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Updated: Mar 31, 2026

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Gastrointestinal microbiome signatures of pediatric patients with irritable bowel syndrome
Delphine M Saulnier1, Kevin Riehle, Toni-Ann Mistretta
1Department of Pathology & Immunology, Baylor College of Medicine, Houston, Texas, USA.
Insights
Pediatric irritable bowel syndrome (IBS) is linked to specific gut bacteria signatures. Identifying these microbiome differences may aid in diagnosing functional bowel disorders in children.
Area of Science:
- Microbiology
- Pediatric Gastroenterology
- Bioinformatics
Background:
- The gut microbiome's role in pediatric irritable bowel syndrome (IBS) is not well understood.
- Adult IBS studies suggest a link between gastrointestinal microbiota and the condition.
Purpose of the Study:
- To define the intestinal microbiomes of healthy children and pediatric IBS patients.
- To identify specific microbial signatures associated with pediatric IBS.
Main Methods:
- Analyzed 71 stool samples from children (ages 7-12) with IBS and healthy controls using 16S rRNA gene sequencing (454 pyrosequencing).
- Employed phylogenetic clustering (Unifrac) and operational taxonomic unit (OTU) analysis with random forest machine learning.
- Utilized 16S rRNA PhyloChip microarray for high-resolution analysis of 8741 bacterial taxa.
Main Results:
- Pediatric IBS microbiomes showed a higher abundance of γ-proteobacteria, including Haemophilus parainfluenzae.
- Supervised learning classified IBS subtypes with 98.5% accuracy using discriminant bacterial species.
- A novel Ruminococcus-like microbe and increased Alistipes genus bacteria were associated with IBS and pain frequency.
Conclusions:
- Specific microbiome signatures were associated with pediatric IBS using 16S metagenomics and PhyloChip analysis.
- Findings highlight the significant link between gut microbes and pediatric IBS.
- These methods show potential for diagnosing functional bowel disorders in children.
Background & Aims:
The intestinal microbiomes of healthy children and pediatric patients with irritable bowel syndrome (IBS) are not well defined. Studies in adults have indicated that the gastrointestinal microbiota could be involved in IBS.
Methods:
We analyzed 71 samples from 22 children with IBS (pediatric Rome III criteria) and 22 healthy children, ages 7-12 years, by 16S ribosomal RNA gene sequencing, with an average of 54,287 reads/stool sample (average 454 read length = 503 bases). Data were analyzed using phylogenetic-based clustering (Unifrac), or an operational taxonomic unit (OTU) approach using a supervised machine learning tool (randomForest). Most samples were also hybridized to a microarray that can detect 8741 bacterial taxa (16S rRNA PhyloChip).
Results:
Microbiomes associated with pediatric IBS were characterized by a significantly greater percentage of the class γ-proteobacteria (0.07% vs 0.89% of total bacteria, respectively; P < .05); 1 prominent component of this group was Haemophilus parainfluenzae. Differences highlighted by 454 sequencing were confirmed by high-resolution PhyloChip analysis. Using supervised learning techniques, we were able to classify different subtypes of IBS with a success rate of 98.5%, using limited sets of discriminant bacterial species. A novel Ruminococcus-like microbe was associated with IBS, indicating the potential utility of microbe discovery for gastrointestinal disorders. A greater frequency of pain correlated with an increased abundance of several bacterial taxa from the genus Alistipes.
Conclusions:
Using 16S metagenomics by PhyloChip DNA hybridization and deep 454 pyrosequencing, we associated specific microbiome signatures with pediatric IBS. These findings indicate the important association between gastrointestinal microbes and IBS in children; these approaches might be used in diagnosis of functional bowel disorders in pediatric patients.
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