Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Aug;178(16):4822–4829. doi: 10.1128/jb.178.16.4822-4829.1996

Metabolic activities of metronidazole-sensitive and -resistant strains of Helicobacter pylori: repression of pyruvate oxidoreductase and expression of isocitrate lyase activity correlate with resistance.

P S Hoffman 1, A Goodwin 1, J Johnsen 1, K Magee 1, S J Veldhuyzen van Zanten 1
PMCID: PMC178263  PMID: 8759844

Abstract

In this study, we compared metronidazole (Mtz)-sensitive and -resistant strains of Helicobacter pylori for metabolic differences that might correlate with drug resistance. Included in this study was an isogenic Mtz(r) strain, HP1107, that was constructed by transforming genomic DNA from Mtz(r) strain HP439 into Mtz(s) strain HP500. Enzyme activities were also measured for Mtz(r) strains grown in the presence or absence of 18 micrograms of metronidazole per ml (ca. one-half of the MIC). These studies confirmed the presence of the Embden-Meyerhof-Parnas, Entner-Doudoroff, and pentose pathways. H. pylori strains expressed enzymatic activities indicative of a complete and active Krebs cycle. All strains expressed pyruvate oxidoreductase (POR) and alpha-ketoglutarate oxidoreductase (KOR) as measured with the redox-active dye benzyl viologen (30 to 96 nmol/min/mg of protein for POR and 30 nmol/min/mg of protein for KOR). When grown in the presence of Mtz at > or = 3.5 micrograms/ml, Mtz(r) strains expressed no detectable POR or KOR activity. The apparent repression of POR and KOR activities by Mtz affected bacterial growth as manifest by extended lag periods and growth yield reductions of > 30%. A dose-dependent relationship was demonstrated between the metronidazole concentration in the growth medium and the specific activity of POR measured in bacterial cell extracts. The observed repression was not due to inactivation of POR by Mtz. In addition to repression of POR and KOR activities, growth in the presence of Mtz also led to decreases in the activities of various Krebs cycle enzymes, including aconitase, isocitrate dehydrogenase and succinate dehydrogenase. All of the Mtz(r) strains examined expressed isocitrate lyase and malate synthase activities indicative of the glyoxylate bypass. No isocitrate lyase activity was detected in Mtz(s) strain HP500. Isocitrate lyase activity was expressed by HP500 following transformation to Mtz resistance (Mtz(r) strain HP1107) with DNA from an Mtz(r) strain. The results of this study suggest that Mtz resistance may be a recessive trait, possibly involving inactivation of a regulatory gene, that results in constitutive expression of isocitrate lyase. Repression of POR and KOR activities in response to low levels of Mtz may be a general response of H. pylori strains to Mtz, but only resistant strains manage to survive via activation of compensatory metabolic pathways.

Full Text

The Full Text of this article is available as a PDF (247.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Britz M. L., Wilkinson R. G. Isolation and properties of metronidazole-resistant mutants of Bacteroides fragilis. Antimicrob Agents Chemother. 1979 Jul;16(1):19–27. doi: 10.1128/aac.16.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chalk P. A., Roberts A. D., Blows W. M. Metabolism of pyruvate and glucose by intact cells of Helicobacter pylori studied by 13C NMR spectroscopy. Microbiology. 1994 Aug;140(Pt 8):2085–2092. doi: 10.1099/13500872-140-8-2085. [DOI] [PubMed] [Google Scholar]
  3. Chang H. C., Lane M. D. The enzymatic carboxylation of phosphoenolpyruvate. II. Purification and properties of liver mitochondrial phosphoenolpyruvate carboxykinase. J Biol Chem. 1966 May 25;241(10):2413–2420. [PubMed] [Google Scholar]
  4. Chen J. S., Blanchard D. K. A simple hydrogenase-linked assay for ferredoxin and flavodoxin. Anal Biochem. 1979 Feb;93(1):216–222. [PubMed] [Google Scholar]
  5. Cover T. L., Blaser M. J. Helicobacter pylori and gastroduodenal disease. Annu Rev Med. 1992;43:135–145. doi: 10.1146/annurev.me.43.020192.001031. [DOI] [PubMed] [Google Scholar]
  6. George H. A., Smibert R. M. Pyruvate oxidation by the Reiter strain of Treponema phagedenis. J Bacteriol. 1982 Dec;152(3):1060–1065. doi: 10.1128/jb.152.3.1060-1065.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goodman T. G., Hoffman P. S. Hydrogenase activity in catalase-positive strains of Campylobacter spp. J Clin Microbiol. 1983 Oct;18(4):825–829. doi: 10.1128/jcm.18.4.825-829.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goodwin C. S., Marshall B. J., Blincow E. D., Wilson D. H., Blackbourn S., Phillips M. Prevention of nitroimidazole resistance in Campylobacter pylori by coadministration of colloidal bismuth subcitrate: clinical and in vitro studies. J Clin Pathol. 1988 Feb;41(2):207–210. doi: 10.1136/jcp.41.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gui L., Sunnarborg A., Pan B., LaPorte D. C. Autoregulation of iclR, the gene encoding the repressor of the glyoxylate bypass operon. J Bacteriol. 1996 Jan;178(1):321–324. doi: 10.1128/jb.178.1.321-324.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. HATHAWAY J. A., ATKINSON D. E. THE EFFECT OF ADENYLIC ACID ON YEAST NICOTINAMIDE ADENINE DINUCLEOTIDE ISOCITRATE DEHYDROGENASE, A POSSIBLE METABOLIC CONTROL MECHANISM. J Biol Chem. 1963 Aug;238:2875–2881. [PubMed] [Google Scholar]
  11. Haas C. E., Nix D. E., Schentag J. J. In vitro selection of resistant Helicobacter pylori. Antimicrob Agents Chemother. 1990 Sep;34(9):1637–1641. doi: 10.1128/aac.34.9.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hof H., Sticht-Groh V. Antibacterial effects of niridazole: its effect on microaerophilic campylobacter. Infection. 1984 Jan-Feb;12(1):36–39. doi: 10.1007/BF01641023. [DOI] [PubMed] [Google Scholar]
  13. Hoffman P. S., Goodman T. G. Respiratory physiology and energy conservation efficiency of Campylobacter jejuni. J Bacteriol. 1982 Apr;150(1):319–326. doi: 10.1128/jb.150.1.319-326.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hoffman P. S., Krieg N. R., Smibert R. M. Studies of the microaerophilic nature of Campylobacter fetus subsp. jejuni. I. Physiological aspects of enhanced aerotolerance. Can J Microbiol. 1979 Jan;25(1):1–7. doi: 10.1139/m79-001. [DOI] [PubMed] [Google Scholar]
  15. Hughes N. J., Chalk P. A., Clayton C. L., Kelly D. J. Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori. J Bacteriol. 1995 Jul;177(14):3953–3959. doi: 10.1128/jb.177.14.3953-3959.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kamel M. Y., Allison D. P., Anderson R. L. Stereospecific D-glucokinase of Aerobacter aerogenes. Purification and properties. J Biol Chem. 1966 Feb 10;241(3):690–694. [PubMed] [Google Scholar]
  17. Kedderis G. L., Argenbright L. S., Miwa G. T. Mechanism of reductive activation of a 5-nitroimidazole by flavoproteins: model studies with dithionite. Arch Biochem Biophys. 1988 Apr;262(1):40–48. doi: 10.1016/0003-9861(88)90166-x. [DOI] [PubMed] [Google Scholar]
  18. Keyer K., Gort A. S., Imlay J. A. Superoxide and the production of oxidative DNA damage. J Bacteriol. 1995 Dec;177(23):6782–6790. doi: 10.1128/jb.177.23.6782-6790.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kletzin A., Adams M. W. Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga maritima. J Bacteriol. 1996 Jan;178(1):248–257. doi: 10.1128/jb.178.1.248-257.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krieg N. R., Hoffman P. S. Microaerophily and oxygen toxicity. Annu Rev Microbiol. 1986;40:107–130. doi: 10.1146/annurev.mi.40.100186.000543. [DOI] [PubMed] [Google Scholar]
  21. Lacey S. L., Moss S. F., Taylor G. W. Metronidazole uptake by sensitive and resistant isolates of Helicobacter pylori. J Antimicrob Chemother. 1993 Sep;32(3):393–400. doi: 10.1093/jac/32.3.393. [DOI] [PubMed] [Google Scholar]
  22. Lascelles J., Calder K. M. Participation of cytochromes in some oxidation-reduction systems in Campylobacter fetus. J Bacteriol. 1985 Oct;164(1):401–409. doi: 10.1128/jb.164.1.401-409.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lindmark D. G., Müller M. Antitrichomonad action, mutagenicity, and reduction of metronidazole and other nitroimidazoles. Antimicrob Agents Chemother. 1976 Sep;10(3):476–482. doi: 10.1128/aac.10.3.476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lockerby D. L., Rabin H. R., Laishley E. J. Role of the phosphoroclastic reaction of Clostridium pasteurianum in the reduction of metronidazole. Antimicrob Agents Chemother. 1985 May;27(5):863–867. doi: 10.1128/aac.27.5.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McQUATE J. T., UTTER M. F. Equilibrium and kinetic studies of the pyruvic kinase reaction. J Biol Chem. 1959 Aug;234(8):2151–2157. [PubMed] [Google Scholar]
  26. Mendz G. L., Hazell S. L., Burns B. P. Glucose utilization and lactate production by Helicobacter pylori. J Gen Microbiol. 1993 Dec;139(12):3023–3028. doi: 10.1099/00221287-139-12-3023. [DOI] [PubMed] [Google Scholar]
  27. Mendz G. L., Hazell S. L., Burns B. P. The Entner-Doudoroff pathway in Helicobacter pylori. Arch Biochem Biophys. 1994 Aug 1;312(2):349–356. doi: 10.1006/abbi.1994.1319. [DOI] [PubMed] [Google Scholar]
  28. Moore R. A., Beckthold B., Bryan L. E. Metronidazole uptake in Helicobacter pylori. Can J Microbiol. 1995 Aug;41(8):746–749. doi: 10.1139/m95-102. [DOI] [PubMed] [Google Scholar]
  29. Müller M. Mode of action of metronidazole on anaerobic bacteria and protozoa. Surgery. 1983 Jan;93(1 Pt 2):165–171. [PubMed] [Google Scholar]
  30. NOLTMANN E. A., GUBLER C. J., KUBY S. A. Glucose 6-phosphate dehydrogenase (Zwischenferment). I. Isolation of the crystalline enzyme from yeast. J Biol Chem. 1961 May;236:1225–1230. [PubMed] [Google Scholar]
  31. NOLTMANN E. A. ISOLATION OF CRYSTALLINE PHOSPHOGLUCOSE ISOMERASE FROM RABBIT MUSCLE. J Biol Chem. 1964 May;239:1545–1550. [PubMed] [Google Scholar]
  32. Narikawa S., Suzuki T., Yamamoto M., Nakamura M. Lactate dehydrogenase activity as a cause of metronidazole resistance in Bacteroides fragilis NCTC 11295. J Antimicrob Chemother. 1991 Jul;28(1):47–53. doi: 10.1093/jac/28.1.47. [DOI] [PubMed] [Google Scholar]
  33. Nedenskov P. Nutritional requirements for growth of Helicobacter pylori. Appl Environ Microbiol. 1994 Sep;60(9):3450–3453. doi: 10.1128/aem.60.9.3450-3453.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Quon D. V., d'Oliveira C. E., Johnson P. J. Reduced transcription of the ferredoxin gene in metronidazole-resistant Trichomonas vaginalis. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4402–4406. doi: 10.1073/pnas.89.10.4402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. RACKER E. Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids. Biochim Biophys Acta. 1950 Jan;4(1-3):211–214. doi: 10.1016/0006-3002(50)90026-6. [DOI] [PubMed] [Google Scholar]
  36. Results of a multicentre European survey in 1991 of metronidazole resistance in Helicobacter pylori. European Study Group on Antibiotic Susceptibility of Helicobacter pylori. Eur J Clin Microbiol Infect Dis. 1992 Sep;11(9):777–781. [PubMed] [Google Scholar]
  37. Reynolds D. J., Penn C. W. Characteristics of Helicobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology. 1994 Oct;140(Pt 10):2649–2656. doi: 10.1099/00221287-140-10-2649. [DOI] [PubMed] [Google Scholar]
  38. Reysset G. Genetics of 5-nitroimidazole resistance in Bacteroides species. Anaerobe. 1996 Apr;2(2):59–69. doi: 10.1006/anae.1996.0008. [DOI] [PubMed] [Google Scholar]
  39. Rosen O. M., Rosen S. M., Horecker B. L. Purification and properties of a specific fructose 1,6-diphosphatase from Candida utilis. Arch Biochem Biophys. 1965 Dec;112(3):411–420. doi: 10.1016/0003-9861(65)90073-1. [DOI] [PubMed] [Google Scholar]
  40. Smith M. A., Edwards D. I. Redox potential and oxygen concentration as factors in the susceptibility of Helicobacter pylori to nitroheterocyclic drugs. J Antimicrob Chemother. 1995 Jun;35(6):751–764. doi: 10.1093/jac/35.6.751. [DOI] [PubMed] [Google Scholar]
  41. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. 1983 Jun 4;1(8336):1273–1275. [PubMed] [Google Scholar]
  42. Veldhuyzen van Zanten S. J., Sherman P. M. Indications for treatment of Helicobacter pylori infection: a systematic overview. CMAJ. 1994 Jan 15;150(2):189–198. [PMC free article] [PubMed] [Google Scholar]
  43. Wang Y., Roos K. P., Taylor D. E. Transformation of Helicobacter pylori by chromosomal metronidazole resistance and by a plasmid with a selectable chloramphenicol resistance marker. J Gen Microbiol. 1993 Oct;139(10):2485–2493. doi: 10.1099/00221287-139-10-2485. [DOI] [PubMed] [Google Scholar]
  44. YOSHIDA A. ENZYMIC PROPERTIES OF MALATE DEHYDROGENASE OF BACILLUS SUBTILIS. J Biol Chem. 1965 Mar;240:1118–1124. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES