Bactericidal efficacy of hydrogen peroxide on Cutibacterium acnes

Abstract

Objectives: The purpose of this study was to examine the bactericidal efficacy of hydrogen peroxide (H₂O₂) on Cutibacterium acnes (C. acnes). We hypothesize that H₂O₂ reduces the bacterial burden of C. acnes.

Methods: The effect of H₂O₂ was assessed by testing bactericidal effect, time course analysis, growth inhibition, and minimum bactericidal concentration. To assess the bactericidal effect, bacteria were treated for 30 minutes with 0%, 1%, 3%, 4%, 6%, 8%, or 10% H₂O₂ in saline or water and compared with 3% topical H₂O₂ solution. For time course analysis, bacteria were treated with water or saline (controls), 3% H₂O₂ in water, 3% H₂O₂ in saline, or 3% topical solution for 5, 10, 15, 20, and 30 minutes. Results were analyzed with a two-way analysis of variance (ANOVA) (p < 0.05).

Results: Minimum inhibitory concentration of H₂O₂ after 30 minutes is 1% for H₂O₂ prepared in saline and water. The 3% topical solution was as effective when compared with the 1% H₂O₂ prepared in saline or water. The controls of both saline and water showed no reduction of bacteria. After five minutes of exposure, all mixtures of H₂O₂ reduced the percentage of live bacteria, with the topical solution being most effective (p < 0.0001). Maximum growth inhibition was achieved with topical 3% H₂O₂.

Conclusion: The inexpensive and commercially available topical solution of 3% H₂O₂ demonstrated superior bactericidal effect as observed in the minimum bactericidal inhibitory concentration, time course, and colony-forming unit (CFU) inhibition assays. These results support the use of topical 3% H₂O₂ for five minutes before surgical skin preparation prior to shoulder surgery to achieve eradication of C. acnes for the skin.Cite this article: P. Hernandez, B. Sager, A. Fa, T. Liang, C. Lozano, M. Khazzam. Bactericidal efficacy of hydrogen peroxide on Cutibacterium acnes. Bone Joint Res 2019;8:3-10. DOI: 10.1302/2046-3758.81.BJR-2018-0145.R1.

Keywords: Cutibacterium acnes; Hydrogen peroxide; Infection; Shoulder; Shoulder arthroplasty; Treatment.

Fig. 1

Growth curve of purified strain of C. acnes. Bacteria were grown in tryptic soy broth–defibrinated sheep blood (TSB-DSB) at 37ºC under anaerobic conditions. Samples were taken at 0, 6, 24, 30, 48, 54, 78, and 97 hours of growth and culture aliquots were plated on TSB-DSB agar. The calculated generation time (gt) is 4.39 hours. Data are presented as the mean (standard deviation) of two measurements. CFUs, colony-forming units.

Minimum bactericidal concentration (MBC) was performed with a dose response of H₂O₂ prepared in saline and in water and compared with a 3% topical solution of H₂O₂. Bacteria were treated for 30 minutes on each solution (0% to 10%) and serial dilutions were seeded on tryptic soy broth–defibrinated sheep blood (TSB-DSB) agar. Colonies were counted after five days of incubation. All treatments are effective in their bactericidal activity. a) Representative image of three independent experiments. b) Graph of percentage of viability of C. acnes versus percentage of H₂O₂ grown in TSB showed a MBC of 1% for H₂O₂ prepared in saline and in water. Topical solution is equally effective. d) C. acnes treated for 30 minutes with 3% H₂O₂ prepared in saline, in water, or 3% topical solution were grown on TSB-DSB agar as lawn to further assess for bacterial growth inhibition. None of the treatments produced growth of colonies, while controls saline and water only show normal bacterial growth.

Time course of H₂O₂ effect. a) Bacteria grown in tryptic soy broth (TSB) showed that a 3% topical solution is superior to the other solutions in its bactericidal effect, starting from five minutes of incubation. 3% H₂O₂ prepared in saline is not significantly different from 3% H₂O₂ prepared in water in any of the timepoints. *p < 0.0001 compared with control saline or water; †p < 0.0001 compared with 3% H₂O₂ prepared in saline or in water. b) Bacteria grown in tryptic soy broth–defibrinated sheep blood (TSB-DSB) agar and treated with each solution of H₂O₂ for five minutes was grown as lawn to further assess for growth inhibition. All treatment solutions inhibited bacterial growth except the controls with water or saline.

Bacterial viability after five minutes of treatment. Viability was tested with Live/Dead BacLight kit after five minutes of incubation with H₂O₂. a) Standard curve used for each experiment was obtained by mixing live bacteria with dead bacteria obtained by incubation at 95°C for ten minutes in saline. Green staining gradually decreased (from 100% to 0% live bacteria) while red increased (from 0% to 100% dead bacteria). b) Both 3% H₂O₂ solution prepared in saline and in water show a similar staining pattern, while 3% topical solution showed a decreased staining for both green and red dyes.