Molecular Phylogenetic Analysis of 16S rRNA Sequences Identified Two Lineages of Helicobacter pylori Strains Detected from Different Regions in Sudan Suggestive of Differential Evolution

Abstract

Background. Helicobacter pylori (H. pylori) is ubiquitous among humans and one of the best-studied examples of an intimate association between bacteria and humans. Phylogeny and Phylogeography of H. pylori strains are known to mirror human migration patterns and reflect significant demographic events in human prehistory. In this study, we analyzed the molecular evolution of H. pylori strains detected from different tribes and regions of Sudan using 16S rRNA gene and the phylogenetic approach. Materials and methods. A total of 75 gastric biopsies were taken from patients who had been referred for endoscopy from different regions of Sudan. The DNA extraction was performed by using the guanidine chloride method. Two sets of primers (universal and specific for H. pylori) were used to amplify the 16S ribosomal gene. Sanger sequencing was applied, and the resulted sequences were matched with the sequences of the National Center for Biotechnology Information (NCBI) nucleotide database. The evolutionary aspects were analyzed using MEGA7 software. Results. Molecular detection of H. pylori has shown that 28 (37.33%) of the patients were positive for H. pylori and no significant differences were found in sociodemographic characteristics, endoscopy series, and H. pylori infection. Nucleotide variations were observed at five nucleotide positions (positions 219, 305, 578, 741, and 763–764), and one insertion mutation (750_InsC_751) was present in sixty-seven percent (7/12) of our strains. These six mutations were detected in regions of the 16S rRNA not closely associated with either tetracycline or tRNA binding sites; 66.67% of them were located in the central domain of 16S rRNA. The phylogenetic analysis of 16S rRNA sequences identified two lineages of H. pylori strains detected from different regions in Sudan. The presence of Sudanese H. pylori strains resembling Hungarian H. pylori strains could reflect the migration of Hungarian people to Sudan or vice versa. Conclusion. This finding emphasizes the significance of studying the phylogeny of H. pylori strains as a discriminatory tool to mirror human migration patterns. In addition, the 16S rRNA gene amplification method was found useful for bacterial identification and phylogeny.

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“…The conditions for amplified gene fragment were initial-denaturation of the target DNA at 94 • C for 5 min followed by final denaturation at 94 • C for one min, primer annealing at 55 • C for one min, initial extension at 70 • C for 2 min, and a final extension at 72 • C for 10 min. The PCR was performed with a total reaction volume of 25 µL containing 196.2 ng of DNA template, 1 µL each of both 10 µM forward (27F) and reverse primer (1492R), 10.5 µL of nuclease-free water, and 12.5 µL of PCR master mix [19]. PCR was set to 30 cycles.…”

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“…11 Another application of molecular tests is to study H. pylori evolution and human migration patterns via molecular phylogenetic analysis of 16S rRNA gene from different populations. 39 NGS such as 16S rRNA sequencing provides not only unbiased culture-independent diagnosis even in formalin-fixed, paraffinembedded gastric biopsies, 40 but also informative microbiome profile, which provides an opportunity to understand how H. pylori interacts with other bacterial species of the gastric microbiota 41 or oral microbiota. 42 Similar to dd-PCR, 16S rRNA sequencing measures the quantitative number of H. pylori as additional information that shows bacterial load variation during disease development.…”

“…Another application of molecular tests is to study H . pylori evolution and human migration patterns via molecular phylogenetic analysis of 16S rRNA gene from different populations 39 …”