Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12145

Differences in Gastric Mucosal Microbiota Profiling in Patients with Chronic Gastritis, Intestinal Metaplasia, and Gastric Cancer Using Pyrosequencing Methods Chang Soo Eun,*,1 Byung Kwon Kim,†,1 Dong Soo Han,* Seon Young Kim,‡ Kyung Mo Kim,§ Bo Youl Choi,¶ Kyu Sang Song,** Yong Sung Kim‡ and Jihyun F. Kim‡‡ *Department of Internal Medicine, Hanyang University Guri Hospital, Gyeonggi-Do, Korea, †Bionano Health Guard Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea, ‡Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea, §Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea, ¶ Department of Preventive Medicine, Hanyang University College of Medicine, Seoul, Korea, **Department of Pathology, Chungnam National University, College of Medicine, Daejeon, Korea, ‡‡Department of Systems Biology and Division of Life Sciences, Yonsei University, Seoul, Korea

Keywords gastric cancer, H. pylori, 16S ribosomal RNA, carcinogenesis. Reprint requests to: Dong Soo Han, Department of Internal Medicine, Hanyang University Guri Hospital, 249-1, Gyomoon-Dong, Guri-Si, Gyeonggi-Do 471-701, Korea. E-mail: [email protected] 1

These authors contributed equally to this work.

Abstract Background: Helicobacter pylori (H. pylori) infection plays an important role in the early stage of cancer development. However, various bacteria that promote the synthesis of reactive oxygen and nitrogen species may be involved in the later stages. We aimed to determine the microbial composition of gastric mucosa from the patients with chronic gastritis, intestinal metaplasia, and gastric cancer using 454 GS FLX Titanium. Methods: Gastric mucosal biopsy samples were collected from 31 patients during endoscopy. After the extraction of genomic DNA, variable region V5 of the 16S rRNA gene was amplified. PCR products were sequenced using 454 high-throughput sequencer. The composition, diversity, and richness of microbial communities were compared between three groups. Results: The composition of H. pylori-containing Epsilonproteobacteria class appeared to be the most prevalent, but the relative increase in the Bacilli class in the gastric cancer group was noticed, resulting in a significant difference compared with the chronic gastritis group. By analyzing the Helicobacter-dominant group at a family level, the relative abundance of Helicobacteraceae family was significantly lower in the gastric cancer group compared with chronic gastritis and intestinal metaplasia groups, while the relative abundance of Streptococcaceae family significantly increased. In a UPGMA clustering of Helicobacter-dominant group based on UniFrac distance, the chronic gastritis group and gastric cancer group were clearly separated, while the intestinal metaplasia group was distributed in between the two groups. The evenness and diversity of gastric microbiota in the gastric cancer group was increased compared with other groups. Conclusions: In Helicobacter predominant patients, the microbial compositions of gastric mucosa from gastric cancer patients are significantly different to chronic gastritis and intestinal metaplasia patients. These alterations of gastric microbial composition may play an important, as-yet-undetermined role in gastric carcinogenesis of Helicobacter predominant patients.

Gastric cancer is a global health problem, with high prevalence in the Far East, especially in Korea and Japan. In Correa’s model of gastric carcinogenesis, chronic Helicobacter pylori (H. pylori) infection of the gastric mucosa progresses through the stages of chronic active gastritis, atrophy, intestinal metaplasia, and dysplasia before the development of gastric cancer [1]. It

© 2014 John Wiley & Sons Ltd, Helicobacter 19: 407–416

has been well known that H. pylori has an important role in gastric cancer development [2,3]. However, eradication of H. pylori does not guarantee the prevention of gastric cancer in patients with premalignant lesions including atrophy and intestinal metaplasia [4]. Persistent colonization of H. pylori can induce chronic inflammation of gastric mucosa that progresses

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into atrophic gastritis, a condition that predisposes the stomach to reduced gastric acid secretion and the overgrowth of non-Helicobacter microbiota [5,6]. It has been postulated that some microbial species colonizing the atrophic stomach may further promote H. pylori-initiated gastritis, genotoxic damage, and the progression to gastric cancer [7,8]. However, their identities remain largely unknown. Characterization of the gastric microbiota has traditionally relied on cultivation of gastric juice or mucosal biopsies. However, a large fraction of the microbes residing in the stomach have not yet been cultivated, suggesting a skewed presentation from culture-based studies. Recent advances in DNA sequence-based technologies allow genetic analysis of complex microbial populations without the need for prior cultivation [9,10]. Although a number of studies using culture-independent metagenomic techniques have been performed to elucidate the relation of dysbiosis (imbalances of gastrointestinal microbiota) with diverse small bowel and colonic diseases [11–13], very little research has investigated the microbial composition of the stomach using metagenomic approaches, particularly in its relation to gastric carcinogenesis [14–16]. In this study, we aimed to determine the microbial composition of gastric mucosa from the patients with chronic gastritis, intestinal metaplasia, and gastric cancer and to analyze any difference in the microbial composition between three groups using high-throughput sequencing platform, 454 GS FLX Titanium.

Materials and Methods Study Population and Samples Collection Thirty-one patients including 11 noncardia gastric cancer patients, 10 intestinal metaplasia patients, and 10 chronic gastritis patients were enrolled prospectively from March 2010 to May 2011. Exclusion criteria were as follows: age under 18 years; the presence of a serious illness such as severe cardiopulmonary, renal, or metabolic disease; prior medication history of antibiotics, acid blockers (proton-pump inhibitor and H2 receptor antagonist), anti-inflammatory agents (aspirin, nonsteroidal anti-inflammatory drugs, and steroids), or probiotics for past 6 months; prior history of any surgical gastric resection; and refusal of consent to the study. Six gastric mucosal biopsies (three for microbial sequencing, two for histopathology, one for rapid urease test) were obtained from the corpus and antrum in each of 31 patients during upper endoscopy at Hanyang University Guri Hospital. Mucosal biopsies in gastric cancer patients were performed on the noncancerous gastric mucosa

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adjacent to the cancer tissue. Three biopsy samples for microbial sequencing were immediately stored at 80 °C. Diagnosis of gastric cancer and intestinal metaplasia were confirmed by histopathology. Chronic gastritis was defined as chronic inflammation of gastric mucosa with no evidence of intestinal metaplasia or atrophy on histopathology based on updated Sydney system [17]. Patients were determined to be H. pylori positive by positive result in one or more of two following conventional tests: rapid urease test and histopathology. All patients provided their written informed consent, and the study was approved by the institutional review board of Hanyang University Guri Hospital.

Bacterial DNA Extraction Bacterial DNA was extracted from a total of 31 gastric mucosal tissues using a phenol/chloroform extraction method combined with physical disruption of bacterial cells and a DNA clean-up kit (Qiagen DNeasy Blood and Tissue extraction kit; Qiagen, Valencia, CA, USA) as previously described [18]. Briefly, 100 mg of frozen gastric mucosal tissues was suspended in 750 lL of sterile bacterial lysis buffer (200 mmol/L NaCl, 100 mmol/L Tris [pH 8.0], 20 mmol/L EDTA, 20 mg/mL lysozyme) and incubated at 37 °C for 30 minutes. Next, 40 lL of proteinase K (20 mg/mL) and 80 lL of 10% SDS were added to the mixture and incubated at 65 °C for 30 minutes. Homogenization was accomplished by adding 300 mg of 0.1 mm zirconium beads (BioSpec Products, Bartlesville, OK, USA) and bead beating for 90 seconds at 2045 9g (TeSeE process 48; Bertin Technologies, Le Bretonneux, France). The homogenized mixture was cooled on ice and then centrifuged at 14,270 9g for 5 minutes. Bacterial DNA was extracted from supernatant by phenol/ chloroform/iso-amyl alcohol (25 : 24 : 1) and then chloroform/iso-amyl alcohol (24 : 1). The supernatant was precipitated by absolute ethanol at 20 °C for 1 hour. The precipitated DNA was suspended in DNase free H2O and then cleaned up using the DNeasy Blood and Tissue extraction kit (Qiagen) according to the manufacturer’s instructions. Prior to microbial characterization, isolated DNA was stored at 80 °C.

PCR Amplification and 16S rRNA Gene Sequencing We amplified a 16S rRNA gene fragment of 280 base pairs, which includes the V5 hypervariable region by 35-cycle PCR. The modified forward primer 784F 50 -CGTATCGCCTCCCTCGCGCCATCAG-MID-AGGATTA GATACCCTGGTA-30 consists of the GS FLX Titanium adapter sequence A (underlined sequence), linker nucleotides (TCAG), multiplex identifiers sequence (MID),

© 2014 John Wiley & Sons Ltd, Helicobacter 19: 407–416

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Cancer Gastritis Cancer Gastritis Metaplasia Cancer Cancer Cancer Metaplasia Gastritis Metaplasia Gastritis Gastritis Cancer Metaplasia Metaplasia

10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 10-9 10-10 10-11 10-12 10-13 10-14 10-15 10-16

68 49 75 75 63 77 78 46 55 49 69 51 44 52 48 64

F M M F F F F F M F M F M M M M

Gender () () (+) (+) (+) (+) () (+) () (+) () (+) (+) () (+) ()

RUT () () () (+) (+) () () () () (+) () (+) (+) () () ()

Histology

test

45.63 0.75 87.68 97.13 86.34 76.23 0.81 70.62 4.55 94.44 0.80 96.90 95.94 0.68 99.78 5.77

Composition of Helicobacteraceae family (%)

H. pylori, Helicobacter pylori; RUT, rapid urease test; M, male; F, female; yrs, years.

Group

Sample

Age (yrs)

Conventional for H. pylori

Table 1 Demographic characteristics and H. pylori status for the enrolled patients

11-1 11-2 11-3 11-4 11-5 11-6 11-7 11-8 11-9 11-10 11-11 11-12 11-13 11-14 11-15

Sample Gastritis Cancer Metaplasia Metaplasia Cancer Cancer Cancer Gastritis Cancer Gastritis Metaplasia Gastritis Gastritis Metaplasia Metaplasia

Group 51 62 63 47 51 74 71 36 69 35 54 58 56 59 53

Age (yrs) F M F M M M M M F M F F F M M

Gender

(+) (+) (+) () () (+) (+) (+) (+) (+) (+) () () () ()

RUT

test

(+) (+) (+) () () () () (+) () (+) (+) () () () ()

Histology

Conventional for H. pylori

94.19 83.19 93.91 87.20 0.10 65.86 18.19 99.41 62.36 66.95 95.74 0.08 0.04 0.01 0.01

Composition of Helicobacteraceae family (%)

Mucosal Microbiota Profiling in Gastric Cancer Eun et al.

© 2014 John Wiley & Sons Ltd, Helicobacter 19: 407–416

410

Cancer Gastritis Cancer Gastritis Metaplasia Cancer Cancer Cancer Metaplasia Gastritis Metaplasia Gastritis Gastritis Cancer Metaplasia Metaplasia

10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 10-9 10-10 10-11 10-12 10-13 10-14 10-15 10-16

68 49 75 75 63 77 78 46 55 49 69 51 44 52 48 64

F M M F F F F F M F M F M M M M

Gender () () (+) (+) (+) (+) () (+) () (+) () (+) (+) () (+) ()

RUT () () () (+) (+) () () () () (+) () (+) (+) () () ()

Histology

test

45.63 0.75 87.68 97.13 86.34 76.23 0.81 70.62 4.55 94.44 0.80 96.90 95.94 0.68 99.78 5.77

Composition of Helicobacteraceae family (%)

H. pylori, Helicobacter pylori; RUT, rapid urease test; M, male; F, female; yrs, years.

Group

Sample

Age (yrs)

Conventional for H. pylori

Table 1 Demographic characteristics and H. pylori status for the enrolled patients

11-1 11-2 11-3 11-4 11-5 11-6 11-7 11-8 11-9 11-10 11-11 11-12 11-13 11-14 11-15

Sample Gastritis Cancer Metaplasia Metaplasia Cancer Cancer Cancer Gastritis Cancer Gastritis Metaplasia Gastritis Gastritis Metaplasia Metaplasia

Group 51 62 63 47 51 74 71 36 69 35 54 58 56 59 53

Age (yrs) F M F M M M M M F M F F F M M

Gender

(+) (+) (+) () () (+) (+) (+) (+) (+) (+) () () () ()

RUT

test

(+) (+) (+) () () () () (+) () (+) (+) () () () ()

Histology

Conventional for H. pylori

94.19 83.19 93.91 87.20 0.10 65.86 18.19 99.41 62.36 66.95 95.74 0.08 0.04 0.01 0.01

Composition of Helicobacteraceae family (%)

Mucosal Microbiota Profiling in Gastric Cancer Eun et al.

© 2014 John Wiley & Sons Ltd, Helicobacter 19: 407–416

Eun et al.

was ranged from 0.82 to 0.98 at all prokaryotic communities. By analyzing the gastric microbial composition at a class level, the composition of Epsilonproteobacteria group that contains H. pylori appeared to be the most prevalent, but the relative increase in the Bacilli group, which contains Streptococci and Lactobacilli, in the patients with gastric cancer was also noticed, resulting in a significant difference compared with the chronic gastritis patients (Fig. 1). In subclass analysis of Epsilonproteobacteria into species level, Epsilonproteobacteria class in most of the enrolled patients (especially in H. pyloripositive patients by conventional methods) were predominantly composed of H. pylori species (see Table S1 in Supporting information). It is well known from previous studies [26,27] that the clinical features and prognosis of gastric cancer may differ according to the H. pylori status; therefore, we have analyzed separately the patients with Helicobacter dominance. Excluding patients with the class Epsilonproteobacteria constituting

Differences in gastric mucosal microbiota profiling in patients with chronic gastritis, intestinal metaplasia, and gastric cancer using pyrosequencing methods.

Helicobacter pylori (H. pylori) infection plays an important role in the early stage of cancer development. However, various bacteria that promote the...
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