Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Synergistic activity of dispersin B and benzoyl peroxide against Cutibacterium acnes/Staphylococcus epidermidis dual-species biofilms

  • Jeffrey B. Kaplan ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    * kaplanjb@american.edu

    Affiliations Department of Biology, American University, Washington, District of Columbia, United States of America, Laboratory for Skin Research, Institute for Medical Research, Galilee Medical Center, Nahariya, Israel

    ⨯
  • Anna Muzaleva,

    Roles Writing – review & editing

    Affiliation Kane Biotech, Inc., Winnipeg, Manitoba, Canada

    ⨯
  • Miloslav Sailer,

    Roles Conceptualization, Formal analysis, Project administration, Supervision, Writing – review & editing

    Affiliation Kane Biotech, Inc., Winnipeg, Manitoba, Canada

    ⨯
  • Robert B. Huizinga,

    Roles Conceptualization, Funding acquisition, Project administration, Software, Writing – review & editing

    Affiliation Kane Biotech, Inc., Winnipeg, Manitoba, Canada

    ⨯
  • Khalaf Kridin

    Roles Funding acquisition, Resources, Writing – review & editing

    Affiliations Laboratory for Skin Research, Institute for Medical Research, Galilee Medical Center, Nahariya, Israel, The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel

    ⨯

Abstract

Cutibacterium acnes plays a key role in the development of acne vulgaris, with biofilm formation contributing to its persistence and resistance to antimicrobial treatments. A critical component of C. acnes biofilms is poly-N-acetylglucosamine (PNAG), an exopolysaccharide that facilitates both biofilm stability and biocide resistance. This study evaluated the efficacy of the PNAG-degrading enzyme dispersin B in enhancing the susceptibility of C. acnes biofilms to benzoyl peroxide (BP), a common anti-acne agent. Dual-species biofilms of C. acnes and Staphylococcus epidermidis, which has been shown to promote C. acnes biofilm growth under aerobic conditions, were cultivated in glass tubes and treated with dispersin B (5–80 µg/mL), BP (0.1–2.5%), or a combination of both. Dispersin B or BP alone reduced C. acnes colony-forming units (CFUs) by 1–2 log units. However, sequential treatment with dispersin B followed by BP achieved a synergistic effect, yielding a >6-log reduction in CFUs. Remarkably, concentrations as low as 5 µg/mL dispersin B combined with 0.5% BP efficiently eradicated C. acnes from the dual-species biofilms. These findings highlight the protective role of PNAG against BP and demonstrate the potential of dispersin B as an adjunctive therapy to enhance the efficacy of BP in acne treatment.

Citation: Kaplan JB, Muzaleva A, Sailer M, Huizinga RB, Kridin K (2025) Synergistic activity of dispersin B and benzoyl peroxide against Cutibacterium acnes/Staphylococcus epidermidis dual-species biofilms. PLoS ONE 20(3): e0320662. https://doi.org/10.1371/journal.pone.0320662

Editor: Nourtan F. Abdeltawab,, Cairo University - Faculty of Pharmacy, EGYPT

Received: December 24, 2024; Accepted: February 23, 2025; Published: March 27, 2025

Copyright: © 2025 Kaplan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript.

Funding: This study was funded by Kane Biotech Inc. (Winnipeg, Manitoba, Canada). The funders played a role in the study design, data collection and analysis, decision to publish, and preparation of the manuscript.

Competing interests: Revised Competing Interests Statement JBK serves as an advisor to Kane Biotech Inc., holds equity in the company, and receives royalties. AM, MS, and RBH are employees of Kane Biotech Inc. The company, which manufactures Dispersin B (DispersinB®), funded this study. These affiliatoons do not alter our adherence to PLOS ONE policies on sharing data and materials. KK has no competing interests to declare.

Abbreviations: BP, benzoyl peroxide; CFU, colony-forming unit; PBS, phosphate buffered saline; PNAG, poly-N-acetylglucosamine; TSA, Tryptic Soy agar; TSB, Tryptic Soy broth.

1. Introduction

The Gram-positive bacterium Cutibacterium acnes was first isolated from a patient with acne vulgaris more than 100 years ago [1]. C. acnes is an aerotolerant anaerobe that utilizes sebum and keratin as nutrient sources, making it well adapted for life inside hair follicles. Although some C. acnes cellular components such as porphyrins, surface proteins, cytotoxic exoenzymes, and biofilms have been shown to trigger inflammation in the later stages of acne [2], the precise role of C. acnes in the early stages of acne remains unknown. For example, C. acnes colonizes hair follicles in all people, and several previous studies found no difference in the abundance of C. acnes in subjects with or without acne [3,4]. Recent studies suggest that acne might be the result of an unbalanced equilibrium between C. acnes and Staphylococcus epidermidis, another major skin resident [3,5–9]. In one study that identified bacteria in hair follicles from acne patients and healthy controls using a highly sensitive cyanoacrylate biopsy method [10], all subjects harbored C. acnes within their follicles, but the follicles of acne patients also harbored S. epidermidis.

Biofilms are defined as densely packed layers of bacterial cells growing attached to a tissue or surface [11]. Bacteria in a biofilm are encased in a sticky, self-synthesized, extracellular polymeric matrix that holds the cells together in a mass, attaches them to the underlying surface, and protects them from killing by antimicrobial agents and host immunity. Biofilms play a role in many chronic infections which are often difficult to treat because of the protective nature of biofilms. In vivo, C. acnes biofilms have been observed in acne lesions [12] and S. epidermidis biofilms have been observed on skin and in sweat glands [13]. Both species have been shown to form biofilms in vitro [1,14] and on the surfaces of implanted medical devices in vivo [15,16].

Poly-N-acetylglucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm formation, antimicrobial resistance, host colonization, immune evasion, and stress tolerance in a wide range of bacterial and fungal pathogens [17,18]. PNAG is an essential virulence factor for Staphylococcus aureus in mouse models of systemic infection [19]; for Aggregatibacter actinomycetemcomitans in a rat model of periodontitis [20]; for Klebsiella pneumoniae in a mouse model of intestinal colonization and systemic infection [21]; and for Actinobacillus pleuropneumoniae during natural porcine pleuropneumonia infection in pigs [22]. Anti-PNAG antibodies have been shown to protect mice against local and/or systemic infections caused by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, and Candida albicans [17], suggesting that PNAG is an important virulence factor in these organisms. Previous studies showed that both C. acnes and S. epidermidis produce PNAG [23,24].

Dispersin B is an enzyme that hydrolyzes PNAG [25]. Dispersin B has been shown to inhibit the attachment of C. acnes cells to surfaces, to inhibit C. acnes biofilm formation, and to sensitize C. acnes biofilms to killing by tetracycline and the common anti-acne agent benzoyl peroxide (BP) in vitro [24]. Dispersin B has also been shown and to inhibit S. epidermidis biofilm formation, detach pre-formed S. epidermidis biofilms, and sensitize pre-formed S. epidermidis biofilm cells to killing by cetylpyridinum chloride and rifampicin in vitro [26,27], and to inhibit attachment of S. epidermidis cells to pig skin in vivo [28].

While C. acnes is an anaerobe whose growth is inhibited by oxygen, previous studies showed that C. acnes can form robust biofilms in air when co-cultured with S. epidermidis [29]. This suggests that aerobic C. acnes/S. epidermidis dual-species biofilms may mimic biofilm formation in the skin’s microenvironments, including the well-oxygenated dermis and the hypoxic but non-anoxic conditions of sebaceous glands and hair follicles [30]. However, the structural components supporting these biofilms and their resistance to antimicrobial agents remain poorly understood. PNAG is a known adhesin in C. acnes and S. epidermidis biofilms, yet its role in maintaining dual-species biofilms under aerobic conditions has not been investigated. Furthermore, while dispersin B has been shown to disrupt single-species biofilms of C. acnes and S. epidermidis, its impact on dual-species biofilms and their susceptibility to BP remains unclear. To address this gap, the present study tested the hypothesis that PNAG is a crucial structural component of aerobic C. acnes/S. epidermidis dual-species biofilms and that enzymatic degradation of PNAG by dispersin B would enhance BP-mediated killing.

2. Materials and methods

2.1. Bacterial strains

The bacterial strains used in this study were C. acnes HL086PA1 [31] and S. epidermidis strain 5 [32]. C. acnes HL086PA1, an erythromycin-resistant strain isolated from a patient with severe acne, was obtained from BEI Resources (Manassas, Virginia, USA) as part of the NIAID NIH Human Microbiome Project. S. epidermidis strain 5 is an erythromycin susceptible, PNAG-producing strain isolated from an implant infection that was previously shown to facilitate growth of C. acnes HL086PA1 biofilms in vitro [29]. The media used were Tryptic Soy agar (TSA) and Tryptic Soy broth (TSB), both purchased in powder form from Becton, Dickinson and Company (BD; Franklin Lakes, New Jersey, USA). All bacteria were cultured at 37 °C. Reference stock cultures were maintained at −80 °C in 20% glycerol/80% TSB. Working stock cultures were restreaked weekly on TSA. S. epidermidis working stocks were incubated for 1 d in air. C. acnes working stocks were incubated for 3 d under anaerobic conditions generated with a BD GasPak EZ Anaerobe sachet system.

2.2. Biofilm culture

Inocula for biofilm cultures were prepared by transferring a 1- µL-size loopful of S. epidermidis cells from a 24-h-old agar plate into 200 µL of phosphate buffered saline (PBS), and then transferring a loopful of C. acnes cells from 72-h-old agar plate into the same tube. The cells were mixed by vortex agitation, diluted 1:1,000 in filter sterilized TSB, and then passed through a 5-µm pore-size syringe filter to remove large clumps of cells. Diluted inocula contained 106–107 CFU/mL of each species. Bacteria were present at a S. epidermidis:C. acnes ratio of 2–10:1. Filtered inocula were aliquoted into sterile 13 ×  100 mm glass tubes (1 mL/tube) and incubated for 24 h statically at 37 °C in air.

2.3. Dispersin B

Two formulations of dispersin B were employed. The first was a dispersin B solution which contained 1 mg/mL recombinant dispersin B protein dissolved in 50 mM phosphate buffer (pH 5.8), 100 mM NaCl, and 50% glycerol. Dispersin B solution was diluted in PBS to achieve a working concentration of 80 µg/mL enzyme. The second formulation was a dispersin B hydrogel which contained 80 µg/mL dispersin B in a mixture of non-ionic surfactant (poloxamer 407), glycerol, levulininc acid, anisic acid, and phosphate buffer. The dispersin B hydrogel was used directly or diluted with blank hydrogel (no enzyme) to achieve working concentrations of 40, 20, 10 and 5 µg/mL of enzyme. Dispersin B solution, dispersin B hydrogel, and blank hydrogel were provided by Kane Biotech (Winnipeg, Manitoba, Canada).

2.4. Benzoyl peroxide

CVS Health brand Maximum Strength Acne Treatment Gel containing 10% benzoyl peroxide (BP) was employed (CVS Corporation, Woonsocket, Rhode Island, USA). This gel contains micronized BP particles evenly dispersed within a polymer-based gelling agent. Although BP has extremely low solubility in water (~0.001%), its effectiveness at higher concentrations in gel formulations is due to its use as a suspension rather than a solution. The gel was diluted in PBS to obtain final working concentrations of 2.5%, 1%, 0.5%, and 0.1% BP.

2.5. Two-step biofilm treatment and enumeration of biofilm CFUs

After growth of biofilms for 24 h, the media was gently aspirated using a finely drawn Pasteur pipette. Each tube was rinsed twice with 3 mL of PBS and gently aspirated after each rinse. For the first treatment, tubes were filled with 1 mL of PBS; 80 µg/mL dispersin B solution; dispersin B hydrogel containing 5, 10, 20, 40 or 80 µg/mL dispersin B; or 1% or 2.5% BP, as indicated. Tubes were incubated for 15 min at 37 °C and then rinsed twice with PBS and aspirated as described above. All treatment and rinse steps involving dispersin B hydrogel were carried out on ice to maintain the liquid state of the hydrogel. For the second treatment, tubes were filled with 1 mL of PBS; 80 µg/mL dispersin B solution; or 0.1%, 0.5%, 1% or 2.5% BP, as indicated. After 15 min at 37 °C, tubes were rinsed three times with 3 mL of PBS and filled with 1 mL of PBS. Biofilm bacteria were detached from the walls of the tubes by sonication for 30 sec using an IKA Labortechnik sonicator set to 50% power and 50% duty cycle. Control experiments showed that this sonication treatment did not affect the viability of C. acnes or S. epidermidis cells. Sonicates were serially diluted in PBS. C. acnes and S. epidermidis CFUs were enumerated independently by plating dilutions on selective media as previously described [29]. To enumerate S. epidermidis CFUs, dilutions were plated on TSA and incubated in air which prevented growth of anaerobic C. acnes. To enumerate C. acnes CFUs, dilutions were plated on TSA supplemented with 20 µg/mL erythromycin and incubated anaerobically which prevented growth of erythromycin-sensitive S. epidermidis.

2.6. Crystal violet binding assay

Biofilms were rinsed vigorously with tap water and stained for 1 min with 1 mL of Gram’s crystal violet. Tubes were then rinsed with tap water to remove the unbound dye and air-dried. To quantitate crystal violet binding, tubes were filled with 1 mL of a 33% acetic acid solution, incubated at room temperature for 30 min, and mixed by vortex agitation. A volume of 200 µL of the dissolved dye was transferred to the well of a 96-well microtiter plate and its absorbance at 620 nm was measured in an automated microplate reader. Tubes containing sterile broth were incubated and processed along with the inoculated tubes to serve as controls. The amount of biofilm detachment was quantitated using the formula 1 − (A620/Dispersin B/A620/No enzyme) ×  100.

2.7. Statistics and reproducibility of results

All experiments were performed in duplicate or triplicate tubes and were repeated three times on different days. All repetitions exhibited a similar range of values and similarly significant differences in mean CFU/tube values among the experimental groups. Representative experiments are shown in the figures. The significance of differences between CFU/tube values was calculated using a two-tailed Student’s t-test for pairwise comparisons and a one-way ANOVA with Tukey’s post hoc analysis for comparison of more than two groups. A p-value <  0.01 was considered significant.

3. Results

3.1. Detachment of C. acnes/S. epidermidis dual-species biofilms by dispersin B solution and dispersin B hydrogel

To confirm that dispersin B causes detachment of C. acnes/S. epidermidis dual-species biofilms grown aerobically in glass tubes, biofilms were treated for 15 min with an 80 µg/mL dispersin B solution, or a hydrogel containing 80 µg/mL dispersin B. The amount of biofilm biomass remaining after treatment was quantitated using a crystal violet binding assay (Fig 1A). Both the dispersin B solution and dispersin B hydrogel treatments caused a significant reduction in biofilm biomass (94% and 78%, respectively). These results are consistent with those of a previous study showing that an 80 µg/mL solution of dispersin B efficiently detached aerobic C. acnes/S. epidermidis dual-species biofilms from glass tubes [29]. The biofilm detaching activity of dispersin B hydrogel was evidently due to the enzyme and not the other components of the hydrogel because a blank hydrogel (no enzyme) did not cause significant detachment of C. acnes/S. epidermidis dual-species biofilms as evidenced by visual inspection of the biofilms after treatment (Fig 1B).

thumbnail
Download:
Fig 1. Detachment of C. acnes/S. epidermidis dual-species biofilms by dispersin B solution and dispersin B hydrogel.

Both agents contained 80 µg/mL of enzyme. (A) Biofilms were rinsed and treated with the indicated agent for 15 min, then stained with crystal violet dye. The amount of bound dye (Absorbance at 620 nm) is proportional to the amount of biofilm biomass. Control tubes were treated with PBS. Blank tubes contained uninoculated broth. Values show mean and range for triplicate tubes for each condition. (B) Detachment of C. acnes/S. epidermidis dual-species biofilms by dispersin B hydrogel and blank hydrogel (no enzyme). Biofilms were rinsed and treated with the indicated agent for 15 min, then rinsed and photographed. Control tubes were treated with PBS. Duplicate tubes for each condition are shown.

https://doi.org/10.1371/journal.pone.0320662.g001

3.2. Pre-treatment of C. acnes/S. epidermidis dual-species biofilms with dispersin B solution renders them susceptible to benzoyl peroxide killing

C. acnes/S. epidermidis dual-species biofilms were subjected to a series of two-step treatments consisting of PBS, 80 µg/mL dispersin B solution, or 1% BP in different combinations. After treatment, biofilms were rinsed to remove loosely adherent cells, and then detached from the walls of the tube by sonication. S. epidermidis and C. acnes CFUs were enumerated independently by plating on selective agar (Fig 2). Dispersin B treatment alone caused a >1 log reduction in S. epidermidis CFUs and a >2 log reduction in C. acnes CFUs. Treatment of biofilms with 1% BP alone caused no reduction in S. epidermidis CFUs and a 1 log reduction in C. acnes CFUs. However, treatment of biofilms with dispersin B followed by BP caused a >2 log reduction in S. epidermidis CFUs and a >5 log reduction in C. acnes CFUs. These results suggest that dispersin B sensitizes both S. epidermidis and C. acnes to BP killing when cultured aerobically in a dual-species biofilm. Treatment of biofilms with BP followed by dispersin B resulted in no significant detachment or killing of S. epidermidis, suggesting that residual BP may partially inactivate or inhibit dispersin B (Fig 2, left panel). BP treatment followed by dispersin B treatment resulted in a 3-log reduction in C. acnes CFUs compared to a >5 log reduction when biofilms were treated with dispersin B followed by BP (Fig 2, right panel).

thumbnail
Download:
Fig 2. Pre-treatment of C. acnes/S. epidermidis dual-species biofilms with dispersin B solution renders them sensitive to killing by benzoyl peroxide.

24-h-old aerobic C. acnes/S. epidermidis dual-species biofilms were rinsed and treated for 15 min with phosphate buffered saline (PBS), 80 µg/ml dispersin B solution (Dsp), or 1% benzoyl peroxide (BP), and then rinsed and subjected to a second 15-min treatment with PBS, Dsp or BP in the combinations indicated at the top. Graphs show CFU/tube values for S. epidermidis (left panel) and C. acnes (right panel) from triplicate tubes for each treatment. Each dot represents one tube. Horizontal bars indicate means. NS, not significantly different.

https://doi.org/10.1371/journal.pone.0320662.g002

In a similar experiment, treatment of dual-species biofilms with dispersin B followed by 1% BP resulted in levels of C. acnes detachment and killing equivalent to those observed with 2.5% BP alone (Fig. 3). These findings suggest that dispersin B treatment may lower the minimum effective dose of BP against C. acnes.

thumbnail
Download:
Fig 3. Dispersin B solution +  1% benzoyl peroxide kills C. acnes biofilms as efficiently as 2.5% benzoyl peroxide alone.

24-h-old aerobic C. acnes/S. epidermidis dual-species biofilms were rinsed and treated for 15 min with phosphate buffered saline (PBS), 80 µg/ml dispersin B solution (Dsp), or 1% or 2.5% benzoyl peroxide (BP1 and BP2.5, respectively), then rinsed and subjected to a second 15-min treatment with PBS or BP1 in the combinations indicated at the top. Graphs show CFU/tube values for S. epidermidis (left panel) and C. acnes (right panel) from triplicate tubes for each treatment. Each dot represents one tube. Horizontal bars indicate means.

https://doi.org/10.1371/journal.pone.0320662.g003

3.3. Dispersin B hydrogel acts synergistically with benzoyl peroxide to kill C. acnes in C. acnes/S. epidermidis dual-species biofilms

To determine whether dispersin B hydrogel can sensitize C. acnes/S. epidermidis dual-species biofilms to BP killing, biofilms were subjected to a two-step treatment where the first step consisted of PBS, or dispersin B hydrogel containing 40 or 80 µg/mL dispersin B, and the second step consisted of PBS, 1% BP, or 2.5% BP. After treatment, biofilms were rinsed to remove dispersed cells, then detached from the walls of the tube by sonication. S. epidermidis and C. acnes CFUs were enumerated independently by plating on selective agar (Fig 4). Dispersin B hydrogel alone at 40 or 80 µg/mL resulted in a >1 log reduction in S. epidermidis CFUs, while BP alone at 1% or 2.5% exhibited no significant killing of S. epidermidis biofilms (Fig 4, left panel). Dispersin B hydrogel at 40 or 80 µg/mL did not sensitize S. epidermidis to 1% or 2.5% BP killing under these conditions (Fig 4, left panel). In contrast, dispersin B hydrogel alone at 40 or 80 µg/mL resulted in a >2 log reduction in C. acnes CFUs, BP alone at 1% or 2.5% caused >1 log killing of C. acnes biofilms, and dispersin B hydrogel at 40 or 80 µg/mL followed by BP at 1% or 2.5% caused >6 log reduction in C. acnes CFUs (Fig 4, right panel). These results suggest that dispersin B hydrogel acts synergistically with BP to kill C. acnes in C. acnes/S. epidermidis dual-species biofilms.

thumbnail
Download:
Fig 4. Dispersin B hydrogel sensitizes C. acnes biofilms to benzoyl peroxide killing.

24-h-old aerobic C. acnes/S. epidermidis dual-species biofilms were rinsed and treated for 15 min with phosphate buffered saline (PBS) or dispersin B hydrogel containing 40 or 80 µg/mL dispersin B (D40 and D80, respectively), then rinsed and subjected to a second 15-min treatment with PBS or benzoyl peroxide at 1% or 2.5% (B1 and B2.5, respectively), in the combinations shown above. Graphs show CFU/tube values for S. epidermidis (left panel) and C. acnes (right panel) from duplicate tubes for each treatment. Dashed line in the right panel indicates the limit of detection (5 ×  101 CFU/tube). Each dot represents one tube. Horizontal bars indicate means. BLD, below limit of detection.

https://doi.org/10.1371/journal.pone.0320662.g004

To determine the lowest concentrations of dispersin B and BP needed to eradicate C. acnes from C. acnes/S. epidermidis dual-species biofilms, biofilms were treated with dispersin B hydrogel containing 20, 10 or 5 µg/mL dispersin B, followed by BP at 0.5% or 0.1% (Fig 5). Dispersin B hydrogel alone at enzyme concentrations of 20, 10 or 5 µg/mL resulted in a >1 log reduction in S. epidermidis CFUs and a 2-log reduction in C. acnes CFUs (Fig 5), while benzoyl peroxide alone at 0.5% or 0.1% exhibited no significant killing of S. epidermidis biofilms (Fig 5, left panel) and a 1–2 log reduction in C. acnes CFUs (Fig 5, right panel). Dispersin B hydrogel at 20, 10 or 5 µg/mL of enzyme did not sensitize S. epidermidis to killing by 0.5% or 0.1% BP (Fig 5, left panel). In contrast, treatment of biofilms with dispersin B hydrogel at enzyme concentrations 20, 10 or 5 µg/mL resulted in a >3 log increase in C. acnes killing by both 0.5% and 0.1% BP compared to 0.5% and 0.1% BP alone (Fig 5, right panel).

thumbnail
Download:
Fig 5. Dispersin B hydrogel sensitizes C. acnes biofilms to benzoyl peroxide killing.

24-h-old aerobic C. acnes/S. epidermidis dual-species biofilms were rinsed and treated for 15 min with phosphate buffered saline (PBS) or dispersin B hydrogel containing 5, 10 or 20 µg/mL of enzyme (D20, D10, and D5 respectively), and then rinsed and treated with PBS or benzoyl peroxide at 0.5% or 0.1% (B.5 and B.1, respectively). Graphs show CFU/tube values for S. epidermidis (left panel) and C. acnes (right panel) from duplicate tubes for each treatment. Each dot represents one tube. Horizontal bars indicate means. BLD, below limit of detection.

https://doi.org/10.1371/journal.pone.0320662.g005

4. Discussion

Benzoyl peroxide (BP) has remained a cornerstone of acne treatment since its introduction in the 1950s. As an oxidizing agent, BP non-specifically oxidizes bacterial surface proteins, leading to structural damage and rapid cell death. BP’s main advantages include its ability to circumvent antibiotic resistance, as it is effective against antibiotic-resistant strains and has not been shown to induce resistance in C. acnes. However, a significant drawback of BP is its skin irritability, which often leads to patients using lower doses to reduce irritation. Unfortunately, these reduced doses may be less effective because they require longer contact times to kill C. acnes [33].

Previous research has demonstrated the microbiocidal effects of BP against C. acnes and S. epidermidis in vitro [34,35]. However, a study by Zhou et al. [36] observed that, following 12 weeks of BP treatment, the prevalence of C. acnes decreased, while that of S. epidermidis increased in the skin microbiota of acne patients. In our study, we observed that BP concentrations ranging from 0.1% to 2.5% effectively killed C. acnes but did not affect S. epidermidis when both bacteria were co-cultured in aerobic biofilms in vitro. This differential susceptibility to BP could be attributed to variations in bacterial strains, experimental conditions, or the complexities of the in vivo environment versus in vitro models.

Dispersin B has been shown to sensitize anaerobic C. acnes mono-species biofilms to BP and tetracycline [24] and to disperse aerobic C. acnes/S. epidermidis dual-species biofilms in glass tubes [29]. In this study, we demonstrated that dispersin B also sensitizes aerobic C. acnes/S. epidermidis dual-species biofilms to BP-induced killing. These findings are consistent with the biocide-sensitizing effects of dispersin B observed against other bacterial species [26,37–39]. Due to its high specificity for PNAG [40], these results suggest that PNAG plays a crucial role in the tolerance of C. acnes to BP in aerobic dual-species biofilms. The use of dispersin B in combination with BP could therefore be a promising strategy for acne treatment and prevention, as it may reduce the minimum effective dose of BP and consequently mitigate skin irritation.

Our study underscores the critical role of PNAG in protecting C. acnes from BP killing in the context of aerobic dual-species biofilms with S. epidermidis. However, a key unresolved question remains: the source of the PNAG in these biofilms. It is well-documented that S. epidermidis strain 5 produces PNAG [32], but it remains unclear whether C. acnes strain HL086PA1 produces PNAG as well. Previous studies have shown that all four C. acnes strains tested to date produce PNAG [24], suggesting that C. acnes PNAG might also contribute to the observed BP resistance. Further experiments are needed to determine whether PNAG production in the aerobic C. acnes/S. epidermidis dual-species biofilms arises from one or both bacterial species, which could provide valuable insights into how PNAG-mediated resistance functions in this context.

A key limitation of the present study is the artificial nature of the in vitro biofilm model used. Biofilms were formed on the surface of glass tubes in Tryptic Soy broth (TSB), which does not fully replicate the lipid-rich environment of the skin, where C. acnes naturally resides. In vivo, C. acnes biofilms are likely influenced by sebum composition, host immune factors, and tissue interactions that are absent in our experimental conditions. Future studies should explore models that incorporate lipid-rich environments and more physiologically relevant conditions to better mimic the complexity of C. acnes biofilms in the skin.

References

  1. 1. Achermann Y, Goldstein EJC, Coenye T, Shirtliff ME. Propionibacterium acnes: from commensal to opportunistic biofilm-associated implant pathogen. Clin Microbiol Rev. 2014;27(3):419–40. pmid:24982315
  2. 2. Mayslich C, Grange PA, Dupin N. Cutibacterium acnes as an opportunistic pathogen: an update of its virulence-associated factors. Microorganisms. 2021;9(2):303. pmid:33540667
  3. 3. Barnard E, Shi B, Kang D, Craft N, Li H. The balance of metagenomic elements shapes the skin microbiome in acne and health. Sci Rep. 2016;6:39491. pmid:28000755
  4. 4. Greydanus DE, Azmeh R, Cabral MD, Dickson CA, Patel DR. Acne in the first three decades of life: an update of a disorder with profound implications for all decades of life. Dis Mon. 2021;67(4):101103. pmid:33041056
  5. 5. Claudel J-P, Auffret N, Leccia M-T, Poli F, Corvec S, Dréno B. Staphylococcus epidermidis: a potential new player in the physiopathology of acne? Dermatology. 2019;235(4):287–94. pmid:31112983
  6. 6. Dreno B, Martin R, Moyal D, Henley JB, Khammari A, Seité S. Skin microbiome and acne vulgaris: Staphylococcus, a new actor in acne. Exp Dermatol. 2017;26(9):798–803. pmid:28094874
  7. 7. Lee YB, Byun EJ, Kim HS. Potential role of the microbiome in acne: a comprehensive review. J Clin Med. 2019;8(7):987. pmid:31284694
  8. 8. McLaughlin J, Watterson S, Layton AM, Bjourson AJ, Barnard E, McDowell A. Propionibacterium acnes and acne vulgaris: new insights from the integration of population genetic, multi-omic, biochemical and host-microbe studies. Microorganisms. 2019;7(5):128. pmid:31086023
  9. 9. O’Neill AM, Gallo RL. Host-microbiome interactions and recent progress into understanding the biology of acne vulgaris. Microbiome. 2018;6(1):177. pmid:30285861
  10. 10. Bek-Thomsen M, Lomholt HB, Kilian M. Acne is not associated with yet-uncultured bacteria. J Clin Microbiol. 2008;46(10):3355–60. pmid:18716234
  11. 11. Penesyan A, Paulsen IT, Kjelleberg S, Gillings MR. Three faces of biofilms: a microbial lifestyle, a nascent multicellular organism, and an incubator for diversity. NPJ Biofilms Microbiomes. 2021;7(1):80. pmid:34759294
  12. 12. Jahns AC, Lundskog B, Ganceviciene R, Palmer RH, Golovleva I, Zouboulis CC, et al. An increased incidence of Propionibacterium acnes biofilms in acne vulgaris: a case-control study. Br J Dermatol. 2012;167(1):50–8. pmid:22356121
  13. 13. Allen HB, Mueller JL. A novel finding in atopic dermatitis: film-producing Staphylococcus epidermidis as an etiology. Int J Dermatol. 2011;50(8):992–3. pmid:21781075
  14. 14. Otto M. Staphylococcus epidermidis--the “accidental” pathogen. Nat Rev Microbiol. 2009;7(8):555–67. pmid:19609257
  15. 15. Bayston R, Ashraf W, Barker-Davies R, Tucker E, Clement R, Clayton J, et al. Biofilm formation by Propionibacterium acnes on biomaterials in vitro and in vivo: impact on diagnosis and treatment. J Biomed Mater Res A. 2007;81(3):705–9. pmid:17187384
  16. 16. Büttner H, Mack D, Rohde H. Structural basis of Staphylococcus epidermidis biofilm formation: mechanisms and molecular interactions. Front Cell Infect Microbiol. 2015;5:14. pmid:25741476
  17. 17. Cywes-Bentley C, Skurnik D, Zaidi T, Roux D, Deoliveira RB, Garrett WS, et al. Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens. Proc Natl Acad Sci U S A. 2013;110(24):E2209-18. pmid:23716675
  18. 18. Soliman C, Walduck AK, Yuriev E, Richards JS, Cywes-Bentley C, Pier GB, et al. Structural basis for antibody targeting of the broadly expressed microbial polysaccharide poly-N-acetylglucosamine. J Biol Chem. 2018;293(14):5079–89. pmid:29449370
  19. 19. Kropec A, Maira-Litran T, Jefferson KK, Grout M, Cramton SE, Götz F, et al. Poly-N-acetylglucosamine production in Staphylococcus aureus is essential for virulence in murine models of systemic infection. Infect Immun. 2005;73(10):6868–76. pmid:16177366
  20. 20. Shanmugam M, Gopal P, El Abbar F, Schreiner HC, Kaplan JB, Fine DH, et al. Role of exopolysaccharide in Aggregatibacter actinomycetemcomitans-induced bone resorption in a rat model for periodontal disease. PLoS One. 2015;10(2):e0117487. pmid:25706999
  21. 21. Chen K-M, Chiang M-K, Wang M, Ho H-C, Lu M-C, Lai Y-C. The role of pgaC in Klebsiella pneumoniae virulence and biofilm formation. Microb Pathog. 2014;77:89–99. pmid:25450884
  22. 22. Subashchandrabose S, Leveque RM, Kirkwood RN, Kiupel M, Mulks MH. The RNA chaperone Hfq promotes fitness of Actinobacillus pleuropneumoniae during porcine pleuropneumonia. Infect Immun. 2013;81(8):2952–61. pmid:23732171
  23. 23. Kaplan JB, Ragunath C, Velliyagounder K, Fine DH, Ramasubbu N. Enzymatic detachment of Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother. 2004;48(7):2633–6. pmid:15215120
  24. 24. Kaplan JB, Cywes-Bentley C, Pier GB, Yakandawala N, Sailer M, Edwards MS, et al. Poly-β-(1→6)-N-acetyl-D-glucosamine mediates surface attachment, biofilm formation, and biocide resistance in Cutibacterium acnes. Front Microbiol. 2024;15:1386017. pmid:38751716
  25. 25. Kaplan JB, Sukhishvili SA, Sailer M, Kridin K, Ramasubbu N. Aggregatibacter actinomycetemcomitans dispersin B: the quintessential antibiofilm enzyme. Pathogens. 2024;13(8):668. pmid:39204268
  26. 26. Ganeshnarayan K, Shah SM, Libera MR, Santostefano A, Kaplan JB. Poly-N-acetylglucosamine matrix polysaccharide impedes fluid convection and transport of the cationic surfactant cetylpyridinium chloride through bacterial biofilms. Appl Environ Microbiol. 2009;75(5):1308–14. pmid:19114520
  27. 27. Lee J-H, Kaplan JB, Lee WY. Microfluidic devices for studying growth and detachment of Staphylococcus epidermidis biofilms. Biomed Microdevices. 2008;10(4):489–98. pmid:18204904
  28. 28. Kaplan JB, Mlynek KD, Hettiarachchi H, Alamneh YA, Biggemann L, Zurawski DV, et al. Extracellular polymeric substance (EPS)-degrading enzymes reduce staphylococcal surface attachment and biocide resistance on pig skin in vivo. PLoS One. 2018;13(10):e0205526. pmid:30304066
  29. 29. Kaplan JB, Assa M, Mruwat N, Sailer M, Regmi S, Kridin K. Facultatively anaerobic staphylococci enable anaerobic Cutibacterium species to grow and form biofilms under aerobic conditions. Microorganisms. 2024;12(12):2601. pmid:39770803
  30. 30. Evans SM, Schrlau AE, Chalian AA, Zhang P, Koch CJ. Oxygen levels in normal and previously irradiated human skin as assessed by EF5 binding. J Invest Dermatol. 2006;126(12):2596–606. pmid:16810299
  31. 31. Fitz-Gibbon S, Tomida S, Chiu B-H, Nguyen L, Du C, Liu M, et al. Propionibacterium acnes strain populations in the human skin microbiome associated with acne. J Invest Dermatol. 2013;133(9):2152–60. pmid:23337890
  32. 32. Chaignon P, Sadovskaya I, Ragunah C, Ramasubbu N, Kaplan JB, Jabbouri S. Susceptibility of staphylococcal biofilms to enzymatic treatments depends on their chemical composition. Appl Microbiol Biotechnol. 2007;75(1):125–32. pmid:17221196
  33. 33. Boonchaya P, Rojhirunsakool S, Kamanamool N, Khunkhet S, Yooyongsatit S, Udompataikul M, et al. Minimum contact time of 1.25%, 2.5%, 5%, and 10% benzoyl peroxide for a bactericidal effect against Cutibacterium acnes. Clin Cosmet Investig Dermatol. 2022;15403–9. pmid:35300432
  34. 34. Cove JH, Holland KT. The effect of benzoyl peroxide on cutaneous micro-organisms in vitro. J Appl Bacteriol. 1983;54(3):379–82. pmid:6874624
  35. 35. Okamoto K, Kanayama S, Ikeda F, Fujikawa K, Fujiwara S, Nozawa N, et al. Broad spectrum in vitro microbicidal activity of benzoyl peroxide against microorganisms related to cutaneous diseases. J Dermatol. 2021;48(4):551–5. pmid:33369759
  36. 36. Zhou L, Chen L, Liu X, Huang Y, Xu Y, Xiong X, et al. The influence of benzoyl peroxide on skin microbiota and the epidermal barrier for acne vulgaris. Dermatol Ther. 2022;35(3):e15288. pmid:34962033
  37. 37. Itoh Y, Wang X, Hinnebusch BJ, Preston JF 3rd, Romeo T. Depolymerization of beta-1,6-N-acetyl-D-glucosamine disrupts the integrity of diverse bacterial biofilms. J Bacteriol. 2005;187(1):382–7. pmid:15601723
  38. 38. Izano EA, Sadovskaya I, Vinogradov E, Mulks MH, Velliyagounder K, Ragunath C, et al. Poly-N-acetylglucosamine mediates biofilm formation and antibiotic resistance in Actinobacillus pleuropneumoniae. Microb Pathog. 2007;43(1):1–9. pmid:17412552
  39. 39. Parise G, Mishra M, Itoh Y, Romeo T, Deora R. Role of a putative polysaccharide locus in Bordetella biofilm development. J Bacteriol. 2007;189(3):750–60. pmid:17114249
  40. 40. Eddenden A, Nitz M. Applications of an inactive dispersin B probe to monitor biofilm polysaccharide production. Methods Enzymol. 2022;665:209–31. pmid:35379435