Antibacterial Activity of the Essential Oil From Litsea cubeba Against Cutibacterium acnes and the Investigations of Its Potential Mechanism by Gas Chromatography-Mass Spectrometry Metabolomics

Abstract

Cutibacterium acnes (C. acnes) is an anaerobic Gram-positive bacterium generally considered as a human skin commensal, but is also involved in different infections, such as acne and surgical infections. Although there are a variety of treatments, the side effects and the problem of bacterial drug resistance still limit their clinical usage. In this study, we found that essential oil (EO) distilled from fresh mature Litsea cubeba possessed promising antibacterial activity against C. acnes. In order to elucidate its potential mechanism, bacteriostatic activity test, Live/Dead kit assay, scanning electron microscope (SEM), transmission electron microscope (TEM), and metabolomics were employed. In addition, the content of adenosine triphosphate (ATP) in bacterium and the activities of key enzymes involved in critical metabolic pathways were detected using a variety of biochemical assays. The results showed that EO exhibited significant antibacterial activity against C. acnes at a minimum inhibitory concentration (MIC) of 400 μg/mL and a minimum bactericidal concentration (MBC) of 800 μg/mL, and EO could destroy C. acnes morphology and inhibit its growth. Moreover, results from our study showed that EO had a significant effect on the C. acnes normal metabolism. In total, 86 metabolites were altered, and 34 metabolic pathways related to the carbohydrate metabolism, energy metabolism, amino acid metabolism, as well as cell wall and cell membrane synthesis were perturbed after EO administration. The synthesis of ATP in bacterial cells was also severely inhibited, and the activities of key enzymes of the glycolysis and Wood-Werkman cycle were significantly affected (Pyruvate Carboxylase, Malate Dehydrogenase and Pyruvate kinase activities were decreased, and Hexokinase was increased). Taken together, these results illustrated that the bacteriostatic effect of EO against C. acnes by breaking the bacterial cell morphology and perturbing cell metabolism, including inhibition of key enzyme activity and ATP synthesis. The results from our study may shed new light on the discovery of novel drugs with more robust efficacy.

Keywords: Cutibacterium acnes; GC-MS untargeted metabolomics; Litsea cubeba; antibacterial; essential oil.

FIGURE 1

Inhibitory effect of EO on the growth of Cutibacterium acnes. (A) Effect of EO on the growth curve of C. acnes within 5 days. CK represented the blank control group and Control represented the solvent control group by 0.1% Tween 80. (B) Representative Live/Dead merged images (green and red) of C. acnes cultured for 8 h with solvent (Control) and different concentrations of EO (1/2 MIC, MIC, 2 MIC and 4 MIC). The positive control was treated with ethanol. Green fluorescence indicated living cells and red fluorescence indicated dead cells. The positive control was treated with ethanol.

FIGURE 2

The effects of EO on the morphology of C. acnes cells were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM), followed by normal bacteria (CK), solvent control bacteria (Control), and bacteria treated with different concentrations of EO (1/2 MIC, MIC, 2 MIC, and 4 MIC). (A) SEM image of C. acnes at 40,000× magnification. (B) SEM image of C. acnes at 10,000× magnification. (C) TEM image of C. acnes at 50,000× magnification.

FIGURE 3

Metabolic profiling of C. acnes before and after EO treated. (A) Typical total ion chromatograms of the metabolic profiles of C. acnes with solvent (Control). (B) Typical total ion chromatograms of the metabolic profiles of C. acnes with EO (MIC). (A,B) Peaks were aligned and internal standard (ribitol) was highlighted. (C) KEGG analysis showed different types of metabolites identified.

FIGURE 4

Metabonomics statistical analysis. (A) PCA score plot; (B) OPLS-DA score plot (R²X = 0.735, R²Y = 1 Q²Y = 0.998). C#-# and M#-# presented the sample name of the control group and MIC group, respectively. (C) Volcano plot; yellow dots represented the increase in metabolite abundance, and blue dots represented the decrease in metabolite abundance (vs. Control). p.adj represented adjusted p-values using FDR. (D) S-plots from the OPLS-DA model, the abscissa indicated the covariance, and the ordinate indicated correlation.

FIGURE 5

Differential metabolite heat map and biomarker identification. (A) The heatmap was constructed based on the differential metabolites in MIC group and Control group (n = 12). The color key indicated metabolite abundance (blue: upregulation; yellow: downregulation). Rows: metabolites; Columns: samples. Control and MIC group were colored yellow and cyan, respectively. (B) The boxplots of biomarkers to show the difference between MIC group and Control group.

FIGURE 6

Metabolic pathway enrichment analysis and critical metabolites involved in the pathway. (A) KEGG enrichment analysis bubble chart of metabolic pathways. The x-axis represented the rich factor (the number of differential metabolites enriched in the pathway). The y-axis represented the name of KEGG pathways. The size of the bubble represented the number of the differential metabolites, and the color represented p-values. (B) KEGG classification of enriched pathways. The number next to the bar represented the number of differential metabolites involved in the pathway. (Percent Of Metabolite: the proportion of differential metabolites involved in this pathway to the total differential metabolites.) (C) Changes in the abundance of critical metabolites before and after administration.

FIGURE 7

Visualization of metabolic pathway and changes of key enzyme activity and ATP content before and after EO treatment. (A) Summary graph of metabolic pathways in MIC group (vs. Control). The black fonts indicated the detected metabolites (blue boxes were significantly down-regulated differential metabolites, yellow boxes were significantly up-regulated metabolites, and the rest are not differential metabolites), while the gray fonts indicated undetected metabolites. The activities of Hexokinase (HK), Pyruvate kinase (PK), Pyruvate Decarboxylase (PC) and Malate Dehydrogenase (MDH) are shown in (B). (C) Effect of EO on ATP synthesis in C. acnes cells.