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. 2024 Sep 9:15:1452127.
doi: 10.3389/fmicb.2024.1452127. eCollection 2024.

Isolation, genomic analysis and functional characterization of Enterococcus rotai CMTB-CA6, a putative probiotic strain isolated from a medicinal plant Centella asiatica

Yunsik Kim  1 , Jin Hee Lee  1 , Jimyeong Ha  2 , Eun-Gyung Cho  1   3   4
Affiliations

Isolation, genomic analysis and functional characterization of Enterococcus rotai CMTB-CA6, a putative probiotic strain isolated from a medicinal plant Centella asiatica

Yunsik Kim et al. Front Microbiol. .

Abstract

Probiotics and their derivatives offer significant health benefits by supporting digestive health, boosting the immune system, and regulating the microbiomes not only of the internal gastrointestinal track but also of the skin. To be effective, probiotics and their derivatives must exhibit robust antimicrobial activity, resilience to adverse conditions, and colonization capabilities in host tissues. As an alternative to animal-derived probiotics, plant-derived lactic acid bacteria (LAB) present promising advantages, including enhanced diversity and tolerance to challenging environments. Our study focuses on exploring the potential of plant-derived LAB, particularly from the medicinal plant Centella asiatica, in improving skin conditions. Through a bacterial isolation procedure from C. asiatica leaves, Enterococcus rotai CMTB-CA6 was identified via 16S rRNA sequencing, whole genome sequencing, and bioinformatic analyses. Based on genomic analysis, antimicrobial-resistance and virulence genes were not detected. Additionally, the potential functions of E. rotai CMTB-CA6 were characterized by its lysates' ability to regulate skin microbes, such as stimulating the growth of Staphylococcus epidermidis while inhibiting that of Cutibacterium acnes, to restore the viability of human dermal fibroblasts under inflammatory conditions, and to demonstrate effective antioxidant activities both in a cell-free system and in human dermal fibroblasts. Our investigation revealed the efficacy of E. rotai CMTB-CA6 lysates in improving skin conditions, suggesting its potential use as a probiotic-derived agent for skin care products. Considering the ecological relationship between plant-inhabited bacteria and their host plants, we suggest that the utilization of E. rotai CMTB-CA6 strain for fermenting its host plant, C. asiatica, could be a novel approach to efficiently enriching bioactive molecules for human health benefits.

Keywords: Centella asiatica; Enterococcus rotai; anti-inflammation; antioxidant; lactic acid bacteria; plant probiotics; skin microbiome; whole genome sequencing.

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Conflict of interest statement

E-GC is an employee of CHA Meditech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Neighbor-joining phylogenetic tree of bacterial isolate strain CMTB-CA6 with the other Enterococcus species. The phylogenetic relationship of E. rotai CMTB-CA6 was assessed with the neighbor-joining method of phylogenetics using the 16S rRNA gene sequences of bacterial isolate strain CMTB-CA6 and NCBI database homologs using the ClustalW alignment. Numbers at the node represent bootstrap values in percent for the node (based on 1,000 bootstrap replicates). The GenBank accession number for each 16S rRNA gene sequence are written after relative species name.
Figure 2
Figure 2
Circular genome map of E. rotai CMTB-CA6. The circular genome map was drawn using circulator tool (v1.5.5; https://github.com/sanger-pathogens/circlator) that uses a complete genome sequence and annotates it using Prokka tool (https://github.com/microbial-bioinformatics/prokka). From outside to inside, information is displayed as follows: the predicted ORFs in the forward strand (forward ORF) and reverse strand (reverse ORF), coding sequences (CDS), rRNA genes (red), tRNA genes (green), GC content, GC skew.
Figure 3
Figure 3
The number of E. rotai CMTB-CA6 genes assigned to functional categories. Predicted proteins based on the CDS were assigned to COG, GO and KEGG functional categories with a bioinformatics software Omicsbox (https://www.biobam.com/omicsbox/). (A) Clusters of orthologous groups (COG) enrichment analysis. Different bar colors represent the further classification of all functional categories into four major classes: poorly characterized (black bar), gene regulation (orange bars), transport & metabolism (blue bars), cellular processes and signaling (gray bars). (B) Gene ontology (GO) enrichment analysis. Different bar colors represent the further classification of all functional categories into three major categories: biological process (blue bars), cellular component (orange bars), molecular function (green bars). (C) Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis. Different bar colors represent the further classification of all functional categories into three major categories: genetic information processing (orange bars), metabolism (blue bars), signaling and cellular processes (gray bars). Among the total 3,532 CDS, 3149 (89.16%), 2,719 (76.98%), and 1984 (56.17%) genes were used for COG, GO, and KEGG analyses, respectively.
Figure 4
Figure 4
Regulatory effect of E. rotai CMTB-CA6 lysates on skin microbes. (A) On agar plates containing Cutibacterium acnes, paper discs loaded with two different concentrations (25, 50 μg/mL) of whole lysates (WL) and supernatants (Sup) from E. rotai CMTB-CA6, or with distilled water (DW) or PBS, were placed (total loaded volume, 15 μL). The agar plates were incubated for 72 h at 37°C. Clear zones formed after loading paper discs were marked with dotted circles. (B) In nutrient broth containing Staphylococcus epidermidis (150 μL), WL or Sup (each at 500 μg/mL; total added volume, 15 μL) from E. rotai CMTB-CA6 were added. After 24 h of culture, the absorbance at 600 nm was measured using a microplate reader. The data are shown as the mean ± standard deviation (n = 3; two-way analysis of variance). **** p < 0.0001.
Figure 5
Figure 5
Anti-cytotoxic and anti-inflammatory effects of E. rotai CMTB-CA6 lysates on human dermal fibroblasts. (A) Human dermal fibroblasts were treated whole lysates (WL) of E. rotai CMTB-CA6 (CA6-WL) with various concentrations. Cell viability was assessed using the water-soluble tetrazolium salt (WST) assay. A dotted line indicates 80% of cell viability, signifying cytotoxicity when cell viability falls below this line. (B) Human dermal fibroblasts were serum starved for 6 h and then treated with 20 ng/mL of TNF-α for 24 h in serum-free conditions. Various CA6-WL concentrations were treated for an additional 24 h in the presence of TNF-α. Cell viability was determined via the WST assay. As a positive control, 10% FBS was used. The data represent the mean ± standard deviation (n = 3; one-way analysis of variance). ** p < 0.01, *** p < 0.001, **** p < 0.0001. TNF, tumor necrosis factor; SF, serum-free control.
Figure 6
Figure 6
Antioxidant activity of E. rotai CMTB-CA6 lysates. (A) The antioxidant activities of whole lysates (WL) of E. rotai CMTB-CA6 (top) and vitamin C (bottom) at various concentrations were assessed using the oxygen radical absorbance capacity (ORAC) assay. Florescent signals were measured every minute during the 60-min incubation period at 37°C. (B) In each group, the net area under the curve (AUC) was calculated by following formula: AUC (sample)-AUC (blank). The data are shown as the mean ± SD (n = 3). Vitamin C was used as a positive control. (C) Intracellular ROS levels were assessed using CM-H2DCFDA, a cellular ROS indicator. HDFs treated with CM-H2DCFDA (1 μM) were incubated with WL of E. rotai CMTB-CA6 at different concentrations (0, 62.5, 125, 250, 500 μg/mL) in the presence of H2O2 for 1 h. Fluorescence signals at 530 nm were measured using a fluorometer. Vitamin C was employed as a positive control. The data represent the mean ± standard deviation (n = 3; two-way analysis of variance). ** p < 0.01, **** p < 0.0001.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Ministry of SMEs and Startups (MSS, Korea) (grant no. S3290362) and supported by the National Research Foundation of Korea(NRF) grant funded by the Ministry of Science and ICT (MSIT, Korea) (No. RS-2023-00273912). This work was supported by an intramural research program from the CHA Advanced Research Institute, grant number CARI-RD-018.

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