Broad spectrum in vitro microbicidal activity of benzoyl peroxide against microorganisms related to cutaneous diseases

Abstract The in vitro microbicidal activity of benzoyl peroxide against Cutibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Malassezia furfur, Malassezia restricta, and Malassezia globosa was investigated. These strains were incubated for 1 h in the presence of 0.25, 0.5, 1, or 2 mmol/L benzoyl peroxide in phosphate buffered saline supplemented with 0.1% glycerol and 2% Tween 80. After exposure to benzoyl peroxide, counts of viable Gram‐positive bacteria and fungi were markedly decreased, whereas counts of Gram‐negative bacteria were unchanged. Transmission electron microscopy images showed a decrease in electron density and the destruction of C. acnes and M. restricta cell walls after exposure to 2 mmol/L benzoyl peroxide. In conclusion, this study showed that benzoyl peroxide has a potent and rapid microbicidal activity against Gram‐positive bacteria and fungi that are associated with various cutaneous diseases. This suggests that the direct destruction of bacterial cell walls by benzoyl peroxide is an essential mechanism of its rapid and potent microbicidal activity against microorganisms.

The microbicidal activity of BPO was measured by an in vitro assay method reported previously. 4 Bacteria and fungi were cultured using the above conditions and suspended at approximately 10 6 colony forming units (CFU)/mL in phosphate buffered saline (PBS) supplemented with 0.1% glycerol and 2% Tween 80. BPO dissolved in dimethyl sulfoxide (DMSO) was added to the bacterial or fungal suspensions at a final concentration ranging from 0.25 to 2 mmol/L (final concentration of DMSO was 3%). After aerobic incubation for 1 h at 35°C, 100 µL of the bacterial or fungal suspensions was removed, serially diluted, plated onto agar, and cultured using the above growth conditions. The number of colonies on agar plates was counted (detection limit, 1.60 log CFU/mL). To investigate the influence of the outer membrane on the bactericidal activity of BPO against E. coli and P. aeruginosa, these strains were preincubated aerobically in PBS with 100 mmol/L ethylenediaminetetraacetic acid (EDTA) for 1.5 h at 35°C. After washing the cells twice with PBS, the bactericidal activity of BPO was measured. Each assay was repeated in triplicate. Statistical analyses were performed by Dunnett's multiple comparison test and Student's t-test using EXSUS ver. 8.0.0 (CAC Croit Corporation, Tokyo, Japan). P-values less than 0.05 were considered significant.
The effect of BPO on cell morphology was evaluated by transmission electron microscopy (TEM). These strains were exposed to 2 mmol/L BPO for 1 h at 35°C under aerobic conditions. The cells were fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer at 4°C overnight. After washing several times with 0.1 mol/L cacodylate buffer, cells were post-fixed with 1.5% potassium permanganate in water at 4°C for 16 h. Following acetone dehydration, samples were embedded in plain resin (Nisshin EM Co., Tokyo, Japan). Ultrathin sections were observed and photographed with a transmission electron microscope (H-7600, Hitachi, Tokyo, Japan) operated at 200 kV. Figure 1 shows the microbicidal activity of BPO against five species of bacteria. The number of colonies of C. acnes, S. aureus, and S. epidermidis were decreased significantly by exposure to 0.5, 1, and 2 mmol/L BPO. However, the number of colonies of P. aeruginosa was only decreased when exposed to 2 mmol/L BPO.

| RE SULTS
Moreover, the number of colonies of E. coli was not decreased by exposure to any concentration of BPO, including 2 mmol/L. The number of EDTA-pretreated E. coli and P. aeruginosa colonies was decreased significantly by exposure to 2 mmol/L BPO for 1 h (Supporting information).  or Mg 2+ present in the outer membrane. 11,12 BPO showed potent bactericidal activity against EDTA-pretreated E. coli and P. aeruginosa.
These results suggest that the bactericidal activity of BPO against E. coli and P. aeruginosa was blocked by their outer membrane.
In TEM observation, decreased electron density and the partial destruction of cell walls in C. acnes and M. restricta were observed after exposure to BPO. These morphological changes were similar to those in E. coli and S. aureus treated with short time ultrasound which is reported to cause a direct damage to the cell walls in a previous study. 13 These findings suggest that BPO directly damaged the cell wall of these strains, causing their death.

F I G U R E 3 Morphological changes
of Cutibacterium acnes and Malassezia restricta after exposure to benzoyl peroxide. C. acnes and M. restricta were exposed to 2 mmol/L benzoyl peroxide (BPO) for 1 h and morphological changes were observed by transmission electron microscopy. Control C. acnes (a), 2 mmol/L BPO-treated C. acnes (magnification: ×60 000) (b), control M. restricta (c), and 2 mmol/L BPO-treated M. restricta (magnification, ×20 000) (d) In conclusion, this study showed that BPO has potent and rapid microbicidal activity against microorganisms that are associated with cutaneous diseases and the direct destruction of bacterial cell walls by BPO is an essential mechanism of its rapid and potent microbicidal activity against microorganisms.

ACK N OWLED G M ENTS
The authors would like to thank Naomasa Gotoh, PhD (Kyoto Pharmaceutical University, Kyoto, Japan) for useful discussion and advice, Yayoi Nishiyama, PhD (Teikyo University, Tokyo, Japan) for technical support regarding TEM.