SpringerLink
Log in

New strategies for Helicobacter pylori isolation and sequencing analysis for virulence genes contributing to its pathogenicity

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

Helicobacter pylori is a fastidious pathogen that is required a complicated medium for growth. Invading epithelial cells of the stomach. H. pylori virulence factors are classified by function, acidic resistivity, adhesion, chemotaxis and motility, molecular mimicry, immunological invasion and modulation, and toxins formation such as cytotoxin-associated genes A (cagA) and vacuolating cytotoxin A (vacA). This study aims to determine a simple and innovative technique to isolate H. pylori from gastric biopsies and assess pathogenicity by virulence factor gene detection.

Methods

A total of 200 patients who were suspected of having H. pylori infection had two antral gastric biopsies undertaken. A rapid urease test (RUT) was used for one, and Brain Heart Infusion broth (BHI) was used to cultivate the other. The molecular study included diagnostics utilizing the 16sRNA housekee** gene along with the identification of the virulence factors genes (cagA, cagT, and vacA) and sequencing,

Result

Of the 200 antral gastric biopsies collected, 135 were positive rapid urease tests, and 17 H. pylori isolates were successfully obtained from 135 biopsies. The 16SrRNA as a housekee** gene is confirmed, and about 53%, 70.5%, and 82.3% of the 17 isolates show carrying cagA, cagT, and vacA genes, respectively. All peptic ulcer isolates have the cagA gene, while Gastroesophageal Reflux Disease (GERD) and non-peptic ulcer disease (NPUD) isolates show the lack of the cagA gene. All bacteria, which were isolated from peptic ulcer, nodular gastritis, and gastritis patients, have a vacA gene.

Conclusion

The effective method for isolating H. pylori is centrifuging the transport broth after 24 h of incubation. The cagA toxin causes peptic ulcer while vacA toxin induces several histopathological changes in the stomach. Three virulence genes were present in all peptic ulcer-causing bacteria, while only one or none were present in the GERD and NPUD biopsy isolates.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data availability

No Data associated in the manuscript.

References

  1. Hooi JK, Lai WY, Ng WK, Suen MM, Underwood FE, Tanyingoh D, Malfertheiner P, Graham DY, Wong VW, Wu JC (2017) Global prevalence of Helicobacter pylori Infection: systematic review and meta-analysis. Gastroenterology 153:420–429

    Article  PubMed  Google Scholar 

  2. Mărginean CO, Meliț, LE, Săsăran MO (2022) Traditional and modern diagnostic approaches in diagnosing Pediatric Helicobacter pylori Infection. Children 9:994

    Article  PubMed  PubMed Central  Google Scholar 

  3. Galoș F, Boboc C, Ieșanu M-I, Anghel M, Ioan A, Iana E, Coșoreanu MT, Boboc AA (2023) Antibiotic Resistance and therapeutic efficacy of Helicobacter pylori Infection in Pediatric Patients—A Tertiary Center Experience. Antibiotics 12:146

    Article  PubMed  PubMed Central  Google Scholar 

  4. Doohan D, Rezkitha YAA, Waskito LA, Yamaoka Y, Miftahussurur M (2021) Helicobacter pylori BabA–SabA key roles in the adherence phase: the synergic mechanism for successful colonization and Disease development. Toxins 13:485

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ansari S, Yamaoka Y (2020) Helicobacter pylori virulence factor cytotoxin-associated gene A (CagA)-mediated gastric pathogenicity. Int J Mol Sci 21:7430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Rezaei F, Alebouyeh M, Mirbagheri SZ, Ebrahimi A, Foroushani AR, Bakhtiari R (2023) Transcriptional analysis of Helicobacter pylori cytotoxic-associated gene-pathogenicity island in response to different pH levels and proton pump inhibitor exposure. Indian J Gastroenterol, 1–8

  7. Lin AS, Dooyema SD, Frick-Cheng AE, Harvey ML, Suarez G, Loh JT, McDonald WH, McClain MS, Peek RM Jr, Cover TL (2020) Bacterial energetic requirements for Helicobacter pylori Cag type IV secretion system-dependent alterations in gastric epithelial cells. Infect Immun 88. https://doi.org/10.1128/iai.00790-19

  8. Chung JM, Sheedlo MJ, Campbell AM, Sawhney N, Frick-Cheng AE, Lacy DB, Cover TL, Ohi MD (2019) Structure of the Helicobacter pylori Cag type IV secretion system. Elife 8, e47644

  9. Zhang X, Li C, Chen D, He X, Zhao Y, Bao L, Wang Q, Zhou J, **e Y (2022) H. Pylori CagA activates the NLRP3 inflammasome to promote gastric cancer cell migration and invasion. Inflamm Res, 1–15

  10. McClain MS, Beckett AC, Cover TL (2017) Helicobacter pylori vacuolating toxin and gastric cancer. Toxins 9:316

    Article  PubMed  PubMed Central  Google Scholar 

  11. Reynolds DJ, Penn CW (1994) Characteristics of Helicobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology 140:2649–2656

    Article  CAS  PubMed  Google Scholar 

  12. Nguyen L, Uchida T, Tsukamoto Y, Kuroda A, Okimoto T, Kodama M, Murakami K, Fujioka T, Moriyama M (2010) Helicobacter pylori dupA gene is not associated with clinical outcomes in the Japanese population. Clin Microbiol Infect 16:1264–1269

    Article  CAS  PubMed  Google Scholar 

  13. Brkić DV, Katičić M, Bedenić B, Stanko, AP, Plečko V (2016) Detection of virulence gene belonging to cag pathogenicity island in Helicobacter pylori isolates after multiple unsuccessful eradication therapy in Northwest Croatia. Periodicum biologorum 118

  14. Nagata R, Sato H, Takenaka S, Yokoyama J, Terai S, Mimuro H, Noiri Y (2023) Analysis of genetic relatedness between gastric and oral Helicobacter pylori in patients with early gastric Cancer using Multilocus sequence ty**. Int J Mol Sci 24:2211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Godbole G, Mégraud F, Bessède E (2020) Diagnosis of Helicobacter pylori Infection. Helicobacter 25:e12735

    Article  CAS  PubMed  Google Scholar 

  16. Seo J-H, Park JS, Rhee KH, Youn H-S (2015) Limitations of urease test in diagnosis of pediatric Helicobacter pylori Infection. World J Clin Pediatr 4:143

    Article  PubMed  PubMed Central  Google Scholar 

  17. Hussen BM, Qader SS, Ahmed HF, Ahmed SH (2013) The prevalence of Helicobacter pylori among university students in Iraq. Indian J Sci Technol 6:5019–5023

    Article  Google Scholar 

  18. Kaplan M, Tanoglu A, Duzenli T, Tozun AN (2019) Helicobacter pylori treatment in Turkey: current status and rational treatment options. North Clin Istanbul 7:87–94

    Google Scholar 

  19. Hakami OA, Alsubaie RA, Albaqami BA, Almutlaq HM, Alqahtani NM, Alkhonezan M, Almuqrin FF, Alghamdi AH, Alaryni AA, Qutob RA (2023) Knowledge and perception of Physicians of different specialties in Saudi Arabia toward Helicobacter pylori. J Multidisciplinary Healthc, 763–771

  20. Khoder G, Muhammad JS, Mahmoud I, Soliman SS, Burucoa C (2019) Prevalence of Helicobacter pylori and its associated factors among healthy asymptomatic residents in the United Arab Emirates. Pathogens 8:44

    Article  PubMed  PubMed Central  Google Scholar 

  21. Patel SK, Mishra GN, Pratap CB, Jain AK, Nath G (2014) Helicobacter pylori is not eradicated after triple therapy: a nested PCR based study. BioMed research international 2014

  22. Malfertheiner P, Megraud F, O’morain C, Gisbert J, Kuipers E, Axon A, Bazzoli F, Gasbarrini A, Atherton J, Graham DY (2017) Management of Helicobacter pylori Infection—the Maastricht V/Florence consensus report. Gut 66:6–30

    Article  CAS  PubMed  Google Scholar 

  23. Metwally M, Sayed MM, Abbas AH, El Shahat ESA, Elkholy H (2023) Helicobacter pylori Culture and Anti-biogram: low yield and beneficial results. Benha Medical Journal

  24. Patel SK, Pratap CB, Jain AK, Gulati AK, Nath G (2014) Diagnosis of Helicobacter pylori: what should be the gold standard? World J Gastroenterology: WJG 20:12847

    Article  CAS  PubMed Central  Google Scholar 

  25. Mégraud F, Lehours P (2007) Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev 20:280–322

    Article  PubMed  PubMed Central  Google Scholar 

  26. Khadangi F, Yassi M, Kerachian MA (2017) Diagnostic accuracy of PCR-based detection tests for Helicobacter Pylori in stool samples. Helicobacter 22, e12444

  27. Moorlag SJ, Coolen JP, van den Bosch B, ** EH-M, Buil JB, Wertheim HF, Melchers WJ (2023) Targeting the 16S rRNA gene by reverse complement PCR next-generation sequencing: specific and sensitive detection and identification of Microbes directly in clinical samples. Microbiol Spectr 11:e04483–e04422

    Article  PubMed  PubMed Central  Google Scholar 

  28. Elnosh M, Altayb H, Hamedelnil Y, Elshareef W, Abugrain A, Osman E, Albasha A, Abdelhamid A, Moglad E, AbdAlla A (2022) Comparison of invasive histological and molecular methods in the diagnosis of Helicobacter pylori from gastric biopsies of Sudanese patients: a cross-sectional study. F1000Research 11

  29. AL-Ouqaili MT, Abdullah EM (2011) Detection of 16S r RNA gene of Helicobacter pylori in patients with Peptic Ulcer and gastric carcinoma: molecular and bacteriological study. Egypt Acad J Biol Sci G Microbiol 3:95–104

    Google Scholar 

  30. Feliciano O, Gutierrez O, Valdés L, Fragoso T, Calderin AM, Valdes AE, Llanes R (2015) Prevalence of Helicobacter pylori vacA, cagA, and iceA genotypes in Cuban patients with upper Gastrointestinal Diseases. Biomed Res Int 2015:1–6

    Article  Google Scholar 

  31. Johnson EM, Gaddy JA, Voss BJ, Hennig EE, Cover TL (2014) Genes required for assembly of pili associated with the Helicobacter pylori cag type IV secretion system. Infect Immun 82:3457–3470

    Article  PubMed  PubMed Central  Google Scholar 

  32. Sallas ML, Melchiades JL, Zabaglia LM, do Prado Moreno JR, Orcini WA, Chen ES, Smith MAC, Payão SLM, Rasmussen LT (2017) Prevalence of Helicobacter pylori vacA, cagA, dupA and oipA genotypes in patients with gastric Disease. Adv Microbiol 7:1–9

    Article  CAS  Google Scholar 

  33. Boonyanugomol W, Kongkasame W, Palittapongarnpim P, Baik S-C, Jung M-h, Shin M-K, Kang H-L, Lee W-K (2020) Genetic variation in the cag pathogenicity island of Helicobacter pylori strains detected from gastroduodenal patients in Thailand. Brazilian J Microbiol 51:1093–1101

    Article  CAS  Google Scholar 

  34. Hussein NR, Mohammadi M, Talebkhan Y, Doraghi M, Letley DP, Muhammad MK, Argent RH, Atherton JC (2008) Differences in virulence markers between Helicobacter pylori strains from Iraq and those from Iran: potential importance of regional differences in H. Pylori-associated Disease. J Clin Microbiol 46:1774–1779

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Sezikli M, Guliter S, Apan T, Aksoy A, Keles H, Ozkurt Z (2006) Frequencies of serum antibodies to Helicobacter pylori CagA and VacA in a Turkish population with various gastroduodenal Diseases. Int J Clin Pract 60:1239–1243

    Article  CAS  PubMed  Google Scholar 

  36. Li Q, Liu J, Gong Y, Yuan Y (2016) Serum VacA antibody is associated with risks of Peptic Ulcer and gastric cancer: a meta-analysis. Microb Pathog 99:220–228

    Article  CAS  PubMed  Google Scholar 

  37. Censini S, Lange C, **ang Z, Crabtree JE, Ghiara P, Borodovsky M, Rappuoli R, Covacci A (1996) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proceedings of the National Academy of Sciences 93, 14648–14653

  38. McMahon B, Bruce M, Koch A, Goodman K, Tsukanov V, Mulvad G, Borresen M, Sacco F, Barrett D, Westby S (2016) The diagnosis and treatment of Helicobacter pylori Infection in Arctic regions with a high prevalence of Infection: Expert Commentary. Epidemiol Infect 144:225–233

    Article  CAS  PubMed  Google Scholar 

  39. Miernyk KM, Bruden D, Rudolph KM, Hurlburt DA, Sacco F, McMahon BJ, Bruce MG (2020) Presence of cagPAI genes and characterization of Vaca s, i and m regions in Helicobacter pylori isolated from alaskans and their association with clinical pathologies. J Med Microbiol 69:218–227

    Article  CAS  PubMed  Google Scholar 

  40. Monroy FP, Brown HE, Sanderson PR, Jarrin G, Mbegbu M, Kyman S, Harris RB (2022) Helicobacter pylori in native americans in northern Arizona. Diseases 10:19

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors gratitude to the members of the RES Laboratory in the physics department and the members of the Molecular Biology Laboratory in the biology department, college of sciences, Mustansiriyah University, Baghdad, Iraq for their efficient collaboration. Utmost thanks and appreciation to the doctors and nursing staff in the Gastroenterology Division, Endoscopy Unit, Baquba Teaching Hospital for their efforts in obtaining gastric biopsies. Also, the authors would like to thank Mustansiriyah University (https://uomustansiriyah.edu.iq/) / Baghdad, Iraq for its support to complete this work.

Funding

This research received no external funding.

Author information

Authors and Affiliations

  1. Department of Biology, College of Science, Mustansiriyah University, Baghdad, 10046, Iraq

    Asma Yahya Al-Jumaily

  2. Department of Physics, College of Science, Mustansiriyah University, Baghdad, 10046, Iraq

    Ahmed Al-Haddad

  3. Branch of Biotechnology, Department of Biology, College of Science, Mustansiriyah University, POX 10244, Baghdad, Iraq

    Sawsan Sajid Al-jubori

Authors
  1. Asma Yahya Al-Jumaily
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ahmed Al-Haddad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sawsan Sajid Al-jubori
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed equally in writing—original draft preparation, all authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Sawsan Sajid Al-jubori.

Ethics declarations

Ethics approval and consent to participate

The study protocol was approved by the ethical committee of the gastroenterology division a Baqubah Teaching Hospital. The study was conducted and samples were collected after receiving approval from the Mustansiriyah University /College of Science’s research tasks and ethics committee on January 1, 2022 (Ref.: BCSMU/1221/0004 M). This study involved the H. pylori’s from patients who are suffering from infections at Gastroenterology Division, Endoscopy Unit, Baqubah Teaching Hospital, Iraq. All authors agree to publish this work.

Research involving human participants and/or animals

This study involved the H. pylori’s from patients who are suffering from infections at Baghdad hospitals, Baghdad, Iraq.

Informed consent

All patients gave their written informed consents before inclusion, written informed consent has been obtained from the patient(s) to publish this paper.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Al-Jumaily, A.Y., Al-Haddad, A. & Al-jubori, S.S. New strategies for Helicobacter pylori isolation and sequencing analysis for virulence genes contributing to its pathogenicity. Mol Biol Rep 51, 95 (2024). https://doi.org/10.1007/s11033-023-09038-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11033-023-09038-4

Keywords

Search

Navigation