Oral probiotics and topical secretome to enhance clinical outcomes and microbiome restoration in acne vulgaris: a double-blind, randomised controlled trial protocol

Abstract

Background

Acne vulgaris is a chronic inflammatory condition primarily caused by Cutibacterium acnes, which disrupts skin homeostasis, thereby triggering immune responses and sebum metabolism. Dysbiosis is an imbalance in the skin and gut microbiota identified as a significant factor contributing to acne progression. Standard therapy often relies on antibiotics, but the long-term use has increased antibiotic resistance, including in Indonesia. Consequently, alternative methods, such as probiotics and mesenchymal stromal cell secretomes, are gaining attention for immunomodulatory and regenerative properties. These novel therapies have shown promising results in modulating the skin and gut microbiota while reducing inflammation.

Methods and analysis

A phase 2 double-blind randomised controlled trial will be conducted using a parallel group design with four arms, namely: (1) standard therapy with oral probiotics and topical secretome (placebo), (2) standard therapy with oral probiotics (placebo) and topical secretome, (3) standard therapy with oral probiotics and topical secretome and (4) standard therapy with oral probiotics (placebo) and topical secretome (placebo). Sixty-four patients with mild to moderate acne vulgaris will be randomly allocated to these groups. Interventions will be administered over a period of 8 weeks, with outcomes to be measured at baseline and post-therapy. This study will be conducted at the Dermatology and Venereology Department of Bali Mandara General Hospital (RSBM). The primary outcome will be the reduction of comedones and inflammatory lesions, assessed using the Yolov8 method. Secondary outcomes will include gut and skin health parameters, such as tryptophan metabolites, collagen, pH, moisture, sebum levels and IL-6, to explore the relationship between microbiome balance, skin condition and inflammation in acne.

Ethics and dissemination

This study will be conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and the International Conference on Harmonisation–Good Clinical Practice guidelines. Ethical approval has been granted by the Health Research Ethics Committee of Bali Mandara Regional Hospital (Approval Reference Number: 060/EA/KEPK.RSBM.DINKES/2024). All participants will provide written informed consent prior to enrolment. Data confidentiality and participant safety will be upheld throughout the trial. The results of this study will be disseminated through journals, scientific conferences and relevant academic platforms to ensure wide accessibility and to support further research and clinical application in the field of dermatology, particularly in addressing antibiotic resistance and microbiome-based acne therapies.

Trial registration number

NCT06925386.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• The double-blind randomised controlled design reduces selection and observer bias.

• The study incorporates Yolov8 AI-based image analysis for objective and standardised assessment of acne lesions, improving data accuracy and reproducibility.

• A multidimensional outcome approach—assessing gut and skin microbiome, biochemical markers (IL-6, tryptophan metabolites) and clinical skin parameters—allows for a holistic evaluation of therapy effects.

• The sample size is relatively small (n=64), which may limit the statistical power to detect smaller effect sizes across all outcome measures.

• The intervention period is limited to 8 weeks, which may not capture the long-term sustainability and relapse rates of acne improvement.

Introduction

Acne vulgaris is a chronic inflammatory condition of the sebaceous glands caused by Cutibacterium acnes, previously known as Propionibacterium acnes, a lipophilic bacterium that produces toxins, adhesions and immunomodulatory metabolites. These metabolites trigger immune responses and sebum metabolism into pro-inflammatory free fatty acids.¹ The severity of acne is influenced by inflammation, immune defence, keratinocyte function and microbiota balance.² The skin microbiome functions similarly to gut microbiota by maintaining homeostasis, protecting against pathogens and regulating immune responses. According to a previous study, dysbiosis disrupts the skin barrier, increasing susceptibility to damage and inflammation.³

Acne patients often show reduced gut microbiota diversity, particularly a decline in Firmicutes, including Lactobacillus and Bifidobacterium, which play critical roles in maintaining gut and immune health.⁴ This imbalance is further increased by stress, affecting gut microbes and increasing systemic inflammation.⁵ Standard therapy for mild-to-moderate acne typically includes topical and oral antibiotics. However, long-term antibiotic use leads to microbiota disruption and increasing resistance, including C. acnes.⁶ In Indonesia, resistance to antibiotics, such as doxycycline, minocycline and azithromycin is common.⁷

Probiotics, consisting of beneficial bacteria, such as Lactobacillus and Bifidobacterium, are gaining attention for their anti-inflammatory and immunomodulatory roles in managing inflammatory conditions.^{8 9} These probiotics help restore gut microbiota, strengthen the intestinal barrier and modulate immune responses.¹⁰ The supernatants have shown antibacterial and antibiofilm effects against acne-causing bacteria, making this method a promising alternative to conventional antibiotics.¹¹

Beyond probiotics, mesenchymal stromal cell (MSC) secretomes are developing as a novel therapy for inflammatory skin conditions, including acne. MSC secretomes contain bioactive factors that stimulate dermal fibroblast migration, promote angiogenesis, and modulate inflammation.¹² These secretomes also have regenerative and antimicrobial properties that can topically reduce inflammation and support skin remodelling.¹³

Considering the need for alternative therapies, this study proposed a novel method combining oral probiotics and topical MSC secretomes to improve clinical outcomes and microbiome balance in mild-to-moderate acne vulgaris. The efficacy of this combination was assessed by a phase 2 double-blind, randomised controlled trial in enhancing clinical improvement and restoring microbiome balance.

Methods and analysis

Study design

A true experimental design, recognised as the strongest method for establishing causal relationships, will be adopted in this study. A parallel group design with four arms will be used: (1) standard therapy with oral probiotics and topical secretome (placebo), (2) standard therapy with oral probiotics (placebo) and topical secretome, (3) standard therapy with oral probiotics and topical secretome and (4) standard therapy with oral probiotics (placebo) and topical secretome (placebo). Participants will be assigned to groups using a random permuted block sampling method (comprising A, B, C, D). This method is commonly applied in clinical trials including small sample sizes with staggered participants’ enrollment. Observation will span 8 weeks. All groups will be subjected to pretest and posttest assessments, measuring acne severity, faecal microbiome metabolites, skin condition and IL-6 before and after intervention.

Study setting and period

This study will be conducted in the Dermatology and Venereology Department of Bali Mandara General Hospital (RSBM). RSBM is a type A hospital equipped with comprehensive and reliable medical treatment facilities. Additionally, the hospital has an Aesthetic Centre, aligning with Bali’s role as a tourist destination with a strategic location near the Special Economic Zone. The presence of this facility is expected to enhance dermatology services, particularly in the treatment of acne vulgaris. Participant recruitment and data collection are planned to commence in June 2025 and conclude in June 2026.

Inclusion and exclusion criteria

Participants eligible for this study include:

• individuals (male or female) aged 13 to 45 years

• diagnosed with acne vulgaris by a certified dermatologist, presenting with mild to moderate facial acne based on the lesion count criteria by Lehmann et al.¹⁴ Mild acne is defined as having fewer than 20 comedones or fewer than 15 papular/pustular lesions, while moderate acne is characterised by 20–100 comedones and 20–50 papular/pustular lesions¹⁴

• must have experienced recurring new acne lesions in the past 6 months must be willing to comply with all protocol requirements must be willing to have standardised facial photographs taken using an imaging system must be able to follow study and adhere to a fixed schedule

• must have ability to provide informed consent for participation in the study (online supplementary file 1)

• must be generally healthy and in good mental condition.

Meanwhile, the exclusion criteria are:

• pregnant patients

• subjects who have undergone hormonal acne treatment within 6 months prior to the study

• subjects who have taken oral isotretinoin within 1 month prior to the study

• simultaneous participation in a different study conducted by an external research institution at the same testing site

• inadequate language proficiency (both spoken and written)

• participation in the study under the influence of alcohol and/or drugs, as well as substance addiction

• severe diseases (cardiovascular, hepatic, renal or pulmonary diseases, severe diabetes mellitus) or chronic infections (Hepatitis, HIV)

• immunodeficiency

• current use of the following topical or systemic medications: corticosteroids, immunosuppressants and antihistamines

• skin conditions such as vitiligo, psoriasis or atopic dermatitis

• any other diseases or medications that may directly interfere with the study or pose a risk to the subject’s health.

Sample size

The sample size will be calculated based on a four-arm randomised controlled trial design comparing continuous outcomes. Using a significance level of α=0.05 (two-tailed) and a statistical power of 80% (1−β=0.8), the required sample size per group will be determined using the following formula:

$$n=2{(\frac{Z_{1-\alpha /2}+Z_{1-\beta }}{d})}^2$$

Where $Z_{1-\alpha /2}=1.96$, $Z_{1-\beta }=0.84$, and the expected effect size (Cohen’s d) will be assumed to be 1.0. Substituting these values:

$$n=2{(\frac{1.96+0.84}{1.0})}^2=15.58\approx 16\mathrm{s}\mathrm{u}\mathrm{b}\mathrm{j}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s}\mathrm{}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}$$

Thus, the total sample size required will be 64 participants, evenly distributed across four groups (16 participants per arm). This calculation will be based on the assumption of a large anticipated effect size (Cohen’s d=1.0), reflecting the expected efficacy of the dual-action intervention. Additionally, the sample size will account for Dunnett’s correction to adjust for multiple comparisons against a single control group. A total of 64 eligible participants, including both male and female patients with inflammatory and non-inflammatory acne lesions, will be enrolled. This selection will be based solely on participants’ willingness to follow the prescribed therapy and attend follow-up visits.

Randomisation

Patients who meet the inclusion and exclusion criteria will be assigned to a study arm through randomisation. The allocation will follow the block permutation results generated by an application. The randomisation process will be centralised and conducted through the Data Entry Programme after the site study team completes the study criteria checklist in the system. Participants will not be allowed to undergo multiple or repeat randomisations.

Outcomes

The primary outcome measured in this study will be the reduction in the number of comedones and/or inflammatory lesions (papules/pustules). This will be assessed using the Yolov8 machine learning detection method, which enables accurate and objective quantification of comedones and lesions while also determining the severity of acne.15,17

Meanwhile, secondary outcomes will include several parameters related to gut and skin health. In the gut, tryptophan metabolites will be measured using the ELISA method to assess the abundance of probiotic bacteria, specifically lactic acid bacteria such as Lactobacillus and Bifidobacterium. Skin health will be evaluated through collagen measurement (skin Elastometer), pH levels (pH metre), moisture content (skin hydrometer), and sebum levels (sebumeter) to determine the balance of the skin microbiome. Additionally, inflammatory responses will be assessed by measuring serum IL-6 levels, which serve as an indicator of systemic inflammation. These parameters will provide a comprehensive understanding of the relationship between microbiome balance, skin condition, and inflammation in acne development.

Interventions

Study protocol flow diagram is presented in figure 1. Participants will be divided into four groups as shown in table 1.

Figure 1. Study protocol flow diagram.

Table 1. Interventions in each group.

Group	Standard therapy	Intervention
1	Face wash contains salicylic acid, non-comedogenic sunscreen, moisturiser, night cream contains clindamycin and tretinoin	Oral probiotics	Topical secretome (placebo)
2		Oral probiotics (placebo)	Topical secretome
3		Oral probiotics	Topical secretome
4		Oral probiotics (placebo)	Topical secretome (placebo)

The standard therapy for participants will include a structured skincare regimen. A face wash will be used once daily before bedtime to cleanse the skin. In the morning, a non-comedogenic sunscreen and a moisturiser will be applied to protect and hydrate the skin. Additionally, a night cream containing clindamycin and tretinoin will be applied once daily at night, specifically on acne-affected areas, to target inflammation and promote skin renewal.

The oral probiotics to be administered will be HEXBIO, an oral probiotic containing multiple beneficial bacterial strains, including Lactobacillus acidophilus BCMC 12130, Lactobacillus casei subsp BCMC 12313, Lactobacillus lactis BCMC 12451, Bifidobacterium bifidum BCMC 02290, Bifidobacterium longum BCMC 02120, and Bifidobacterium infantis BCMC 02129. This probiotics product will be administered in granule form and swallowed directly. Participants will consume the probiotic twice daily, after breakfast and dinner, for 8 weeks to support gut microbiome balance and potential skin health benefits.

Furthermore, the topical secretome to be used will be a hydrogel-based serum containing secretome components, including cytokines, growth factors, exosomes, microvesicles and cell differentiation factors. The serum will be formulated with a hydrogel base consisting of xanthan gum, sclerotium gum, 1% alginate, 2% glycerin, 1% caprylyl glycol and 96% aqua. It will be applied after cleansing the face with a facial cleanser, twice daily, in the morning and evening, for 8 weeks.

Compliance assessment and monitoring

Participants who meet the inclusion and exclusion criteria will be enrolled in a dedicated WhatsApp group together with the research team to facilitate ongoing communication and adherence monitoring. Compliance will be evaluated monthly using a structured questionnaire that will assess medication adherence, occurrence of adverse events, dietary patterns, physical activity, sleep patterns, and other relevant behaviours. The information obtained from these forms will be used to determine each participant’s level of adherence to the intervention. Participants will be considered compliant if they achieved at least 80% adherence to the prescribed regimen. Those with adherence below 80% will be excluded from the study (dropout). To support adherence, participants will be instructed at the beginning of the intervention to set daily alarms on their mobile phones as medication reminders. In addition, the research team will provide weekly reminders through the WhatsApp group to encourage consistent medication use and maintain participant engagement throughout the study period.

Statistical analysis

The data obtained will come from acne score measurements using the Yolov8 method and microbiome tests. Measurements will be conducted repeatedly to assess the impact of each intervention over time. The results from each study arm will then be analysed using analysis of variance (ANOVA) repeated measures to determine the specific time point at which the intervention has a significant effect. Subsequently, the effectiveness of the three interventions will be compared using one-way ANOVA to identify any significant differences between them. To determine which intervention has the most significant effect, a post hoc test with Duncan’s test will be performed, and correlations will be analysed using Spearman’s rank correlation coefficient.

Data and safety monitoring

In this study, the ownership of the data obtained will be held by the analysts, participants, and relevant institutions, with a focus on ensuring the protection and confidentiality of participants’ data. At every visit, participants will be questioned about any symptoms that showed adverse reactions to the therapy. Any dermatological side effects reported will be recorded and assessed. Safety monitoring and analysis will be carried out by the study in conjunction with the Head of the Dermatology and Venereology Department at RSUD (Regional General Hospital) Bali Mandara. Any adverse events will be documented and reported to the nearest healthcare facility. When the nearest facility is not equipped to handle the adverse event, participants will be referred to a more comprehensive healthcare facility for further therapy.

Discussion

In Indonesia, acne cases are quite high, and most therapies using antibiotics are not clinically effective, as shown by recurring symptoms, acne spread, and frequent doctor visits (87%).⁷ This is particularly evident in adolescents, with psychological impacts.^{18 19} Currently, acne vulgaris therapy includes antibiotics, but long-term or inappropriate use can lead to antibiotic resistance and microbiota imbalance, causing dysbiosis.³ All antibiotics in Indonesia have become resistant to acne-causing bacterium, C. acnes, including doxycycline, minocycline, tetracycline, gentamicin, erythromycin and azithromycin.⁷ A 60.1% increase was observed for macrolides, and other studies reported high resistance levels for C. acnes.⁷ This growing resistance and reduced effectiveness of antibiotics have shifted the focus towards new therapy strategies.

Dysbiosis, an imbalance in the composition of microbial communities, has been increasingly implicated in the pathophysiology of acne vulgaris. Gut dysbiosis, particularly an altered Firmicutes to Bacteroidetes ratio, may contribute to systemic inflammation and elevated levels of insulin-like growth factor-1 (IGF-1), which can stimulate sebaceous gland activity and promote C. acnes colonisation.^{20 21} Oral probiotics are proposed to modulate gut microbiota, restore microbial balance, reduce IGF-1 levels, and subsequently downregulate inflammatory pathways related to acne development. Studies have demonstrated that individuals with acne may exhibit a less diverse gut microbiota, characterised by reduced abundance of beneficial genera such as Lactobacillus and Bifidobacterium, which are commonly found in healthy individuals.²²

Skin dysbiosis is another critical factor in acne pathogenesis. Healthy human skin is typically colonised by commensal bacteria such as Staphylococcus epidermidis and C. acnes in balanced proportions, which contribute to immune homeostasis and protection against pathogenic microbes. In acne-affected skin, however, there is an overgrowth of virulent C. acnes phylotypes and a reduction in microbial diversity, leading to increased inflammation and disruption of the skin barrier.^{23 24} Topical application of secretome has emerged as a promising approach to address skin dysbiosis by selectively inhibiting pro-inflammatory strains of C. acnes, restoring microbial balance, and supporting skin regeneration. The secretome may exert its effect through antimicrobial peptides, anti-inflammatory metabolites, and enzymes that degrade biofilms or regulate keratinocyte differentiation.²⁵

Probiotics have developed as a promising solution to balance microbiota and reduce acne symptoms, with potential anti-inflammatory and immunomodulatory effects. Furthermore, such as MSC and the secretome are showing promise in treating inflammatory skin conditions, including acne vulgaris. MSCs, known for their role in tissue repair and immune modulation, have shown antibacterial properties.^{26 27} The secretome of MSC promotes fibroblast migration, angiogenesis, and immune system modulation, and has proven effective in combating antibiotic-resistant biofilms and stimulating skin cell immune responses.²⁷

Several studies have shown the potential of probiotics in alleviating acne symptoms through antibacterial and anti-inflammatory mechanisms.²⁸ It was reported that Lactobacillus rhamnosus SP1 supplementation could alter skin gene expression involved in insulin signalling pathways and improve acne conditions in adult subjects, with a significant impact on the expression of IGF-1 and FOXO1.²⁸ This study shows the role of probiotics in improving skin conditions through genetic mechanisms related to inflammation and hormonal regulation. Furthermore, Cha et al showed that Lactobacillus paraplantarum THG-G10 had the highest antibacterial activity against C. acnes, the primary bacterium targeted in medical therapies and acne prevention, supporting the use of probiotics as an effective anti-acne agent.²⁹ Another study by Kim et al confirmed that Lactobacillus rhamnosus GG inhibited the growth of C. acnes and modulated inflammatory responses. This result shows that probiotics can reduce pathogenic colonisation of the skin.³⁰

Secretome derived from MSC has the potential as an adjunctive therapy. MSC-derived secretome is known to contain immunomodulatory factors that inhibit the proliferation of inflammatory immune cells, which are crucial in inflammatory processes occurring in acne. Several studies have shown that MSC secretomes accelerate skin regeneration by promoting angiogenesis and modulating tissue repair processes, thereby playing a role in improving the condition of skin infected or inflamed by acne.³¹ MSC secretomes also exhibit natural antimicrobial effects that support the control of C. acnes and other pathogenic bacteria on the skin, further reinforcing the potential as an anti-acne therapy.³²

In a study conducted by, Dapkevicius et al probiotics were proven to be an alternative to antibiotics in acne therapy, reducing the risk of adverse effects associated with long-term use.³³ Brandi et al also showed that probiotics modulate immune responses by inhibiting IL-8 in keratinocytes, which plays a role in reducing skin inflammation.³⁴ Furthermore, Abdu-Allah et al confirmed that probiotic bacterial supernatants, such as Lactobacillus acidophilus and Bacillus subtilis, reduced biofilm production by Staphylococcus aureus, which was also included in acne development.³⁵

In general, the combination of oral probiotics and topical secretome therapy presents a highly effective method for managing acne vulgaris, with dual mechanisms that include modulation of the skin microbiome, reduction of inflammation, and prevention of pathogenic bacterial colonisation. Therefore, further development of probiotic-based and secretome therapies could provide an innovative, safer, and more effective solution for managing acne vulgaris, reducing reliance on antibiotics and promoting skin recovery.

Ethics and dissemination

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and the International Conference on Harmonisation–Good Clinical Practice guidelines. Ethical approval was granted by the Health Research Ethics Committee of Bali Mandara Regional Hospital (Approval Reference Number: 060/EA/KEPK.RSBM.DINKES/2024). All participants provided written informed consent prior to enrolment. Data confidentiality and participant safety were upheld throughout the trial. The results of this study will be disseminated through journals, scientific conferences, and relevant academic platforms to ensure wide accessibility and to support further research and clinical application in the field of dermatology, particularly in addressing antibiotic resistance and microbiome-based acne therapies.

The funder had no role in the study design; data collection, analysis, or interpretation; manuscript preparation; or the decision to submit the paper for publication