Objective: To investigate the distribution of helicobacter pylori-related genotypes of oipA, babA2, and babB in patients with gastrointestinal diseases.
Method: The retrospective study was conducted at the Jiamusi College, Heilongjiang University of Traditional Chinese Medicine, Harbin, China, and comprised data from February 2017 to May 2020 of patients of either gender 20-80 years who underwent gastroscopy. An instrument based on polymerase chain reaction was used to amplify oipA, babA2 and babB genes, and their distribution in terms of gender, age and pathological types was analysed.
Results: Among the 116 patients, 52(44.8%) had oipA genotype, 48(41.2%) babA2, and 72 (62.1%) babB, respectively, and the size of amplified products of 486bp, 219bp and 362bp, respectively. The infection rate of oipA and babB genotypes was highest [26(50.0%) and 31(43.1%)]in those aged 61-80 years, and lowest [9(17.3%) and 15(20.8%)]in those aged 20-40 years. The infection rate of babA2 genotype was highest [23(47.9%)] in those aged 41-60 years, and lowest [12(25.0%)] in those aged 61-80 years. Male patients were under a higher [28(53.9%) and 26(54.2%)] rate of infection with oipA and babA2, and female patients has a higher [40(55.6%)] rate of infection with babB. Among Hp-infected patients with digestive diseases, babB genotype was mainly found in patients with chronic superficial gastritis[17(58.6%)], duodenal ulcer[17(85.0%)], chronic atrophic gastritis[19(59.4%)] and gastric ulcer[16(72.7%)], while oipA genotype was mainly found in patients with gastric cancer[8(61.5%)].
Conclusion: Chronic superficial gastritis, duodenal ulcer, chronic atrophic gastritis, and gastric ulcer may have a close bearing on babB genotype infection, while oipA genotype infection may be associated with gastric cancer.
Keywords: Helicobacter pylori, oipA, babA2, babB, Chronic superficial gastritis, Chronic atrophic gastritis, Duodenal ulcer, Gastric ulcer, Gastric cancer. (JPMA 72: 249; 2022)
Submission completion date: 16-02-2022 - Acceptance date: 14-09-2022
Helicobacter pylori (Hp) may be colonised on the epithelium of the gastric mucosa (Figure-1), and most patients with Hp infection show no symptoms.1 It has been shown in relevant studies that Hp infection is a vital pathogenic factor for gastrointestinal (GI)diseases, such as chronic superficial gastritis (CSG), chronic atrophic gastritis (CAG), duodenal ulcer (DU), gastric ulcer (GU) and gastric cancer (GC), while host susceptibility, environmental factors and differences in virulence gene expression can affect the clinical outcome after Hp infection.2 Differences in Hp virulence may be determined by Hp virulence gene polymorphism.3 To date, Hp gene polymorphism has been studied mainly on the two genes of cytotoxin-related protein cagA and vacuolar toxin vacA, and Hp is divided into type I strains (cagA-positive and vacA-positive) and type II strains (cagA-negative and vacA-negative).4 However, a few studies have been carried out on the expression of virulence genes, such as oipA, babA2 and babB, in various types of gastrointestinal disorders (GIDs). The current study was planned to investigate the distribution of oipA, babA2 and babB genotypes in various GIDs.
Materials and Methods
The retrospective study was conducted at the Jiamusi College, Heilongjiang University of Traditional Chinese Medicine, Harbin, China, and comprised data from February 2017 to May 2020 of patients who underwent gastroscopy. Data related to patients of either gender aged 20-80 years who met the diagnostic criteria of various GIDs and the diagnoses were confirmed through gastroscopic biopsy5-7 Patients gave voluntary consent for their data to be used for research purposes provided a study was approved by the institutional ethics review committee, which the current study obtained. Data was excluded for patients with missing information, those who had taken antibiotics, non-steroidal antacids, gastric mucosal protective agents, and anti-inflammatory drugs within 2 weeks prior to specimen collection, patients with immune system diseases, and those with gastroesophageal reflux.
Deoxyribonucleic acid (DNA) samples were generated through an extraction kit (Article No.: K368-50RXN; Qingdao Jieskang Biotechnology Co., Ltd.), amphotericin B (Article No.: K0018; Shanghai Baoman Biotechnology Co., Ltd.), vancomycin (Article No.: V76062; Shanghai Acmec Biochemical Co., Ltd.), trimethoprim (Article No.: SND-1171; Chuzhou Shinuoda Biological Technology Co., Ltd.), polymyxin (Article No.: P8350; Shanghai Zhenyu Biological Technology Co., Ltd.), agar (Article No.: A111; Shanghai Yanhui Biological Technology Co., Ltd.), electrophoresis instrument (Model: 81-0478-01; Shanghai Fuze Trading Co., Ltd.), low-temperature high-speed centrifuge (Model: TG-16; Thermo Fisher Scientific, United States), polymerase chain reaction (PCR_ instrument (Model: Veriti 96-Well Thermal Cycler; Thermo Fisher Scientific, USA), and a gel imaging system (Model: Quick Gel 6200; Monad ]Suzhou] Biotech Co., Ltd).
For sample collection, the gastroscope was moved into the patient's digestive tract to search for the location of the lesion. The oesophagus, stomach and duodenum were explored. After finding the suspected lesion, gastric mucosa specimens were taken and stored at -80°C for testing and genotyping.
All the specimens were inoculated in prepared Hp medium (10% glucose, 7% defibrinated sheep blood, amphotericin B 10mg/L, vancomycin 10mg/L, trimethoprim 5mg/L, polymyxin 2500U/L, 2% agar) for 4d under micro-oxygen conditions (85% nitrogen gas [N2], 37°C, 10% carbon dioxide [CO2], 5% oxygen [O2]). Subsequently, biochemical reactions (urease, thiase, oxidase) and colony morphology were observed. Hp strains were confirmed by gram stain smears and stored at -80°C for later use.
The preserved Hp strains were resuscitated and the bacteria were scraped after 48h of culture, and then washed with phosphate-buffered saline (PBS) 1-2 times. Hp nucleic acid DNA was extracted with the extraction kit and stored at -80°C for later use.
A PCR instrument was utilised to amplify oipA, babA2, and babB genes, and the primer sequences were noted
(Table-1). PCR was performed in 25µL of the reaction system, containing 1µL of Hp template (50 ng/µL), 2µL each of forward and reverse primers (10µM), 2µL of deoxynucleoside triphosphate [dNTP], 2.5µL of 10×Buffer, 1µL of Thermus aquaticus DNA [TaqDNA], plus double-distilled water [ddH2O] 25µL. Each sample was repeated 3 times. After amplification, agarose gel electrophoresis was performed. A gel imager was used for observation, and ddH2O was used as the negative control.
Data was analysed using SPSS 25. Data was expressed as mean±standard deviation and frequencies and percentages, as appropriate. To compare the differences, t test and chi-square test were used, as appropriate. P<0.05 was taken as statistically significant.
Among the 116 patients, no significant difference was observed related to age or gender among the five disease groups (Table-2).
There were 52 (44.8%) cases of oipA genotype and the size of amplified product 486 bp (Figure-2). There were 48 (41.2%) cases of babA2 and the size of amplified product 219bp (Figure-3). There were 72 (62.1%) cases with babB and the size of amplified product 362bp (Figure-4).
The distribution of oipA, babA2 and babB genotypes was noted in relation to age and gender (Table-3), and with respect to different pathologies (Table-4).
Gastric ailments and chronic infections of the duodenum are common diseases of the digestive tract. Gastric cancer is a common malignant tumour of the digestive tract, and is also among the top three tumours with a low 5-year survival rate.8 The aetiology of gastric cancer has remained unclear to date. However, in recent years, many studies have proposed the development model of “chronic gastritis-atrophic gastritis-intestinal metaplasia-gastric cancer”, and studies have shown that Hp infection affects gastric mucosal inflammation and immunity of patients, which will increase the incidence of gastric cancer.9,10 It is currently believed that the principal pathogenic factors of GIDs caused by Hp are adhesion, flagella, reactive oxygen species (ROS), heat shock protein, urease, cytotoxin, etc., all of which are involved in gastric mucosal damage.11
Epidemiological investigations have revealed that only a small proportion of Hp-infected people will develop GIDs, such as CSG and CAG, which may be mainly caused by differences in Hp strains. On the other hand, differences in Hp strains were in turn associated with virulence gene polymorphism.12 In recent years, babA has likewise gradually become a research hotspot among Hp virulence genes. Gene babA, as one of the outer membrane proteins of Hp, can mediate the adhesion of Hp and has two genotypes, babA1 and babA2. Both are sequentially homologous, but only babA2 is biologically active because it has a 10bp insertion sequence that produces a translation starting point.13 As shown in the current study, among 116 Hp-infected patients with GIDs, the positive rate of babA2 was 41.4%, which was higher than the detection rate of babA2 in an earlier study.14 The positive rate of babA2 gene in Brazil was 44%, which was related to peptic ulcer and gastric cancer.15 The positive rate of babA2 in Japan was 84.9%, but it had nothing to do with the clinical outcome.16
Moreover, in the current study, the infection rate of babA2 genotype was highest in patients aged 41-60 years, and lowest in patients aged 61-80 years; The detection rate of babA2 in patients with GU, DU and GC was higher than that in patients with CAG and CSG, though not statistically significant, which is basically consistent with earlier results,17 suggesting that babA2 genotype may be associated with various GIDs. Genotype oipA is a pro-inflammatory outer membrane protein, which has a close bearing on neutrophil infiltration, clinical symptoms of patients and bacterial colonisation density after Hp infection, contributing to the colonisation of Hp in gastric mucosa.18 A study confirmed that the opiA gene is closely related to inflammation.19 As shown in the current study, the positivity rate of oipA was 44.83%, and Hp in GC patients was dominated by oipA genotype, while patients with DU and GU had a lower infection rate of oipA genotype, which was basically consistent with earlier findings,20 suggesting that oipA infection may cause more severe gastric mucosal damage and is more likely to induce GC compared to babA2 and babB genotypes. Also babB itself acts as a lectin that binds to an unknown receptor on host epithelial cells or modulates the binding of other adhesins.18 As shown in the current study, the positivity rate of babB was 62.07%, which was consistent with earlier reports.21 Furthermore, the infection rate of babB genotype was the highest in patients aged 61-80 years, and the lowest in patients aged 20-40 years. Patients with CSG, DU, CAG, and GU are mainly infected with babB genotype Hp, while patients with GC have a low babB infection rate,21 suggesting that babB genotype may be highly correlated with elderly patients and patients with CSG, DU, CAG and GU, but may have relatively little relationship with GC.
The current study has certain limitations as patients from only one region were enrolled. The mechanism of oipA, babA2 and babB genotypes involved in GC needs to be further studied via cell model experiments or animal model experiments, and other studies need to be combined to further explore the polymorphism and diversity of Hp virulence genes.
CSG, DU, CAG and GC were found to be mainly babB genotype infections, and oipA genotype infections were found to be associated with GC.
Conflict of Interest: None.
Source of Funding: None.
1. Li C, Huang P, Wong K, Xu Y, Tan L, Chen H, et al. Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process. J Enzyme Inhib Med Chem 2018;33:1362-75. doi: 10.1080/14756366.2018. 1501044.
2. Park JY, Forman D, Waskito LA, Yamaoka Y, Crabtree JE. Epidemiology of Helicobacter pylori and CagA-Positive Infections and Global Variations in Gastric Cancer. Toxins (Basel) 2018;10:163. doi: 10.3390/toxins10040163.
3. Lin DS, Xia L, Fang L. Relevance analysis of vacA virulence gene of helicobacter pylori and its drug resistance. J Med Res 2018;47:124-6.
4. Carlosama-Rosero YH, Bolaños-Bravo H, Sierra-Tórres CH, Rosero EA. Association of the Helicobacter pylori cagA, vacA, and iceA genotypes with chronic follicular gastritis in a Colombian population at high risk for gastric cancer. Rev Gastroenterol Mex (Engl Ed) 2019;84:158-64. doi: 10.1016/j.rgmx.2018.03.004.
5. Song HB, Wen CB. Research progress on the objectification of Chinese medicine four diagnosis methods information of chronic gastritis. Asia-Pacific Tra Med 2019;15:182-4.
6. Wang PZ. Gastroscopic manifestations and pathological diagnosis of the peptic ulcer. J Baotou Med 2017;41:78-80.
7. Yu M, Tang HH. Diagnosis and surgical treatment of early gastric cancer. J Imaging Res and Med Applications 2017;1:220-222.
8. Cheung KS, Chan EW, Wong AYS, Chen L, Wong ICK, Leung WK. Long-term proton pump inhibitors and risk of gastric cancer development after treatment for Helicobacter pylori: a population-based study. Gut 2018;67:28-35. doi: 10.1136/gutjnl-2017-314605
9. Polakovicova I, Jerez S, Wichmann IA, Sandoval-Bórquez A, Carrasco-Véliz N, Corvalán AH. Role of microRNAs and Exosomes in Helicobacter pylori and Epstein-Barr Virus Associated Gastric Cancers. Front Microbiol 2018;9:e636. doi: 10.3389/fmicb. 2018.00636.
10. Rong Y, Liu MH, Deng ZX. Analysis of the risk factors causing gastric stump cancer for peptic ulcer patients after accepting subtotal gastrectomy as well as related intervention measures. Pro Modern Bio 2019;19:79-82.
11. Jafarzadeh A, Larussa T, Nemati M, Jalapour S. T cell subsets play an important role in the determination of the clinical outcome of Helicobacter pylori infection. Microb Pathog 2018;116:227-36. doi: 10.1016/j.micpath.2018.01.040.
12. Shi M. Advances in virulence genes and pathogenicity of Helicobacter pylori. J Cli Ped 2019;37:233-6.
13. Román-Román A, Martínez-Carrillo DN, Atrisco-Morales J, Azúcar-Heziquio JC, Cuevas-Caballero AS, Castañón-Sánchez CA, et al. Helicobacter pylori vacA s1m1 genotype but not cagA or babA2 increase the risk of ulcer and gastric cancer in patients from Southern Mexico. Gut Pathog 2017;9:e18. doi: 10.1186/s13099-017-0167-z.
14. Meng W, Jian H, Wei Z, YeHua L, FuHua Z, Hong M. Relationship between genotypes of Helicobacter pylori iceA, babA2 and diseases in Hexi Corridor. Chongqing Medicine 2019;48:3126-33.
15. Oliveira AG, Santos A, Guerra JB, Rocha GA, Rocha AM, Oliveira CA, et al. babA2- and cagA-positive Helicobacter pylori strains are associated with duodenal ulcer and gastric carcinoma in Brazil. J Clin Microbiol 2003;41:3964-6. doi: 10.1128/JCM.41.8.3964-3966.2003.
16. Mizushima T, Sugiyama T, Komatsu Y, Ishizuka J, Kato M, Asaka M. Clinical relevance of the babA2 genotype of Helicobacter pylori in Japanese clinical isolates. J Clin Microbiol 2001;39:2463-5. doi: 10.1128/JCM.39.7.2463-2465.2001.
17. Abdi E, Latifi-Navid S, Yazdanbod A, Zahri S. Helicobacter pylori babA2 Positivity Predicts Risk of Gastric Cancer in Ardabil, a Very High-Risk Area in Iran. Asian Pac J Cancer Prev 2016;17:733-8. doi: 10.7314/apjcp.2016.17.2.733.
18. Farzi N, Yadegar A, Aghdaei HA, Yamaoka Y, Zali MR. Genetic diversity and functional analysis of oipA gene in association with other virulence factors among Helicobacter pylori isolates from Iranian patients with different gastric diseases. Infect Genet Evol 2018;60:26-34. doi: 10.1016/j.meegid.2018.02.017.
19. Yamaoka Y, Kita M, Kodama T, Imamura S, Ohno T, Sawai N, et al. Helicobacter pylori infection in mice: Role of outer membrane proteins in colonization and inflammation. Gastroenterology 2002;123:1992-2004. doi: 10.1053/gast.2002.37074.
20. Su YL, Huang HL, Huang BS, Chen PC, Chen CS, Wang HL, et al. Combination of OipA, BabA, and SabA as candidate biomarkers for predicting Helicobacter pylori-related gastric cancer. Sci Rep 2016;6:36442. doi: 10.1038/srep36442.
21. Dabiri H, Jafari F, Baghaei K, Shokrzadeh L, Abdi S, Pourhoseingholi MA, et al. Prevalence of Helicobacter pylori vacA, cagA, cagE, oipA, iceA, babA2 and babB genotypes in Iranian dyspeptic patients. Microb Pathog 2017;105:226-30. doi: 10.1016/j.micpath.2017.02.018.