JOURNAL OF CLINICAL MICROBIOLOGY, May 2011, p. 1987–1989 Vol. 49, No.

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0095-1137/11/$12.00 doi:10.1128/JCM.02630-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Role of Helicobacter Species in Chinese Patients with

Inflammatory Bowel Disease䌤
Shenghong Zhang,1† Bihui Zhong,1† Kang Chao,1 Yingliang Xiao,1 Yi Cui,1 Xiang Gao,1
Baili Chen,1 Yao He,1 Pinjin Hu,1 Minhu Chen,1* and Hazel M. Mitchell2*
Division of Gastroenterology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China,1
and School of Biotechnology and Biomolecular Sciences, the University of New South Wales, Sydney, NSW 2052, Australia2
Received 28 December 2010/Accepted 12 February 2011

Based on 16S rRNA gene PCR, no significant difference was observed in rates of detection of Helicobacter
species in intestinal biopsy specimens from 160 Chinese inflammatory bowel disease (IBD) patients (10%) and
80 controls (6.3%). By using a [13C]urea breath test, the H. pylori infection rate in 208 Chinese IBD patients
(19.7%) was found to be significantly lower than that in 416 controls (48.8%).

Inflammatory bowel disease (IBD), including ulcerative coli-
tis (UC) and Crohn’s disease (CD), is a chronic relapsing
idiopathic inflammatory disorder of the gastrointestinal tract
with unknown etiology (20). While it is well accepted that
microorganisms play an essential role in the development of
IBD, the specific microorganisms involved in the disease re-
main unclear. Although a number of recent studies in Western
countries have reported increased prevalence of enterohepatic
Helicobacter species in the intestinal tracts of IBD patients, the
role of these organisms remains controversial (1–3, 5, 7, 10,
12–14, 18, 21). To date, such studies have not been conducted
in China.
Studies showing lower prevalence rates of gastric Helicobac-
ter pylori infection in IBD patients than controls have suggested
that gastric H. pylori infection may protect against IBD devel-
opment (11). This concept seems to be supported by the find-
ing that the prevalence of IBD has been increasing in China
over the last 2 decades (19) while the prevalence of H. pylori
infection has been decreasing. However, studies investigating
H. pylori prevalence in Chinese IBD patients are limited, due
partly to the low prevalence of IBD among the Chinese pop-
ulation (19).
This study used two separate arms to investigate Helicobac-
ter in Chinese IBD patients. In one arm, we used ribosomal
PCR to broadly investigate the prevalence of Helicobacter spe-
cies in the intestinal mucosae of IBD patients. In a separate
arm, we used the [13C]urea breath test (UBT) to compare the
prevalence rates of gastric H. pylori infection between IBD
patients and the general population.
Using a Helicobacter-specific 16S rRNA gene PCR, we de-
* Corresponding author. Mailing address for Minhu Chen: Division
of Gastroenterology, the First Affiliated Hospital, Sun Yat-sen Uni-
versity, No. 58 Zhongshan Road 2, Guangzhou 510080, People’s Re-
public of China. Phone: (8620) 87755766, ext. 8172. Fax: (8620)
87332916. E-mail: chenminhu@vip.163.com. Mailing address for
Hazel M. Mitchell: School of Biotechnology and Biomolecular
Sciences, University of New South Wales, Sydney, NSW 2052,
Australia. Phone: (612) 9385 2040. Fax: (612) 9385 1483. E-mail:
H.Mitchell@unsw.edu.au.
† Shenghong Zhang and Bihui Zhong contributed equally to the
work.
䌤
Published ahead of print on 23 February 2011.
termined whether Helicobacter species were present in intesti-
nal biopsy specimens from 240 patients (88 CD patients, 72 UC
patients, and 80 controls) undergoing colonoscopy at the en-
doscopy center of the First Affiliated Hospital of Sun Yat-sen
University, Guangzhou, People’s Republic of China, between
September 2008 and November 2010. According to recent
consensus guidelines, diagnosis of IBD was based on the com-
bination of clinical presentation and endoscopic, histological,
and radiological findings (4). Patients who had received anti-
biotics 3 months prior to colonoscopy or had a history of
previous H. pylori eradication therapy were excluded. Controls
were randomly recruited from among patients who underwent
colonoscopy for various reasons, such as hematochezia, polyp
follow-up, constipation, and cancer screening, but had no mu-
cosal lesions found upon colonoscopy and no abnormal histo-
pathological findings detected by microscopy. In the IBD
group, two biopsy specimens per patient were collected, one
each from endoscopically involved and noninvolved areas. In
the control group, two biopsy specimens were collected from
locations on the normal intestinal mucosa matched with the
IBD group. Biopsy specimens were used for DNA extraction
and histological staining. DNA samples were extracted from
intestinal biopsy specimens (about 5 mg per biopsy sample) by
using the QIAamp DNeasy tissue kit (Qiagen, Hilden, Ger-
many). Two different Helicobacter genus-specific primer sets,
pair 1 (H276f and H676r) and pair 2 (C97 and C98) (14, 21),
were used to amplify a fragment of approximately 400 bp
within the 16S rRNA gene that is conserved among members
of the Helicobacter genus. Positive (H. pylori DNA) and nega-
tive (water) controls were included in all PCR runs. All 16S
rRNA gene PCR-positive samples were sequenced.
A second cohort was recruited to investigate the gastric H.
pylori status as evaluated by UBT. A total of 208 IBD patients
and 416 age- and sex-matched healthy controls participated in
a regular check-up program in the hospital over the same
period. Details of previous treatment history (i.e., use of sul-
fasalazine, 5-aminosalicylic acid, corticosteroids, antibiotics,
and immunosuppressants) were obtained from medical records
and verified by face-to-face interview. Patients were excluded if
they had current or past use of proton pump inhibitor therapy
or histamine blockers 1 month prior to study entry, if they had

1987
1988 NOTES J. CLIN. MICROBIOL.

TABLE 1. Demographic and clinical features of IBD patients and controls who underwent intestinal biopsies
Value for:
Characteristic P value
CD patients UC patients Controls

No. of subjects 88 72 80
Gender (no. of males/females) 62/26 46/26 51/29 ⬎0.05
Mean age ⫾ SD (yr) 29.2 ⫾ 12.4 42.3 ⫾ 14.1 35.1 ⫾ 14.6 ⬍0.05
Mean disease duration ⫾ SD (mos) 52.9 ⫾ 33.9 52.3 ⫾ 49.1 ⬎0.05

Drug history
No. treated with sulfasalazine or 5-aminosalicylic 56 60
No. treated with corticosteroid 42 21
No. treated with immunosuppressant 44 14
No. treated with no medication 17 12

used antibiotics 3 months prior to study entry, or if they had a
history of H. pylori eradication therapy. A positive history of
antibiotic usage was defined as accumulated treatment dura-
tion of more than 1 week (17). The study protocol was ap-
proved by the human ethics committee of the First Affiliated
Hospital, Sun Yat-sen University. All patients provided written
informed consent.
The demographic and clinical characteristics of 240 patients
who underwent colonoscopy are presented in Table 1. Based
on the two Helicobacter genus-specific PCRs, eight CD patients
(9.1%), eight UC patients (11.1%), and five controls (6.3%)
were positive for Helicobacter spp. Subsequent sequencing of
the PCR products showed all to be 100% similar to H. pylori.
Fifteen PCR-positive patients (71.4%) had known gastric H.
pylori infection, while the status of H. pylori infection for the
remaining six patients was unknown. Examination of biopsy
tissues stained with hematoxylin and eosin or with modified
Giemsa stain showed no evidence of Helicobacter species in
intestinal tissue from any group.
The demographic and clinical characteristics of IBD patients
and controls who underwent the UBT are presented in Table
2. The prevalence of H. pylori infection in IBD patients was
19.7% (18.3% for CD patients and 21.2% for UC patients) and
was significantly lower than that in the age- and sex-matched
controls (48.8%) (␹2 ⫽ 49.267; P ⬍ 0.001) (Table 2). Patients
with UC had a higher H. pylori infection rate than patients with
CD, although the difference was not statistically significant
(␹2 ⫽ 0.273; P ⫽ 0.601). The H. pylori infection rate in IBD
patients who had previously used metronidazole or ciprofloxa-
cin was significantly lower (8.7%) than the rate in those who
had not (22.8%) (␹2 ⫽ 4.529; P ⫽ 0.033). However, the prev-
alence of H. pylori infection in nonusers of metronidazole or
ciprofloxacin (22.8%) remained significantly lower than that in
age- and sex-matched controls (48.5%) (␹2 ⫽ 29.551; P ⬍
0.001). Intake of other drugs (i.e., sulfasalazine, 5-aminosali-
cylic acid, corticosteroids, and immunosuppressants) had no
significant influence on the H. pylori infection rate (P ⬎ 0.05).
In this study, which consisted of the largest number of IBD
patients included in a study to date, we employed for the first
time two pairs of highly sensitive and specific primers to detect
Helicobacter species in Chinese IBD patients and controls (14,
21). In our study, no significant difference in the prevalence of
Helicobacter species DNA in the intestinal mucosa was found
between patients with IBD and controls. The sequences of all
positive samples were found to be identical to the H. pylori 16S
rRNA gene, which may reflect H. pylori gastric infection given
that 15 of the PCR-positive patients were known to have H.
pylori gastric infection. Currently, the data on whether the
presence of Helicobacter species is increased in patients with
IBD are inconclusive. While some studies have found an in-
creased prevalence rate (3, 10, 12, 14, 18, 21), other studies
have found otherwise (1, 2, 5, 7, 13). Discrepancies among
studies may be explained by geographical and genetic differ-
ences among countries and populations and/or differences in
disease stage, the length of time since initial diagnosis, and
previous antibiotic or other treatments received. Further stud-
ies including newly diagnosed patients, without any history of
intervention, are required to confirm this result.
We found a significantly lower prevalence of gastric H. pylori
infection in both Chinese UC and CD patients than in matched
controls, consistent with the result of a recent meta-analysis
(11). Our study also indicated that a history of treatment with
metronidazole or ciprofloxacin might, to some extent, lower
the gastric H. pylori infection rate, as these antibiotics have the
potential to eradicate H. pylori. However, after adjusting for
the impact of metronidazole or ciprofloxacin, we still found a

TABLE 2. Demographic and clinical characteristics of IBD patients and controls undergoing the 关13C兴urea breath test
Value for:
Characteristic P value
CD patients UC patients Controls

No. of subjects 104 104 416

Gender (no. of males/females) 69/35 60/44 258/158 ⬎0.05
Mean age ⫾ SD (yr) 31.0 ⫾ 12.2 40.9 ⫾ 13.6 36.0 ⫾ 13.8 ⬍0.05
Mean disease duration ⫾ SD (mos) 59.6 ⫾ 58.4 48.3 ⫾ 45.8 0.124
No. of smokers/nonsmokers 10/94 7/97 35/381 0.749
Prevalence of H. pylori infection (%) 18.3 21.2 48.8 ⬍0.001
VOL. 49, 2011
significantly lower prevalence of H. pylori infection in IBD
patients than in matched controls. Usage of other drugs such as
sulfasalazine, 5-aminosalicylic acid, steroids, and azathioprine
did not influence the prevalence of H. pylori infection, in agree-
ment with the results of most previous studies (11, 16, 17). In
support of this finding, a recent study conducted on a small
scale in eastern China also reported a lower prevalence of
gastric H. pylori infection in Chinese IBD patients than in
controls (15). The researchers in that study also found signif-
icantly increased prevalence of gastric H. pylori infection in UC
patients compared to that in CD patients, while such a signif-
icant increase was absent in our study. Although the design of
that study was similar to our study design, results from the
previous study are questionable in several ways. First, it in-
volved a small number of subjects. Second, it did not clearly
address the criteria for diagnosing IBD and it did not clearly
define exclusion and inclusion criteria in relation to treatment
intervention, both of which are factors that could lead to false-
negative results. Furthermore, only serological testing (for IgG
antibodies) was used to diagnose H. pylori infection, which may
result in false-positive findings, given the low specificity of
serological testing (11). In contrast, our study involved a large
number of patients and used consensus diagnostic criteria,
clear inclusion/exclusion criteria, and UBT for diagnosis of H.
pylori infection. Overall, results from our study provide a more
accurate prevalence of gastric H. pylori infection among IBD
patients.
The decline in the prevalence of H. pylori infection accom-
panied by a simultaneous rise in the occurrence of IBD in the
20th century had led to the hypothesis of the protective role of
H. pylori against IBD (9). Our results are in concordance with
this hypothesis. However, the mechanism behind this hypoth-
esis is unknown. It was reported previously that gastric infec-
tion with H. pylori alters the dendritic cell-polarized Th17/Treg
balance toward a Treg-biased response, which may influence
the immunological response in the intestine (8). Another study
reporting that prior H. pylori infection influences distal muco-
sal immune responses, as indicated by increased interleukin-10
expression measured in the mesenteric lymph nodes of mice
infected with H. pylori, also confirmed that H. pylori infection in
the stomach alters the immunological environment of the
lower gastrointestinal tract, providing mechanistic support for
the epidemiological observation of a negative association be-
tween H. pylori status and the risk of IBD (6). Further studies
investigating the effect of H. pylori eradication on the risk of
development of IBD are warranted to justify the beneficial role
of H. pylori for IBD prevention.
In conclusion, Helicobacter species are not found to play an
important role in the etiology of IBD in Chinese adult patients.
The lower rate of gastric H. pylori infection found in our study
in Chinese patients with IBD than in the controls suggests that
gastric H. pylori infection may protect against the development
of IBD. Future studies addressing this protective mechanism
will be of great interest.
This work is sponsored by the Guangdong Provincial Foundation of
Science & Technology (grants 2008B03031298 and 2009B030801174),
Yat-sen Innovative Talents’ training program for outstanding supervi-
NOTES 1989
sors of Sun Yat-sen University, and the International Program Fund of
the 985 Project of Sun Yat-sen University.
We appreciate Tracy Zhu from the Chinese University of Hong
Kong for her kind critical reading and linguistic revisions of the man-
uscript. We acknowledge Lihong Che and Zijin Weng from the De-
partment of Pathology for their assistance in histological sectioning
and staining and Wei Chen from the Division of Gastroenterology for
help with the UBT. We also acknowledge all the participants for their
considerable time and effort contributed toward this study.
We have no conflict of interest to declare.
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