LETTERS to the EDITOR

Metronidazole-resistant Helicobacter pylori

The growth-inhibiting activity of Tabebuia impetiginosa Martius ex DC dried inner bark-derived constituents against Helicobacter pylori ATCC 43504 was examined using paper disc diffusion and minimum inhibitory concentration (MIC) bioassays. The activity of the isolated compounds was compared to that of the commercially available anti-Helicobacter pylori agents, amoxicillin, metronidazole, and tetracycline. The biologically active components of Tabebuia impetiginosa dried inner bark (taheebo) were characterized by spectroscopic analysis as 2-(hydroxymethyl)anthraquinone, anthraquinone-2-carboxylic acid, and 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone (lapachol). With the paper disc diffusion assay 2-(hydroxymethyl)anthraquinone exhibited strong activity against Helicobacter pylori ATCC 43504 at 0.01 mg/disc. Anthraquinone-2-carboxylic acid, lapachol and metronidazole were less effective, exhibiting moderate anti-Helicobacter pylori activity at 0.1 mg/disc. Amoxicillin and tetracycline were the most potent compounds tested, displaying very strong activity at 0.005 mg/disc. 2-(Hydroxymethyl)anthraquinone exhibited moderate activity at this dose. Tetracycline still had strong activity at 0.001 mg/disc while amoxicillin had little activity at this dose. In the MIC bioassay, 2-(hydroxymethyl)anthraquinone (2 μg/mL), anthraquinone-2-carboxylic acid (8 μg/mL), and lapachol (4 μg/mL) were more active than metronidazole (32 μg/mL) but less effective than amoxicillin (0.063 μg/mL) and tetracycline (0.5 μg/mL). The anti-Helicobacter pylori activity of seven 1,4-naphthoquinone derivatives (structurally related to lapachol), 1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone (naphthazarin), 2-methyl-1,4-naphthoquinone (menadione), 2-hydroxy-1,4-naphthoquinone (lawsone), 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), 5-hydroxy-1,4-naphthoquinone (juglone), and 2,3-dichloro-1,4-naphthoquinone (dichlone) was also evaluated using the paper disc assay. Menadione and plumbagin were the most potent compounds tested with the later still exhibiting very strong activity at 0.001 mg/disc. Menadione, juglone and tetracycline had strong activity at this low dose while the latter two compounds and amoxicillin had very strong activity at 0.005 mg/disc. Lawsone was unusual in that it had very strong activity at 0.1 and 0.05 mg/disc but weak activity at doses of 0.01 mg/disc and lower. Naphthazalin, lapachol and dichlone had similar activities while metronidazole had the lowest activity of all compounds tested. These results may be an indication of at least one of the pharmacological actions of taheebo. The Tabebuia impetiginosa dried inner bark-derived materials, particularly 2-(hydroxymethyl)anthraquinone, merit further study as potential Helicobacter pylori eradicating agents or lead compounds.

The effect of ether, alcoholic and water extracts of black myrobalan (Teminalia chebula Retz) on Helicobactor pylori were examined using an agar diffusion method on Columbia Agar. Water extracts of black myrobalan showed significant antibacterial activity and had a minimum inhibitory concentration (MIC) and minimum bacteriocidal concentration (MBC) of 125 and 150 mg/l, respectively. The extract was active after autoclaving for 30 min at 121°C. Plant powder (incorporated in agar) gave higher MIC and MBC values (150 and 175 mg/l, respectively). Water extracts of the black myrobalan at a concentration of 1–2.5 mg/ml inhibited urease activity of H. pylori. The results show that black myrobalan extracts contain a heat stable agent(s) with possible therapeutic potential. Other bacterial species were also inhibited by black myrobalan water extracts.

Helicobacter pylori is very susceptible in vitro to most antibiotics, but when they are used in the clinical setting, eradication of the bacteria from the gastric mucosa is not obtained. Dual or triple therapy including two of the following antibiotics: amoxicillin, tetracycline, metronidazole or clarithromycin, plus a proton pump inhibitor, bismuth salt or ranitidine bismuth citrate is the most frequently used. Various in vitro susceptibility methods have been used: disk diffusion, agar dilution and Epsilometer test (E-test). Metronidazole resistance among H. pylori strains is now found worldwide, and resistance rates vary according to the population studied. It is higher in developing than in developed countries and it could reach 80–90% in Africa. The prevalence on clarithromycin resistance is much lower, usually below 10%, although very high values are reported in Peru. Infection with metronidazole- or clarithromycin-resistant H. pylori strains is correlated with treatment failure when using regimens including these antibiotics.

Fifty-five strains of Helicobacter pylori isolated from November 1997 until October 1998 from 33 female and 22 male adults attending for endoscopy at the Evangel Hospital, Jos, Nigeria were assayed for antibiotic susceptibility to amoxycillin, clarithromycin, metronidazole and tetracycline by the E-test strip method. Minimum inhibitory concentration (MIC) within the attainable peak serum concentrations for each drug was used as the parameter to determine the susceptibility of H. pylori. The results showed 100% susceptibility for amoxycillin, 89·0% for tetracycline, 87·3% for clarithromycin and 60% for metronidazole. The MIC₅₀ and MIC₉₀ values were: 0·016 μg/mL and 0·75 μg/mL for amoxycillin, 0·016 μg/mL and 2 μg/mL for clarithromycin, 0·094 μg/mL and 12 μg/mL for tetracycline, and 2 μg/mL and >48 μg/mL for metronidazole. The MIC₉₀ values for metronidazole (>48 μg/mL) and tetracycline (12 μg/mL) were in each case higher than the break-point value (peak serum concentrations) of 8 μg/mL for metronidazole and 3 μg/mL for tetracycline. This pattern of resistance to metronidazole and tetracycline has to be considered when therapeutic regimens against H. pylori contain either or both drugs.

We examined the molecular mechanism of metronidazole resistance by constructing a λ-Zap II phagemid expression library with genomic DNA from a metronidazole-resistance strain ofHelicobacter pylori.Twenty-two clones were found to have elevated MTZ resistances in XLOLR strain ofE. coli.Phagemids belonging to the twenty two clones were extracted and then retransformed into the XLOLR strain ofE. coli.After MTZ selection, five clones could confer metronidazole resistance consistently. According to Southern hybridization and DNA sequencing, the five clones contained a same locus,recA.In addition, transforming the five clones into BL21 strain ofE. coliproduced a higher resistance to MTZ. Interestingly, electroporation of one of the five phagemid clones into two MTZ sensitiveH. pyloriyielded MTZ resistant strains. Comparing amino acid sequence in MTZ resistant with sensitive isolates revealed two point mutations at this locus. Above results suggest that mutation inrecAmay be associated with metronidazole resistance ofH. pylori.