Högskolan i Skövde

The tendency of Helicobacter pylori (H. pylori) to flourish in an acidic gastric environment and its virulence nature may result in a fatal clinical outcome such as gastric cancer. Early diagnosis and prompt eradication of H. pylori infection may help in decreasing the risk of gastric cancer development. Around 40-90% of the adult population in Iran is infected by H. pylori and there is a rising trend in antimicrobial resistance (AMR). So far, no previous study has been published where next-generation sequencing (NGS) has been applied on H. pylori isolates from Iran. In this project, whole-genome sequencing was done for bacterial isolates from 329 individuals from Tehran, Iran using the Illumina MiSeq platform. Phenotypic antimicrobial susceptibility testing results were obtained for H. pylori against tetracycline, metronidazole, clarithromycin, levofloxacin, ciprofloxacin, rifampicin, and amoxicillin from Iran. The NGS analysis included quality control (QC), de novo assembly of obtained fastq files, and annotation using the nextflow based BACTpipe pipeline (v 3.0), with the tools Fastp, Shovill, Sendsketch, Prokka, and MultiQC. Generated output files were further used for variant calling (for AMR) and phylogenetic analysis. H. pylori assemblies were obtained from 258 samples. Phenotypic resistance was the highest for metronidazole (79%) and the lowest for tetracycline (10%). Known genomic variants in genes rdxA, frxA, infB, rplV, pbp, gyrA, and rpoB, were observed in 198, 181, 128, 109, 52, 28, and 5 of isolates, respectively. However, there were no known variants identified in the whole length of 23S rRNA, 16S rRNA, and gyrB responsible for clarithromycin, tetracycline, and fluoroquinolone resistance, respectively. Isolates with genomic variants were then compared to susceptible or resistant phenotypes. The most frequently found variants in the frxA gene were frameshift mutation at amino acid (AA) position 18 followed by missense mutations at 7, 169, 85, 16, 126, 41, 106, 76, 58, 110, 70, 66, 36 and stop codons (at 137, 86, and 5). Frequently found variants in the rdxA gene were at AA positions 97 followed by 90, 16, 108, 163, 56, 80, 111, 27, 163, 148, 176 and stop codon (Gln50*). For the pbp gene substitutions at AA positions 414, 556, 562, and 414. Only one variant observed for the rpoB gene was Pro636Leu. Variants in gyrA were observed at AA positions 91, 130, 63, 87, and 172. In the infB gene frequently found variants were at nucleotide position C133G followed by G547A, A403G, and 297_298 delAAinsGG. In rplV, only one variant, G20A was found. Sixty-nine percent of the genomes were positive for the CagA gene and EPIYA motifs A, B and C were present in 68% of H. pylori isolates. Multiple EPIYA-C motifs were present in 17% of CagA positive genotypes. The East Asian type EPIYA-D variants were not found in this study. Using phylogenetic analysis, the majority of CagA sequences were found to be of South/Central Asian origin. In conclusion, the resistance against metronidazole was high in Iranian isolates. With NGS analysis it was found that mutations associated with resistance in literature were also found in susceptible phenotypes. Therefore, a study at a large scale of approximately 1000 or more in size is required to identify the significant association of these variants with AMR.