Evaluation of the antibacterial activity of Elsholtzia ciliate essential oil against halitosis-related Fusobacterium nucleatum and Porphyromonas gingivalis

Abstract

The broad-spectrum antimicrobial activity of Elsholtzia ciliate essential oil (ECO) has been previously reported, but its effectiveness against halitosis-causing bacteria such as Fusobacterium nucleatum and Porphyromonas gingivalis is not well understood. In this study, we investigated the bacteriostatic activity of ECO against planktonic cells and biofilms of F. nucleatum and P. gingivalis, as well as its ability to inhibit bacterial metabolism and production of volatile sulfur compounds (VSCs) at sub-lethal concentrations. Our findings revealed that ECO exhibited comparable activities to chlorhexidine against these oral bacteria. Treatment with ECO significantly reduced the production of VSCs, including hydrogen sulfide, dimethyl disulfide, and methanethiol, which are major contributors to bad breath. As the major chemical components of ECO, carvacrol, p-cymene, and phellandrene, were demonstrated in vitro inhibitory effects on F. nucleatum and P. gingivalis, and their combined use showed synergistic and additive effects, suggesting that the overall activity of ECO is derived from the cumulative or synergistic effect of multiple active components. ECO was found to have a destructive effect on the bacterial cell membrane by examining the cell morphology and permeability. Furthermore, the application of ECO induced significant changes in the bacterial composition of saliva-derived biofilm, resulting in the elimination of bacterial species that contribute to halitosis, including Fusobacterium, Porphyromonas, and Prevotella. These results provide experimental evidence for the potential clinical applications of ECOs in the prevention and treatment of halitosis.

Keywords: Elsholtzia ciliate; Fusobacterium nucleatum; Porphyromonas gingivalis; antibacterial activity; biofilm; essential oil; halitosis.

Figure 1

Activity of ECO against Fusobacterium nucleatum and Porphyromonas gingivalis planktonic bacterial and inhibition of their VSCs production. (A) MICs and MBCs of ECO and CHX against F. nucleatum planktonic cells, and their inhibition of F. nucleatum VSCs production at 1/2× MIC. (B) 12 h-Growth curves of F. nucleatum in BHI broth at different concentrations of ECO or CHX. (C) MICs and MBCs of ECO and CHX against P. gingivalis planktonic cells, and their inhibition of P. gingivalis VSCs production at 1/2× MIC. (D) 12 h-Growth curves of P. gingivalis in BHI broth at different concentrations of ECO or CHX.

Figure 2

Elimination F. nucleatum and P. gingivalis mixed biofilm and inhibition of bacterial metabolism by ECO. (A) Determination of the biofilm elimination and metabolic inhibition by CHX (left) and ECO (right). Grey bars indicate the biofilm elimination rate and white bars indicate the metabolic inhibition rate. (B) CLSM observation of the mixed biofilms stained by the combination of SYTO9 (green) and PI (red) with or without the treatment (left, bar indicates 0.2 μm in the merged images). In the right panel, grey columns show the ratio of dead cells in the corresponding groups.

Figure 3

GC-MS analysis of the chemical composition of the prepared ECO sample. (A) TIC of the GC-MS. (B) Mass spectrum of peak 1 in panel (A), which was identified as carvacrol. (C) Mass spectrum of peak 2 in panel (A), which was identified as p-cymene. (D) Mass spectrum of peak 3 in panel (A), which was identified as phellandrene.

Figure 4

Antibacterial activity of major chemical components in ECO. (A) MICs and MBCs of carvacrol, p-cymene, and phellandrene against F. nucleatum (left) and P. gingivalis (right) planktonic cells. (B) The checkerboard results show the antibacterial effects against F. nucleatum (left) and P. gingivalis (right) by the combination of carvacrol, p-cymene, and phellandrene in pairs.

Figure 5

VSCs profiles produced by F. nucleatum and P. gingivalis with or without ECO treatment. (A) The production of hydrogen sulfide (black column) and methanethiol (white column) by F. nucleatum (left) and P. gingivalis (right) in the stimulation test. (B) VSCs profiles of F. nucleatum biofilm without (left) or with (right) the presence of 2× MIC ECO, which are shown as TICs, and the mass spectra of major peaks in the TIC are indicated in the small panels. (C) VSCs profiles of P. gingivalis biofilm without (left) or with (right) the presence of 2× MIC ECO, which are shown as TICs, and the mass spectra of major peaks in the TIC are indicated in the small panels.

Figure 6

Effect of ECO on bacterial cell morphology and membrane integrity. (A) Effect of ECO on F. nucleatum (upper) and P. gingivalis (bottom) cell morphology observed through SEM, and bars indicate 2 μm. (B) Cell membrane permeability F. nucleatum (left) and P. gingivalis (right) of with or without the presence of CHX or ECO determined by PI staining.

Figure 7

Effect of ECO treatment on microbial diversity of the saliva-derived bacterial biofilm. (A) Shannon’s index representing alpha-diversity of microbial communities in the saliva-derived bacterial biofilms with or without treatment. (B) Principal coordinates analysis (PCoA) plot based on the Bray–Curtis distance showing the relatedness of the bacterial community composition between different samples. (C) Combining the UPGMA clustering tree and the taxonomic composition histogram (at the genus level) shows the similarity of bacterial community structures among the different samples. (D) Relative abundance of main and halitosis-related (small panel) bacterial genus in different groups. In all panels, group 1 represents the untreated control group, group 2 represents the CHX-treated control group, and group 3 represents the ECO-treated control group.