Article Figures & Data
Figures
- FIG. 1.
POR assay and competition with benzyl viologen. (A) Competitive inhibition of POR activity (benzyl viologen reduction) in H. pylori extracts as a function of NTZ concentration was monitored spectrophotometrically at 546 nm. The specific activity at each concentration of nitazoxanide was recorded. (B) POR was assayed in cell extracts of H. pylori by monitoring the pyruvate-dependent reduction of nitazoxanide at 412 nm (A) as described in the text.
- FIG. 2.
Lack of NTZ- or nitrofuran-induced DNA fragmentation in H. pylori. The MTZs strain H. pylori (Hp) 26695 was challenged with various concentrations of NTZ or nitrofurazone for 30 min as described in the text. The bacteria were suspended and lysed in agarose plugs, and agarose gels were run under alkaline conditions to display the extent of DNA fragmentation of denatured genomic DNA. Bacteria were treated with hydrogen peroxide (20 mM) for 15 min (positive controls). wt, wild type.
- FIG. 3.
Lack of drug-induced DNA fragmentation of E. coli strain CC104 carrying pBSK. The bacteria were grown in the presence of the nitrofuran drugs as described in the text. The preparation of agarose plugs is as described in Fig. 2. Hydrogen peroxide was added at a 20 mM concentration as a positive control. The distinct bands noted in each of the lanes are pBSK plasmid DNA.
- FIG. 4.
Lack of nitazoxanide-induced DNA fragmentation of E. coli strains carrying rdxA of H. pylori. E. coli strain CC104 containing either pBSK (control) or pGS950 (rdxA+) was grown in the presence of NTZ, and bacteria were suspended and lysed in agarose plugs and electrophoresed as described for Fig. 2 and detailed in the text. Hydrogen peroxide was added at a 20 mM concentration as a positive control. The distinct bands noted in the various lanes are plasmid DNA.
Tables
- TABLE 1.
POR activity of PORGDAB expressed in E. colia
Compound Sp act (nmol/min/mg of protein) JVQ2:pBSKPOR AB1157:pPBSKPOR NTZ 3,325 ± 428 2,233 ± 117 Nitrofurazone 41.2 ± 5.4 37.9 ± 1.5 Nitrofurantoin 29.2 ± 4.1 21.4 ± 0.6 Furazolidone 25.6 ± 1.7 18.1 ± 0.4 MTZ <0.1 <0.1 ↵ a The E. coli strains used in this study are AB1157 (wild type) and JVQ2nfsAnfsB (deficient in nitroreductase activity). Plasmid pPORGDAB contains the POR operon in pBSK. Enzyme activities were determined under anaerobic conditions at the appropriate wavelength for each compound as detailed in the text.
- TABLE 3.
Mutagenic action of sublethal concentrations of MTZ, NTZ, and furazolidone on Mtzs and MtzrH. pylori
Drug concn (μg/ml) Mutation frequency (no. Rifr/108) Metronidazole Nitazoxanide Furazolidone WT (EOP)a rdxA (EOP) WT (EOP) rdxA (EOP) WT (EOP) rdxA (EOP) 0 12 (1) 8 (1) 2.27 (1) 6.10 (1) 1.0 (1) 4.35 (1) 0.25 NTb NT 1.27 (1) NT 2.64 (0.1) 16.43 (1) 0.5 NT NT 1.50 (1) 2.94 (1) 6.90 (0.1) 64.91 (0.1) 1.0 14 (1) 5 (1) 1.41 (0.5) 4.46 (1) 0 (10−3) 0 2.0 69 (10−2) 10 (1) 0.97 (0.1) 2.47 (1) NT NT 3.0 144 (10−2) 5 (1) NT NT NT NT 5.0 0 (0) 12 (1) 0 (10−5) 5.26 (0.5) NT NT 10.0 NT NT NT 0 (10−8) NT NT 25.0 NT 213 (10−2) NT NT NT NT - TABLE 4.
Frequency of rifampin resistance in E. coli tester strain CC104
Strain, compound, and concn (μg/ml)a Survivalb Rifr frequency (per 108)c Fold CC104 Nitazoxanide 0 1 4.8 5 1 6.7 10 1 2.6 CC104(pGS950) Nitazoxanide 0 1 7.0 5 1 5.5 10 1 6.3 CC104 Nitrofurazone 0 1 3.36 1 1 26.4 7.9 2 0.7 58.6 17.4 5 10−3 75.4 22.4 Nitrofurantoin 0 1 3.36 1 0.9 6.14 1.8 2 0.9 8.59 2.6 5 2.5 × 10−2 32.58 9.7 Furazolidone 0 1 3.36 0.1 10−2 64.0 19 1 10−3 0 Controld Metronidazole 0 1 0.75 5 10−2 53 70 10 10−4 378 504 ↵ a The bacteria (E. coli CC104 + pBSK or CC104 + pGS950rdxA+) were grown on LB agar with the appropriate concentration of drug and then plated on medium containing rifampin as indicated in the text.
↵ b EOP − efficiency of plating (EOP of 1 = no loss of viability).
↵ c The Rifr frequencies represent the means of three determinations and are reported as the number of Rifr colonies per 108 bacteria plated.
↵ d Similar to data of Sisson et al. (29); included for comparison. Control, CC104(pGS950).
