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Updated: Jul 4, 2025
Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Chronic intestinal pseudo-obstruction: associations with gut microbiota and genes expression of intestinal
Giulia Radocchia1, Massimiliano Marazzato1, Karim Ben Harbi1
1Department of Public Health and Infectious Diseases, Microbiology section, Sapienza University of Rome, Rome, Italy.
Insights
Pediatric chronic intestinal pseudo-obstruction (PIPO) is linked to altered gut microbiota and serotonin pathway gene expression. This pilot study suggests potential new biomarkers and therapies targeting these pathways for PIPO patients.
Area of Science:
- Gastroenterology
- Microbiology
- Genetics
Background:
- Pediatric chronic intestinal pseudo-obstruction (PIPO) is a rare disorder mimicking intestinal obstruction without physical blockage, stemming from severe motility impairment.
- The intestinal endocrine system and enteric nervous system regulate gut function, with serotonin (5-HT) playing a key role in peristalsis and secretion.
- The interaction between gut microbiota and the enteric nervous system influences serotonin pathways, but its role in PIPO is poorly understood.
Purpose of the Study:
- To investigate the correlation between mucosa-associated microbiota (MAM) and the expression of serotonin-related genes in pediatric chronic intestinal pseudo-obstruction (PIPO).
- To explore potential links between gut microbiota composition, serotonin pathway alterations, and clinical parameters in PIPO patients.
Main Methods:
- Biopsies from the colon, ileum, and duodenum were collected from 7 PIPO patients and 7 healthy controls.
- Mucosa-associated microbiota (MAM) was analyzed using next-generation sequencing (NGS) of the 16S rRNA gene.
- The expression of key serotonin pathway genes (TPH1, SLC6A4, 5-HTR3, 5-HTR4) was quantified using quantitative PCR (qPCR).
Main Results:
- PIPO patients displayed a distinct MAM composition characterized by reduced biodiversity and altered species interconnections (dysbiosis) compared to controls.
- Significant modifications in the expression of intestinal serotonin-related genes were observed in PIPO patients.
- Correlation analyses did not reveal any direct connections between MAM, serotonin gene expression, and clinical parameters in this cohort.
Conclusions:
- This study provides the first evidence of a specific MAM and an altered intestinal serotonin pathway in PIPO patients.
- A potential link between gut dysbiosis and serotonin pathway dysfunction may contribute to the severe dysmotility seen in PIPO.
- These findings lay the groundwork for developing novel biomarkers and therapeutic strategies targeting the microbiota or serotonin pathways for PIPO management.
Background:
Pediatric chronic intestinal pseudo-obstruction (PIPO) is a rare disease characterized by symptoms and radiological signs suggestive of intestinal obstruction, in the absence of lumen-occluding lesions. It results from an extremely severe impairment of propulsive motility. The intestinal endocrine system (IES) jointly with the enteric nervous system (ENS) regulates secreto-motor functions via different hormones and bioactive messengers/neurotransmitters. The neurotransmitter 5-hydroxytryptamine (5-HT) (or serotonin) is linked to intestinal peristalsis and secretory reflexes. Gut microbiota and its interplay with ENS affect 5-HT synthesis, release, and the subsequent serotonin receptor activation. To date, the interplay between 5-HT and gut microbiota in PIPO remains largely unclear. This study aimed to assess correlations between mucosa associated microbiota (MAM), intestinal serotonin-related genes expression in PIPO. To this purpose, biopsies of the colon, ileum and duodenum have been collected from 7 PIPO patients, and 7 age-/sex-matched healthy controls. After DNA extraction, the MAM was assessed by next generation sequencing (NGS) of the V3-V4 region of the bacterial RNA 16 S, on an Illumina Miseq platform. The expression of genes implicated in serotoninergic pathway (TPH1, SLC6A4, 5-HTR3 and 5-HTR4) was established by qPCR, and correlations with MAM and clinical parameters of PIPO have been evaluated.
Results:
Our results revealed that PIPO patients exhibit a MAM with a different composition and with dysbiosis, i.e. with a lower biodiversity and fewer less connected species with a greater number of non-synergistic relationships, compared to controls. qPCR results revealed modifications in the expression of serotonin-related intestinal genes in PIPO patients, when compared to controls. Correlation analysis do not reveal any kind of connection.
Conclusions:
For the first time, we report in PIPO patients a specific MAM associated to underlying pathology and an altered intestinal serotonin pathway. A possible dysfunction of the serotonin pathway, possibly related to or triggered by an altered microbiota, may contribute to dysmotility in PIPO patients. The results of our pilot study provide the basis for new biomarkers and innovative therapies targeting the microbiota or serotonin pathways in PIPO patients.
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