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. 2021 Sep 1;26(3):130-138.
doi: 10.1089/apb.20.0060. Epub 2021 Sep 13.

Mechanisms of Sporicidal Activity Induced by Ionized Hydrogen Peroxide in the Spores of Bacillus atrophaeus

Miguel A Grimaldo  1   2 , Nicole L Mendell  2 , Nathen E Bopp  2 , Donald H Bouyer  2 , Claudio Mafra  1
Affiliations

Mechanisms of Sporicidal Activity Induced by Ionized Hydrogen Peroxide in the Spores of Bacillus atrophaeus

Miguel A Grimaldo et al. Appl Biosaf. .

Abstract

Introduction: Ionized hydrogen peroxide (iHP) is a new technology used for the decontamination of surfaces or laboratory areas. It utilizes a low concentration of hydrogen peroxide (H2O2) mixed with air and ionized through a cold plasma arc. This technology generates reactive oxygen species as a means of decontamination. Objectives: The purpose of this study is to review the effects of iHP on the structure of the spores of Bacillus atrophaeus by observing its effects using transmission electron microscopy (TEM) and also by evaluating the existence of DNA damage by fluorescence-based quantitative polymerase chain reaction (qPCR). Methods: Spore samples of B. atrophaeus decontaminated using iHP at different exposure times (Control, 1, 2, 6, and 12 h) were fixed for TEM. In addition, DNA was extracted for evaluation of DNA damages using fluorescence-based qPCR assays. Results: Damages to the spore structures of B. atrophaeus caused by the decontamination process with iHP at different exposure times (Control, 1, 2, 6, and 12 h) can be observed in micrographs. The effects of the decontamination to short DNA segment (132 base pairs [bp]) of the yaaH gene using qPCR present a linear degradation, and for the long DNA segment (680 bp), it presents a biphasic mode. Conclusion: The results of the qPCR analysis show two initial stages of damage to DNA with very noticeable damage at 12 h contact time, which confirms the observations of the TEM micrographs for the B. atrophaeus spores. The study demonstrates damage to the spore core DNA.

Keywords: Bacillus atrophaeus; DNA damage; decontamination; ionized hydrogen peroxide; transmission electron microscopy.

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Conflict of interest statement

No competing financial interests exist.

Figures

Figure 1.
Figure 1.
Progression of decontamination effects of iHP in the spores of Bacillus atrophaeus. (A) Nonexposed, (B) 1 h exposure, (C) 2 h exposure, (D) 6 h exposure, (E) 12 h exposure. Arrows indicate the location of the damage. CO, core; CT, cortex; IC, inner spore coat; iHP, ionized hydrogen peroxide; IM, inner membrane; OC, outer spore coat.
Figure 2.
Figure 2.
Progression of decontamination effects of iHP in the spore coat of Bacillus atrophaeus spores. (A) Nonexposed control, (B) 1 h exposure, (C) 2 h exposure, (D) 6 h exposure, (E) 12 h exposure. Arrows indicate the location of the damage.
Figure 3.
Figure 3.
Progression of decontamination effects of iHP in the spore coat and inner membranes of Bacillus atrophaeus spores. (A) Nonexposed control, (B) 1 h exposure, (C) 2 h exposure, (D) 6 h exposure, (E) 12 h exposure. Arrows indicate the location of the damage.
Figure 4.
Figure 4.
Results of the qPCR analysis to the damage to the yaaH gene of the Bacillus atrophaeus spores. Data are presented as the median with interquartile range. qPCR, quantitative polymerase chain reaction.
Figure 5.
Figure 5.
t-Test analysis result for each decontamination time for the qPCR results of the BA yaaH Primer Set 3 (132 bp).
Figure 6.
Figure 6.
t-Test analysis result for each decontamination time for the qPCR results of the BA yaaH Primer Set 2 (680 bp).
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