Assays of Sensitivity of Antibiotic-Resistant Bacteria to Hydrogen Peroxide and Measurement of Catalase Activity

  • Mirjana Macvanin
  • Diarmaid Hughes
Part of the Methods in Molecular Biology book series (MIMB, volume 642)


Bacteria, in common with other organisms that take advantage of aerobic respiration, generate and accumulate reactive oxygen species (ROS) that damage DNA, fatty acids, and proteins. In addition, intracellular pathogens like Salmonella enterica are exposed to an oxidate burst produced by host macrophages. The relative ability of aerobically growing bacteria to withstand oxidative stress and eliminate ROS has a large impact of their fitness in vitro and in vivo. Methods are described here to measure the viability and relative fitness of bacteria in the presence of hydrogen peroxide. A protocol for the determination of catalase activity, an important part of the ROS detoxification process, is also described.

Key words

Antibiotic resistance Fitness cost Reactive oxygen species Hydrogen peroxide Catalase Salmonella typhimurium 


  1. 1.
    Janssen R, van der Straaten T, van Diepen A, van Dissel JT (2003) Responses to reactive oxygen intermediates and virulence of Salmonella typhimurium. Microbes Infect 5:527–534CrossRefPubMedGoogle Scholar
  2. 2.
    Gonzalez-Flecha B, Demple B (1997) Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli. J Bacteriol 179:382–388PubMedGoogle Scholar
  3. 3.
    Pomposiello PJ, Bennik MH, Demple B (2001) Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate. J Bacteriol 183:3890–3902CrossRefPubMedGoogle Scholar
  4. 4.
    Zheng M, Wang X, Templeton LJ, Smulski DR, LaRossa RA, Storz G (2001) DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide. J Bacteriol 183:4562–4570CrossRefPubMedGoogle Scholar
  5. 5.
    Imlay JA (2002) How oxygen damages microbes: oxygen tolerance and obligate anaerobiosis. Adv Microb Physiol 46:111–153CrossRefPubMedGoogle Scholar
  6. 6.
    Ferenci T, Spira B (2007) Variation in stress responses within a bacterial species and the indirect costs of stress resistance. Ann N Y Acad Sci 1113:105–113CrossRefPubMedGoogle Scholar
  7. 7.
    Giuliodori AM, Gualerzi CO, Soto S, Vila J, Tavio MM (2007) Review on bacterial stress topics. Ann N Y Acad Sci 1113:95–104CrossRefPubMedGoogle Scholar
  8. 8.
    Claiborne A, Fridovich I (1979) Purification of the o-dianisidine peroxidase from Escherichia coli B. Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities. J Biol Chem 254:4245–4252PubMedGoogle Scholar
  9. 9.
    Claiborne A, Malinowski DP, Fridovich I (1979) Purification and characterization of hydroperoxidase II of Escherichia coli B. J Biol Chem 254:11664–11668PubMedGoogle Scholar
  10. 10.
    Storz G, Tartaglia LA, Ames BN (1990) The OxyR regulon. Antonie Van Leeuwenhoek 58:157–161CrossRefPubMedGoogle Scholar
  11. 11.
    Heimberger A, Eisenstark A (1988) Compartmentalization of catalases in Escherichia coli. Biochem Biophy Res Commun 154:392–397CrossRefGoogle Scholar
  12. 12.
    Sak BD, Eisenstark A, Touati D (1989) Exonuclease III and the catalase hydroperoxidase II in Escherichia coli are both regulated by the katF gene product. Proc Natl Acad Sci U S A 86:3271–3275CrossRefPubMedGoogle Scholar
  13. 13.
    Macvanin M, Ballagi A, Hughes D (2004) Fusidic acid-resistant mutants of Salmonella enterica serovar typhimurium have low levels of heme and a reduced rate of respiration and are sensitive to oxidative stress. Antimicrob Agents Chemother 48:3877–3883CrossRefPubMedGoogle Scholar
  14. 14.
    Macvanin M, Bjorkman J, Eriksson S, Rhen M, Andersson DI, Hughes D (2003) Fusidic acid-resistant mutants of Salmonella enterica serovar Typhimurium with low fitness in vivo are defective in RpoS induction. Antimicrob Agents Chemother 47:3743–3749CrossRefPubMedGoogle Scholar
  15. 15.
    Winquist L, Rannug U, Rannug A, Ramel C (1984) Protection from toxic and mutagenic effects of H2O2 by catalase induction in Salmonella typhimurium. Mutat Res 141:145–147CrossRefPubMedGoogle Scholar
  16. 16.
    Paul KG, Ohlsson PI, Jonsson NA (1982) The assay of peroxidases by means of dicarboxidine on enzyme-linked immunosorbent assay level. Anal Biochem 124:102–107CrossRefPubMedGoogle Scholar
  17. 17.
    Elgrably-Weiss M, Park S, Schlosser-Silverman E, Rosenshine I, Imlay J, Altuvia S (2002) A Salmonella enterica serovar typhimurium hemA mutant is highly susceptible to oxidative DNA damage. J Bacteriol 184:3774–3784CrossRefPubMedGoogle Scholar
  18. 18.
    Choi P, Wang L, Archer CD, Elliott T (1996) Transcription of the glutamyl-tRNA reductase (hemA) gene in Salmonella typhimurium and Escherichia coli: role of the hemA P1 promoter and the arcA gene product. J Bacteriol 178:638–646PubMedGoogle Scholar
  19. 19.
    Imlay JA, Linn S (1986) Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide. J Bacteriol 166:519–527PubMedGoogle Scholar
  20. 20.
    Testerman TL, Vazquez-Torres A, Xu Y, Jones-Carson J, Libby SJ, Fang FC (2002) The alternative sigma factor sigmaE controls antioxidant defences required for Salmonella virulence and stationary-phase survival. Mol Microbiol 43:771–782CrossRefPubMedGoogle Scholar
  21. 21.
    Gonzalez-Flecha B, Demple B (2000) Genetic responses to free radicals. Homeostasis and gene control. Ann N Y Acad Sci 899:69–87CrossRefPubMedGoogle Scholar
  22. 22.
    Kaul N, Forman HJ (1996) Activation of NF kappa B by the respiratory burst of macrophages. Free Radic Biol Med 21:401–405CrossRefPubMedGoogle Scholar
  23. 23.
    Vazquez-Torres A, Jones-Carson J, Mastroeni P, Ischiropoulos H, Fang FC (2000) Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro. J Exp Med 192:227–236CrossRefPubMedGoogle Scholar
  24. 24.
    Youmans GP, Karlson AG (1947) Streptomycin sensitivity of tubercle bacilli – studies on recently isolated tubercle bacilli and the development of resistance to streptomycin in vivo. Am Rev Tuberc 55:529–535PubMedGoogle Scholar
  25. 25.
    Welshimer HJ (1963) Vitamin requirements of Listeria monocytogenes. J Bacteriol 85:1156–1159PubMedGoogle Scholar
  26. 26.
    Premaratne RJ, Lin WJ, Johnson EA (1991) Development of an improved chemically defined minimal medium for Listeria monocytogenes. Appl Environ Microbiol 57:3046–3048PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Mirjana Macvanin
    • 1
  • Diarmaid Hughes
    • 2
  1. 1.Laboratory of Molecular Biology, National Cancer InstituteNational Institutes of HealthBethesdaUSA
  2. 2.Microbiology Programme, Department of Cell and Molecular BiologyUppsala UniversityUppsalaSweden

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