High Prevalence of hefA Efflux Pump Overexpression in Isolates of Helicobacter pylori Resistant to Clarithromycin

<b>Background/Objectives:</b> <i>Helicobacter pylori</i> 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 <i>23S rRNA</i> mutations, secondary mechanisms such as efflux pumps remain understudied. The present study reports a high prevalence of <i>hefA</i> efflux pump overexpression as a main molecular basis of clarithromycin resistance in <i>H. pylori</i> isolates from southern Chile. <b>Materials and Methods:</b> A total of 102 <i>H. pylori</i> isolates were obtained from gastric biopsy cultures. Isolates were analyzed for clarithromycin susceptibility by MIC, the <i>23S rRNA</i> mutations A2142G/A2143G by PCR-RFLP followed by sequencing, and <i>hefA</i> relative expression by qPCR. <b>Results:</b> 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 <i>hefA</i> expression was observed in resistant strains without <i>23S rRNA</i> mutations, indicating efflux pump dysregulation as a resistance mechanism. <b>Conclusions:</b> Our findings reveal a shift in epidemiology of clarithromycin resistance mechanisms in <i>H. pylori</i> that extends beyond classical <i>23S rRNA</i> mutations to include efflux-mediated adaptive resistance as a contributing mechanism. The positive correlation between <i>hefA</i> 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.