GUT MICROBIOME AND CHILDHOOD STUNTING: A CORRELATION STUDY FOR BETTER FUTURE THERAPY

• Main Author: Rinanda, Tristia
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/79991
• Institution: Institut Teknologi Bandung

Stunting is a chronic nutritional disorder which has become an alarming global health problem. Several established stunting eradication programs have been mainly focused on the supplementation of macronutrients and micronutrients. The success of supplementation relies significantly on the gut absorption function, which declines due to Environmental Enteric Dysfunction (EED) in stunting. EED is reversible. Hence, balancing the state of gut microbiota can potentially improve growth. The gut microbiota is also known to induce the secretion of Short Chain Fatty Acid (SCFA), Glucagon-like Peptide-1 (GLP-1), and Insulin-like Growth Factor-1 (IGF-1), which play a role in supporting growth and metabolic processes. This study aimed to examine the correlation between gut microbiome (archaea and bacteria), and growth in stunted children in Pidie District, Aceh Province. This case-control study involved 42 subjects aged 24 to 59 months, comprising 21 stunted children for the case and 21 normal children for the control group. The Next Genertaion Sequencing (NGS) for gut archaea was conducted using V54-V5 primer of the 16S rDNA archaeal gene, whereas the gut bacteria composition was determined using V3-V4 primer or 16S rDNA bacterial gene. The DNA extraction from fecal specimens and the NGS were conducted by PT. Genetika Science Indonesia and Novogene as the metagenomics service providers. The raw data of 16S rDNA amplicon sequencing was analyzed using the QIIME2. Further bioinformatics analyses were conducted to present the gut microbiota composition, alpha and beta diversities, and functional metagenomics prediction. Gene expression study was conducted using the Quantitative Polymerase Chain Reaction (qPCR) assay. This study observed the expression of the mcrA gene that encodes the Methyl co-enzyme reductase A, which plays a role in methanogenesis. This study also observed the expression of pathogens virulence genes as follows: aaiC (Enteroaggregative Escherichia coli/EAEC), eaeA (Enteropathogenic E. coli/EPEC), estA (Enterotoxigenic E. coli/ETEC), ipaH3 (Shigella/Enteroinvasive E. coli/EIEC) and ompC (Salmonella enterica). The present study also measured the levels of SCFA, GLP-1, and IGF-1. The fecal SCFA level was determined using Gas Chromatography-Mass Spectrometry (GC-MS) by the National Prodia Referral Laboratory, Jakarta. The GLP-1 and IGF-1 sera levels were measured using Enzyme-linked Immunosorbent Assay (ELISA). vi The variables associated with stunting were abnormality of stool consistency, history of diarrhea, incomplete basic immunization status, infant formula feeding at the age of less than 6 months and low consumption of vegetables and animal source proteins. There were no differences between SCFA and GLP-1 levels in stunted and normal children. Meanwhile, IGF-1 levels in stunted children were lower than those in normal children. The 16S rDNA amplicon sequencing results revealed a lower relative abundance of methanogens in stunted children compared to normal children. Methanobrevibacter smithii and Methanosphaera stadmanae were the identified genera of methanogens. Alpha diversity analysis showed that the presence of methanogens increased gut microbiota diversity in stunted children. Metagenomic functional prediction also showed that the archaea metabolic pathway was the active metabolic pathway in normal children. The methanogenesis was found to be lower in stunted children compared to normal children. Methanogenesis was also found to be positively correlated to SCFA, GLP-1, and IGF-1 levels, as well as to anthropometric parameters such as height and Body Mass Index (BMI), although statistically insignificant. The composition of gut bacteria in stunted children showed a dysbiosis with an increased of Firmicutes to Bacteroides ratio. Stunted children demonstrated a high relative abundance of genera associated with inflammation, metabolic abnormalities and high fat-low fiber diets, such as Blautia, Dorea, Collinesella, Clostridium sensu stricto and Streptococcus. Interestingly, this study found a higher abundance of water biological contaminants, such as Aeromonas, Stappiaceae, and Synechococcus CC9902 in stunted children than in normal children. Beta diversity analysis demonstrated a significant difference of gut microbiota diversity within observed groups. Alpha diversity of gut bacteria in stunted children was lower than that in normal children, which might increase susceptibility to enteric infections. This vulnerability was confirmed by the presence of enteric pathogen virulence genes, which was higher in stunted children. The expression of the virulence genes was negatively correlated to IGF-1 levels, height, weight, and BMI. Correlation analysis also showed the co-infection between various gut pathogens. This study confirmed the essential role of gut microbiota in supporting linear growth in stunted children in Pidie District, Aceh. To our knowledge, gut microbiota composition in stunted children from Aceh Province as well as the role of gut archaea (methanogens) in stunting have never been reported. In addition, the virulence gene expression study in stunted children has never been conducted. These aspects were the novelties proposed by this study. The findings of the present study highlighted a number of critical points that must be improved from established stunting eradication programs. This study also recommended the use of prebiotics and probiotics to restore the balance of gut microbiota in stunted children. Further research involving more subjects and a longitudinal study design is expected to provide more conclusive results and contribute significantly to stunting eradication programs in Aceh and Indonesia.