Antibacterial Investigation of Thyme Essential Oil and Its Main Constituents in Combination with Tetracycline

Abstract

The chemical composition and antibacterial activity of Thymus glabrescens Willd. (thyme) essential oil were examined, as well as its association with tetracycline. The antibacterial activities of geraniol and thymol, the main constituents of T. glabrescens oil, were also determined. Gas chromatography and gas chromatography/mass spectrometry were used to analyze the chemical composition of the oil. The antibacterial activities of the oil, geraniol, and thymol were investigated by the broth microdilution method. The interactions of the essential oil, geraniol, and thymol with tetracycline, toward five selected strains, were evaluated using the microdilution checkerboard assay. Oxygenated monoterpenes were the most abundant compound class in the oil (57.14%), with geraniol (22.33%) as the major compound. The essential oil exhibited in vitro antibacterial activity against all tested bacterial strains. The combinations, essential oil–tetracycline and thymol–tetracycline, produced synergistic interaction to a greater extent compared with geraniol–tetracycline association. All synergistic combinations reduced the minimum effective dose of the antibiotic and, consequently, minimized its adverse side effects.

In recent work, we have investigated Thymus glabrescens Willd. essential oil in combination with chloramphenicol, trying to find an appropriate antibacterial combination, that would be successful to overcome bacterial resistance.¹ In the present work, we have chosen to investigate the association between thyme oil and tetracycline. The antibacterial activities of geraniol and thymol, the main active constituents of thyme oil, in combination with tetracycline were also determined.

The aerial parts of T. glabrescens Willd. (Lamiaceae) were collected in June 2011 from natural populations at the village of Kravlje, Serbia. A voucher specimen, with the accession number 16642, is deposited at the Herbarium of the Department of Botany, Faculty of Biology, University of Belgrade-Herbarium Code BEOU. All chemicals, reagents, and standards were of analytical reagent grade and were purchased from the Sigma-Aldrich Chemical Company (Germany). Oil isolation, GC, GC-MS analyses, and identification of oil compounds were performed as previously described.¹

Activities of the essential oil, geraniol, thymol, and tetracycline samples were tested toward five different bacteria. Gram-negative bacteria were represented by Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa ATCC 27853, while the researched gram-positive strain was Staphylococcus aureus ATCC 29213. The inocula of the bacterial strains were prepared from overnight broth cultures and the suspensions were adjusted to 0.5 McFarland standard turbidity (corresponding to 10⁸ CFU/mL, depending on genera-consensus standard by the Clinical and Laboratory Standards Institute).² Microwell dilution assay and microdilution checkerboard assay were used to determine the minimum inhibitory concentration (MIC), minimum bactericidal concentration, and to assess antimicrobial combinations in vitro, as previously described.¹

Chemical analysis of the tested essential oil is presented and commented in the previous work.¹ The results from the antibacterial assay showed that thyme essential oil, geraniol, and thymol possessed antimicrobial activities against all the tested microorganisms.¹ The reference antibiotic was active in the range of concentration 128–1024 μg/mL.

The results of the possible interactions among the essential oil, geraniol, and thymol with the reference antibiotic are given in Table 1. Of the 45 combinations, T. glabrescens essential oil–tetracycline, 17 (37.8%) showed synergism, 17 (37.8%) had an additive, and 11 (24.4%) had an antagonistic effect. From all tested geraniol–tetracycline combinations, 15 (33.3%) showed synergism, 14 (31.1%) had an additive, and 16 (35.6%) had the antagonistic effect. Of the 45 combinations of thymol with the tetracycline, synergy was best noted for 22 (48.9%) ratios, an additive effect was recorded for 12 (26.7%) ratios, and 11 combinations (24.4%) exhibited the antagonistic effect.

Table 1.

Antibacterial Activity of Tetracycline-Studied Combinations, Represented by FIC and FIC_A Indices of Examining Substances

		FIC_A indices of T. glabrescens oil, geraniol, and thymol
Bacteria		0.10	0.20	0.30	0.40	0.50	0.60	0.70	0.80	0.90
E. coli ATCC 25922	^1*	0.21	0.32	0.43	0.54	0.65	0.76	0.87	0.98	1.09
	^2*	0.32	0.32	0.43	0.54	0.80	0.76	0.87	0.98	1.28
	^3*	0.21	0.32	0.43	0.54	0.65	0.76	0.87	0.98	1.09
K. pneumonia ATCC 700603	^1*	0.76	0.80	0.95	1.10	1.10	1.08	1.21	1.16	1.47
	^2*	0.76	0.80	0.95	1.10	1.25	1.24	1.38	1.34	1.66
	^3*	0.65	0.80	0.95	1.10	1.10	1.24	1.21	1.34	1.66
P. mirabilis ATCC 12453	^1*	0.43	0.56	0.69	0.96	0.95	1.08	1.04	1.16	1.66
	^2*	0.65	0.80	0.82	0.96	0.95	0.92	1.04	0.98	1.47
	^3*	0.54	0.68	0.69	0.54	0.80	0.76	0.87	0.98	1.09
P. aeruginosa ATCC 27853	^1*	0.54	0.80	1.08	1.24	1.10	1.08	1.04	1.16	1.66
	^2*	0.76	0.92	1.08	1.38	1.10	1.08	1.21	1.16	1.47
	^3*	0.76	0.92	1.21	1.24	1.10	0.76	0.87	0.98	1.47
S. aureus ATCC 29213	^1*	0.65	0.68	0.82	0.96	1.10	1.24	1.04	1.16	1.47
	^2*	0.76	0.80	0.95	1.10	1.25	1.24	1.21	1.34	1.85
	^3*	1.20	1.40	1.60	1.80	0.65	0.76	0.87	0.98	1.09

Examined combinations: oil–tetracycline (^1*); geraniol–tetracycline (^2*); thymol–tetracycline (^3*).

In the present study, the combination of thyme oil and tetracycline against all the tested bacteria exhibited predominantly synergistic and additive effects and decreased the MIC value of tetracycline ranging from 2-fold for K. pneumoniae ATCC 700603 to 10-fold for E. coli ATCC 25922. Based on the present analyses, it can be assumed that in research of the antibacterial effects of essential oil–antibiotic combinations, the choice of gram-negative or gram-positive bacterial species is not decisively significant. In other words, the proper essential oil–antibiotic association will act as equally stronger or weaker against all gram-positive and gram-negative bacterial strains.³ There are some generally accepted mechanisms of antibacterial interaction that produce synergism, including inhibition of protective enzymes, combination of membrane-active agents, sequential inhibition of common biochemical pathways, and the use of membranotropic agents to enhance the diffusion of other antimicrobials.⁴

The results of antibacterial activity of geraniol–tetracycline combination are different compared with the results of the thyme oil–tetracycline combination. The difference is in the increased percentage of interactions that produce antagonistic effects. The combination of geraniol and tetracycline against all the tested bacteria decreased the MIC value of tetracycline ranging from 1.5-fold for P. aeruginosa ATCC 27853 to 10-fold for E. coli ATCC 25922.

The results of antibacterial activity of the thymol–tetracycline combination are very similar to the results of the thyme oil–tetracycline association. The antagonistic effect was noted for the same number of combinations (11), and the sum of synergistic and additive effects is also the same (34). The combination of thymol and tetracycline against all the tested bacteria decreased the MIC value of tetracycline ranging from 2-fold for K. pneumoniae ATCC 700603 to 10-fold for E. coli ATCC 25922. The results obtained with the combinations of thymol–penicillin toward MRSA were antagonistic, while the association between thymol and penicillin against E. coli showed synergistic activity with FIC values of 0.15.⁵ It could be argued that these results correspond to the results of the present research.

If all combinations of the examined essential oil, geraniol, and thymol with tetracycline toward the five bacterial strains are taken into consideration, it could be stated that examined associations against E. coli ATCC 25922 produced strong synergistic interaction. In addition, the maximum effect of decreasing the MIC value of tetracycline, essential oil, geraniol, and thymol (10-fold) was noted for E. coli ATCC 25922. On the other side, thymol–tetracycline combinations against S. aureus ATCC 29213 were characterized by ratios (FIC_A≤0.4) of extremely strong antagonistic interactions.

The mechanisms of interaction that produce antagonistic effects have been studied less. Some of the studies included combinations of bactericidal and bacteriostatic agents, use of compounds that act on the same target of the microorganism, and chemical interactions among compounds.⁶ A significant percentage of antagonistic activity recorded for the geraniol–tetracycline combination can indicate the fact that the presence of trace components in essential oil could possibly influence the antibacterial activity of the oil-selected antibiotic combination to a significant extent. In this case, changes of varying intensity of the antibacterial activity of the essential oil can be expected, with regard to dominant component.⁷

Footnotes

Acknowledgments

This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant Nos. OI 171025 and TR 31079).

Author Disclosure Statement

No competing financial interests exist.

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