Chemical Composition and Antibacterial Effect of Clove and Thyme Essential Oils on Growth Inhibition and Biofilm Formation of Arcobacter spp. and Other Bacteria

Abstract

Background: In recent years, significant resistance of microorganisms to antibiotics has been observed. A biofilm is a structure that significantly aids the survival of the microbial population and also significantly affects its resistance. Methods: Thyme and clove essential oils (EOs) were subjected to chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with a flame ionization detector (GC-FID). Furthermore, the antimicrobial effect of these EOs was tested in both the liquid and vapor phases using the volatilization method. The effect of the EOs on growth parameters was monitored using an RTS-8 bioreactor. However, the effect of the EOs on the biofilm formation of commonly occurring bacteria with pathogenic potential was also monitored, but for less described and yet clinically important strains of Arcobacter spp. Results: In total, 37 and 28 compounds were identified in the thyme and clove EO samples, respectively. The most common were terpenes and also derivatives of phenolic substances. Both EOs exhibited antimicrobial activity in the liquid and/or vapor phase against at least some strains. The determined antimicrobial activity of thyme and clove oil was in the range of 32-1024 µg/mL in the liquid phase and 512-1024 µg/mL in the vapor phase, respectively. The results of the antimicrobial effect are also supported by similar conclusions from monitoring growth curves using the RTS bioreactor. The effect of EOs on biofilm formation differed between strains. Biofilm formation of Pseudomonas aeruginosa was completely suppressed in an environment with a thyme EO concentration of 1024 µg/mL. On the other hand, increased biofilm formation was found, e.g., in an environment of low concentration (1-32 µg/mL). Conclusions: The potential of using natural matrices as antimicrobials or preservatives is evident. The effect of these EOs on biofilm formation, especially Arcobacter strains, is described for the first time.

Keywords: Arcobacter; antimicrobial effect; biofilm formation; clove; essential oil; thyme.

Figure 1

Growth curves of selected bacteria in a thyme essential oil environment. (A) Pseudomonas aeruginosa CCM 1961; (B) Staphylococcus aureus CCM 4223; (C) Escherichia coli CCM 2024; (D) Enterococcus faecalis CCM 4222. negative control; positive control; 32 µg/mL; 64 µg/mL; 128 µg/mL; 256 µg/mL; 512 µg/mL; 1024 µg/mL.

Figure 2

Growth curves of selected bacteria in a thyme essential oil environment. (A) Arcobacter butzleri CCUG 30484; (B) Arcobacter cryaerophilus CCM 7050; (C) Arcobacter skirrowii LMG 6621; (D) Arcobacter defluvii LMG 25694. negative control; positive control; 32 µg/mL; 64 µg/mL; 128 µg/mL; 256 µg/mL; 512 µg/mL; 1024 µg/mL.

Figure 3

Growth curves of selected bacteria in a clove essential oil environment. (A) Pseudomonas aeruginosa CCM 1961; (B) Staphylococcus aureus CCM 4223; (C) Escherichia coli CCM 2024; (D) Enterococcus faecalis CCM 4222. negative control; positive control; 32 µg/mL; 64 µg/mL; 128 µg/mL; 256 µg/mL; 512 µg/mL; 1024 µg/mL.

Figure 4

Growth curves of selected bacteria in a clove essential oil environment. (A) Arcobacter butzleri CCUG 30484; (B) Arcobacter cryaerophilus CCM 7050; (C) Arcobacter skirrowii LMG 6621; (D) Arcobacter defluvii LMG 25694. negative control; positive control; 32 µg/mL; 64 µg/mL; 128 µg/mL; 256 µg/mL; 512 µg/mL; 1024 µg/mL.

Figure 4

Growth curves of selected bacteria in a clove essential oil environment. (A) Arcobacter butzleri CCUG 30484; (B) Arcobacter cryaerophilus CCM 7050; (C) Arcobacter skirrowii LMG 6621; (D) Arcobacter defluvii LMG 25694. negative control; positive control; 32 µg/mL; 64 µg/mL; 128 µg/mL; 256 µg/mL; 512 µg/mL; 1024 µg/mL.

Figure 5

Effect of thyme EO on biofilm formation of common bacteria (A) and Arcobacter species (B). Data are presented as mean value of optical density (OD) ± SD. Pseudomonas aeruginosa CCM 1961, Arcobacter butzleri CCUG 30484; Staphylococcus aureus CCM 4223, Arcobacter cryaerophilus CCM 7050; Enterococcus faecalis CCM 4224, Arcobacter skirrowii LMG 6621; Escherichia coli CCM 2024, Arcobacter defluvii LMG 25694; positive control.

Figure 6

Effect of clove EO on biofilm formation of common bacteria (A) and Arcobacter species (B). Data are presented as mean value of optical density (OD) ± SD. Pseudomonas aeruginosa CCM 1961, Arcobacter butzleri CCUG 30484; Staphylococcus aureus CCM 4223, Arcobacter cryaerophilus CCM 7050; Enterococcus faecalis CCM 4224, Arcobacter skirrowii LMG 6621; Escherichia coli CCM 2024, Arcobacter defluvii LMG 25694; positive control.