H.T.Pham[1]*, T.K.T.Le[1,2], D.K.Tran[1,2], T.H.T.Nguyen[1,2], V.H.Pham[1,3]
[1] Vietnam Research and Development Institute of Clinical Microbiolgy, Vietnam
[2] Nam Khoa Biotek Co. Ltd., Vietnam
[3] Phan Chau Trinh University, Vietnam

Background

In Vietnam, H. pylori causing gastric and duodenal ulcers exhibits high resistance to Clarithromycin and Levofloxacin, the primary treatment antibiotics. However, there is no large-scale study regarding the cultivation and antibiotic susceptibility testing of H. pyloriisolated from gastric biopsy specimens of patients suspected of H. pylori-induced gastric and duodenal ulcers, and limited research on mutations related to resistance to these two antibiotics.

Aims of the study

Investigate the isolation rate and resistance status of H. pylori cultured from gastric biopsy specimens taken from patients clinically suspected of gastric and duodenal ulcers caused by H. pylori. Simultaneously, identify mutations in two genes – 23sRNADNA linkedto Clarithromycin resistance, and gyrA associated with Levofloxacin resistance.

Materials and methods

Samples were gastric biopsy specimens obtained through endoscopy from adults suspected of H. pylori-induced gastric ulcers. The samples were cultured within 4 hours of collection and maintained in a preservation medium, following NK-BIOTEK laboratory’s standard protocol. Antibiotic susceptibility testing was performed using Amoxicillin, Clarithromycin, Levofloxacin, Tetracycline, and Metronidazole with Minimum Inhibitory Concentration (MIC) determination. Isolated strains were preserved in TSB medium with 20% glycerol at -80°C. Strains resistant to both Clarithromycin and Levofloxacin underwent PCR amplification of specific sequences containing mutations related to Clarithromycin resistance (23sRNADNA gene) and Levofloxacin resistance (gyrA gene). Subsequently, SANGER sequencing was performed on these PCR products to detect and document mutations.

Results

From 2021 to 2022, we collected a total of 1391 samples for culture and antibiotic susceptibility testing. The results revealed 807 positive H. pylori cultures, 0.5% of which exhibited resistance to Tetracycline, 11.3% to Amoxicillin, 52.2% to Metronidazole, 94.5% to Clarithromycin, and 63.8% to Levofloxacin. Notably, 60.1% displayed resistance to both Clarithromycin and Levofloxacin. 200 strains resistant to both antibiotics were randomly selected for PCR and sequencing of 23sRNADNA and GyrA genes to identify mutations. On the 23sRNADNA gene, 100% of the strains contained mutations with the following percentages: Asn87Lys (64.71%), Asp91Gly (11.76%), Asp91Asn (2.94%), Asp91Tyr (17.65%), Ala88Val (2.94%). Regarding the GyrA gene, 100% of the strains had mutations with percentages as follows: C1953T (27.12%), T1965C (3.39%), A2142G (1.69%), A2143G (91.53%), T2182C (100.00%), G2212A (1.69%), A2223G (74.58%), T2295C (1.69%). Unlike the single mutation found on the 23sRNADNA gene for each strain, the GyrA gene exhibited 9 combinations of mutations with percentages such as T2182C (5.08%), T2182C+A2223G (1.69%), A2142G+T2182C (1.69%), A2143G+T2182C (13.56%), A2143G+T2182C+G2212A (1.69%), A2143G+T2182C+T2295C (1.69%), C1953T+A2143G+T2182C+A2223G(27.12%), A2143G+T2182C+A2223G (44.07%), T1965C+A2143G+T2182C+A2223G (3.39%).

Conclusions

This study, involving a large sample size sent for culture and antibiotic susceptibility testing of H. pylori, reveals seriousClarithromycin and Levofloxacin resistance situation and potentially impacts future treatment guidelines. Analysis of resistancemutations suggests that the origin of resistance lies in mutations within responsible genes. The detection rates of these mutationscan aid in designing molecular biology tests, such as PCR or real-time PCR, as alternatives to the clinically impractical antibiotic susceptibility testing, which often takes up to 7 days for results.

Keywords: Helicobacter pylori, Clarithromycin and Levofloxacin resistance, 23sRNADNA mutations, gyrA mutations