Abstract

Background/Objectives: Helicobacter pylori is a cause of chronic gastric infections and gastrointestinal carcinogenesis, with a prevalence of 20–90% around the world. Its eradication is increasingly challenged by clarithromycin resistance, particularly in regions with high rates of antibiotic resistance. While clarithromycin resistance is primarily attributed to 23S rRNA mutations, secondary mechanisms such as efflux pumps remain understudied. The present study reports a high prevalence of hefA efflux pump overexpression as a main molecular basis of clarithromycin resistance in H. pylori isolates from southern Chile. Materials and Methods: A total of 102 H. pylori isolates were obtained from gastric biopsy cultures. Isolates were analyzed for clarithromycin susceptibility by MIC, the 23S rRNA mutations A2142G/A2143G by PCR-RFLP followed by sequencing, and hefA relative expression by qPCR. Results: Clarithromycin resistance was detected in 38% of isolates. Resistance was significantly associated with therapeutic failure and urban residence. While 44% of resistant isolates harbored A2142G/A2143G mutations, 56% did not, suggesting alternative resistance mechanisms. Mutation C2182T was identified in 11% resistant isolates, and increased hefA expression was observed in resistant strains without 23S rRNA mutations, indicating efflux pump dysregulation as a resistance mechanism. Conclusions: Our findings reveal a shift in epidemiology of clarithromycin resistance mechanisms in H. pylori that extends beyond classical 23S rRNA mutations to include efflux-mediated adaptive resistance as a contributing mechanism. The positive correlation between hefA overexpression and MIC elevation underscores its role in resistance. These findings have important implications for the efficacy of clarithromycin-based therapies and highlight the need to reassess empirical treatment strategies in response to emerging resistance patterns. © 2025 by the authors.