Analysis of antimicrobial resistance patterns and genetic mutations in Helicobacter pylori from West Bengal, India depicting escalating clarithromycin and high levofloxacin resistance.

Abstract

Background: The growing resistance of Helicobacter pylori (H. pylori) to antibiotics poses a significant global health challenge, particularly in developing regions where infection rates are high, and routine antibiotic susceptibility surveillance is limited. This study aimed to evaluate the antimicrobial resistance patterns and associated genetic mutations in H. pylori strains isolated from patients in West Bengal (WB), India.

Methods: A total of 88 H. pylori strains were isolated from gastric biopsy samples collected between 2018 and 2020 from patients diagnosed with gastritis, duodenal ulcer, or gastric cancer. Antimicrobial susceptibility was determined using the agar dilution method against six antibiotics: metronidazole, tetracycline, clarithromycin, furazolidone, levofloxacin, and amoxicillin. Resistance-associated mutations in gyrA (levofloxacin), frxA and rdxA (metronidazole), and pbp1A (amoxicillin) were characterized via Sanger sequencing. Mutations in the 23SrRNA gene, implicated in clarithromycin resistance, were identified through allele-specific PCR, validated by Sanger sequencing and transformation assays. Additionally, BsaI and BbsI restriction enzyme digestion was used to confirm specific point mutations in 23SrRNA gene of clarithromycin-resistant isolates.

Results: Of 210 biopsies analyzed, 79 (37.6%) were H. pylori-positive, yielding 88 distinct strains-some patients harbored multiple genetically diverse isolates, identified by differences in cagA/vacA genotypes and antibiotic resistance profiles. Although, no association was found between virulence gene profile and resistance pattern. Resistance rates were highest for levofloxacin (69.3%), followed by metronidazole (61.4%) and clarithromycin (19.3%). Nearly all clarithromycin-resistant isolates carried the A2143G mutation in the 23SrRNA gene, capable of getting naturally transmitted into the next generations also. Levofloxacin-resistant strains harbored mutations in GyrA at codons 63, 87, 88, 91, 130, and 150, and a novel Gly85Ala substitution was identified. Metronidazole resistance correlated more strongly with rdxA mutations than with frxA. No resistance was detected against furazolidone and tetracycline, while amoxicillin resistance was rare (1.1%). Multidrug resistance was observed in 10.2% of isolates, with 32.9% displaying dual resistance to metronidazole + levofloxacin, and 6.8% to clarithromycin + levofloxacin.

Conclusions: High clarithromycin, substantial metronidazole and levofloxacin resistance in H. pylori strains from WB underscore the urgent need to revise empirical treatment strategies. These findings advocate for localized, resistance-guided therapy protocols and reinforce the importance of continuous antimicrobial surveillance to optimize treatment outcomes and mitigate resistance development.