INVESTIGATION OF Helicobacter pylori VIRULENCE GENOTYPE IN GASTRIC BIOPSIES BY PCR

Abstract

  Background: Helicobacter pylori infections has been associated with the genetic diversity of their virulence factors, the virulence genotypes are valuable as molecular marker in the diagnosis of patients with bacterial infections . Our main objective was to analyze the frequency and allelic genotype of vacA , cagA also investigate another virulence genes of H. pylori. Methods: 75 biopsies of  patients with gastritis and peptic ulcer diseases were selected to investigate the presences of H. pylori and collected from them antrum biopsies, then  genomic DNA was extracted from  antrum biopsies using genomic DNA kit .Subsequently, the virulence genes of H. pylori   were amplified using specific primers including vacA , cagA, cagE and oipA and iceA by PCR in 49 cases that positive to 16SrRNA which previously investigated. Results: A high prevalence of genes cagA  (28.6%), vacAs1bm2 (56.8%), iceA2 (30.6%)  and oipA  (42.9%) was found, while  vacA s2m1  and iceA1 genotypes  was not found in our study.  There was significant correlation between the presence of cagA and cagE genotypes (p = 0.02), suggesting that these two genes almost used together as cag PAI integrity marker. The  presence of cagA gene was significantly associated with peptic ulceration (p ≤ 0.001), whereas different vacA genotypes or iceA2 genotype were no statistically significant  with clinical outcome.  Patients with peptic ulcer disease more likely to have oipA gene (61.9% ) than those with gastritis (38.1%), P = 0.037, also the presence oipA gene was statistically significant with presence iceA2. Conclusion : Most H. pylori genotypes which associated with peptic ulcer and gastritis were moderate virulent strains.  

Author Biography

Rabab Omran Al-jelawi-, babylon university
biology deparament , biotechnology-genetic engineering

References

1. Thorell K, Hosseini S, Palacios Gonzáles RV, Chaotham C, Graham DY, Paszat L, et al. Identification of a Latin American-specific BabA adhesin variant through whole genome sequencing of Helicobacter pylori patient isolates from Nicaragua. BMC Evol Biol 2016;16:53.
2. Kao CY, Sheu BS, Wu JJ. Helicobacter pylori infection: An overview of bacterial virulence factors and pathogenesis. Biomed J 2016;39(1):14-23.
3. Hemmatinezhad B, Momtaz H, Rahimi E. VacA, cagA, iceA and oipA genotypes status and antimicrobial resistance properties of Helicobacter pylori isolated from various types of ready to eat foods. Ann Clin Microbiol Antimicrob 2016;15:2.
4. Essawi T, Hammoudeh W, Sabri I, Sweidan W, Farraj MA. Determination of Helicobacter pylori virulence genes in gastric biopsies by PCR. ISRN Gastroenterol 2013;2013:606258.
5. Zhou J, Wang W, Xie Y, Zhao Y, Chen X, Xu W, et al. Proteomics - Based identification and analysis of proteins associated with Helicobacter pylori in gastric cancer. PLoS One 2016;11(1):e0146521.
6. Ganguly M, Sarkar S, Ghosh P, Sarkar A, Alam J, Karmakar BC, et al. Helicobacter pylori plasticity region genes are associated with the gastroduodenal diseases manifestation in India. Gut Pathog 2016;8:10.
7. da Costa DM, Pereira Edos S, Rabenhorst SH. What exists beyond cagA and vacA? Helicobacter pylori genes in gastric diseases. World J Gastroenterol 2015;21(37):10563-72.
8. Keilberg D, Ottemann KM. How Helicobacter pylori senses, targets and interacts with the gastric epithelium. Environ Microbiol 2016;18(3):791-806.
9. Espinoza MG, Vazquez RG, Mendez IM, Vargas CR, Cerezo SG. Detection of the glmM gene in Helicobacter pylori isolates with a novel primer by PCR. J Clin Microbiol 2011;49(4):1650-2.
10. Ramis IB, Vianna JS, Gonçalves CV, von Groll A, Dellagostin OA, da Silva PE. Polymorphisms of the IL-6, IL-8 and IL-10 genes and the risk of gastric pathology in patients infected with Helicobacter pylori. J Microbiol Immunol Infect 2015. pii: S1684-118200721-5.
11. Johnson EM, Gaddy JA, Voss BJ, Hennig EE, Cover TL. Genes required for assembly of pili associated with the Helicobacter pylori cag type IV secretion system. Infect Immun 2014;82(8):3457-70.
12. Haley KP, Gaddy JA. Helicobacter pylori: Genomic insight into the host-pathogen interaction. Int J Genomics 2015;2015:386905.
13. Pajavand H, Alvandi A, Mohajeri P, Bakhtyari S, Bashiri H, Kalali B, et al. High frequency of vacA s1m2 genotypes among Helicobacter pylori isolates from patients with gastroduodenal disorders in Kermanshah, Iran. Jundishapur J Microbiol 2015;8(11):e25425.
14. Souod N, Kargar M, Doosti A, Ranjbar R, Sarshar M. Genetic analysis of cagA and vacA genes in Helicobacter pylori isolates and their relationship with gastroduodenal diseases in the West of Iran. Iran Red Crescent Med J 2013;15(5):371-5.
15. Ashwini P, Sumana MN, Shilpa U, Mamatha P, Manasa P, Dhananjaya BL, et al. A review on Helicobacter pylori: Its biology, complications and management. Int J Pharm Pharm Sci 2015;7 Suppl 1:14-20.
16. Figueiredo C, Quint WG, Sanna R, Sablon E, Donahue JP, Xu Q, et al. Genetic organization and heterogeneity of the iceA locus of Helicobacter pylori. Gene 2000;246(1-2):59-68.
17. Shiota S, Watada M, Matsunari O, Iwatani S, Suzuki R, Yamaoka Y. Helicobacter pylori iceA, clinical outcomes, and correlation with cagA: A meta-analysis. PLoS One 2012;7(1):e30354.
18. Shiota S, Suzuki R, Yamaoka Y. The significance of virulence factors in Helicobacter pylori. J Dig Dis 2013;14(7):341-9.
19. Liu J, He C, Chen M, Wang Z, Xing C, Yuan Y. Association of presence/absence and on/off patterns of Helicobacter pylori oipA gene with peptic ulcer disease and gastric cancer risks: A meta-analysis. BMC Infect Dis 2013;13:555.
20. Oleastro M, Ménard A. The role of Helicobacter pylori outer membrane proteins in adherence and pathogenesis. Biology (Basel) 2013;2(3):1110-34.
21. Dossumbekova A, Prinz C, Mages J, Lang R, Kusters JG, Van Vliet AH, et al. Helicobacter pylori HopH (OipA) and bacterial pathogenicity: Genetic and functional genomic analysis of hopH gene polymorphisms. J Infect Dis 2006;194(10):1346-55.
22. Gehlot V, Mahant S, Das K, Das R. Most of the Helicobacter pylori isolates are resistant to levofloxacin in north India. Int J Pharm Pharm Sci 2016;8(1):454-56.
23. Gehlot V, Mahant S, Vijayraghwan P, Das K, Hoda S, Das R. Therapeutic potential of lichen Parmelia perlata against daul drag - resistant Helicobacter pylori isolates. Int J Pharm Pharm Sci 2016;8(1):205-8.
24. Omran R, Al-Khafagee AH. Multiple resistance to antibiotics in Helicobacter pylori isolated from peptic and duodenal ulcers. J Babylon Univ 2013;21(6):2050-7.
25. Al-Khafagee AH. Genetic study of Helicobacter pylori. M.Sc. Thesis College of Science, Babylon University; 2012.
26. Sambrook J, Fritschi E, Maniatis T. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1989.
27. Omran R, Al-Zaag A. Ultrastructure and genetic analysis of mucogenicity in Klebseilla pneumoniae. Iraqi J Biotechnol 2005;4(2):85-98.
28. Al-Naji HA, Omran O, Al-Sherify A. Molecular detection of Helicobacter pylori infection in gastric biopsy specimens by PCR. J Babylon Univ 2016;25(3):1016-28.
29. Atherton JC, Cao P, Peek RM Jr, Tummuru MK, Blaser MJ, Cover TL. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem 1995;270(30):17771-7.
30. Ben Mansour K, Fendri C, Zribi M, Masmoudi A, Labbene M, Fillali A, et al. Prevalence of Helicobacter pylori vacA, cagA, iceA and oipA genotypes in Tunisian patients. Ann Clin Microbiol Antimicrob 2010;9:10.
31. Souod N, Sarshar M, Dabiri H, Momtaz H, Kargar M, Mohammadzadeh A, et al. The study of the oipA and dupA genes in Helicobacter pylori strains and their relationship with different gastroduodenal diseases. Gastroenterol Hepatol Bed Bench 2015;8 Suppl 1:S47-53.
32. Erdogdu C, Saribas Z, Akyön Yilmaz Y. Detection of cagA and vacA genotypes of Helicobacter pylori isolates from a university hospital in Ankara region, Turkey. Turk J Med Sci 2014;44(1):126-32.
33. Amer FA, El-Sokkary RH, Elahmady M, Gheith1 T, Abdelbary EH, Elnagar Y, et al. Helicobacter pylori genotypes among patients in a university hospital in Egypt: Identifying the determinants of disease severity. J Microb Infect Dis 2013;3(3):109-15.
34. Yakoob J, Abid S, Abbas Z, Jafri W, Ahmad Z, Ahmed R, et al. Distribution of Helicobacter pylori virulence markers in patients with gastroduodenal diseases in Pakistan. BMC Gastroenterol 2009;9:87.
35. Aziz F, Chen X, Yang X, Yan Q. Prevalence and correlation with clinical diseases of Helicobacter pylori cagA and vacA genotype among gastric patients from Northeast China. Biomed Res Int 2014;2014:142980.
36. Yamazaki S, Yamakawa A, Okuda T, Ohtani M, Suto H, Ito Y, et al. Distinct diversity of vacA, cagA, and cagE genes of Helicobacter pylori associated with peptic ulcer in Japan. J Clin Microbiol 2005;43(8):3906-16.
37. Atherton JC. The pathogenesis of Helicobacter pylori-induced gastro-duodenal diseases. Annu Rev Pathol 2006;1:63-96.
38. van Doorn LJ, Figueiredo C, Rossau R, Jannes G, van Asbroek M, Sousa JC, et al. Typing of Helicobacter pylori vacA gene and detection
of cagA gene by PCR and reverse hybridization. J Clin Microbiol 1998;36(5):1271-6.
39. Honarmand-Jahromy S, Siavoshi F, Malekzadeh R, Nejad Sattari T, Latifi-Navid S. Reciprocal impact of host factors and Helicobacter pylori genotypes on gastric diseases. World J Gastroenterol 2015;21(31):9317-27.
40. Wong BC, Yin Y, Berg DE, Xia HH, Zhang JZ, Wang WH, et al. Distribution of distinct vacA, cagA and iceA alleles in Helicobacter pylori in Hong Kong. Helicobacter 2001;6(4):317-24.
41. Sugimoto M, Zali MR, Yamaoka Y. The association of vacA genotypes and Helicobacter pylori - Related gastroduodenal diseases in the Middle East. Eur J Clin Microbiol Infect Dis 2009;28(10):1227-36.
42. Hussein NR. Helicobacter pylori and gastric cancer in the Middle East: A new enigma? World J Gastroenterol 2010;16(26):3226-34.
43. Alikhani MY, Arebestani MR, Sayedin Khorasani M, Majlesi A, Jaefari M. Evaluation of Helicobacter pylori vacA and cagA genotypes and correlation with clinical outcome in patients with dyspepsia in hamadan province, Iran. Iran Red Crescent Med J 2014;16(11):e19173.
44. González CA, Figueiredo C, Lic CB, Ferreira RM, Pardo ML, Ruiz Liso JM, et al. Helicobacter pylori cagA and vacA genotypes as predictors of progression of gastric preneoplastic lesions: A long-term follow-up in a high-risk area in Spain. Am J Gastroenterol 2011;106(5):867-74.
45. Rhead JL, Letley DP, Mohammadi M, Hussein N, Mohagheghi MA, Hosseini ME, et al. A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer. Gastroenterology 2007;133:926-36.
46. Salehi Z, Abadi AS, Ismail PB, Kqueen CY, Jelodar MH, Kamalidehghan B. Evaluation of Helicobacter pylori vacA genotypes in Iranian patients with peptic ulcer disease. Dig Dis Sci 2009;54(11):2399-403.
47. Sugimoto M, Yamaoka Y. The association of vacA genotype and Helicobacter pylori-related disease in Latin American and African populations. Clin Microbiol Infect 2009;15(9):835-42.
48. Duncan SS, Valk PL, McClain MS, Shaffer CL, Metcalf JA, Bordenstein SR, et al. Comparative genomic analysis of East Asian and non-Asian Helicobacter pylori strains identifies rapidly evolving genes. PLoS One 2013;8(1):e55120.
49. Hussein NR, Mohammadi M, Talebkhan Y, Doraghi M, Letley DP, Muhammad MK, et al. Differences in virulence markers between Helicobacter pylori strains from Iraq and those from Iran: Potential importance of regional differences in Helicobacter pylori - Associated disease. J Clin Microbiol 2008;46:1774-9.
50. Saribasak H, Salih BA, Yamaoka Y, Sander E. Analysis of Helicobacter pylori genotypes and correlation with clinical outcome in Turkey. J Clin Microbiol 2004;42(4):1648-51.
51. Chen XJ, Yan J, Shen YF. Dominant cagA/vacA genotypes and coinfection frequency of Helicobacter pylori in peptic ulcer or chronic gastritis patients in Zhejiang province and correlations among different genotypes, co-infection and severity of the diseases. Chin Med J (Engl) 2005;118(6):460-7.
52. Ghose C, Perez-Perez GI, van Doorn LJ, Domínguez-Bello MG, Blaser MJ. High frequency of gastric colonization with multiple Helicobacter pylori strains in Venezuelan subjects. J Clin Microbiol 2005;43(6):2635-41.
53. Khayat AE, Soweid AM, Kattar MM, Tawil A, Gold B, Matar GM. Prevalence and clinical relevance of Helicobacter pylori cagA and vacA genes in Lebanese patients with gastritis and peptic ulcer disease. J Infect Dev Countries 2007;1:55-61.
54. Rizzato C, Torres J, Plummer M, Muñoz N, Franceschi S. Camorlinga-Ponce M, et al. Variations in Helicobacter pylori cytotoxin-associated genes and their influence in progression to gastric cancer: Implications for prevention. PLoS One 2012;7(1):e29605.
55. Lima VP, Silva-Fernandes IJ, Alves MK, Rabenhorst SH. Prevalence of Helicobacter pylori genotypes (vacA, cagA, cagE and virB11) in gastric cancer in Brazilian’s patients: An association with histopathological parameters. Cancer Epidemiol 2011;35(5):e32-7.
56. Jones KR, Joo YM, Jang S, Yoo YJ, Lee HS, Chung IS, et al. Polymorphism in the CagA EPIYA motif impacts development of gastric cancer. J Clin Microbiol 2009;47(4):959-68.
57. Monstein HJ, Karlsson A, Ryberg A, Borch K. Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs. BMC Res Notes 2010;3:35.
58. Ozbey G, Aygun C. Prevalence of genotypes in Helicobacter pylori isolates from patients in eastern Turkey and the association of these genotypes with clinical outcome. Braz J Microbiol 2012;43(4):1332-9.
59. Kidd M, Peek RM, Lastovica AJ, Israel DA, Kummer AF, Louw JA. Analysis of iceA genotypes in South African Helicobacter pylori strains and relationship to clinically significant disease. Gut 2001;49:629-35.
60. Yamaoka Y. Mechanisms of disease: Helicobacter pylori virulence factors. Nat Rev Gastroenterol Hepatol 2010;7(11):629-41.
61. Zhong Y, Anderl F, Kruse T, Schindele F, Jagusztyn-Krynicka EK, Fischer W, et al. Helicobacter pylori HP0231 influences bacterial virulence and Is essential for gastric colonization. PLoS One 2016;11(5):e0154643.
Statistics
288 Views | 291 Downloads
Citatons
How to Cite
Al-jelawi-, R. O. “INVESTIGATION OF Helicobacter Pylori VIRULENCE GENOTYPE IN GASTRIC BIOPSIES BY PCR”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 9, no. 6, Nov. 2016, pp. 106-14, doi:10.22159/ajpcr.2016.v9i6.13795.
Section
Original Article(s)