Chemical Composition and Antibacterial Activity of Liquid and Volatile Phase of Essential Oils against Planktonic and Biofilm-Forming Cells of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening, hard-to-heal infections associated with the presence of a biofilm. Essential oils (EOs) are promising agents to combat pseudomonal infections because of the alleged antimicrobial activity of their volatile fractions and liquid forms. Therefore, the purpose of this paper was to evaluate the antibacterial efficacy of both volatile and liquid phases of seven EOs (thyme, tea tree, basil, rosemary, eucalyptus, menthol mint, lavender) against P. aeruginosa biofilm and planktonic cells with the use of a broad spectrum of analytical in vitro methods. According to the study results, the antibacterial activity of EOs in their liquid forms varied from that of the volatile fractions. Overall, liquid and volatile forms of rosemary EO and tea tree EO displayed significant antibiofilm effectiveness. The outcomes indicate that these particular EOs possess the potential to be used in the therapy of P. aeruginosa infections.

Keywords: EOs in liquid form; Pseudomonas aeruginosa; antimicrobial activity; biofilm; essential oil; volatile fractions.

Figure 1

The ability of Pseudomonas aeruginosa clinical (PA 1–7, PA 13–19) and the reference (ATCC 15442) strains to form biofilm. (A) Biofilm biomass level assessed with the crystal violet method. (B) Metabolic activity of biofilm-forming cells, determined with resazurin staining. Ab, absorbance. The average and standard deviations are marked.

Figure 2

Changes in the biofilm-forming cells viability (%) of P. aeruginosa clinical (PA 1–7, PA 13–19) and the reference (ATCC 15442) strains after treatment with emulsified essential oils in their liquid forms in the concentration of 25.0% (v/v). Results of microdilution methodology with (A,B) TTC and (C–G) resazurin staining. Standard deviations are marked. The negative values indicate an increase in biofilm-forming cells viability after their treatment with EOs in comparison to the growth control (untreated cells).

Figure 2

Changes in the biofilm-forming cells viability (%) of P. aeruginosa clinical (PA 1–7, PA 13–19) and the reference (ATCC 15442) strains after treatment with emulsified essential oils in their liquid forms in the concentration of 25.0% (v/v). Results of microdilution methodology with (A,B) TTC and (C–G) resazurin staining. Standard deviations are marked. The negative values indicate an increase in biofilm-forming cells viability after their treatment with EOs in comparison to the growth control (untreated cells).

Figure 2

Changes in the biofilm-forming cells viability (%) of P. aeruginosa clinical (PA 1–7, PA 13–19) and the reference (ATCC 15442) strains after treatment with emulsified essential oils in their liquid forms in the concentration of 25.0% (v/v). Results of microdilution methodology with (A,B) TTC and (C–G) resazurin staining. Standard deviations are marked. The negative values indicate an increase in biofilm-forming cells viability after their treatment with EOs in comparison to the growth control (untreated cells).

Figure 3

Impact of R-EO on P. aeruginosa ATCC 15442 biofilm. (A,B) Pseudomonal biofilm untreated and treated with R-EO in its liquid form, assessed with the modified A.D.A.M. (antibiofilm dressing’s activity measurement) method. (C,D) Pseudomonal biofilm untreated and treated with R-EO volatiles, assessed with the AntiBioVol (antibiofilm activity of volatile compounds) assay. The red/orange color shows pseudomonal cells altered/damaged as the result of exposure to R-EO, while green-colored cells are non-altered, viable cells. Moreover, the darker (less green) picture shows that fewer live cells are captured in this particular field of vision.