H. pylori clinical isolates and biopsy specimens
Gastric biopsy specimens were obtained from 70 patients who underwent upper gastroduodenal endoscopy at Research Institute for Gastroenterology and Liver Diseases in Tehran between January 2017 and May 2019. Three antral biopsies were taken from each patient and examined for culture and histopathology. The biopsy specimens were immediately placed in transport medium containing Thioglycolate supplemented with 3% yeast extract (Oxoid Ltd., Basingstoke, UK) and 1.3 g/L agar (Merck, Germany). All patients provided written informed consent. The study was approved by the Institutional Ethical Review Committee of Research Institute for Gastroenterology and Liver Diseases at Shahid Beheshti University of Medical Sciences (Project No. IR.SBMU.RIGLD.REC.1398.023).
H. pylori culture and identification
Biopsy specimens were carefully homogenized and inoculated onto the surface of Brucella agar plates (Merck, Germany) supplemented with 7% (v/v) horse blood, 10% fetal calf serum (FCS), Campylobacter-selective supplement (vancomycin 2.0 mg, polymyxin 0.05 mg, trimethoprim 1.0 mg), and amphotericin B (2.5 mg/l). The incubation was performed at 37°C for 3-7 days under a microaerophilic atmosphere (5% O2, 10% CO2 and 85% N2) in a CO2 incubator (Innova® CO-170; New Brunswick Scientific, USA). The suspected colonies were identified as H. pylori based on colony morphology, Gram staining, positive reaction for oxidase, catalase and urease tests, and also by H. pylori gene-specific PCR following the previously described protocols [27, 28]. Pure cultures from confirmed isolates were kept in 0.5 ml of Brain heart infusion (BHI) medium (Merck, Germany) containing 15% glycerol plus 20% FCS, and stored at -80°C until further analysis.
Genomic DNA extraction
Genomic DNA was extracted from freshly harvested colonies on agar plates, using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The quality of DNA was checked by using NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). The extracted DNA samples were stored at -20°C until PCR assay.
Genotyping of H. pylori virulence-associated genes
PCR analysis was performed to detect virulence target genes including cagL, cagA, vacA alleles (s1/s2 and m1/m2), babA2, sabA and dupA genes using specific primers (Table S1). Briefly, PCR mixtures in a volume of 25 µl consisted of 2 µl of template DNA (approximately 200 ng), 0.1 mM of each primer, 2.5 µl of a 10-fold concentrate PCR buffer, 100 mM of deoxynucleotide triphosphates, 1 mM MgCl2, and 1.5 U of Super-TaqTM DNA polymerase (HT Biotechnology Ltd., Cambridge, UK). PCR amplifications were performed in a thermocycler (Eppendorf, Hamburg, Germany) under the following conditions: initial denaturation at 94°C for 4 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at the indicated temperature for each reaction in Table S1 for 45 s, extension at 72°C for 1 min. A final extension step was performed at 72°C for 10 min to ensure full extension of the PCR products. PCR amplicons were electrophoresed on a 1.2% TBE agarose gel, stained with ethidium bromide, and examined under a UV transilluminator. H. pylori J99 (CCUG 47164) and a no-template mixture served as positive and negative controls in each PCR experiment, respectively.
Primer designation for cagI and cagN genotyping
The NCBI GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) and the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/) were searched for all available complete and partial cagI and cagN sequences of H. pylori strains. Based on pairwise and multiple nucleotide sequence alignments of cagI and cagN genes from different H. pylori strains and using the complete relevant sequence of H. pylori P12 (CP001217.1) as the reference strain, two pairs of specific primers were designed from the conserved regions for detection of complete related sequences using CLC Sequence Viewer 8 software (https://www.qiagenbioinformatics.com/). The selected primer target sites were compared to all available complete and partial cagI and cagN sequences of H. pylori strains with the Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Analysis of cagI and cagN diversity by PCR sequencing
For DNA sequencing of cagI and cagN, PCR amplification was carried out in a final volume of 25 µl using designed specific primers including 5΄-CATTTGACTTACCTTGATTAC-3΄ (cagI-F) and 5΄-TTTGAGCACTTGTTGGTTGG-3΄ (cagI-R), 5΄-GAGCGACAAAACAACTATGC-3΄ (cagN-F) and 5΄-GATCCCTAGAACAAAGTAAGC-3΄ (cagN-R) yielding DNA fragments of about 1377 and 1192 bp in length, respectively. The PCR products were purified using the Silica Bead DNA Gel Extraction Kit (Thermo Scientific, Fermentas, USA) followed by sequencing on both strands using an automated sequencer (Macrogen, Seoul, Korea). DNA sequences were edited by Chromas Lite version 2.5.1 (Technelysium Pty Ltd, Australia) and BioEdit version 7.2.5 . The cagI and cagN nucleotide and amino acid sequences were aligned to H. pylori strain P12 as a reference strain (GenBank: CP001217.1). The single nucleotide variations and codon usage of the sequences were examined using BioEdit version 7.2.5.
Phylogenetic trees were generated for CagI and CagN nucleotide and amino acid sequences using Molecular Evolutionary Genetics Analysis version 7.0 (MEGA7) . Evolutionary history was inferred by the Maximum Likelihood trees using Tamura 3-parameter model and Poisson correction method for nucleotide and amino acid sequences, respectively.
Nucleotide sequence accession numbers
The complete and partial nucleotide sequences of cagI and cagN genes from H. pylori strains determined in this study were deposited in the NCBI GenBank database under the accession numbers MG573078-MG573107 (cagI) and MG559675-MG559720 (cagN).
The statistical associations between H. pylori virulence genotypes and different clinical status were determined by the Chi-square and Fisher’s exact tests. A two-sided P value of less than 0.05 was regarded statistically significant. The IBM SPSS Statistics for Windows version 21.0 (Armonk, NY: IBM Corp.) was used for all statistical analyses.