Anti- Helicobacter pylori activity of ethoxzolamide

Abstract

Ethoxzolamide (EZA), acetazolamide, and methazolamide are clinically used sulphonamide drugs designed to treat non-bacteria-related illnesses (e.g. glaucoma), but they also show antimicrobial activity against the gastric pathogen Helicobacter pylori. EZA showed the highest activity, and was effective against clinical isolates resistant to metronidazole, clarithromycin, and/or amoxicillin, suggesting that EZA kills H. pylori via mechanisms different from that of these antibiotics. The frequency of single-step spontaneous resistance acquisition by H. pylori was less than 5 × 10^-9, showing that resistance to EZA does not develop easily. Resistance was associated with mutations in three genes, including the one that encodes undecaprenyl pyrophosphate synthase, a known target of sulphonamides. The data indicate that EZA impacts multiple targets in killing H. pylori. Our findings suggest that developing the approved anti-glaucoma drug EZA into a more effective anti-H. pylori agent may offer a faster and cost-effective route towards new antimicrobials with a novel mechanism of action.

Keywords: MIC/MBC; Mutation frequency; ethoxzolamide; genome sequencing.

Figure 1.

The chemical structures of acetazolamide, methazolamide and ethoxzolamide.

Figure 2.

The antimicrobial effects of carbonic anhydrase inhibitors EZA, AAZ, and MZA on various H. pylori strains. Sensitivity of the laboratory strains P12, 26695, SS1, and J99 to (A) EZA, (C) AAZ, and (E) MZA and sensitivity of the clinical strains CH425, CH426, CH427 to (B) EZA, (D) AAZ, and (F) MZA are represented as percentage survival after 24 h (CFU survival (%)=(CFU_24h/CFU_0h)*100). The CFU survival levels corresponding to MIC (100.1% survival), MBC (0.1% survival) and limit of detection (LOD) are indicated by horizontal lines. Error bars represent the standard error of the mean for three independent biological replicates.

Figure 3.

Analysis of the time and dose dependency of the antimicrobial action of AAZ, MZA, and EZA on H. pylori P12. (A) Bactericidal kinetics for 1 × MBC and 2 × MBC of the respective sulphonamide, measured at neutral pH (B) Bactericidal kinetics for 2 × MBC of the respective sulphonamide under neutral (pH 6.8) and acidic (pH 4.5) conditions. The horizontal dashed line represents the limit of detection (100 cells) and the horizontal solid line corresponds to 99.9% cell death. Error bars represent the standard error of the mean for three independent biological replicates.

Figure 4.

Growth curves for H. pylori P12 WT and MutE measured over 36 h. Error bars represent the standard error of the mean for three independent biological replicates. Significant differences compared to wild type P12 are indicated; *p < .05, **p < .01. All other differences are not significant.

Figure 5.

Sensitivity of H. pylori P12 WT and its sulphonamide-resistant mutants MutE to clinically used antibiotics amoxicillin, clarithromycin, tetracycline, and metronidazole. Error bars represent the standard error of the mean for two independent biological replicates. (A) The MIC values of both the WT strain and MutE for amoxicillin were 0.0075 µg/ml, with the MBC value for MutE (0.015 µg/ml) being 2-fold lower than for the WT strain. (B) The P12 WT strain and MutE showed very close sensitivities to clarithromycin (MIC = 0.015 µg/ml, MBC = 0.06 µg/ml). (C) The sensitivity assay for tetracycline yielded the MIC and MBC values of 0.25 µg/ml and 1 µg/ml, respectively for both the WT strain and MutE. (D) The P12 WT strain and MutE showed the same sensitivity pattern for metronidazole (MIC = 0.5 µg/ml, MBC = 1 µg/ml).