The Bactericidal Action of Peroxides; An E.P.R. Spin-Trapping Study

Abstract

E.P.R. spin trapping has been employed to study radical production during the bactericidal action of three peroxide compounds (peracetic acid, 4-percarboxy-N-isobutyltrimellitimide and magnesium monoperoxyphthalate) upon both Gram negative (Escherichia Coli) and Gram positive (Staphylococcus Aureus) bacteria. Use of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has allowed direct detection of both carbon-centred and hydroxyl radicals, which are produced at varying rates for the different bacteria/peracid systems studied. The inhibition of bactericidal action, by DMPO and two antioxidants, Vitamin C and Trolox C, indicates that radicals are the lethal species and evidence is presented which suggests that radical production is internal to the bacterial cell. Hydroxyl radicals are believed to be the lethal species. The effect of added iron chelators and haem protein inhibitors indicates that iron species and haem proteins in particular are involved. A marked variation is found in observed hydroxyl-radical adduct signals with both the nature and concentration of peracid. A strong inverse correlation is found between the concentration of the observed radical adduct signal and the relative strength of the peroxide as a bactericide; use of a stable nitroxide as a radical scavenger confirms that strong bactericides produce radicals at a much faster rate than weak bactericides. Plots of radical generation versus time are correlated with % bacterial kill, offering further evidence that hydroxyl radicals are the lethal species.

Comparative Transcriptomic and Phenotypic Analysis of the Responses ofBacillus cereusto Various Disinfectant Treatments

“…HP is commonly used as a disinfectant and is considered environmentally friendly because it breaks down into oxygen and water (9). It induces a burst of free OH ⅐ radicals that can damage DNA and may ultimately result in cell death (15,18). PAA is formed by the reaction of HP and acetic acid and is considered a more potent biocide than HP, because it shows antibacterial activity at lower concentrations than HP alone (9).…”

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

“…S. aureus cells do not produce detectable levels of hydroxyl radicals. Since the ESR signal recorded by the PBN probe in bacterial cultures appeared to be highly influenced by the radicals formed in the broth medium itself, we attempted to detect cellular free radicals using the cell-permeative spin trap probe, DMPO, applied to bacterial cells suspended in sodium sulfate buffer (36). Compared with PBN, the DMPO probe is relatively hydrophilic, with a partitioning coefficient of 0.15 in octanal/water (32).…”

“…In the case of L. acidophilus, production of hydroxyl radicals was shown to take place extracellularly (34). In an early study, the presence of intracellular radicals in bacterial cells was inferred from the ability of the DMPO probe to reduce killing by peracetic acid but direct detection of hydroxyl radicals was complicated by the presence of differing degrees of membrane permeability and large background signals (36). Our data reveal additional challenges in detection of free radicals in bacterial cells, namely, the formation of free radicals in the broth medium and the hydrogen peroxide-scavenging enzymes produced by some bacteria.…”

“…Importantly, catalase production by S. aureus was reduced by treatment with the bactericidal antibiotics vancomycin and ciprofloxacin and this reduction correlated with the increased PBN spin-adduct signal that was observed compared to the level seen for untreated cells. ESR (34,36,43). All of those studies focused on the detection of hydroxyl radicals, which mostly have been claimed to be present intracellularly in the bacteria.…”

“…The rest of the culture was centrifuged at 1.2 ϫ 10 4 ϫ g at room temperature for 30 s, and then the supernatant and the cell pellet resuspended with fresh medium were measured by ESR as well. The second protocol was modified from a previous study (36) with the following changes. Cells were collected when the OD 600 reached 0.1 and were resuspended in 50 mM sodium sulfate buffer to a final concentration of 2.5 ϫ 10 9 CFU/ml.…”