Background
Helicobacter pylori (
H. pylori) is a chronic infectious pathogen that can lead to gastroduodenal diseases such as chronic gastritis, peptic ulcer disease (PUD), gastric carcinoma (GC) and mucosa associated lymphoid tissue (MALT) lymphoma [
41]. Owing to the carcinogenicity of
H. pylori, it was classified as a grade I carcinogen by the World Health Organization [
3]. It has been proved that more than half of the world’s population are infected with
H. pylori and the prevalence of
H. pylori has been declining in Western countries, whereas the prevalence has plateaued at a high level in developing countries [
21].
H. pylori is characterized by genetic diversity, but the clinical symptoms caused by different strains are variable and considered to be related to the genetic susceptibility and living environment of the host, mainly due to the bacterial virulence factors [
40].
Several
H. pylori virulence factors, such as
cagA,
vacA,
iceA,
oipA and
dupA have been identified to play an important role in the pathogenicity of
H. pylori [
24]. The CagA (cytotoxin-associated gene A) has been considered as an important carcinogen and
cagA-positive strains can increase the risk of PUD or GC. There are EPIYA segments in the CagA C-terminal region, which are the tyrosine phosphorylation sites of CagA protein. According to the difference of the amino acid sequences flanking the EPIYA motifs, CagA C-terminal region can be divided into four different segments: EPIYA-A, EPIYA-B, EPIYA-C and EPIYA-D [
20].
VacA (vacuolating cytotoxin A), which can induce vacuolation and multiple cellular activities, is encoded by
vacA gene, which has distinct alleles [
13]. Although
vacA is present in all
H. pylori strains, it shows allelic variation in three main regions: the signal (s) region (s1a, s1b and s1c, s2), the intermediate (i) region (i1 and i2) and the middle (m) region (m1 and m2) [
37]. The different combination of s and m regions determines the production of cytotoxic activity and constitutes mosaic gene structure. Strains with the genotype s1m1 produce high levels of toxin in vitro, followed by s1m2, while s2m1 strains produce low toxicity and s2m2 strains produce little or no toxin [
8]. It has been shown that s1m2 strains that contain the i1 allele are vacuolating, whereas strains that contain the i2 allele are non-vacuolating [
17]. Studies have shown that s1m1 subtype is highly correlated with PUD and GC [
19,
31,
38]. Additional studies also found strains containing the
vacA i1 allele can increase the risk of GC [
32]. There are geographic variations in the distribution of
vacA genotypes in different regions. Many researches have shown that
vacA s1a and s1c are predominant in Asia and northern Europe, whereas s1b is common in South America, Southern Europe and South Africa [
14,
47]. These differences may lead to diversity in prevalence of gastroduodenal diseases in different geographic regions.
The
iceA (induced by contact with epithelium) has two main allelic variants:
iceA1 and
iceA2, which also has a particular geographic distribution [
22]. The
iceA1 was common in Japan and Korea while the
iceA2 was predominant in the America, Colombia and Europe [
35]. The OipA (outer inflammatory protein A) increases inflammatory response by affecting interleukin 8 (IL-8) production. The
oipA functional status is regulated by slipped-strand mispairing that is based on the number of CT dinucleotide repeats in the signal sequences of the gene (switch “on” = functional and switch “off” = nonfunctional) [
45]. Studies have shown that the prevalence of
oipA in duodenal ulcer (DU) and GC is higher, suggesting that
oipA is not only associated with inflammation, but also the development of GC [
46]. The
dupA (duodenal ulcer promoting gene A), first recognized as a marker of
H. pylori specific disease, can induce DU and inhibit GC [
2].
China is a country with large population, wide area and high incidence of
H. pylori infection. In addition, the incidence rate of GC in China is higher than that in the Western countries. Heilongjiang and Qinghai provinces are high risk areas of GC, which are located in the northeast and northwest of China respectively, the mortality rate of GC ranges from 40 to 70 per 100,000 persons, compared to 10 to 20 per 100,000 persons in Guangxi and Hunan, low risk areas of GC, which are located in the South and Central South of China respectively [
10]. Shandong province is located in the east of China and the crude mortality rate of GC was 49 per 100,000 persons, accounting for 21% of all malignant cancers [
28]. At present, there are many studies focusing on the relationship between
H. pylori virulence factors and clinical outcomes in China. However, only a few studies regarded information on the relationship between
H. pylori virulence genotypes and different geographic regions. We therefore investigated the distribution of
vacA,
cagA,
iceA,
oipA and
dupA genotypes in different regions of China and their association with clinical outcomes.
iceA status
Overall,
iceA1 was detected in 187 (69.5%) of all 269 isolates examined and
iceA2 was found in 54 isolates (20.1%) (Table
1). The
iceA1 frequency was significantly more prevalent in Hunan (82.6%) than in the other four regions (χ
2 = 11.358, P < 0.05). The
iceA2 was present in 25.6% and 21.1% of
H. pylori strains isolated from Guangxi and Heilongjiang, respectively, whereas only 16.2% of isolates from Shandong were infected with
iceA2-positive strains. However, the difference was not statistically significant (χ
2 = 3.204, P > 0.05). There was also no association between the
iceA status and clinical outcomes in the five regions of China (Table
3).
oipA status
242 (90%) isolates were positive with
oipA set primers, and, overall, 88.1% had a functional status “on” (Table
1). A total of 10
oipA CT repeat patterns were identified (Table
4). The pattern containing (3 + 1) CT repeats was the most frequently associated with the “on” status (125/237, 52.7%), and the pattern with 5 CT repeats was the most prevalent for a nonfunctional (“off”)
oipA gene (3/5, 60%). The
oipA functional status “on” was more prevalent in Shandong isolates than in Heilongjiang isolates (χ
2 = 8.060, P < 0.05). Overall, 87.1% of NUD patients and 100% of PUD patients were infected with
oipA functional status “on” strains, the difference was not statistically significant (χ
2 = 3.561, P > 0.05) (Table
3). When the analyses were carried out in each geographic region, the differences were also not statistically significant.
Table 4
Frequency of the oipA CT repeat patterns
“On” status | | |
ATGAAAAAAGCTCTCTTACTAACTCTCTTTTTCTCGTTTTGGCTCCACGCTGAA | 3 + 1 | 125 |
M K K A L L L T L F F S F W L H A E | | |
ATGAAAAAAACCCTTTTACTCTTTCTGTCTTTCTCGTTTTGGCTCCACGCTGAA | 2 + 1 + 1 | 82 |
M K K T L L L F L S F S F W L H A E | | |
ATGAAAAAAGCTCTCTTACTAACTCTCTTTCTCTCGTTTTGGCTCCACGCTGAA | 3 + 2 | 14 |
M K K A L L L T L F L S F W L H A E | | |
ATGAAAAAAACCCTTTTACTCACTCTTTCTCTCTCGTTTTGGCTCCACGCTGAA | 2 + 3 | 1 |
M K K T L L L T L S L S F W L H A E | | |
ATGAAAAAAACCCTTTTACTCTTTCTGTCTCTCTCGTTTTGGCTCCACGCTGAA | 1 + 3 | 5 |
M K K T L L L F L S L S F W L H A E | | |
ATGAAAAAAGCTCTCTTACTAATTCTCTTTTTCTCGTTTTGGCTCCACGCTGAA | 2 + 1 | 1 |
M K K A L L L I L F F S F W L H A E | | |
ATGAAAAAAGCTCTCTTACTAACTCTCTCTCTCTCGTTCTGGCTCCACGCTGAA | 6 | 9 |
M K K A L L L T L S L S F W L H A E | | |
“Off” status | | |
ATGAAAAAAGCTCTCTTACTAACTCTCTCTCTCGTTTTGGCTCCACGCTGA | 5 | 3 |
M K K A L L L T L S L V L A P R * | | |
ATGAAAAAAGCTCTCTTACTCTCTCTCTCTCTCGTTCTGGCTCCATGCTGA | 7 | 1 |
M K K A L L L S L S L V L A P C * | | |
ATGAAAAAAGCTCTCTTACTAACTCTCTCTCTCTCTCTCGTTTTGGCTCCACGCTGA | 8 | 1 |
M K K A L L L T L S L S L V L A P R* | | |
dupA status
121 (45%) patients were infected with
dupA-positive strains (Table
1). The
dupA-positive strains were present in 73.9% of Hunan, 81.8% of Qinghai and 65.8% of Heilongjiang. In contrast, only 31.1% of Shandong and 15.4% of Guangxi strains were infected with
dupA-positive
H. pylori (χ
2 = 72.497, P < 0.001). Overall, 43.5% of patients with NUD and 61.9% of those with PUD were infected with
dupA-positive strains (Table
3). The
dupA-positive strains were significantly more common in PUD patients than in NUD patients in Shandong (χ
2 = 6.830, P < 0.01) and Guangxi (χ
2 = 4.254, P < 0.05). In contrast, the
dupA-positive strains were more common in NUD patients (76.2%) than in PUD patients (50%) in Hunan, but the difference was not statistically significant (χ
2 = 1.299, P > 0.05).
Materials and methods
Study subjects
A total of 348 patients were involved in this study including 89 patients from Rushan People’s Hospital (Weihai, Shandong Province, China), 91 from the Second Nanning People’s Hospital (Nanning, Guangxi Province, China), 57 from Yiyang Central Hospital (Yiyang, Hunan Province, China), 58 from the People’s Hospital of Huzhu Tu Ethnic Autonomous County (Haidong, Qinghai Province, China) and 53 from The First Affiliated Hospital of Jiamusi University (Jiamusi, Heilongjiang Province, China). Their gastric biopsy specimens were obtained during upper gastrointestinal endoscopy with informed consent. This study was approved by Ethical Committee of National Institute for Communicable Disease Control and Prevention Chinese Center for Disease Control and Prevention (approval No. ICDC-2013001).
H. pylori culture and DNA extraction
Gastric biopsy specimens were homogenized thoroughly in brain heart infusion (BHI) broth and then streaked onto the Karmali blood agar base plates under a biological safety cabinet (Thermo Scientific). The Karmali Agar base (Oxoid, CM 0935) was supplemented with 5% defibrinated sheep blood and 1% combined antibiotics comprising of trimethoprim (150 mg/L), vancomycin (125 mg/L), amphotericin B (100 mg/L) and polymyxin B (100 mg/L). The plates were incubated at 37 °C under microaerobic conditions (5% O2, 10% CO2 and 85% N2) for 3–5 days. H. pylori colonies were identified according to its morphological characteristics, negative Gram staining and positive for catalase, oxidase, and urease. The identified H. pylori was subcultured to single colonies and then preserved in sterile BHI broth with 20% glycerol and frozen at − 80 °C until the genomic DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted DNA was stored at − 20℃ and used directly for PCR.
PCR amplification
The PCR reaction was carried out in a total volume of 25 μL containing forward and reverse primers (0.2 μM each), 2 ng/μL DNA template, 12.5 μL Go Taq® Green Master Mix (Promega, USA) and 9.5 μL nuclease-free water. The amplification was as follows: initial denaturation at 94 °C for 5 min and then denaturation at 94 °C for 30 s, primer annealing at 54, 56, 60, 56 and 62 °C for
cagA,
iceA,
dupA,
vacA (s1/s2, s1a, s1b, s1c, m1, m2 and i1, i2) and
oipA, respectively, for 30 s and extension at 72 °C for 40 s. All reactions were performed through 35 cycles. The final cycle included a final extension at 72 °C for 10 min. The presence of the
cagA,
iceA and
dupA genes was determined by PCR as previously described [
9,
23,
30]. The genotypes of
vacA s1/s2, s1a, s1b, s1c, m1, m2 and i1, i2 were also determined by PCR as previously described [
6,
16,
29,
37]. The
oipA gene was detected by PCR, which was additionally sequenced in order to define its functional status as either “on” or “off”. The signal sequences of
oipA gene including the CT repeats were amplified by using primer pairs as described previously [
25]. The primers used to amplify the targeted genes were summarized in Table
5. The amplified products were analyzed in 1% agarose gel containing 1 × TAE, stained with GelStain and visualized by electrophoresis at 110 V for 30 min using the gel documentation system (Bio-Rad, USA).
Table 5
Primers used for PCR amplification of cagA, vacA, iceA, oipA and dupA genes
cagA | cagA3'-F | TGCGTGTGTGGCTGTTAGTAG | 593–752 |
| cagA3'-R | CCTAGTCGGTAATGGGTTGT | |
vacA | | | |
s1/s2 | Vs-F | ATGGAAATACAACAAACACAC | 259/286 |
| VA-R | CTGCTTGAATGCGCCAAAC | |
s1a | Vs1a-F | GTCAGCATCACACCGCAAC | 190 |
| VA-R | CTGCTTGAATGCGCCAAAC | |
s1b | Vs1b-F | AGCGCCATACCGCAAGAG | 187 |
| VA-R | CTGCTTGAATGCGCCAAAC | |
s1c | Vs1c-F | CTCTCGCTTTAGTGGGGYT | 213 |
| VA-R | CTGCTTGAATGCGCCAAAC | |
m1 | Vm1-F | GGCCCCAATGCAGTCATGGAT | 240 |
| Vm1-R | GCTGTTAGTGCCTAAAGAAGCAT | |
m2 | Vm2-F | GGAGCCCCAGGAAACATTG | 352 |
| Vm2-R | CATAACTAGCGCCTTGCAC | |
i1 | Vi-F | GTTGGGATTGGGGGAATGCCG | 495 |
| Vi1-R | TTAATTTAACGCTGTTTGAAG | |
i2 | Vi-F | GTTGGGATTGGGGGAATGCCG | 495 |
| Vi2-R | GATCAACGCTCTGATTTGA | |
iceA1 | iceA1-F | GTGTTTTTAACCAAAGTATC | 246 |
| iceA1-R | CTATAGCCAGTCTCTTTGCA | |
iceA2 | iceA2-F | GTTGGGTATATCACAATTTAT | 229 |
| iceA2-R | TTGCCCTATTTTCTAGTAGGT | |
oipA | oipA-F | CGCGGAAAGGAACGGGTTTT | 519 |
| oipA-R | TTAGCGTCTAGCGTTCTGCC | |
dupA | dupA-F | GACGATTGAGCGATGGGAATAT | 971 |
| dupA-R | CTGAGAAGCCTTATTATCTTGTTGG |
Positive PCR products were sent to the Beijing Genomics Institute (BGI) for purification and sequencing. The nucleotide sequences of the
cagA 3’ variable region and
oipA were submitted to China National Microbiological Data Center. The accession numbers are NMDCN0000M60 to NMDCN0000ME4 and NMDCN0000ME5 to NMDCN0000MLM, respectively. DNA sequences were edited by EditPlus version 5.3.0 and the edited nucleotide sequences were subjected to translation using BioEdit version 7.2.5. The EPIYA segment types of CagA were analyzed using the program WebLogo 3 (
http://weblogo.threeplusone.com/). Neighbor-Joining phylogenetic tree was constructed from
cagA 3’ variable region nucleotide sequences using MEGA version 7.0.18 and bootstrap analysis was performed with 1000 replications. The Western strain 26695 (GenBank No. CP003904) and the East Asian strain GZ27 (GenBank No. KR154756) were used as reference sequences.
Statistical analysis
Statistical data were analyzed by SPSS software version 20. The chi-square test and Fisher’s exact test were used to assess the relationship between specific genotype and geographic origins and clinical outcomes. P-value < 0.05 was considered of a statistically significant difference.
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