Journal of Cancer Prevention 2016; 21(1): 60-65
Published online March 30, 2016
© Korean Society of Cancer Prevention
Jihee Sung1, Nayoung Kim1,2, Jaeyeon Kim2, Hyun Jin Jo1, Ji Hyun Park2, Ryoung Hee Nam1, Yeong-Jae Seok3, Yeon-Ran Kim3, Dong Ho Lee1,2, and Hyun Chae Jung1
1Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, 2Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea, 3Department of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul, Korea
Correspondence to :
Nayoung Kim, Department of Internal Medicine, Seoul National University Bundang Hospital, 82 Gumi-ro 173beon-gil, Bundang-gu, Seongnam 13620, Korea, Tel: +82-31-787-7008, Fax: +82-31-787-4051, E-mail: firstname.lastname@example.org, ORCID: Nayoung Kim, http://ordid.org/0000-0002-9397-0406
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Not much is known about the role of gastric microbiota except for Gastric biopsies and stomach juices were collected from 4 subjects who underwent standard endoscopy at Seoul National University Bundang Hospital. Gastric microbiota of antral mucosa, corpus mucosa samples, and gastric fluids were analyzed by barcoded 454-pyrosequencing of the 16S rRNA gene. The analysis focused on bacteria, such as Gastric fluid samples showed higher diversity compared to that of gastric mucosa samples. The mean of operational taxonomic units was higher in gastric fluid than in gastric mucosa. The samples of gastric fluid and gastric mucosa showed different composition of phyla. The composition of Even though these samples were small, gastric mucosa could be more effective than gastric fluid in the detection of meaningful gastric microbiota by pyrosequencing.
Not much is known about the role of gastric microbiota except for
Gastric biopsies and stomach juices were collected from 4 subjects who underwent standard endoscopy at Seoul National University Bundang Hospital. Gastric microbiota of antral mucosa, corpus mucosa samples, and gastric fluids were analyzed by barcoded 454-pyrosequencing of the 16S rRNA gene. The analysis focused on bacteria, such as
Gastric fluid samples showed higher diversity compared to that of gastric mucosa samples. The mean of operational taxonomic units was higher in gastric fluid than in gastric mucosa. The samples of gastric fluid and gastric mucosa showed different composition of phyla. The composition of
Even though these samples were small, gastric mucosa could be more effective than gastric fluid in the detection of meaningful gastric microbiota by pyrosequencing.
Human gut, colonized by complex communities of microorganisms, plays essential roles in digestion, absorption of nutrients,1 stimulation of intestinal epithelial regeneration,2 and immune reactions.3 Keeping these microbial communities in balance with host is important for health maintenance and disease prevention.4 Before the discovery of
This study was approved by the ethics committee of Seoul National University Bundang Hospital (B-1112/141-007). Gastric biopsies and fluid samples were collected from 4 subjects who underwent standard endoscopy to screen for premalignant or malignant gastric mucosal lesions or received endoscopy due to dyspepsia. Gastric mucosal (antrum and corpus) biopsies and blood samples were obtained from each patient during endoscopy from October 2008 to March 2013 at Seoul National University Bundang Hospital. Ten biopsy specimens per subjects were obtained to perform
The positivity of
The mucosal and gastric fluid samples from 4 subjects were subjected to pyrosequencing. Total genomic DNA was separated using a commercial kit (iNtRON Biotechnology, Seongnam, Korea). PCR amplification was done by taking primers targeting the V1 to V3 regions of the 16S rRNA gene with extracted DNA. For bacterial amplification, barcoded primer of 9F (5’-CCTATCCCC-TGTGTGCCTTGGCAGTC-TCAG-AC-
The primary analysis was conducted as described above. Reads taken from different samples were classified by unique barcodes of each PCR product. After identifying the target region in barcoded primers (9F or 541R), all of the linked sequences including adapter, barcode, and linker were eliminated. Low quality sequences such as reads containing two or more indefinite nucleotides, reads with a low quality score (average score < 25), or reads shorter than 300 bp, were eliminated. Potential chimeric sequences were confirmed by the Bellerophon formula, which compares the BLASTN search conclusions between the forward half and reverse half sequences.16 After removing the chimeric sequences, the taxonomic sorting of each read was assigned against the EzTaxon-e database (
To compare species richness between samples of different sizes, rarefaction curve, and diversity indices such as abundance-based coverage estimator, Chao1 estimator, and Jackknife estimator. Simpson diversity index and Shannon diversity index were estimated in the CLcommunity program (Chunlab). Random subsampling was conducted to equalize the read size of samples to compare the different read size within samples. To compare the OTUs between samples, shared OTUs were obtained with the XOR analysis of the CLcommunity program.
Descriptive statistics were reported as mean ± SD, and confidence intervals were computed as two-tailed using 95% coverage. Categorical variables were described as frequencies and proportions. Comparisons between continuous parameters were performed by the
Baseline characteristics of clinical and pyrosequencing results of gastric antral mucosal and fluid samples are shown in Table 1. Four subjects (2 gastric cancer, 1 gastritis, and 1 control) were included in this study. The mean age of subjects was 48.7 years (38?59 years). Subject 1 and 3 were male and subject 2 and 4 were female. Sample pH values varied between 1.0 and 7.4 (mean: 2.79). Three samples were found to contain
Though there was no statistical significance, the mean of total number of reads was lower in gastric fluid samples than gastric mucosa samples. However, the mean of OTUs was higher in gastric fluid (Fig. 1A). Generally, gastric fluid samples show higher diversity compared to gastric mucosa samples (Fig. 1B). At the phylum level, members of
Stomach plays an important role in maintaining gastrointestinal (GI) health, as a barrier against ingested infectious disease agents of the lower GI tract.18 In healthy subjects, swallowed pathogens are inactivated by gastric fluid, which contains both hydrochloric acid and proteolytic enzymes.19 Atrophic gastritis, gastric surgery, or drugs that inhibit acid secretion can cause hypochlorhydria.18 Decreased gastric acid secretion is responsible for an increased risk of infection.20 There were few studies related to gastric juice. von Rosenvinge et al.21 reported the microbiota composition of gastric fluid in relation to various human host parameters, including immune status, gastric fluid pH, use of proton pump inhibitors, and antibiotic medications.
Previous research has primarily focused on the microbiota of gastric mucosal biopsies and applied only-DNA-based methodologies; yet it was unable to distinguish between transcriptionally active, inactive, or dead bacteria. Analysis by the 16S rRNA gene contents of microbial samples after amplification by PCR has changed the characterization of microbial communities.5,11,22 Using a 16S rRNA transcript amplicon sequencing strategy, we can differentiate transcriptionally active RNA microbiota from the total DNA microbiota compositions.23
Our results reveal that human gastric fluid harbors a diverse microbiota dominated by
Contrary to previous studies about stomach environment based on biopsy samples from the mucosa,5,6
In contrast to that, in gastric mucosa, the proportion of the
Higher microbioal diversity of gastric fluid compared to gastric mucosa was observed in
This work was supported by the National Research Foundation of Korea (NRF) grant for the Global Core Research Center (GCRC) funded by the Korea government (MSIP) (No. 2011-0030001).
No potential conflicts of interest were disclosed.
Baseline characteristics of 4 subjects.
|Subject No.||Sex/Age (yr)||Disease||Gastric pH||Site||Intesinal metaplasia (n)||Neutrophil infiltration (n)||Monocyte infiltration (n)||Atrophy (n)||CLO||H&E||Culture||HP IgG||Eradication history||PG I/II ratio|
CLO, Campylobacter-like organism; HP IgG, Helicobacter pylori immunoglobulin G; PG, pepsinogen; M, male; F, female; EGC, early gastric cancer; AGC; advancedgastric cancer; Ina, inadequate to assess atrophy; N/A, not assessed..
The phylum composition between gastric mucosa samples and gastric fluid samples.
Values are presented as percentage only..
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