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

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.

INTRODUCTION

Benzoyl peroxide (BPO) is widely used as a topical drug for acne vulgaris, and has keratolytic/comedolytic, anti‐inflammatory effects and antimicrobial activity against Cutibacterium acnes. , We also previously demonstrated the rapid and potent bactericidal activity of BPO against C. acnes. BPO is rapidly degraded to benzoic acid to generate free radicals, and it is thought to cause damage to the bacterial cell walls. Therefore, BPO may be effective against microorganisms other than C. acnes. Akaza et al. reported the possibility that not only C. acnes but also other cutaneous resident microorganisms such as Staphylococcus species and Malassezia species are related to acne. In this study, we investigated the in vitro microbicidal activity of BPO against various bacteria and fungi.

METHODS

Microorganisms used in this study were C. acnes ATCC11827, Staphylococcus aureus ATCC29213, S. epidermidis ATCC12228, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, Candida albicans ATCC90028, Malassezia furfur ATCC14521, M. restricta ATCC MYA‐4611, and M. globosa ATCC MYA‐4612. C. acnes was cultured on Anaero Columbia agar supplemented with 5% rabbit blood (Nippon Becton Dickinson Company Ltd., Tokyo, Japan) for 72 h at 35°C under anaerobic conditions using an AnaeroPack system (Mitsubishi Gas Chemical Company Inc, Tokyo, Japan). S. aureus, S. epidermidis, E. coli and P. aeruginosa were cultured on Mueller‐Hinton agar (Nippon Becton Dickinson Company Ltd.) for 24 h at 35°C under aerobic conditions. C. albicans was cultured on Sabouraud agar (Nippon Becton Dickinson Company Ltd.) for 24 h at 32°C under aerobic conditions. M. furfur and M. globosa were cultured on modified Leeming and Notman agar for 3 or 7 days at 32°C under aerobic conditions and M. restricta was cultured on modified Leeming and Notman agar 5–7 days at 32°C under microaerobic conditions using an AnaeroPack system (Mitsubishi Gas Chemical Company Inc.). BPO was purchased from Sigma‐Aldrich (St. Louis, MO, USA).

The microbicidal activity of BPO was measured by an in vitro assay method reported previously. Bacteria and fungi were cultured using the above conditions and suspended at approximately 106 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.

RESULTS

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. 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 ([Link], [Link], [Link]).

Figure 1

Microbicidal activity of benzoyl peroxide against various bacterial strains. Cutibacterium acnes ATCC11827 (a), Staphylococcus aureus ATCC29213 (b), Staphylococcus epidermidis ATCC12228 (c), Escherichia coli ATCC25922 (d), and Pseudomonas aeruginosa ATCC27853 (e) were incubated for 1 h with 0.25, 0.5, 1, or 2m mol/L benzoyl peroxide (BPO). After incubation, the bacterial suspensions were collected and plated onto agar plates to calculate viable counts. Data indicate the mean ± standard deviation of three repeated experiments. CFU, colony forming unit; C, control; BDL, below the detection limit (<1.60 log CFU/mL). *P < 0.05, **P < 0.01 compared with the control group (Dunnett's multiple comparison test, 2‐sided)

Figure 2 shows the microbicidal activity of BPO against four species of fungi. The number of colonies of C. albicans was only decreased significantly when exposed to 2 mmol/L BPO. In contrast, the number of colonies of M. furfur, M. restricta, and M. globosa was decreased significantly when exposed to 0.25, 0.5, 1, and 2m mol/L BPO.

Figure 2

Microbicidal activity of benzoyl peroxide against various fungal strains. Cutibacterium albicans ATCC90028 (a), Malassezia furfur ATCC14521 (b), Malassezia restricta ATCC MYA‐4611 (c), and Malassezia globosa ATCC MYA‐4612 (d) were incubated for 1 h with 0.25, 0.5, 1, or 2 mmol/L benzoyl peroxide (BPO). After incubation, the fungal suspensions were collected and plated onto agar plates to calculate the viable counts. Data indicate the mean ± standard deviation of three repeated experiments. CFU, colony forming unit; C, control; BDL, below the detection limit (<1.60 log CFU/mL). **P < 0.01, compared with the control group (Dunnett's multiple comparison test, 2‐sided)

Figure 3 shows the influence of BPO on the cell morphology of C. acnes and M. restricta by TEM C. acnes and M. restricta after exposure of BPO (Figure 3B,D) showed a rough and blurry cell wall. In C. acnes, cell wall was disrupted partly, cytoplasmic materials were released to the extracellular (Figure 3B). In M. restricta, cell wall was expanded and electron density of it was decreased obviously (Figure 3D). These findings suggest that BPO directly damaged the cell wall of these strains, causing their death.

Figure 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)

DISCUSSION

Benzoyl peroxide had a potent and rapid microbicidal activity against Gram‐positive bacteria and fungi within 1 h. That is, significant decrease of viable counts of C. acnes, S. aureus, S. epidermidis and Malassezia species was observed at 15, 30 and 60 min after exposure of 2 mmol/L BPO ([Link], [Link], [Link]). Especially, Malassezia species were more sensitive to BPO than other bacteria. The cell wall of Malassezia is surrounded by a lipid‐rich outer layer. BPO is a highly lipophilic compound and therefore might more easily penetrate the cell wall of Malassezia than bacterial cell walls. It has been reported that Malassezia species, especially M. globosa and M. restricta are involved in the pathogenesis of acne vulgaris. The microbicidal activity of BPO against those Malassezia species may contribute to the effectiveness of the acne treatment. Various Malassezia species present on human skin as commensals are also associated with multiple skin diseases including pityriasis versicolor, Malassezia folliculitis, seborrheic dermatitis, atopic dermatitis, and psoriasis. A previous study reported that M. globosa and M. restricta were the predominating species isolated from patients with these diseases. However, BPO may not be used for these diseases, especially atopic dermatitis or psoriasis, because skin irritation, scales or erythema may occur during the use of this drug.

Benzoyl peroxide had a weaker bactericidal activity against Gram‐negative bacteria within 1 h. Gram‐negative bacteria have an outer membrane that functions as a permeability barrier against macromolecules and hydrophilic substances. , EDTA is a chelating agent that damages the outer membrane of Gram‐negative bacteria by forming a chelate complex with a divalent metal ion such as Ca2+ or Mg2+ present in the outer membrane. , 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. These findings suggest that BPO directly damaged the cell wall of these strains, causing their death.

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.

CONFLICT OF INTEREST

Kazuaki Okamoto, Shoji Kanayama, Fumiaki Ikeda, Naoki Hayashi, Koki Fujikawa, Shiori Fujiwara, Naoki Nozawa, Sachi Mori, Tatsumi Matsumoto are employees of Maruho Co., Ltd.. Masataka Oda is collaborator of Maruho Co., Ltd.

Figure S1

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Table S1

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Table S2

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