Physiological changes induced in four bacterial strains following oxidative stress
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In order to study the behavior and resistance of bacteria under extreme conditions, physiological changes associated with oxidative stress were monitored using flow cytometry. The study was conducted to assess the maintenance of membrane integrity and potential as well as the esterase activity, the intracellular pH and the production of superoxide anions in four bacterial strains (Ralstonia metallidurans, Escherichia coli, Shewanella oneidensis and Deinococcus radiodurans). The strains were chosen for their potential use in bioremediation. Suspensions of R. metallidurans, E. coli, S. oneidensis and D. radiodurans were submitted to 1 h of oxidative stress (H2O2 at various concentrations from 0 to 880 mM). Cell membrane permeability (propidium iodide) and potential (rhodamine-123,3,3’-dihexyloxacarbocyanine iodide), intracellular esterase activity (fluorescein diacetate), intracellular-reactive oxygen species concentration (hydroethidine) and intracellular pH (carboxy-fluorescein diacetate succinimidyl ester 5-(6)) were monitored to evaluate the physiological state and the overall fitness of individual bacterial cells under oxidative stress. The four bacterial strains exhibited varying sensitivities towards H2O2. However, for all the bacterial strains, some physiological damage could already be observed from 13.25 mM H2O2 onwards, in particular with regard to their membrane permeability. Depending on the bacterial strains, moderate to high physiological damage could be observed between 13.25 mM and 220 mM H2O2. The membrane potential, esterase activity, intracellular pH and production of superoxide anion production were in all four strains considerably modified at high H2O2 concentrations. In conclusion, we show that a range of significant physiological alterations occur when bacteria are challenged with H2O2 and fluorescent staining methods coupled with flow cytometry are used for monitoring the changes induced not only by oxidative stress, but also by other stresses like temperature, radiation, pressure, pH, etc.
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