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Article

Management of Scalp Biofilm in Local Dermatosis: From In Vivo Visualization to Optimal Treatments

by
Jang-Ho Joo
,
Jaeyoon Kim
,
Jae-Young Shin
,
Yun-Ho Choi
,
Heena Rim
,
Seung-Hyun Jun
and
Nae-Gyu Kang
*
R&I Institute, LG H&H, Seoul 07795, Republic of Korea
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(21), 10008; https://doi.org/10.3390/app142110008
Submission received: 5 August 2024 / Revised: 29 October 2024 / Accepted: 31 October 2024 / Published: 1 November 2024
(This article belongs to the Section Applied Microbiology)
Browse Figures
Versions Notes

Abstract

:
The fungi and bacteria on the human scalp play important roles in both health and disease. Scalp biofilms have pathogenic effects on cutaneous tissues, such as seborrheic dermatitis. However, investigations into scalp biofilms and their physiological effects on scalp skin are limited. In this study, we suggest an evaluation method through which the scalp is stained a reddish color using erythrosine to visualize scalp biofilms, which strongly depends on the presence of bacteria and fungi. We found that the physiological properties of the scalp significantly differed between high and low levels of stained red areas (sRAs) on the scalp. The sRA levels showed a strong positive correlation with IL-8 levels and sebum production. It is worth noting that the production of sebum has a dominant effect on the scalp microbiome via the growth of microbes, leading to the formation of a biofilm, as evidenced by changes in the sRA levels. Furthermore, the sRA levels could be reduced through the use of antimicrobial agents, such as climbazole and hexamidine diisethionate (HD), as well as the manipulation of the physical properties of the scrubs used in scalp care products. These scalp care products could potentially disrupt the formation and accumulation of a biofilm on the scalp, providing strong evidence for the importance of considering the scalp microbiome and its interactions with sebum in the development of biofilms. Consequently, we suggested that the administration of anti-microbial agents, such as climbazole and HD, could be an effective strategy to alleviate biofilm accumulation for the maintenance of scalp health.

1. Introduction

Biofilms are widely observed when fungi and bacteria adhere to surfaces, secreting a sticky substance in environments with high moisture levels. These biofilms can also be detected on human tissues in the form of slimy layers on teeth, referred to as plaque, which can lead to tooth decay [1]. The scalp provides a distinct microenvironment compared to other areas of the skin, characterized by unique physiological conditions, such as sebum content, moisture, pH, and topography [2]. It is hypothesized that the scalp exhibits unique features related to biofilm formation for two reasons: the high moisture condition created by hair fibers and the high sebum production that promotes the growth of Malassezia and Cutibacterium [3]. Dandruff is a common scalp condition that many people experience at some point in their lives. It is characterized by the presence of flakes on the scalp and hair, and it can cause itching [4]. While dandruff is widely recognized as a symptomatic problem, there has been limited research on its pathophysiology [5].
One popular theory is that dandruff is caused by the overgrowth of a fungus called Malassezia, which thrives on the lipids present in sebum [6]. When sebum is degraded by Malassezia, this leads to inflammation, irritation, and the flaking of the scalp. This process is associated with an increase in pro-inflammatory mediators like interleukin-8 [7]. Evidence supporting the role of Malassezia in dandruff includes the fact that antifungal agents like ketoconazole can alleviate symptoms, and inoculating animals with Malassezia can induce dandruff-like symptoms [8,9].
In natural environments, biofilms commonly consist of multiple species or poly-microbial biofilms, coexisting as part of a larger community structure [10]. Similar to single-species biofilms, the formation of poly-microbial biofilms is affected by various factors, such as the physiochemical properties of the surface environment, host receptors, nutrient availability, aggregation patterns, and local immune system responses [11]. The presence of different species in a particular habitat can involve different forms of communication between species, including quorum sensing [12]. Biofilms can also form within human organs, such as the middle ear and upper respiratory tract, oral cavity, cardiovascular system, lung, stomach, colon, urogenital system, bone, and soft tissue wounds [13]. Among them, the most extensively studied biofilms are oral biofilms, mainly due to the ease of sampling accessibility and the ubiquity of dental plaque, which is a type of multispecies biofilm. The oral bacterial diversity in the human spans hundreds of species across nine phyla. Different compositions of these communities have been associated with healthy and diseased states, such as periodontitis. In particular, erythrosine is widely used for detecting biofilms in the mouth owing to its sensitive cell viability and non-toxicity with respect to humans. Previously, we took advantage of erythrosine and developed a quick and convenient method for staining biofilms in the scalp [14].
Herein, we investigated the microbial properties of the scalp using a biofilm staining method. There are two distinctive groups: the staining-positive and staining-negative groups. Various properties of the scalp were measured, including TEWL, sebum production, and IL-8 levels. Among them, we identified sebum production as the dominant factor controlling biofilm formation in the scalp owing to its strong correlation with fungi and bacteria growth. To determine the effect of fungi and bacteria on biofilm formation in the human scalp, the composition and distribution of Cutibacterium, Staphylococcus, and Malassezia were analyzed. Based on our results, we suggest that climbazole and hexamidine diisethionate can effectively disturb biofilm formation as antimicrobial agents. This scalp biofilm visualization and assessment method could be of assistance in understanding the physiological properties of the scalp, particularly in the initial stage of its development.

2. Materials and Methods

2.1. Subjects

The present study adhered to the tenets of the Declaration of Helsinki. The clinical studies were reviewed and approved by the Review Board of the LG Household and Healthcare Ltd. (approval number LGHH-20240314-AB-02-01). Written informed consent was obtained from all subjects prior to any study-related procedures. Volunteers with an overall healthy physical condition (62 Koreans: 46 females and 16 males) were recruited. It should be noted that the number of female subjects is higher than male subject owing to the consideration of scalp conditions. All subjects washed their hair 24 h prior to examination and did not use any antifungal haircare products for 2 weeks.

2.2. Analysis of Scalp Physiological Conditions

The subjects’ scalps were wiped using paper towels and acclimated for 20 min in an air-conditioned room (temperature: 22 ± 2 °C; relative humidity: 50 ± 10%). The measurements were performed across the midline of the scalp at the four sites, including the frontal, mid, vertex, and occipital sites. Each measurement was taken using Folliscope® (LeedM Corporation, Seoul, Republic of Korea) using a 50-fold magnification lens. The skin barrier was evaluated based on transepidermal water loss (TEWL). Transepidermal water loss (TEWL) values were recorded in triplicate using a Tewameter® TM Nano with MAP-systems (Courage + Khazaka electronic GmbH, Cologne, Germany). The measurements were performed after blow-drying and resting for 30 min in an air-controlled room. The sebum levels were measured with a Meibometer® MB 560 (Courage-Khazaka electronic GmbH, Cologne, Germany) by pressing slightly onto the scalp for 30 s.

2.3. Staining and Analysis of Biofilms on the Human Scalp

The biofilm staining was performed at four scalp sites: the temporal recession site (right and left), midline of vertex site, and occipital site. First, a wet cotton swab with erythrosine solution (Trace® Disclosing Solution, Earth City, MO, USA) was used to scrub the human scalp twice (staining process). After a 15-min incubation, to check that the solution had sufficiently stained the biofilm, the same scalp area was swabbed with deionized water twice to remove residual erythrosine and sebum from the scalp’s surface (washing process). At this stage, it is important not to rub the cotton swab on the scalp but rather to rotate it with a consistent force to absorb the remaining solution thoroughly. After the washing process, scalp images of each subject were taken using Folliscope® (LeedM Corporation) with a 50-fold magnification lens. The obtained images were analyzed using Celleste 6 Image Analysis Software (Invitrogen, Waltham, MA, USA) to compare the intensity of the redness (105 Lum·pix2). In detail, the stained red area was designated as the object, and the clean area of the scalp and hair is designated as the background against which we calculate the red intensity of objects. In addition, subjects who participated in the experiment regularly used the climbazole (0.3%) shampoo, salt scrub (35%) shampoo, or hexamidine diisethionate (0.1%) hair tonic (full ingredient list provided in Table S2).

2.4. Bacteria Analysis in Scalp Corneocytes

Scalp corneocytes were obtained by stripping the surface of the midline of the vertex site with D-Squame® skin sampling discs (CuDerm, Dallas, TX, USA). The discs were soaked with 1 mL of PBS solution (1% BSA, 5% FBS, 0.01% Tween20) for 1 h to prevent undesired bindings between antibodies and bacteria in a 6-well plate. After that, the discs were washed with 1 mL of the PBS solution 3 times and incubated with 1.5 mL of antibody solution containing rabbit polyclonal S. aureus antibody (1:500 dilution, ab20920, Abcam, Cambridge, MA, USA) and mouse monoclonal C. acnes antibody (1:500 dilution, D371-3, MBL, Nagoya, Japan) overnight at 4 °C. This was followed by incubation with goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:1500 dilution, Alexa Fluor™ 488, ThermoFisher, Waltham, MA, USA) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody (1:1500 dilution, Alexa Fluor™ 594, ThermoFisher, Waltham, MA, USA) for 1 h.

2.5. Immunoassay for the Expression of IL-8 on Scalps

The scalp tape strips were collected from a hairless area in the midline of the vertex area for both control and dandruff participants. Five D-Squame tape strip samples (22-mm diameter; CuDerm, Dallas, TX, USA) were collected from dandruff sites on the vertex area. Tape stripping was performed by one investigator, who tried to apply similar pressure during the tape stripping of each subject.
The D-Squame tape strip samples from the human scalp were extracted using phosphate-buffered saline (PBS), containing an additional 0.25 M NaCl. The samples were centrifuged for 5 min at 2100× g to remove skin solids. Following the manufacturer’s instruction, the total protein of the extracted samples was analyzed using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Immunoassays to detect IL-8 (R&D Systems, Minneapolis, MN, USA) were also performed, following on the manufacturer’s instructions. IL-8 levels were calculated as pg/g of total protein.

2.6. Bacteria and Fungi Analysis on Scalps

The area from the crown vertex to the mid-frontal point of the scalp was gently swabbed with Copan ESwab (Copan Diagnostic Inc, Murrieta, CA, USA) and soaked in 1 × PBS to obtain bacteria and fungi. The swab with bacteria was preserved immediately in an ESwab transport tube containing transport medium and stored at −80 °C before processing. DNA was extracted from swab samples with a QIAamp DNA mini kit (Qiagen, Hilden, Germany), using a modified user-developed protocol. To evaluate the relative abundance of bacteria (Cutibacterium acnes and Staphylococcus epidermidis) and fungi (Malassezia restricta and Malassezia globosa), quantitative real-time PCR (qPCR) was performed with primer sets (Table S1) and Power SYBR Green Master mix (Thermo Scientific, Waltham, MA, USA). The relative amount of bacteria and fungi was separately normalized using Unibac and ITS primer sets [15,16].

2.7. Statistical Analysis

Statistical analysis was conducted using SPSS 20.0 (SPSS, Chicago, IL, USA) and Prism 10.0.2 (GraphPad Software, Boston, MA, USA). Data are presented as mean ± standard error of mean (SEM). We assessed the data both for the normal distribution and the non-normal distribution groups using a Shapiro–Wilk test. For the physiological parameters following a normal distribution, statistical significance was evaluated using a two-tailed unpaired Student’s t test for comparisons between two parameters. For non-normally distributed parameters, statistical significance was evaluated using the Mann–Whitney U test with two groups. The correlation was evaluated using Pearson’s correlation test.

3. Results

3.1. Staining Type Depends on the Stained Red Area (sRA)

The scalp showed a stained red area (sRA) produced by erythrosine (Trace® Disclosing Solution), which was caused by properties of erythrosine such as its red color and high binding affinity for dead cells [17]. The initial red intensity of the sRA did not significantly differ among the subjects. After the washing process, the majority of subjects had a clean scalp (staining-negative group), but some subjects still had a high sRA (staining-positive group). We plotted the sRA as a function of the cumulative percentage to distinguish the staining-negative and staining-positive groups. As a result, almost 30% of subjects—those who exhibited a greater than 40% sRA—were assigned to the staining-positive group, and the others were assigned to the staining-negative group (Figure 1A). More specifically, the sRA increased significantly in individuals with oily scalp conditions. Interestingly, in cases of dry scalp, the presence of sRA was still observed around the scalp pores owing to two reliable factors: (1) structural hindrance and (2) high quantities of hyperkeratotic corneous layers and biofilms (Figure 1B) [18]. To validate the effectiveness of the staining and washing process, we altered the number of staining and washing cycles. Modifying the duration of staining and washing times in the same subject did not have a significant impact on the change in the sRA.

3.2. Physiological Properties of the Scalp

To explore the physiological characteristics distinguishing the individuals in the staining-positive and staining-negative groups, we further investigated markers of scalp physiology such as the sebum secretion rate, scalp barrier, and elasticity. As a result, we found two parameters, sebum secretion and IL-8, which have strong correlations with the presence of the sRA (Table 1).
In Figure 2, the results of Pearson’s correlation analysis indicate that there is a significant medium-level positive relationship between the sRA and IL-8 (r(24) = 0.496, p = 0.010). However, we focused on the other factor, namely, sebum production, because an imbalance of fungi or bacteria in the scalp caused by high sebum secretion could result in itchiness, redness, dandruff, and inflammation. Consequently, these troubles lead to an increase in IL-8 levels. Therefore, an increase in IL-8 levels means an imbalance in fungi and bacteria, which is caused by high sebum production.

3.3. Staining Types and sRA Dependent the Scalp

Based on the correlation between the sRA and sebum production presented in Table 1, we hypothesized that high sebum production accelerates the growth of microbes, which leads to the formation of a biofilm on the scalp (Scheme 1) [3].
To test the hypothesis, we analyzed representative fungi and bacteria, such as Malassezia, Cutibacterium, and Staphylococcus, on the scalp using qPCR analysis. As expected, the sRA-positive group showed a much higher relative amount than the sRA-negative group (Table 2).
Moreover, we plotted the relative total amount of Malassezia as a function of sebum secretion in Figure 3, and the Pearson’s correlation results indicate that there is a significant medium-level positive relationship between sebum production and the relative total amount of Malassezia.
In addition, to visualize bacteria in the scalp, we conducted immunohistochemistry staining of scalp corneocytes to detect C. acnes and S. aureus using the tape-stripping method for both staining groups (Figure 4). In the staining-negative group (Figure 4A), as expected, bacteria were rarely observed (red for C. acnes and green for S. aureus). Among the few that were observed, C. acnes had the highest proportion. Meanwhile, in the staining-positive group, a large amount of bacteria was observed. Interestingly, the proportion of S. aureus was significantly increased, resulting in an even greater distribution of the two species of bacteria than in the staining-negative group (Figure 4B).

3.4. Effect of Antimicrobial Agents on the sRA

Based on the results, we decided to try using an antibacterial agent to reduce sRA. Climbazole (CBZ) is widely known for its powerful antifungal and antibacterial activity against Malassezia, C. acnes, and S. aureus; therefore, it is commonly used in antidandruff hair care products [19]. In addition, hexamidine diisethionate (HD) has also demonstrated in vivo antifungal and antibacterial effects [20]. To decrease sRA without causing chemical or physical damage to the scalp, we administered a wash-off scalp care product containing 0.3% CBZ (Figure 5A).
After two weeks of using the scalp care product, it was observed that 87% of subjects (n = 27) experienced a significant reduction in the sRA. However, 13% of subjects (n = 4) still continued to exhibit an sRA that was not easily removed, which suggests that not only a chemical agent but also a physical agent should be used. Therefore, to overcome this hurdle, we applied a scalp care product with a scrub to physically reduce the biofilm formed on the outer stratum corneum layer of the scalp (Figure 5B). The average sRA in the non-scrub group (n = 31) was 44.9%; however, the salt scrub group (n = 31) had an average sRA of 24.6%, indicating that physically removing the stratum corneum layer is very effective in decreasing the sRA.
Although scalp care products applied with a scrub have shown a dramatic effect in reducing he sRA, they should be used with caution, especially for individuals with severe symptoms like itchiness, psoriasis, dandruff, or seborrheic dermatitis. This is because the salt scrub could cause minor physical damage to the scalp with continuous administration with shampooing. In addition, among the severe sRA group, with higher levels of dandruff and itchiness, scalp symptoms were not alleviated with treatment using wash-off products.
We further investigated the efficacy of the scalp leave-on product with 0.1% HD with respect to subjects with severe seborrheic dermatitis and itchiness (n = 6). After a 2-week administration period, the subjects’ sRA values decreased from 70.6% to 54.6%, which means that HD exhibits effective antimicrobial activities with respect to reducing the sRA in the scalp (Figure 5C). Therefore, the administration of antibacterial and antifungal agents such as CBZ and HD, along with physical assessment, could alleviate scalp symptoms and biofilm accumulation.

4. Discussion

The interactions between pathogenic microbes and the host’s skin are central to maintaining homeostasis and the initiation of disease [21]. Bacteria undergo metabolic changes to adapt to scalp skin surfaces, which involve producing a substance to bind bacterial communities and controlling specific genes [22]. This leads to the formation of networks that support multicellular functions, ultimately resulting in the development of biofilms, a community-like living state. Biofilms provide survival benefits to bacterial community members, such as increased virulence, pathogenesis, and resistance to antibiotic agents, when viewed from the host’s perspective [23]. The visualization and removal strategies of pathogenic biofilms provide opportunities for studies of the various host−microbiome interactions and maintenance of scalp health.
We evaluated the bacteria on the surface of the scalp, and Staphylococcus aureus (S. aureus) and Cutibacterium acnes (C. acnes) were observed in high levels in the first layer of coenocytes with aggregative features (Figure 4). However, to advance the evaluation of the biofilm’s physiological effect on scalp skin, analyses of scalp biofilm components such as peptidoglycan, analyses of the population of bacteria and fungi, and examinations of skin irritation biomarkers are needed [22]. The examination of cultivable microorganisms associated with the skin, such as Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Propionibacterium acnes (P. acnes), Malassezia spp., Candida albicans, and various others, has occurred over a number of years and has revealed intricate molecular processes crucial to their interactions with the skin [24,25,26,27,28].
Numerous studies in microbiology and dermatology have consistently indicated strong connections between P. acnes and acne vulgaris, S. aureus and atopic dermatitis (AD), and Malassezia species and dandruff. Furthermore, there has been extensive research on the biofilm-forming abilities of many skin-associated microbes, and there are ongoing efforts to prevent such biofilm formation [29]. In this study, the levels of S. epidermis, C. acnes, and Malassezia were increased in the high sRA-positive group. In particular, the relative total amount of Malassezia is significantly different compared with that of the sRA-negative group, which means that Malassezia has a dominant effect in controlling biofilm staining on the scalp.
The skin microbiota’s single-species biofilms, such as S. aureus, S. epidermidis, and P. acnes, have been extensively studied in vitro, but there is relatively limited research on Malassezia spp. biofilms. Furthermore, important interactions between different species have been discovered among the skin prokaryotes, including S. epidermidis, inhibiting the growth of P. acnes and S. aureus biofilms [30,31]. However, these interactions between species have not yet been linked to fungal skin commensals, particularly Malassezia [22]. Following attachment to scalp skin, the bacteria secrete a range of substances to enhance the attachment and growth capabilities of the emerging community, collectively known as the extracellular matrix [32]. The extracellular components of the bacterial biofilm comprise various biopolymers, such as polysaccharides, DNA, proteins, and lipids [33]. Mineral scaffolds have been demonstrated to have an impact on the formation of the extracellular matrix [34]. Diverse bacterial and fungal skin microbiomes, such as S. aureus, C. acnes, and Malassezia species, contribute to the formation of scalp skin biofilms [22,33]. The compositions of homogenous biofilms from each bacterial and fungi species were investigated.
The structure and matrix composition of Cutibacterium acnes biofilms were reported depending on C. acnes subtypes [35]. There were variations in the ability of previous cutaneous Propionibacterium species to generate extracellular polysaccharides or pili-like structures, likely resulting from disparities in the bacteria’s genomes [36]. P. acnes strains 266 and KPA171202, cultivated in liquid medium, lacked extracellular structures based on polysaccharides, while P. avidum strains were characterized by the presence of abundant polysaccharide coatings surrounding the cells [36]. A novel extracellular antioxidant protein, RoxP, was recently identified in C. acnes, and its expression seems to be specific to certain strains. RoxP is extensively released into the liquid culture medium and may play a beneficial role for both host cells and C. acnes in the skin [37,38]. In addition, the biofilm matrix of the acne-causing C. acnes RT5, one of the pathogenic subtypes of acne, seems to consist primarily of polysaccharides. The main matrix components are present in the following proportions: 62.6% polysaccharides, 9.6% proteins, 4.0% DNA, and 23.8% other compounds (including porphyrin precursors and others) [35].
The planktonic form of S. aureus does not appear to be resistant to disinfectants, compared to other bacteria, but it can be one of the most resistant when adhered to a surface [39]. S. aureus has the ability to generate a multi-layered biofilm enclosed within a glycocalyx with varied protein expression throughout. This results in the formation of at least two types of biofilms: ica-dependent biofilms, facilitated by polysaccharide intercellular adhesin (PIA)/poly-N-acetyl-1,6-β-glucosamine (PNAG), and ica-independent biofilms, facilitated by proteins [40]. Additionally, the extracellular matrix of clinical S. aureus biofilms is composed of cytoplasmic proteins that interact with the cell surface in response to a declining pH. Sensitive adaptation to the environment enhanced S. aureus’s resistance and biofilm formation capabilities [41].
Malassezia is a genus known for yeasts with a thick cell wall [42]. Dandruff and seborrheic dermatitis are among the main skin disorders attributed to Malassezia furfur [43]. It has also been documented in the literature that M. furfur has the ability to form biofilms [44]. Moreover, in more severe cases, this species can cause fungemia associated with intravenous catheters in immunocompromised patients [45]. The biofilm exhibits distinct physiological and phenotypic traits compared to planktonic cells, including increased efflux pump activity and alterations in membrane/matrix composition [46]. The arrangement of blastoconidia into clusters within the Malassezia and Candida biofilm, forming multi- or monolayers and generating the extracellular matrix, is likely to create a barrier against drugs. However, the biofilm’s resistance to drugs cannot solely be attributed to this physiological barrier. For instance, the natural selection of biofilm cells through the apoptosis of sub-lethally damaged cells, as well as the increased expression of efflux pumps encoded by CDR1, CDR2, and MDR1 genes, may also contribute to the biofilm’s resistance [28,47,48].
Investigation into scalp biofilm properties are limited. The biofilms of C. acnes, S. aureus, and Malassezia typically form in distinctive environments, such as on prosthetic joint implants or in skin glands and hair follicles, which have different physiological features from other cutaneous tissues [49,50]. In addition, the biofilm phenotype is the result of changes in the expression of numerous genes [51], and the process begins when microbial cells adhere to the surface [52]. Therefore, it is essential to study natural biofilms to comprehend their extracellular matrix. Indeed, it is still questionable as to whether we can extrapolate the molecular mechanism of planktonic cells to biofilms.
Scalp skin biofilms grow predominantly in lipid-rich areas around hair follicles and associated skin folds. They can cause severe damage to the scalp epidermis via partial hypoxia states [53]. Epidermal cells require oxygen to produce ATP; however, because the epidermis lacks blood circulation, the process depends on oxygen diffusion from the atmosphere [54,55]. The reliance of keratinocytes on direct oxygen diffusion from the atmosphere results in a constant state of mild hypoxia within the epidermis [56]. The presence of bacteria on the epidermis could potentially worsen the level of hypoxia in this tissue because of the oxygen consumption by bacteria, even if it is highly concentrated in one area [57]. Biofilms develop quickly on the scalp skin and use up oxygen in the tissues below [58]. As a result, the colonization of the skin leads to the formation of localized biofilm communities, which then deplete oxygen from the underlying skin tissue, leading to apparent damage to the keratinocytes throughout the skin [53].
Recent progress in the analysis of microbial gene sequences on normal skin has led to an extensive understanding of the types of microbes residing in different locations on the skin and their diversity [59]. Pioneering research has delved into shotgun sequencing methods to further investigate the taxonomic diversity and geographical distribution of microbes in small groups of healthy individuals [60]. Commensal skin microbes maintain an adaptive skin immune balance by interacting with specific subsets of antigen-presenting dendritic cells (DCs) and effector T-cell populations, consequently supporting their own survival and safeguarding against the proliferation of pathogens [61,62]. For example, commensal skin microbes are linked to disease flare-ups in AD, which serves as a model of an atopic/allergic inflammatory disease [63]. However, the association of distinct microbial classes with biofilms on the scalp affects host physiology and causes inflammatory skin diseases, such as seborrheic dermatitis, atopic dermatitis, and psoriasis [64].
In this study, we developed an in vivo method to visualize biofilms on the scalp using erythrosine; these biofilms strongly depend on the bacteria and fungi present on human scalp. The physiological properties of the scalp significantly differ with respect to staining-positive subjects, for whom the sRA rarely decreases, and staining-negative subjects, for whom it does not. The sRA shows a medium-to-strong positive correlation with IL-8 levels and sebum production. In particular, we conclude that sebum production has a dominant effect on the scalp microbes, which leads to the formation of biofilms, as observed by changes in the sRA. Moreover, we decreased the sRA using antimicrobial agents such as CBZ and HD and the physical properties of scrubs in scalp care products. We suggest that the evaluation of scalp biofilms could be of assistance in understanding scalp physiology and investigating new approaches for the development of therapeutic products for scalp health.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/app142110008/s1, Table S1. RT-PCR primer sets, Table S2. Ingredient for hair care product and Figure S1. Individual images for S. aureus and C. acnes.

Author Contributions

Conceptualization, J.-H.J., Y.-H.C. and N.-G.K.; methodology, J.-H.J., J.K. and J.-Y.S.; software, J.-H.J. and H.R.; validation, J.-H.J., H.R. and S.-H.J.; formal analysis, J.-H.J. and S.-H.J.; investigation, J.-H.J., J.K., Y.-H.C. and S.-H.J.; resources, J.-Y.S. and N.-G.K.; data curation, J.K. and Y.-H.C.; writing—original draft preparation, J.-H.J. and J.K.; writing—review and editing, J.-H.J., J.K., S.-H.J. and N.-G.K.; visualization, J.-H.J.; supervision, S.-H.J. and N.-G.K.; project administration, N.-G.K.; funding acquisition, S.-H.J. and N.-G.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of LG Household and Healthcare Ltd. (Approval number LGHH-20220811-AB-03-01).

Informed Consent Statement

Informed consent was obtained from all subjects involved in this study.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the corresponding author upon request.

Conflicts of Interest

All Authors, Jang-Ho Joo, Jaeyoon Kim, Jae-Young Shin, Yun-Ho Choi, Heena Rim, Seung-Hyun Jun, and Nae-Gyu Kang are employed by the company LG Household and Healthcare, Ltd.

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Figure 1. (A) Staining-positive and staining-negative groups depending on the sRA value. (B) Representative images of the scalp for staining-positive (top) and staining-negative (bottom) subjects.
Figure 1. (A) Staining-positive and staining-negative groups depending on the sRA value. (B) Representative images of the scalp for staining-positive (top) and staining-negative (bottom) subjects.
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Figure 2. IL-8 was plotted as a function of the sRA. Results of Pearson’s correlation analysis indicate that there is a significant medium-level positive relationship between the sRA and IL-8 (r(24) = 0.496, p = 0.010).
Figure 2. IL-8 was plotted as a function of the sRA. Results of Pearson’s correlation analysis indicate that there is a significant medium-level positive relationship between the sRA and IL-8 (r(24) = 0.496, p = 0.010).
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Scheme 1. Scalp biofilm formation in the staining-positive (left) and staining-negative (right) groups.
Scheme 1. Scalp biofilm formation in the staining-positive (left) and staining-negative (right) groups.
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Figure 3. Relative amount of total Malassezia depending on sebum production. There is a significant medium-level positive relationship between sebum production and the relative total amount of Malassezia (r(21) = 0.414, p = 0.050).
Figure 3. Relative amount of total Malassezia depending on sebum production. There is a significant medium-level positive relationship between sebum production and the relative total amount of Malassezia (r(21) = 0.414, p = 0.050).
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Figure 4. Representative images of SC obtained from the (A) staining-positive and (B) staining-negative groups (red: C. acnes; green: S. aureus).
Figure 4. Representative images of SC obtained from the (A) staining-positive and (B) staining-negative groups (red: C. acnes; green: S. aureus).
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Figure 5. The sRA decreased after the administration of scalp care products: (A) shampoo with CBZ; (B) shampoo with salt scrub; (C) tonic with HD (* p < 0.05, **** p < 0.0001).
Figure 5. The sRA decreased after the administration of scalp care products: (A) shampoo with CBZ; (B) shampoo with salt scrub; (C) tonic with HD (* p < 0.05, **** p < 0.0001).
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Table 1. Scalp physiologic properties dependent on sRA staining types.
Table 1. Scalp physiologic properties dependent on sRA staining types.
PropertysRA-Positive Samples (13)sRA-Negative Samples (14)
sRA (%)44.6 ± 17.5 ***9.9 ± 5.5
Scalp skin barrier (TEWL) (g/m2h)35.3 ± 5.6736.3 ± 5.62
Sebum production (a.u.)560.1 ± 48.6 **427.9 ± 54.7
IL-8 (pg/g)0.33 ± 0.87 **0.22 ± 0.04
Significantly different results compared with sRA-negative samples were evaluated using a Mann–Whitney U test (** p < 0.01, *** p < 0.001).
Table 2. Relative amount (fold) of fungi and bacteria with the sRA negative group set at a value of 1.
Table 2. Relative amount (fold) of fungi and bacteria with the sRA negative group set at a value of 1.
SpeciessRA-Positive Samples
(n = 13)		sRA-Negative Samples
(n = 14)
C. acnes1.34 ± 0.321.00 ± 0.21
S. epidermis16.72 ± 6.891.00 ± 0.25
M. restricta5.82 ± 1.081.00 ± 0.47
M. globosa51.16 ± 14.141.00 ± 0.42
M. dermatis36.15 ± 10.801.00 ± 0.39
Malassezia (total)31.04 ± 6.01 *1.00 ± 0.42
Significantly different values compared with sRA-negative samples were evaluated using a Mann–Whitney U test (* p < 0.05).
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Joo, J.-H.; Kim, J.; Shin, J.-Y.; Choi, Y.-H.; Rim, H.; Jun, S.-H.; Kang, N.-G. Management of Scalp Biofilm in Local Dermatosis: From In Vivo Visualization to Optimal Treatments. Appl. Sci. 2024, 14, 10008. https://doi.org/10.3390/app142110008

AMA Style

Joo J-H, Kim J, Shin J-Y, Choi Y-H, Rim H, Jun S-H, Kang N-G. Management of Scalp Biofilm in Local Dermatosis: From In Vivo Visualization to Optimal Treatments. Applied Sciences. 2024; 14(21):10008. https://doi.org/10.3390/app142110008

Chicago/Turabian Style

Joo, Jang-Ho, Jaeyoon Kim, Jae-Young Shin, Yun-Ho Choi, Heena Rim, Seung-Hyun Jun, and Nae-Gyu Kang. 2024. "Management of Scalp Biofilm in Local Dermatosis: From In Vivo Visualization to Optimal Treatments" Applied Sciences 14, no. 21: 10008. https://doi.org/10.3390/app142110008

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