]. In one study, Akkermansia was reduced in obese children aged 4-5 years living in Sweden, which was analyzed by quantitative PCR [31]. However, the current reports including our study, using 16S rRNA next generation sequencing analysis, revealed no significant difference in Akkermansia levels between normal-weight and obese children [32, 33]. This disparity can be explained with the differences in the methodology, ethnicity, and extent of its colonization which starts from early childhood and reaches a similar level to adults [34].

To investigate the relationships between gut microbial community composition, BMI, and selected variables from the biochemical markers and diet intake, CCA analysis was performed. The gut microbial community in the obesity group revealed a strong correlation with BMI -score, which was in line with previous reports [35, 36]. Inflammatory markers, including hs-CRP, neutrophil count, and ferritin, were related to microbial composition in the obese group, suggesting that obesity is closely linked to inflammation [37]. Evidence for a relationship between inflammation and the microbiota continues to be revealed. Bacterial products, such as lipopolysaccharide and short-chain fatty acids (SCFAs), can induce inflammation through immune cell activation and fat accumulation in adipocytes [38, 39]. These findings suggest the role of the gut microbiota in the development of inflammation in the pathogenesis of pediatric obesity. Among dietary intakes, niacin, Na, and fat seemed to affect gut microbial composition in the obese group. Higher fat and Na intake is associated with obesity and metabolic syndrome [40, 41]. Moreover, a recent study indicated the possible association of chronic niacin overload on pediatric obesity [42]. In our study, dietary intake of vitamin B6 seemed to be important in the microbial community of normal-weight children. Prior work has suggested that the gut microbiota of lean adolescents seems to be more involved in vitamin B6 synthesis [25]. Further research is needed to corroborate the present results. The correlation network of the gut microbiota in this study showed that the normal-weight group had less clustering of genera than the obese group. This finding is consistent with prior studies by Riva et al. [32], showing that the gut microbiota in the obese group has a different correlation network structure than the gut microbiota in the normal-weight group.

The mechanisms by which the microbiota affects energy balance in the human body are not clear. Our results from the PICRUSt analysis showed that gut microbial function in obese children involves energy metabolism, such as photosynthesis and nitrogen metabolism, which can stimulate lipogenesis or gluconeogenesis [43]. Recent research has revealed that Bacteroides ovatus can release monosaccharides from cellulose and hemic cellulose for further metabolism by a wide variety of gut commensals via glycolytic pathways [44]. Our results showed that carbohydrate metabolism was more predicted in normal-weight children than in obese children, which can be speculated to be related to the positive association of Bacteriodes ovatus in normal-weight children. Our functional analysis also showed a significantly greater presentation of genes involved in amino acid metabolism, such as cysteine and methionine metabolism and tyrosine and phenylalanine biosynthesis, in obese children than in normal-weight children. The fermentation pathways of cysteine and methionine are included in sulfur metabolism, which leads to the production of hydrogen sulfide, which has been known to have detrimental effects on colonic epithelial energy metabolism [45, 46]. Tyrosine and phenylalanine biosynthesis are known to be associated with obesity, diabetes, and metabolic syndrome by reducing the activation of alkaline phosphatase [47, 48]. This result in our study may have important long-term implications for bowel health in the context of the consumption of excessive protein diets. In addition, higher abundances of microbial communities related to lipid metabolism were observed in normal-weight children than in obese children [49]. These changes in predicted metabolic pathways caused by intestinal microbiota can induce an imbalance between energy production and absorption. The specific mechanism associated with this functional analysis will need to be studied further.

In our study, hs-CRP, NLR, and MPV were higher in the obese group than in the normal-weight group. hs-CRP and NLR are well-known inflammatory markers that are associated with obesity because adipose tissue can be the major source of proinflammatory cytokines [50]. Increased MPV, a biomarker of platelet activity, is known to be associated with acute myocardial infarction, stroke, and thrombosis in individuals with morbid obesity [50]. The percentage of children born by cesarean section was significantly higher in the obese group than in the normal-weight group. This finding is the same as those of previous studies, which are explained by the disruption of mother-to-child transmission of gut microbiota associated with cesarean section [52, 53]. In regard to lifestyle, the use of electronic devices is becoming a major problem in the context of pediatric obesity rather than a lack of physical activity [54]. The present study also indicated that not only the lack of exercise but also the use of electronic devices for more than two hours a day was significantly higher in the obesity group. A recent study reported that electronic device usage close to bedtime can disrupt sleep patterns, which could lead to obesity [55]. Obesity is not only microbiota-driven; thus, a careful evaluation of all factors, including delivery mode, diet, and lifestyle, should be taken into account [56].

One of the strengths of this study is that it included only young Korean children, which makes the study population less influenced by ethnicity and environmental factors such as smoking, drugs, and alcohol use. Instead of simply comparing the microbiota between groups, we analyzed the relationship of multivariate factors in the microbiota, biochemical markers, and diet intake through a correlogram and CCA. Additionally, we showed the results of a network analysis and functional analysis of the gut microbiota in obese and normal-weight young children. The limitations of this study had to do with the small number of participants and the cross-sectional design, which prevents the determination of causality. Therefore, a prospective large-scale study is required to clarify the relationship between the microbiota and childhood obesity.

In conclusion, the microbial communities of obese children exhibited significant differences in beta diversity and a significantly elevated F : B ratio compared to those in normal-weight children. The phylum Bacteroidetes was significantly reduced in the obese group and was negatively correlated with BMI -score. The LEfSe biomarker discovery analysis also suggested that the Bacteroidetes population was the key phylotype differentiating the two groups. These findings suggest the importance of Bacteroidetes in pediatric obesity. The gut microbial community in the obese group was linked to BMI -score; blood biomarkers associated with inflammation and metabolic syndrome; and intakes of niacin, Na, and fat. In the microbial network analysis, the gut microbiota in the obese group showed more clustering of genera than the gut microbiota in the normal-weight group. PICRUSt analysis revealed that the functions related to carbohydrate and lipid metabolism were more enriched in the microbiota of the normal-weight group than in that of the obese group. Our data may contribute to the understanding of the gut microbial structure of young Korean children in relation to obesity. Further studies are required to target Bacteroidetes as a new therapeutic intervention for pediatric obesity.

Data Availability

All raw 16S rRNA gene sequencing data were deposited in the NCBI Sequence Read Archive (SRA) under accession number SUB7560699 (BioProject PRJNA637782). Data related to the current study are available from the corresponding author on reasonable request.

Disclosure

The manuscript was presented at the 69th Fall Congress of Korean Pediatrics and Youth Science Society. The funding bodies had no role in the study design; in the collection, analysis, or interpretation of the data; or in the writing of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

Research in this publication was supported by a Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (NRF-2018R1D1A1B07046799) and by the Hallym University Research Fund (HURF-2017-81), Anyang si, Korea.

Supplementary Materials

Supplementary Figure S1: box plots for comparisons of Firmicutes (a), Actinobacteria (b), Proteobacteria (c), and Firmicutes-to-Bacteroidetes (F : B) ratio (d) between the normal and obese groups. There were no significant differences in the relative abundances of Firmicutes, Actinobacteria, or Proteobacteria. The F : B ratio revealed a significant difference between the normal and obese groups (). Supplementary Figure S2: differences in the relative abundance of the family Lachnospiraceae (a), the species Bacteroides ovatus (b), and the genus Akkermansia (c) between the normal and obese groups. Supplementary Table S1: name and classification of the genera shown in Figure 4. Supplementary Table S2: properties of correlation networks generated from the normal and obese groups. Supplementary Table S3: PICRUSTt predicted functions of KEGG categories presented in the obese group compared to those in the normal group. (Supplementary Materials)

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Copyright © 2020 Saeam Shin and Ky Young Cho. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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