Unravelling the Complex Antimicrobial Interactions of Essential Oils — The Case of Thymus vulgaris (Thyme)
Abstract
:1. Introduction
2. Results and Discussion
2.1. Essential Oil Composition
2.2. Minimum Inhibitory Concentrations (MIC)
Compounds | RRI | % of oil |
---|---|---|
α-Pinene | 1016 | 0.63 |
α-Thujene | 1019 | 0.50 |
Camphene | 1057 | 0.61 |
β-Pinene | 1104 | 0.17 |
Myrcene | 1159 | 1.17 |
α-Terpinene | 1174 | 1.01 |
Limonene | 1194 | 0.35 |
1,8 Cineole | 1202 | 0.24 |
β-Phellandrene | 1203 | 0.13 |
γ-Terpinene | 1242 | 6.39 |
p-Cymene | 1270 | 15.44 |
Camphor | 1521 | 0.39 |
Linalool | 1541 | 4.22 |
β-Caryophyllene | 1596 | 1.31 |
Terpinen-4-ol | 1602 | 0.93 |
Thymol methyl ether | 1607 | 0.51 |
Boroneol | 1702 | 1.76 |
α-Terpineol | 1707 | 0.32 |
δ-Cadinene | 1763 | 0.09 |
Isothymol | 2171 | 0.12 |
Thymol | 2225 | 60.18 |
Carvacrol | 2228 | 2.88 |
Total: | 99.35 |
Pathogen | Minimum Inhibitory Concentrations (mg mL−1) | ||||||||
---|---|---|---|---|---|---|---|---|---|
TvEO | Thymol | Carvacrol | Linalool | p-Cymene | Borneol | α-Terpinene | γ-Terpinene | Controls * | |
Escherichia coli ATCC8739 | 0.500 | 1 | 1 | 4 | >8 | >8 | 8 | >8 | 0.0005 |
Morexella cattarhalis ATCC23246 | 0.500 | 1 | 1 | 2 | >8 | 8 | 8 | >8 | 0.001 |
Staphylococcus aureus ATCC126000 | 0.500 | 1 | 0.500 | 2 | >8 | 8 | 4 | 8 | 0.0005 |
Enterococcus faecalis ATCC29212 | 0.125 | 0.500 | 0.500 | 1 | >8 | 4 | 8 | >8 | 0.001 |
Bacillus cereusATCC11778 | 0.125 | 0.500 | 0.250 | 2 | >8 | 8 | 4 | 8 | 0.0002 |
Candida albicans ATCC10231 | 0.062 | 0.125 | 0.125 | 2 | >8 | 1 | 1 | 8 | 0.001 |
Candida tropicalisATCC201380 | 0.062 | 0.125 | 0.125 | 0.250 | >8 | 1 | 0.250 | 4 | 0.001 |
EO compoundsmg mL−1 | E. coli | INT | M. cattarhalis | INT | S. aureus | INT | E. faecalis | INT | B. cereus | INT | C. albicans | INT | C. tropicals | INT | ||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
THY + CARV | FICA | 0.500 | ADD | 0.500 | ADD | 1.000 | IND | 1.000 | IND | 1.000 | IND | 0.500 | ADD | 0.500 | ADD | |||||||||||||||||||||||||||||
FICB | 0.500 | 0.500 | 2.000 | 1.000 | 2.000 | 0.500 | 0.500 | |||||||||||||||||||||||||||||||||||||
∑FIC | 1.000 | 1.000 | 3.000 | 2.000 | 3.000 | 1.000 | 1.000 | |||||||||||||||||||||||||||||||||||||
THY + LIN | FICA | 0.500 | ADD | 1.000 | IND | 0.500 | ADD | 0.500 | ADD | 2.000 | IND | 0.500 | ADD | 0.500 | ADD | |||||||||||||||||||||||||||||
FICB | 0.250 | 0.500 | 0.250 | 0.250 | 0.500 | 0.031 | 0.250 | |||||||||||||||||||||||||||||||||||||
∑FIC | 0.750 | 1.500 | 0.750 | 0.750 | 2.500 | 0.531 | 0.750 | |||||||||||||||||||||||||||||||||||||
THY + CYM | FICA | 1.000 | IND | 0.250 | SYN | 0.250 | SYN | 1.000 | IND | 1.000 | IND | 0.500 | SYN | 0.500 | SYN | |||||||||||||||||||||||||||||
FICB | 0.063 | 0.031 | 0.031 | 0.031 | 0.031 | 0.004 | 0.004 | |||||||||||||||||||||||||||||||||||||
∑FIC | 1.063 | 0.281 | 0.281 | 1.031 | 1.031 | 0.504 | 0.504 | |||||||||||||||||||||||||||||||||||||
THY + BOR | FICA | 1.000 | IND | 4.000 | ANT | 0.500 | ADD | 2.000 | IND | 0.250 | SYN | 0.500 | ADD | 0.500 | ADD | |||||||||||||||||||||||||||||
FICB | 0.063 | 1.000 | 0.062 | 0.250 | 0.016 | 0.063 | 0.250 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 1.063 | 5.000 | 0.562 | 2.250 | 0.261 | 0.563 | 0.750 | |||||||||||||||||||||||||||||||||||||
THY + α-TER | FICA | 1.000 | IND | 0.500 | ADD | 1.000 | IND | 1.000 | IND | 0.500 | ADD | 2.000 | IND | 0.500 | ADD | |||||||||||||||||||||||||||||
FICB | 0.120 | 0.063 | 0.250 | 0.063 | 0.063 | 0.250 | 0.250 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 1.125 | 0.563 | 1.250 | 1.063 | 0.563 | 2.250 | 0.750 | |||||||||||||||||||||||||||||||||||||
THY + γ -TER | FICA | 1.000 | IND | 1.000 | IND | 1.000 | IND | 2.000 | IND | 2.000 | IND | 1.000 | IND | 1.000 | IND | |||||||||||||||||||||||||||||
FICB | 0.063 | 0.063 | 0.125 | 0.063 | 0.125 | 0.015 | 0.031 | |||||||||||||||||||||||||||||||||||||
∑FIC | 1.063 | 1.063 | 1.125 | 2.063 | 2.125 | 1.015 | 1.031 | |||||||||||||||||||||||||||||||||||||
CARV + LIN | FICA | 0.500 | ADD | 0.500 | ADD | 0.500 | ADD | 1.000 | IND | 0.250 | SYN | 0.500 | ADD | 0.500 | ADD | |||||||||||||||||||||||||||||
FICB | 0.250 | 0.250 | 0.125 | 0.500 | 0.030 | 0.031 | 0.250 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 0.750 | 0.750 | 0.625 | 1.500 | 0.281 | 0.531 | 0.750 | |||||||||||||||||||||||||||||||||||||
CARV + CYM | FICA | 0.125 | SYN | 0.125 | SYN | 0.500 | SYN | 0.500 | SYN | 0.250 | SYN | 1.000 | IND | 1.000 | IND | |||||||||||||||||||||||||||||
FICB | 0.032 | 0.008 | 0.008 | 0.008 | 0.008 | 0.008 | 0.008 | |||||||||||||||||||||||||||||||||||||
∑FIC | 0.157 | 0.133 | 0.508 | 0.508 | 0.258 | 1.008 | 1.008 | |||||||||||||||||||||||||||||||||||||
CARV + BOR | FICA | 2.000 | IND | 0.500 | ADD | 0.250 | SYN | 0.500 | ADD | 0.500 | ADD | 0.500 | ADD | 0.500 | ADD | |||||||||||||||||||||||||||||
FICB | 0.125 | 0.125 | 0.015 | 0.125 | 0.016 | 0.063 | 0.250 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 2.125 | 0.625 | 0.265 | 0.625 | 0.516 | 0.563 | 0.750 | |||||||||||||||||||||||||||||||||||||
α-TER + CARV | FICA | 0.500 | ADD | 0.500 | ADD | 0.250 | SYN | 1.000 | IND | 0.250 | SYN | 0.250 | SYN | 0.500 | ADD | |||||||||||||||||||||||||||||
FICB | 0.625 | 0.063 | 0.062 | 0.063 | 0.250 | 0.063 | 0.250 | |||||||||||||||||||||||||||||||||||||
FIC | 0.562 | 0.563 | 0.312 | 1.063 | 0.500 | 0.313 | 0.750 | |||||||||||||||||||||||||||||||||||||
CARV + γ -TER | FICA | 0.500 | ADD | 0.500 | ADD | 0.250 | ANT | 1.000 | IND | 0.500 | ADD | 0.250 | SYN | 0.500 | ADD | |||||||||||||||||||||||||||||
FICB | 0.063 | 0.031 | 4.000 | 0.031 | 0.030 | 0.015 | 0.015 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 0.563 | 0.531 | 4.250 | 1.031 | 0.530 | 0.265 | 0.515 | |||||||||||||||||||||||||||||||||||||
LIN + CYM | FICA | 2.000 | IND | 0.250 | SYN | 1.000 | IND | 0.500 | ADD | 1.000 | IND | 1.000 | IND | 0.125 | SYN | |||||||||||||||||||||||||||||
FICB | 0.250 | 0.062 | 0.125 | 0.063 | 0.125 | 0.125 | 0.016 | |||||||||||||||||||||||||||||||||||||
FIC | 2.250 | 0.312 | 1.125 | 0.563 | 1.125 | 1.125 | 0.141 | |||||||||||||||||||||||||||||||||||||
LIN + BOR | FICA | 0.500 | ADD | O.500 | ADD | 0.250 | SYN | 0.500 | ADD | 0.250 | SYN | 0.250 | ADD | 1.000 | IND | |||||||||||||||||||||||||||||
FICB | 0.125 | 0.250 | 0.062 | 0.125 | 0.063 | 0.500 | 1.000 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 0.625 | 0.750 | 0.312 | 0.625 | 0.312 | 0.750 | 2.000 | |||||||||||||||||||||||||||||||||||||
LIN +α-TER | FICA | 0.250 | SYN | 0.500 | ADD | 1.000 | IND | 0.250 | SYN | 0.500 | ADD | 0.500 | IND | 0.250 | SYN | |||||||||||||||||||||||||||||
FICB | 0.063 | 0.125 | 0.500 | 0.031 | 0.250 | 1.000 | 0.250 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 0.312 | 0.625 | 1.500 | 0.281 | 0.750 | 1.500 | 0.500 | |||||||||||||||||||||||||||||||||||||
LIN + γ -TER | FICA | 1.000 | IND | 0.500 | ADD | 1.000 | IND | 2.000 | IND | 0.500 | ADD | 0.500 | ADD | 1.000 | IND | |||||||||||||||||||||||||||||
FICB | 0.120 | 0.063 | 0.250 | 0.125 | 0.125 | 0.125 | 0.063 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 1.120 | 0.563 | 1.250 | 2.125 | 0.625 | 0.625 | 1.063 | |||||||||||||||||||||||||||||||||||||
CYM + BOR | FICA | 0.500 | ADD | 0.063 | SYN | 0.250 | SYN | 0.250 | IND | 0.250 | ADD | 0.125 | IND | 1.000 | IND | |||||||||||||||||||||||||||||
FICB | 0.500 | 0.125 | 0.125 | 1.000 | 0.500 | 2.000 | 0.015 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 1.00 | 0.188 | 0.375 | 1.250 | 0.750 | 2.125 | 1.015 | |||||||||||||||||||||||||||||||||||||
CYM + α-TER | FICA | 0.500 | IND | 0.250 | ADD | 0.500 | IND | 0.250 | ADD | 0.250 | IND | 0.125 | IND | 1.000 | IND | |||||||||||||||||||||||||||||
FICB | 1.000 | 0.500 | 2.000 | 0.500 | 1.000 | 2.000 | 0.015 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 1.500 | 0.750 | 2.500 | 0.750 | 1.250 | 2.125 | 1.015 | |||||||||||||||||||||||||||||||||||||
CYM + γ -TER | FICA | 0.500 | ADD | 0.063 | SYN | 0.500 | IND | 0.500 | ADD | 0.250 | ADD | 0.500 | IND | 0.500 | IND | |||||||||||||||||||||||||||||
FICB | 0.500 | 0.125 | 1.000 | 0.500 | 0.500 | 1.000 | 2.000 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 1.000 | 0.188 | 1.500 | 1.000 | 0.750 | 1.500 | 2.500 | |||||||||||||||||||||||||||||||||||||
BOR + α-TER | FICA | 0.250 | SYN | 0.500 | ADD | 0.250 | ADD | 0.500 | ADD | 0.500 | IND | 0.500 | ADD | 0.124 | SYN | |||||||||||||||||||||||||||||
FICB | 0.250 | 0.250 | 0.500 | 0.250 | 1.000 | 0.500 | 0.124 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 0.500 | 0.750 | 0.750 | 0.750 | 1.500 | 1.000 | 0.248 | |||||||||||||||||||||||||||||||||||||
BOR + γ -TER | FICA | 0.250 | SYN | 0.500 | ADD | 0.500 | ADD | 0.500 | ADD | 0.500 | ADD | 0.250 | SYN | 2.000 | IND | |||||||||||||||||||||||||||||
FICB | 0.250 | 0.250 | 0.500 | 0.125 | 0.500 | 0.031 | 0.125 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 0.500 | 0.750 | 1.000 | 0.625 | 1.000 | 0.281 | 2.125 | |||||||||||||||||||||||||||||||||||||
α-TER + γ -TER | FICA | 0.500 | ADD | 0.500 | ADD | 0.500 | ADD | 0.125 | SYN | 0.500 | ADD | 1.000 | IND | 1.000 | IND | |||||||||||||||||||||||||||||
FICB | 0.250 | 0.250 | 0.250 | 0.125 | 0.250 | 0.125 | 0.016 | |||||||||||||||||||||||||||||||||||||
ƩFIC | 0.750 | 0.750 | 0.750 | 0.250 | 0.750 | 1.125 | 1.016 |
3. Experimental
3.1. Strains, Media and Chemicals
3.2. Gas Chromatography-Mass Spectrometry Analysis
3.3. Antimicrobial Susceptibility Tests
3.3.1. Determination of Minimal Inhibitory Concentration (MIC)
3.3.2. Assessment of the FIC Index
3.3.3. Varied Ratio Combinations and Isobolograms
4. Conclusions
Acknowledgments
Author Contributions
Conflictts of Interest
References
- Wagner, H. Multitarget therapy-The future of treatment for more than just functional dyspepsia. Phytomedicine 2006, 13, 122–129. [Google Scholar] [CrossRef]
- Bakkali, F.; Averbeck, S.; Averbeck, D.; Idaomar, M. Biological effects of essential oils-A review. Food Chem. Toxicol. 2008, 46, 446–475. [Google Scholar] [CrossRef]
- De Rapper, S.; Kamatou, G.; Viljoen, A.; van Vuuren, S. The in vitro antimicrobial activity of Lavandula angustifolia essential oil in combination with other aroma-therapeutic oils. Evid. Based Complement. Alternat. Med. 2013, 2013, 852049. [Google Scholar]
- Zarzuelo, A.; Crespo, E. The medicinal and non-medicinal uses of thyme. In Thyme: The Genus Thymus; Stahl-Biskup, E., Saez, F., Eds.; Taylor & Francis: London, UK, 2003; pp. 263–292. [Google Scholar]
- De Lira, M.K.S.; de Oliveira, P.F.; de Oliveira, W.A.; Lima, I.O.; de Oliveira Lima, E. Antifungal activity of Thymus vulgaris L. essential oil and its constituent phytochemicals against Rhizopus oryzae: Interaction with ergosterol. Molecules 2012, 17, 14418–14433. [Google Scholar]
- Marino, M.; Bersani, C.; Comi, G. Antimicrobial activity of the essential oils of Thymus vulgaris L. measured using a bioimpedometric method. J. Food Prot. 1999, 62, 1017–1023. [Google Scholar]
- Rota, M.C.; Herrera, A.; Martinez, R.A.; Sotomayor, J.A.; Jordán, M.J. Antimicrobial activity and chemical composition of Thymus vulgaris, Thymus zygis and Thymus hyemalis essential oils. Food Control 2008, 19, 681–687. [Google Scholar] [CrossRef]
- Viljoen, A.M.; van Vuuren, S.F.; Ernst, E.; Klepser, M.; Demirci, B.; Başer, H.; van Wyk, B.E. Osmitopsis asteriscoides (Asteraceae)-the antimicrobial activity and essential oil composition of a Cape-Dutch remedy. J. Ethnopharmacol. 2003, 88, 137–143. [Google Scholar] [CrossRef]
- Van Vuuren, S.F.; Viljoen, A.M. Antimicrobial activity of limonene enantiomers and 1,8-cineole alone and in combination. Flavour Fragr. J. 2007, 22, 540–544. [Google Scholar] [CrossRef]
- Keefover-Ring, K.; Thompson, J.D.; Linhart, Y.B. Beyond six scents: Defining a seventh Thymus vulgaris chemotype new to southern France by ethanol extraction. Flavour Fragr. J. 2009, 24, 117–122. [Google Scholar] [CrossRef]
- Thompson, J.D.; Chalchat, J.C.; Michet, A.; Linhart, Y.B.; Ehlers, B. Qualitative and quantitative variation in monoterpene co-occurrence and composition in the essential oil of Thymus vulgaris chemotypes. J. Chem. Ecol. 2003, 29, 859–880. [Google Scholar] [CrossRef]
- Shabnum, S.; Wagay, M.G. Essential oil composition of Thymus vulgaris L. and their uses. J. Res. Dev. 2011, 11, 83–94. [Google Scholar]
- Hyldgaard, M.; Mygind, T.; Meyer, R.L. Essential oils in food preservation: Mode of action, synergies, and interactions with food matrix components. Front Microbiol. 2012, 3, 12. [Google Scholar]
- Ahmad, A.; Khan, A.; Khan, L.A.; Manzoor, N. In vitro synergy of eugenol and methyl eugenol with fluconazole against clinical Candida isolates. J. Med. Microbiol. 2010, 59, 1178–1184. [Google Scholar] [CrossRef]
- Bagamboula, C.F.; Uyttendaele, M.; Debevere, J. Inhibitory effect of thyme and basil essential oils, carvacrol, thymol, estragol, linalool and p-cymene towards Shigella sonnei and S. flexneri. Food Microbiol. 2004, 21, 33–42. [Google Scholar] [CrossRef]
- Koutsoudaki, C.; Krsek, M.; Rodger, A. Chemical composition and antibacterial activity of the essential oil and the gum of Pistacia lentiscus Var. chia. J. Agric. Food Chem. 2005, 53, 7681–7685. [Google Scholar] [CrossRef]
- Rao, A.; Zhang, Y.; Muend, S.; Rao, R. Mechanism of antifungal activity of terpenoid phenols resembles calcium stress and inhibition of the TOR pathway. Antimicrob. Agents Chemother. 2010, 54, 5062–5069. [Google Scholar] [CrossRef]
- Cristani, M.; D’Arrigo, M.; Mandalari, G.; Castelli, F.; Sarpietro, M.G.; Micieli, D.; Venuti, V.; Bisignano, G.; Saija, A.; Trombetta, D. Interaction of four monoterpenes contained in essential oils with model membranes: Implications for their antibacterial activity. J. Agric. Food Chem. 2007, 55, 6300–6308. [Google Scholar] [CrossRef]
- Oyedemi, S.O.; Okoh, A.I.; Mabinya, L.V.; Pirochenva, G.; Afolayan, A.J. The proposed mechanism of bactericidal action of eugenol, α-terpineol and g-terpinene against Listeria monocytogenes, Streptococcus pyogenes, Proteus vulgaris and Escherichia coli. Afr. J. Biotechnol. 2009, 8, 1280–1286. [Google Scholar]
- Xu, J.; Zhou, F.; Ji, B.P.; Pei, R.S.; Xu, N. The antibacterial mechanism of carvacrol and thymol against Escherichia coli. Lett. Appl. Microbiol. 2008, 47, 174–179. [Google Scholar] [CrossRef]
- Ultee, A.; Bennik, M.H.; Moezelaar, R. The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Appl. Environ. Microbiol. 2002, 68, 1561–1568. [Google Scholar] [CrossRef]
- Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. In Approved Standard M7-A6, 6th ed.; National Committee for Clinical Laboratory Standards: Fort Wayne, IN, USA, 2003. [Google Scholar]
- Berenbaum, M.C. What is synergy? Pharmacol. Rev. 1989, 41, 93–141. [Google Scholar]
- Canton, E.; Peman, J.; Gobernado, M.; Viudes, A.; Espinel, I.A. Synergistic activities of fluconazole and voriconazole with terbinafine against four Candida species determined by checkerboard, time-kill, and E-test methods. Antimicrob. Agents Chemother. 2005, 49, 1593–1596. [Google Scholar] [CrossRef]
- Van Vuuren, S.; Viljoen, A. Plant-based antimicrobial studies—methods and approaches to study the interaction between natural products. Planta Med. 2011, 77, 1168–1182. [Google Scholar] [CrossRef]
- Suliman, S.; van Vuuren, S.F.; Viljoen, A.M. Validating the in vitro antimicrobial activity of Artemisia afra in polyherbal combinations to treat respiratory infections. S. Afr. J. Bot. 2010, 76, 655–661. [Google Scholar] [CrossRef]
- Sample Availability: Samples of the Samples of the The essential oil used in this study is available from the corresponding author.
© 2014 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).
Share and Cite
Ahmad, A.; Van Vuuren, S.; Viljoen, A. Unravelling the Complex Antimicrobial Interactions of Essential Oils — The Case of Thymus vulgaris (Thyme). Molecules 2014, 19, 2896-2910. https://doi.org/10.3390/molecules19032896
Ahmad A, Van Vuuren S, Viljoen A. Unravelling the Complex Antimicrobial Interactions of Essential Oils — The Case of Thymus vulgaris (Thyme). Molecules. 2014; 19(3):2896-2910. https://doi.org/10.3390/molecules19032896
Chicago/Turabian StyleAhmad, Aijaz, Sandy Van Vuuren, and Alvaro Viljoen. 2014. "Unravelling the Complex Antimicrobial Interactions of Essential Oils — The Case of Thymus vulgaris (Thyme)" Molecules 19, no. 3: 2896-2910. https://doi.org/10.3390/molecules19032896