The antimicrobial activity of plant oils and extracts has been recognized for many years. However, few investigations have compared large numbers of oils and extracts using methods that are directly comparable. In the present study, 52 plant oils and extracts were investigated for activity against Acinetobacter baumanii, Aeromonas veronii biogroup sobria, Candida albicans, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhimurium, Serratia marcescens and Staphylococcus aureus, using an agar dilution method. Lemongrass, oregano and bay inhibited all organisms at concentrations of ≤2·0% (v/v). Six oils did not inhibit any organisms at the highest concentration, which was 2·0% (v/v) oil for apricot kernel, evening primrose, macadamia, pumpkin, sage and sweet almond. Variable activity was recorded for the remaining oils. Twenty of the plant oils and extracts were investigated, using a broth microdilution method, for activity against C. albicans, Staph. aureus and E. coli. The lowest minimum inhibitory concentrations were 0·03% (v/v) thyme oil against C. albicans and E. coli and 0·008% (v/v) vetiver oil against Staph. aureus. These results support the notion that plant essential oils and extracts may have a role as pharmaceuticals and preservatives.
Plant oils and extracts have been used for a wide variety of purposes for many thousands of years ( Jones 1996). These purposes vary from the use of rosewood and cedarwood in perfumery, to flavouring drinks with lime, fennel or juniper berry oil ( Lawless 1995), and the application of lemongrass oil for the preservation of stored food crops ( Mishra & Dubey 1994). In particular, the antimicrobial activity of plant oils and extracts has formed the basis of many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies ( Reynolds 1996; Lis-Balchin & Deans 1997).
While some of the oils used on the basis of their reputed antimicrobial properties have well documented in vitro activity, there are few published data for many others ( Morris et al. 1979 ; Ross et al. 1980 ; Yousef & Tawil 1980; Deans & Ritchie 1987; Hili et al. 1997 ). Some studies have concentrated exclusively on one oil or one micro-organism. While these data are useful, the reports are not directly comparable due to methodological differences such as choice of plant extract(s), test micro-organism(s) and antimicrobial test method ( Janssen et al. 1987 ).
The aim of this study was to test a large number of essential oils and plant extracts against a diverse range of organisms comprising Gram-positive and Gram-negative bacteria and a yeast. The purpose of this was to create directly comparable, quantitative, antimicrobial data and to generate data for oils for which little data exist.
Materials and methods
Organisms and growth conditions
Micro-organisms were obtained from the culture collections of the Department of Microbiology at The University of Western Australia and the Western Australian Centre for Pathology and Medical Research. Organisms were as follows: Acinetobacter baumanii NCTC 7844, Aeromonas veronii biogroup sobria ATCC 9071 (Aer. sobria), Candida albicans ATCC 10231, Enterococcus faecalis NCTC 8213, Escherichia coli NCTC 10418, Klebsiella pneumoniae NCTC 11228, Pseudomonas aeruginosa NCTC 10662, Salmonella enterica subsp. enterica serotype typhimurium ATCC 13311 (Salm. typhimurium), Serratia marcescens NCTC 1377 and Staphylococcus aureus NCTC 6571. Organisms were maintained on blood agar (BA) (Unipath). Overnight cultures were prepared by inoculating approximately 2 ml Mueller Hinton broth (MHB) (Unipath) with 2–3 colonies of each organism taken from BA. Broths were incubated overnight at 35 °C with shaking. Inocula were prepared by diluting overnight cultures in saline to approximately 108 cfu ml−1 for bacteria and 107 cfu ml−1 for C. albicans. These suspensions were further diluted with saline as required.
Details of the sources of extracts, as provided by Sunspirit Oils Pty Ltd, are given in Table 1.Plant oils and extracts were derived from a total of 37 genera. All oils were diluted v/v in both agar and broth dilution methods.
Agar dilution method
The agar dilution method followed that approved by the NCCLS with the following modification: a final concentration of 0·5% (v/v) Tween-20 (Sigma) was incorporated into the agar after autoclaving to enhance oil solubility. Briefly, a series of twofold dilutions of each oil, ranging from 2% (v/v) to 0·03% (v/v), was prepared in Mueller Hinton agar with 0·5% (v/v) Tween-20. Plates were dried at 35 °C for 30 min prior to inoculation with 1–2 μl spots containing approximately 104 cfu of each organism, using a multipoint replicator. Mueller Hinton agar, with 0·5% (v/v) Tween-20 but no oil, was used as a positive growth control. Inoculated plates were incubated at 35 °C for 48 h. Minimum inhibitory concentrations (MICs) were determined after 24 h for the bacteria and after 48 h for C. albicans. The MICs were determined as the lowest concentration of oil inhibiting the visible growth of each organism on the agar plate. The presence of one or two colonies was disregarded.
Broth microdilution method
The broth microdilution assay was performed as described previously ( Hammeret al. 1996 ) with the following modifications: MHB was used instead of heart infusion broth and, in tests with C. albicans, sub-cultures were performed after 48 h incubation. For most oils, the highest concentration tested was 4·0% (v/v), although for some this was 8·0% (v/v). The lowest concentration tested was 0·008% (v/v).
Lemongrass, oregano and bay inhibited all organisms at ≤2·0% (v/v). Rosewood, coriander, palmarosa, tea tree, niaouli, peppermint, spearmint, sage and marjoram inhibited all organisms except Ps. aeruginosa at ≤2·0% (v/v). Six oils, comprising the five fixed oils (pumpkin, macadamia, evening primrose, apricot kernel and sweet almond) and the essential oil clary sage, failed to inhibit any organisms at the highest concentration, which was 2·0% (v/v). Myrrh and cypress inhibited Gram-positive organisms only, while carrot, patchouli, sandalwood and vetiver inhibited Gram-positive bacteria and C. albicans only. Mandarin oil inhibited C. albicans at 2·0% (v/v), while bacteria were not inhibited at ≤2·0% (v/v). None of the oils inhibited Gram-negative bacteria only.
Pseudomonas aeruginosa was inhibited by the lowest number of extracts (three), significantly less susceptible than Salm. typhimurium (17). Candida albicans and Staph. aureus were the most susceptible organisms, inhibited at ≤2·0% (v/v) by 41 and 40 extracts, respectively.
Table 2 shows MICs and minimum cidal concentrations (MCCs) of 20 plant oils and extracts obtained by the broth microdilution method. Thyme had the lowest MIC of 0·03% (v/v) against C. albicans and E. coli, and vetiver had the lowest MIC of 0·008% (v/v) against Staph. aureus. Comparison of MICs obtained by agar and broth methods showed that differences exceeding two serial dilutions were seen with peppermint, patchouli, sandalwood, thyme and vetiver. The greatest difference was for C. albicans and sandalwood, where the MIC obtained by agar dilution was 0·06% (v/v) compared with the MIC by broth microdilution of >8·0% (v/v).