High-throughput 16S rRNA gene sequencing reveals alterations of mouse intestinal microbiota after radiotherapy

doi:10.1016/j.anaerobe.2015.01.004

Anaerobe

Volume 33, June 2015, Pages 1-7

https://doi.org/10.1016/j.anaerobe.2015.01.004 Get rights and content

Highlights

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The gut microbiota in mice receiving irradiation was characterized by high-throughput sequencing method using 16S rRNA gene.
•
Gut microbial abundances showed that significant alterations in the both intestines affected by radiation.
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Alistipes and Mucisprillum might be associated with gamma irradiation exposure and host gut health.

Abstract

The mammalian gastrointestinal tract harbors a highly complex microbial community that comprises hundreds of different types of bacterial cells. The gastrointestinal microbiota plays an important role in the function of the host intestine. Most cancer patients undergoing pelvic irradiation experience side effects such as diarrhea; however, little is currently known about the effects of irradiation on the microorganisms colonizing the mucosal surfaces of the gastrointestinal tract. The aim of this study was to investigate the effects of gamma irradiation on the compositions of the large and small intestinal microbiotas. The gut microbiotas in control mice and mice receiving irradiation treatment were characterized by high-throughput sequencing of the bacterial 16S rRNA gene. Irradiation treatment induced significant alterations in the bacterial compositions of the large and small intestines at the genus level. Unexpectedly, irradiation treatment increased the number of operational taxonomic units in the small intestine but not the large intestine. In particular, irradiation treatment increased the level of the genera Alistipes in the large intestine and increased the level of the genus Corynebacterium in the small intestine. By contrast, compared with that in the corresponding control group, the level of the genera Prevotella was lower in the irradiated large intestine, and the level of the genera Alistipes was lower in the irradiated small intestine. Overall, the data presented here reveal the potential microbiological effects of pelvic irradiation on the gastrointestinal tracts of cancer patients.

Introduction

Recent studies have shown that the human body is inhabited by at least ten times more microorganisms than the number of somatic and germ-line cells it contains [1]. The host gut and its microbiota have co-evolved intricate relationships, and the mammalian gastrointestinal tract is colonized by 10–100 trillion microorganisms that are essential to host cell maintenance in health and disease; therefore, the human gut microbiota has attracted increasing interest from medical researchers. Comprehensive 16S rRNA gene-based analyses of fecal microbial communities have demonstrated that the gut microbiota is highly diverse between individuals; however, Bacteroidetes and Firmicutes are the predominant phyla in most individuals [2], [3]. The majority of studies examining the beneficial and/or pathogenic influences of microbes on intestinal diseases have focused on irritable bowel or inflammatory bowel diseases [4], [5]; however, recent evidence has linked the composition of the gut microbiota to gastrointestinal responses to irradiation [6], [7], [8], [9].

Radiotherapy is a common treatment for cancers, especially gynecological and colorectal cancers. Approximately 70% of all cancer patients receive radiotherapy treatment [6], [10], [11] and these patients typically present clinical symptoms of gastrointestinal irradiation injury [8], [12], [13]. Pelvic radiotherapy of gynecological cancer patients causes changes in the microbial composition of the gut; Nam et al. [14] demonstrated that radiation therapy changes the intestinal levels of the Firmicutes and Fusobacterium phyla significantly. Furthermore, Crawford and Gordon [7] used germ-free mice models to demonstrate microbial regulation of intestinal radio-sensitivity. These two studies focused on the large intestine microbiota and cell function, respectively; however, several studies [6], [8], [15], [16] have suggested that injury to the small intestine in cancer patients receiving pelvic irradiation leads to adverse side effects such as diarrhea. Nevertheless, analyses of the small intestine microbiota are lacking because sampling of this region is challenging. Therefore, the objective of this study was to investigate and compare the microbial compositions of fecal samples from irradiated mouse large and small intestines using Illumina MiSeq high-throughput sequencing.

Section snippets

Animal preparation

Male 8–10-week-old C57BL/6 mice were purchased from Orient Bio (Seongnam, Republic of Korea) and housed with access to irradiated food and water. All mouse experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee of Jeju National University. The mice were irradiated with a single 8 Gy dose using a Cobalt 60 source irradiator [17]. The small and large intestinal contents were collected three days after irradiation. During the

Clustering of the mouse gut microbiotas

Table 1 summarizes the sequencing reads, diversity indices, and sample coverages of the mouse intestinal samples included in the study. The control group comprised mice 1 and 2, and the irradiation group comprised mice 7, 8, and 9. After quality control processing and removal of chimeric reads, a total of 51,505 sequences (from 60,394 raw reads) were used for abundance and diversity analyses, as well as taxonomic comparisons. Based on unweighted pair group method with arithmetic mean clustering

Discussion

It was reported recently that the human body possesses at least ten times more intestinal microbial cells than human cells [27]. The composition of the gut microbiota can have a profound impact on human health and disease. To date, most studies have focused on pathogenic or beneficial microorganisms that influence gastrointestinal disorders, such as irritable bowel and inflammatory bowel diseases [4], [5]; however, the relationships between the human and microbial cell communities remain

Acknowledgments

This work was supported by a research grant from the Jeju National University Hospital, received in 2014.

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¹: Y.S.K. and J.K. contributed equally to this work.

View full text

High-throughput 16S rRNA gene sequencing reveals alterations of mouse intestinal microbiota after radiotherapy

Highlights

Abstract

Introduction

Section snippets

Animal preparation

Clustering of the mouse gut microbiotas

Discussion

Acknowledgments

Clin. Oncol. R. Coll. Radiol.

Dig. Liver Dis.

Micron

Anaerobe

Cell. Host Micro

Int. J. Antimicrob. Agents

Anaerobe

The NIH human microbiome project

Genome Res.

Diversity of the human intestinal microbial flora

Science

Metagenomic analysis of the human distal gut microbiome

Science

Intestinal microbes in inflammatory bowel diseases

Am. J. Gastroenterol

From meta-omics to causality: experimental models for human microbiome research

Microbiome

Microbial influences on the small intestinal response to radiation injury

Curr. Opin. Gastroenterol.

Microbial regulation of intestinal radiosensitivity

Proc. Natl. Acad. Sci. U. S. A.

Gastrointestinal radiation injury: prevention and treatment

World J. Gastroenterol.

Intestinal microbiota reduces genotoxic endpoints induced by high-energy protons

Radiat. Res.

Significance of endothelial dysfunction in the pathogenesis of early and delayed radiation enteropathy

World J. Gastroenterol.

Cancer: Principles & Practice of Oncology

Impact of pelvic radiotherapy on gut microbiota of gynecological cancer patients revealed by massive pyrosequencing

PLoS One

Gastrointestinal radiation injury: symptoms, risk factors and mechanisms

World J. Gastroenterol.

Increased superoxide formation induced by irradiation preconditioning triggers kidney resistance to ischemia-reperfusion injury in mice

Am. J. Physiol. Ren. Physiol.

Ultra-high-throughput microbial community analysis on the illumina HiSeq and MiSeq platforms

ISME J.

Practical innovations for high-throughput amplicon sequencing

Nat. Methods

Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq illumina sequencing platform

Appl. Environ. Microbiol.

The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data

Nucleic Acids Res.

Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities

Appl. Environ. Microbiol.

UniFrac–an online tool for comparing microbial community diversity in a phylogenetic context

BMC Bioinforma.

FastTree 2 – approximately maximum-likelihood trees for large alignments

PloS One

On a test of whether one of two random variables is stochastically larger than the other

Ann. Math. Stat.

Honor thy gut symbionts redux

Science

Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors

Proc. Natl. Acad. Sci. U. S. A.