Clinical Potential of Novel Microbial Therapeutic LP51 Based on Xerosis-Microbiome Index

Abstract

Xerosis, characterized by dry, rough skin, causes discomfort and aesthetic concerns, necessitating effective treatment. Traditional treatments often show limited efficacy, prompting the need for innovative therapies. This study highlights the efficacy of microbiome therapeutic LP51, derived from a healthy vaginal microbiome, in improving xerosis. A double-blind clinical trial involving 43 subjects with dry inner arm skin compared the effects of a 2.9% LP51 extract formulation to a placebo over 4 weeks. The LP51 group exhibited a significant increase in stratum corneum hydration (10.0 A.U.) compared to the placebo group (4.8 A.U.) and a 21.4% decrease in transepidermal water loss (TEWL), whereas the placebo group showed no significant change. LP51 also demonstrated benefits in enhancing skin hydration, improving the skin barrier, and exhibited anti-atopic, anti-inflammatory, and antioxidant properties. Safety was confirmed through in vitro cytotoxicity tests. These effects are attributed to the microbiome-safe component in LP51 and its role in improving xerosis, reflected by an increase in the xerosis-microbiome index, defined by the Firmicutes/Actinobacteria ratio. These findings position microbiome therapeutic LP51 as a promising novel treatment for xerosis.

Keywords: Firmicutes/Actinobacteria ratio; microbiome therapeutic LP51; vaginal microbiota; xerosis; xerosis-microbiome index.

Figure 1

Analysis of whole-genome sequencing results of candidate strain LP51. (A) The genome of the strain revealed a single circular chromosome with a length of 3,003,557 bp. The antisense and sense strands, color-coded according to COG categories, are displayed from the outer edge to the center. The circular map also shows tRNA and rRNA, indicated by red and blue, respectively. The inner circles of the map represent the GC skew, with yellow and blue indicating positive and negative values, and the GC content is highlighted in red and green. (B) Functional classification of the genes revealed the relative abundance of 2441 proteins assigned to COG (Clusters of Orthologous Groups) families. Biological functions were identified for 1761 proteins, while the roles of 680 CDSs remain unknown. (C) The genome similarity of LP51 was analyzed using the OrthoANI (Orthologous Average Nucleotide Identity) method and compared to other Lacticaseibacillus and non-Lacticaseibacillus strains. The data showed that LP51 had a close similarity of 99.74% with Lacticaseibacillus rhamnosus CP-1. It exhibited similarities of 79.58%, 77.21%, and 66.68% with other Lacticaseibacillus strains, such as LC130, HL182, and YH-lac23, respectively. (D) The chromosomal characteristics of different L. rhamnosus strains were compared, and the results strongly suggested that the newly discovered strain, LP51, represents a novel strain of L. rhamnosus. The NCBI accession number for the candidate strain L. rhamnosus LABIO-PMC-51 (LP51) in this study is PRJNA1123863, and the strains used for genome comparison, along with their NCBI RefSeq assemblies, are Hsryfm 1301 (GCF_008727835.1), TK-F8B (GCF_015377485.1), LR-B1 (GCF_004010975.1), LDTM7511 (GCF_017795605.1), SN21-1 (GCF_033802705.1), and VSI43 (GCF_029011275.1).

Figure 2

Optimization of LP51 growth in a laboratory-scale fermenter using FGM. (A,B) The growth ability of the candidate probiotic strain was optimized over 43 h using a laboratory-scale fermenter based on FGM (food-grade medium). (C) Bacterial growth was monitored periodically, showing optimal growth in the FGM containing glucose. (D) Additionally, pH levels were measured during bacterial growth, revealing lower pH in the glucose medium compared to the sucrose medium. Results are presented as mean values with corresponding standard deviations. Statistical significance between the groups was determined using the Student’s t-test (***, p < 0.001).

Figure 3

In vitro test results of LP51 culture filtrate on various skin-related parameters. The effects of LP51 on skin hydration, skin barrier strengthening, and anti-atopic, anti-inflammatory, antioxidant, and cytotoxic effects were validated through various in vitro tests. (A) The impact of LP51 culture filtrate on skin barrier strength and moisturizing ability was assessed through measurement of HAS3 (hyaluronan synthase 3) transcription levels in HaCaT (human keratinocyte) cells, showing significant increases at concentrations of 0.75% or higher, with 3% achieving results similar to those of hEGF (human epidermal growth factor, 1 µg/mL). The effect on skin barrier strengthening was evaluated through measurement of (B) FLG (filaggrin), (C) IVL (involucrin), and (D) LOR (loricrin) transcription levels in the same cell line, with significant, concentration-dependent increases at 1.5% and 3% LP51. (E) The anti-atopic effect was evaluated through an analysis of changes in TARC (thymus and activation-regulated chemokine) production, where 1% LP51 significantly reduced TARC levels in cells treated with TNF-α (10 ng/mL) and IFN-γ (20 ng/mL), demonstrating superior results compared to those of the immunosuppressive corticosteroid dexamethasone (10 µg/mL). (F) The anti-inflammatory effect was explored using RAW 264.7 macrophages, where LP51 significantly reduced NO (nitric oxide) levels induced by LPS (lipopolysaccharide, 1 µg/mL) in a concentration-dependent manner, starting at 0.25%. (G) LP51 demonstrated strong antioxidant activity, as shown by DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging, with effects similar to those of L-ascorbic acid (50 μg/mL) at concentrations of 15% or higher. (H) Additionally, LP51 played a role in restoring cell viability under UVB (ultraviolet B) exposure in HaCaT cells. Results are presented as mean values with corresponding standard deviations. Statistical significance between the groups was determined using the Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 4

Results of the double-blind clinical trial on the effect of lp51 formulation on dry skin. For efficacy, a 2.9% LP51 formulation was applied to the flexor area of the arms twice daily (morning and evening) for 4 weeks. Evaluations were performed at baseline (week 0), week 2, and week 4 and compared with a placebo group that received the formulation without LP51. (A,B) Skin hydration levels significantly increased at weeks 2 and 4 compared to baseline, with statistically significant increases compared to placebo. (C,D) Transepidermal water loss (TEWL) significantly decreased at week 4 compared to baseline, with a significant reduction compared to the placebo. (E,F) The severity of itching, measured by the visual analog scale (VAS), and the visual appearance assessed by the ESIF scale (erythema, scaling, induration, fissures) showed a decreasing trend, although no statistically significant difference was observed compared to the placebo. The results are presented as means with standard deviations, and the statistical significance between groups was determined using repeated measures ANOVA for VAS scores, while the Mann–Whitney U test using delta values was applied for other comparisons (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (G,H) Photographs of the treatment area were taken at baseline (week 0), week 2, and week 4 using a digital camera and a Folliscope with a 40× magnification lens, and images for all subjects are provided in the Supplementary Materials. (I,J) The stability of the formulation was tested, and “N” indicates normal, with corresponding images provided to illustrate the results.

Figure 5

Skin metagenomics analysis based on the xerosis-microbiome index (XMI) for LP51 compared to placebo. (A) Over the course of the 4-week clinical trial, skin metagenomic analysis was conducted to examine changes in microbial composition before and after administration. LP51 notably decreased Actinobacteria and prominently increased Firmicutes. Significant differences in microbial communities were determined using the Wilcoxon signed-rank test (**, p < 0.01; ***, p < 0.001). (B) As a result, LP51 significantly increased the xerosis-microbiome index (XMI), a novel metric proposed in this study based on the Firmicutes/Actinobacteria ratio, compared to the placebo group. Over the 4-week period, LP51 demonstrated a substantial effect in increasing the fold change of XMI compared to the placebo. (C) In the linear regression analysis, XMI showed a positive correlation with skin hydration levels, and LP51 increased the slope of skin hydration relative to XMI. (D) Transepidermal water loss (TEWL) exhibited a negative correlation with XMI, and LP51 effectively lowered the y-intercept for TEWL, suggesting a reduction in water loss. (E) The in vitro antimicrobial assay results demonstrated that LP51 exhibited superior inhibitory effects on Propionibacterium acnes (Actinobacteria) compared to Staphylococcus aureus (Firmicutes), further supporting the observed XMI results. (F) The modulation of this XMI is considered to be attributed to the production of short-chain fatty acids (SCFAs) by the LP51 strain. (G) Additionally, the alpha and beta diversity analyses indicated that LP51 did not induce dysbiosis in the skin microbiome, showcasing its microbiome-safe properties.

Figure 6

Safety profile of LP51. (A–C) In the WST-1 assay using mouse macrophagic RAW 264.7 cells and immortalized human keratinocytes HaCaT cells, as well as the LDH assay using human colorectal adenocarcinoma HT-29 cells, LP51 did not exhibit cytotoxicity under any conditions, similarly to L. rhamnosus (KCTC 5033). This contrasts with the significant cytotoxicity observed in the positive control (lysis buffer) and pathogenic E. coli strain (NCCP 14780). (D) Furthermore, D-lactate production did not significantly increase in LP51-treated cells, confirming the safety of LP51, similarly to the aforementioned reference strain L. rhamnosus. (E) Additionally, LP51 demonstrated γ-hemolysis (no hemolysis) on blood agar, similarly to the L. rhamnosus strain mentioned above, in contrast to the β-hemolysis observed with S. aureus (ATCC 6538). (F) LP51, similarly the reference strain L. rhamnosus (KCTC 5033), did not grow on bile salt agar and showed no taurodeoxycholic acid (TDCA) hydrolysis activity unlike the positive control L. plantarum (KCTC 3105). (G) LP51 was also evaluated for antibiotic susceptibility according to the EFSA (European Food Safety Authority) cut-off criteria. Statistical significance compared to the control group was determined using the Student’s t-test (** p < 0.01; *** p < 0.001).

Figure 7

Graphical summary: LP51-mediated modulation of the skin microbiome—enhancing hydration and barrier function according to the xerosis-microbiome index (XMI). This figure presents the key findings of the study, including the effects of LP51 on the skin microbiome, improvements in xerosis, changes in microbial composition, and the correlation between the xerosis-microbiome index (XMI) and skin hydration and barrier function. The summary visually encapsulates how LP51 reduces Actinobacteria and increases Firmicutes, positively impacting skin hydration and integrity while maintaining microbial diversity and safety. This reinforces the potential of LP51 as a therapeutic agent for treating skin xerosis.