GC-MS Analysis and Microbiological Evaluation of Caraway Essential Oil as a Virulence Attenuating Agent against Pseudomonas aeruginosa

Abstract

The emergence of resistant microbes threatens public health on our planet, and the emergence of resistant bacteria against the most commonly used antibiotics necessitates urgent alternative therapeutic options. One way to fight resistant microbes is to design new antimicrobial agents, however, this approach takes decades of research. An alternative or parallel approach is to target the virulence of bacteria with natural or synthetic agents. Active constituents from medicinal plants represent a wide library to screen for natural anti-virulence agents. Caraway is used as a traditional spice and in some medicinal applications such as carminative, antispasmodic, appetizer, and expectorant. Caraway essential oil is rich in terpenes that were previously reported to have antimicrobial activities. In our study, we tested the caraway essential oil in sub-inhibitory concentration as a virulence agent against the Gram-negative bacteria Pseudomonas aeruginosa. Caraway essential oil in sub-inhibitory concentration dramatically blocked protease activity, pyocyanin production, biofilm formation, and quorum sensing activity of P. aeruginosa. The gas chromatography-mass spectroscopy (GC-MS) profile of caraway fruit oil identified 13 compounds representing 85.4% of the total oil components with carvone and sylvestrene as the main constituents. In conclusion, caraway essential oil is a promising virulence-attenuating agent that can be used against topical infections caused by P. aeruginosa.

Keywords: GC-MS; Pseudomonas aeruginosa; bacterial biofilm; caraway; essential oil; quorum sensing.

Figure 1

GC-MS profile of caraway fruits essential oil in (a) and major components (carvone and sylvestrene) in (b).

Figure 2

Mass spectrum of sylvestrene in (a) and chemical structures of sylvestrene and limonene in (b).

Figure 3

Effect of caraway oil on P. aeruginosa growth and virulence. (a): Minimum inhibitory concentration (MIC) of the oil, (b,c): Pyocyanin inhibition by caraway oil, (d,e): Protease production inhibition by caraway oil using skim milk agar method. (f): Protease inhibition assay using modified skimmed milk broth, supernatants from different cultures with or without caraway essential oil were incubated with skimmed milk 1.25% as a negative control for no protease activity, one tube with no supernatants was prepared and denoted as (-). **, ***, and ****: significant change in caraway oil-treated group relative to untreated and Tween-treated groups at p < 0.05, ns denotes no significance.

Figure 4

Effect of caraway oil on P. aeruginosa biofilm formation and in vitro biofilm eradication (a,b). qPCR analysis of quorum sensing genes expression in presence or absence of caraway essential oil (c), RopD was employed as a housekeeping gene. *, **, ***, and ****: significant change in caraway oil-treated group relative to untreated group at p < 0.05, ns denotes no significance.