Anti-Helicobacter pylori activity of ethoxzolamide

Figures & data

Figure 1. The chemical structures of acetazolamide, methazolamide and ethoxzolamide.

Table 1. Sensitivity profiles of H. pylori clinical isolate strains CH425, CH426, and CH427 to clinically used antibiotics.

Antimicrobial	H. pylori clinical isolates
	CH425		CH426		CH427
	MIC (μg/mL)	Interpretation	MIC (μg/mL)	Interpretation	MIC (μg/mL)	Interpretation
Metronidazole	>256	Resistant	12	Resistant	0.047	Sensitive
Clarithromycin	0.38	Intermediate	>256	Resistant	0.032	Sensitive
Amoxicillin	0.064	Sensitive	>256	Resistant	128	Resistant
Tetracycline	0.125	Sensitive	0.125	Sensitive	0.19	Sensitive

Table 2. MIC and MBC values of three sulphonamide drugs against four laboratory strains and three clinical isolates.

H. pylori strains	Sulphonamide drugs
	Ethoxzolamide (mM)		Acetazolamide (mM)		Methazolamide (mM)
	MIC	MBC	MIC	MBC	MIC	MBC
Lab strains
P12	0.2	0.4	2	8	0.5	3
26695	0.3	0.5	8	15	>5	>5
SS1	0.2	0.4	4	8	0.5	3
J99	0.3	0.5	8	15	>5	>5
Clinical strains
CH425	0.25	0.4	2	8	1	4
CH426	0.25	0.4	2	8	1	4
CH427	0.15	0.4	2	8	1	4

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.

Table 3. Nucleotide changes in H. pylori P12 EZA-resistant mutants generated by transformation of WT P12 with MutE chromosomal DNA.

Position^a	Type	Reference	Allele	Locus tag	Amino acid substitution	Gene product
H. pylori P12 EZA-resistant transformant MutETF1
1275233	SNV	C	T	HPP12_RS06100the	Glu173Lys	UPP pyrophosphate synthase
1573139	SNV	T	C	HPP12_RS07625	Cys29Arg	transcription termination factor NusA
294832	Deletion	A	–	HPP12_RS01490	Gln113fs^b	inner membrane protein
312794	SNV	G	T	HPP12_RS01540	Asp56Tyr	peptide ABC transporter substrate binding protein
H. pylori P12 EZA-resistant transformant MutETF2
1275233	SNV	C	T	HPP12_RS06100	Glu173Lys	UPP pyrophosphate synthase
1573139	SNV	T	C	HPP12_RS07625	Cys29Arg	transcription termination factor NusA
294832	Deletion	A	–	HPP12_RS01490	Gln113fs	inner membrane protein
509788	Deletion	A	–	HPP12_RS02505	Lys303fs	DNA methyltransferase
H. pylori P12 EZA-resistant transformant MutETF3
1275233	SNV	C	T	HPP12_RS06100	Glu173Lys	UPP pyrophosphate synthase
1573139	SNV	T	C	HPP12_RS07625	Cys29Arg	transcription termination factor NusA
294832	Deletion	A	–	HPP12_RS01490	Gln113fs	membrane protein
312794	SNV	G	T	HPP12_RS01540	Asp56Tyr	peptide ABC transporter substrate binding protein
H. pylori P12 EZA-resistant transformant MutETF4
1275233	SNV	C	T	HPP12_RS06100	Glu173Lys	UPP pyrophosphate synthase
1573139	SNV	T	C	HPP12_RS07625	Cys29Arg	transcription termination factor NusA
294832	Deletion	A	–	HPP12_RS01490	Gln113fs	inner membrane protein
312794	SNV	G	T	HPP12_RS01540	Asp56Tyr	peptide ABC transporter substrate binding protein
509788	Deletion	A	–	HPP12_RS02505	Lys303fs	DNA methyltransferase

^a Nucleotide positions are indicated with reference to the published H. pylori P12 genome.

^b fs: frameshift mutation.

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).