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

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 housekeeping 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 housekeeping 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.

Keywords: Helicobacter pylori; PCR; Rapid urease test; cagA; vacA.