Abstract
We have applied techniques that enable us to measure quantitatively the action of an antimicrobial agent upon the five characteristic growth parameters of a microbial culture. The methods were used to study the action of Eucalyptus globulus (Myrtaceae) essential oil upon a suspension of Staphylococcus aureus in a standard growth medium. We showed that the Eucalyptus oil produced a cultivable population reduction during both the adaptation phase and the exponential growth phase with the same specific reduction rate. This reduction rate was determined as an explicit function of the oil concentration. The oil does not affect the adaptation time. Our results suggest that the oil inactivates the bacteria via a “target” that is accessible to the oil while the internal cell transformation prepares the cell for its exponential growth period, but later, as the cells transform themselves into the stationary phase, this target ceases to be “accessible” to the oil. This process is reversible; the change from “accessible” to “inaccessible” occurs when the bacteria are transforming into the stationary phase.
Introduction
Many plant oils and extracts have been traditionally used as topical antiseptics or have been reported to have antimicrobial properties. With the resurgence of interest in natural therapies and increasing consumer demand for effective, safe, natural products, quantitative data on the antimicrobial effects of plant extracts are required. This interest is reflected by the large number of articles concerned with the screening of potential antimicrobial agents published in this journal such as CitationDa Silva et al. (2006), CitationDob et al. (2006), CitationDulger (2006), CitationKuçuk et al. (2006), and CitationMahid (2006) to cite a few. These and many similar articles all use either the agar disk diffusion technique or the broth dilution method to test the possible antimicrobial activity via the determination of a minimum inhibitory concentration (MIC). The results obtained by the disk diffusion and dilution methods for the same microbe and antimicrobial agent may vary between assays, and there is often only a weak correlation between the results obtained by the two methods (CitationDonaldson et al., 2005). The usefulness of these methods is limited to the generation of preliminary qualitative data, and they cannot give any more precise information than the estimation of a concentration threshold that delimits an “inhibition—no inhibition” effect. It is very important to screen a large number of potential antimicrobial agents before undertaking more sophisticated investigations.
In order to progress in our understanding of the action of an antimicrobial agent, we must have quantitative information about how the agent modifies the principal parameters that characterize the growth of a microbial suspension in a medium containing the potential antimicrobial agent:
The adaptation time of the microbe to the medium.
A specific reduction rate of the cultivable cell population during the adaptation phase due to the action of the antimicrobial agent.
A specific growth rate of the cell population during the exponential growth phase.
If the antimicrobial agent does not totally inhibit growth, the maximum cell concentration attained at the beginning of the stationary growth phase.
A specific reduction rate of the cultivable cell population during the stationary phase due to the action of the antimicrobial agent.
Certain agents, depending upon their concentration, might modify one or more parameters, others might only affect the adaptation time, and yet others might only act upon dividing cells and so affect only the exponential growth phase. An antimicrobial agent is deemed to have an action if it modifies at least one of the above parameters.
In naturally occurring situations when antimicrobial action is required, the bacteria are in one of two forms: as a planktonic suspension or as a sessile biofilm. The study of antimicrobial action against biofilm bacteria is certainly very important, but it also requires very specific techniques. Biofilm bacteria proffer unexpected resistance to standard prophylaxis, due partly to the protection afforded by the extracellular polymeric substances that form the biofilm see CitationCaubet et al. (2004) for a technique using antimicrobial molecules combined with electric fields and for further references. Here, we restrict our attention uniquely to the investigation of the action of antimicrobial agents against microbes in suspension.
We have developed new original methods (CitationEllison et al., 2007) that can be used with automatic multicell conductance or multicell light absorbance techniques. Compared with the usual applications of these techniques, our methods give precise quantitative information about the action of an antimicrobial agent upon the five suspension growth parameters as a function of the antimicrobial concentration. Our methods have a light workload, avoid most of the traditional CFU plate-counts, and can provide real-time specific growth rates. This latter possibility is useful when investigating the effects of physical and chemical stresses upon microbial growth.
As an illustration of these methods, we present a study of the antimicrobial activity of a commercially available Eucalyptus (Myrtaceae) essential oil against Staphylococcus aureus. Eucalyptus extracts are often used in oral hygiene products (mouth rinses, toothpastes, antiplaque chewing gums), surface cleaning “wipes,” and so forth. Many screening reports, using disk diffusion and dilution techniques, have established an antimicrobial activity of Eucalyptus extracts from various species against a number of pathogens; these reports include CitationInouye et al. (2001) (Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus), CitationFarah et al. (2001) (Escherichia coli, Staphylococcus aureus, Aspergillus niger), CitationTakarada et al. (2004) (Porphyromonas gingivalis, Streptococcus sobrinus, Streptococcus mutans), CitationWilkinson and Cavanagh (2005) (Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa), CitationMounchid et al. (2005) (Escherichia coli), and CitationSchelz et al. (2006) (Escherichia coli, Staphylococcus epidermidis). To our knowledge, there is no published investigation of the effect of Eucalyptus oil on any of the parameters characterizing the growth phases of a microbial suspension.
Our growth paradigm is derived from the “three linear phase” model described by Buchannan et al. (1997), in which TAdapt(c) and μExp(c) are precisely defined. We have the following possible actions when Staphylococcus aureus cells are inoculated in a medium containing a concentration c of Eucalyptus oil:
The oil modifies the adaptation time, TAdapt(c), of the cells to the medium.
The oil causes a cultivable population reduction (CPR) during the adaptation phase (CPRAdapt). Thus, during the adaptation time, the cell concentration will decrease with a specific rate μAdapt(c).
The oil modifies the specific exponential growth rate μExp(c) of the suspension.
The oil modifies the maximum cell concentration, CFUMax(c), of the suspension.
The oil causes a cultivable population reduction during the stationary phase (CPRStat). Thus, during the stationary phase, the cell concentration will decrease with a specific rate μStat(c).
We shall determine TAdapt(c), μAdapt(c), μExp(c), CFUMax(c), μStat(c) for a suspension of S. aureus in a standard growth medium containing a concentration c, 0 ≤ c ≤ 1600 ppm, of Eucalyptus oil and discuss the implications of these data and directions for further studies designed to elucidate the mode of action of Eucalyptus essential oil against these bacteria.
Materials and Methods
Apparatus
The conductance data were obtained with the Rapid Automatic Bacterial Impedance Technique (RABIT: Don Whitley Scientific, Shipley, UK).
Conductance growth medium
Whitley Impedance Broth (W.I.B.), a nonselective medium designed to have relatively large conductivity changes as a function of microbial growth (Don Whitley Scientific), was used.
Bacteria and temperature conditions
Staphylococcus aureus (C.I.P. 103429) was stored in a glycerol peptone medium at −80°C. Freshly thawed aliquots were used for each experiment. The growth temperature was 37°C.
Suspended bacteria culture
A preculture was made by inoculating frozen cells in 10 mL Müller-Hinton broth. After incubation for 24 h at 37°C, 2 mL of the preculture was inoculated in 150 mL Müller-Hinton broth contained in a 250-mL Erlenmeyer flask and shaken at 120 rpm for 24 h in a water bath (type SW23; Julabo, Seelbach, Germany) at 37°C. Samples (0.1 mL) of different cell concentrations were then inoculated into 4 mL W.I.B. (Shipley) medium at 37°C containing a known concentration of antimicrobial agent.
CFU/mL counts
The CFU/mL counts were made using the spiral colony counting method (Whitley Automatic Spiral Plater; Don Whitley Scientific). The inoculated plates of Plate Count Agar (PCA; Difco, Lawrence, Kensas, USA), were incubated for 24 h at 37°C before counting the colonies.
The antimicrobial agent
The Eucalyptus (Eucalyptus globulus) was a commercial essential oil. No further refinement or chemical analysis was performed. It was stored at 4°C. A 10% (volume-volume) mother solution was prepared using DMSO. Eucalyptus oil concentrations in the W.I.B. medium were in the range 0–1600 ppm.
The suspension growth parameters protocol
The protocols and the mathematical analysis behind our methods are fully described in CitationEllison et al. (2007). We recall here the minimum explanation necessary to understand how the results are obtained. We use an automatic conductance method to monitor the microbial growth. With modern devices, one can continuously monitor several hundred suspension samples simultaneously. The principle of the conductance method is that a growing microbial suspension, by the excretion of ionic metabolic substances into the surrounding liquid medium, modifies the conductivity of the growth medium. We have shown that, in the case under consideration, the conductance changes for S. aureus are proportional to the cell concentration. Thus, automatic monitoring of the conductance of the suspension gives real-time information about the microbial cell concentration. For very low cell concentrations, the conductance change is not detectable; as growth continues, the cell concentration increases to a certain value NDet, at which point the apparatus does detect a conductance change. The time, TDet, after inoculation of the microbes in the growth medium at which this occurs is called the “detection time.” For suspension growing in a growth medium containing a concentration c of antimicrobial agent, there is a relationship between the initial cell concentration C0, the adaptation time TAdapt(c), the CPR reduction rate μAdapt(c), the specific exponential growth rate μExp(c), and the detection time:
where the adaptation factor β (c),
The quantities μExp(c), β (c), TAdapt(c), μAdapt(c), CFUMax(c), and μStat(c) can be determined by a series of experimental protocols involving the automatic measurement of detection times. They are described in detail in CitationEllison et al. (2007). The only plate-counting necessary is for the determination of the initial suspension cell concentrations. The first step is to determine μExp(c) and β (c) for a series of inhibitor concentrations. The second step is to determine the adaptation time TAdapt(c) for the same concentrations. Once the adaptation time is known one can determine μAdapt(c) from the above formula, as NDet is known. The penultimate step consists in the determination of CFUMax(c) and whether or not there is a CPR during the stationary phase for each concentration. If there is a CPR, then one must determine μStat(c).
Acquired microbial resistance protocol
We shall see that S. aureus cells in contact with Eucalyptus oil and grown until a stationary phase is reached are resistant to inactivation by the oil. The resistance may be due to a temporary adaptation or to a more permanent modification of the cells. To distinguish between these two possibilities, we evaluate this resistance by growing suspensions of S. aureus in W.I.B. or W.I.B + Eucalyptus oil until the stationary phase is reached, then inoculating stationary phase bacteria into either pure W.I.B. or into W.I.B. + Eucalyptus oil and recording the conductance data. The cycle inoculation, growth to stationary phase is repeated several times. The degree of acquired microbial resistance to the Eucalyptus is reflected in the detection times.
Statistical analysis
Linear regressions were performed using a maximum likelihood analysis rather than a least squares method, as the latter is very sensitive to the occasional and inevitable outlaying points. Nonparametric significance tests for differences between mean values were used, as the necessary hypotheses required for using Student's t-test could not be verified.
Results
The relationship between the initial cell concentration C0 and the conductance detection time TDet for a S. aureus suspension in pure W.I.B. is given by:
where μExp(0) = 1.97, β (0) = 21.18, with r2 = 0.997.
The adaptation time is given by the formula TAdapt(0) = (β (0) − LogNDet)/μExp(0), where NDet is the cell concentration that produces a conductance change detection. For this species of S. aureus, NDet = 5.2× 106 CFU/mL. This gives an adaptation time of TAdapt(0) = 2.9 h for stationary phase S. aureus, cultured in Müller-Hinton broth inoculated into pure W.I.B. medium.
Determination of μExp(c) and β (c)
The specific exponential phase growth rate μExp(c) and the factor β (c) for Eucalyptus oil concentrations in the range 0 ≤ c ≤ 1600 ppm are shown in . A maximum likelihood regression analysis gives μExp(c) = 1.97–0.0007c, with a linear correlation coefficient r2 = 0.98 and β (c) is constant and equal to 22.267, with SD = 0.392. Thus, β (c) is independent of the oil concentration. This is a special situation and, as shown in CitationEllison et al. (2007), suggests that for the oil concentration range 0–1600 ppm, there is a CPR during both the adaptation and the exponential phases with the same specific reduction rate = μExp(0)−μExp(c) = 0.0007c and that the adaptation time TAdapt(c) of S. aureus from the stationary phase in the Müller-Hinton broth to the W.I.B. + Eucalyptus mixture does not depend upon the oil concentration. Thus, TAdapt(c) should be equal to 2.9 h for all c up to 1600 ppm.
Figure 1. Specific growth rate μExp(c) (♦) and β (c) (▪) as functions of Eucalyptus oil concentration.
Adaptation phase results
In , we have the results of the adaptation phase experiments for 500, 1000, and 1500 ppm oil concentrations. After inoculation, the detection time differences between samples with and without oil decrease; this corresponds with the CPR during the adaptation phase. Once the bacteria have adapted to the medium, the exponential growth phase begins with a linear relationship between contact time and detection time difference. The increasing straight line begins when the bacteria have been in contact with the Eucalyptus oil for slightly less than 3 h for each concentration. This represents the adaptation time, which is in very good agreement with the 2.9 h obtained from the previous experiments and confirms that the Eucalyptus oil concentration has no notable effect upon adaptation time.
Figure 2. Contact time versus difference in detection times between growth in pure W.I.B. and for bacteria in contact with Eucalyptus oil (♦ = 500 ppm, ▴ = 1000 ppm, ▪ = 1500 ppm).
As an independent check upon the estimation of TAdapt(c), we carried out CFU plate-counts every 30 min during the period from inoculation up to detection time for an oil concentration of 1000 ppm. This enabled us to determine independently the CPR during the adaptation phase and so determine TAdapt(1000). The results are shown in . Again, the slope change occurs slightly less than 3 h after the inoculation.
Figure 3. CFU/mL counts versus contact time for a concentration of 1000 ppm Eucalyptus oil.
Stationary phase results
The conductance changes as a function of time and Eucalyptus concentration are shown in . In our conditions, the CFU/mL cell concentration is proportional to the conductance change and so the relative cell concentrations are the same as the relative conductance changes (CitationEllison et al., 2007). The maximum CFU concentration is always the same. The only variability is the time taken to reach this maximum value. Thus, CFUMax(c) does not depend upon c.
Figure 4. The conductance changes of the S. aureus suspension as a function of the contact time for each Eucalyptus oil concentration.
The detection times of suspension samples taken during the first 24 h of the stationary phase and inoculated into pure W.I.B. are shown in . The detection times show no significant differences between all the oil concentrations and all stationary phase samples taken over the 24 h period. This means that the stationary phase cell concentrations are not significantly different. Hence, there is no CPR during the first 24 h of the stationary phase.
Figure 5. Detection times of samples taken during the stationary phase and inoculated into pure growth medium.
The microbial resistance persistence
The results for a Eucalyptus oil concentration of 1600 ppm are shown in . The significance of the inoculation growth cycle notation is as follows: For example, “W+E → W → W+E” means:
Samples are inoculated in W.I.B. containing a 1600 ppm Eucalyptus concentration and allowed to grow to a stationary phase.
Samples are then inoculated in pure W.I.B. and allowed to grow to a stationary phase.
Samples are inoculated in W.I.B. containing a 1600 ppm Eucalyptus concentration and allowed to grow until a conductance change is detected.
The detection time of the final suspension is recorded. Each of the cycle experiments was performed with n = 8 samples.
Table 1. Detection times for the various growth to stationary phase cycles.
The detection time for S. aureus growing in pure W.I.B. is 1.4 h. When the cells reach a stationary phase and are re-inoculated into W.I.B. containing 1600 ppm Eucalyptus oil, the detection time is 6.4 h. Cells cultivated in W.I.B. + 1600 ppm Eucalyptus until they reach a stationary phase and then re-inoculated into pure W.I.B. again have a detection time of 1.4 h. Thus, Eucalyptus resistant cells, when inoculated into pure W.I.B. have a normal growth pattern. S. aureus cells that have undergone two or three growth cycles in W.I.B. + 1600 ppm Eucalyptus oil do not show any change in the corresponding detection times. Thus, the acquired resistance of the stationary phase cells is not modified by successive contacts with Eucalyptus oil.
In all samples, there was no significant difference in the microbial growth characteristics from the detection time up to the resulting stationary phase. This means that once the exponential growth phase begins, the past history of the cells does not influence the subsequent microbial growth.
Discussion
It is obviously important to have some information about Eucalyptus oil composition, but large variations exist, both in extraction methods and in the plants themselves. For example, CitationDella Port et al. (1999) analyzed the composition of Eucalyptus globulus. They found that with one type of extraction method, the principal components were 1,8-cineole 62.6%, α -pinene 10.5%, aromadendrene 8%, and p-mentha-1,3,5-triene 1.6%. A different extraction method, using the same source of Eucalyptus leaves, gave the components: 1,8-cineole 48.2%, α -pinene 6.9%, p-mentha-1,3,5-triene 3.8%, and aromadendrene 13.7%. Other published composition analyses naturally vary in the percentages of each component. This illustrates one of the major difficulties working with essential oils, namely the difficulty of obtaining reproducible samples. In our case, this is not a problem.
The principal component is not necessarily the active antimicrobial component. One cannot draw hasty conclusions about the active component of the oil uniquely from a standard chromatography analysis. The antimicrobial effect of Eucalyptus essential oils has sometimes been ascribed to the presence of 1,8-cineole, its principal component. This is not necessarily true. CitationLis-Balchin and Deans (1997) tested Eucalyptus globulus, Eucalyptus radiata, and Eucalyptus citriodora against 20 different strains of Listeria monocytogenes. The major component of Eucalyptus globulus was 1,8-cineole (90.8%), in Eucalyptus radiata it was present at 84%, and in Eucalyptus citriodora it was only present at 0.005%. However, the Listeria strains used in the study did not correlate with the 1,8-cineole concentration. The Eucalyptus globulus inhibited 6 of 20 strains, and the E. radiata and E. citriodora inhibited all 20 strains. The above results indicate that 1,8-cineole is not necessarily the principal active agent, at least for Listeria strains. CitationBelaiche et al. (1995) studied the antimicrobial effects of three components of Eucalyptus oil (1,8-cineole, linalool, and eugenol) upon Staphylococcus aureus. They found that 1,8-cineole had no effect, but the other two terpenes, used individually, had significant effects, but when used at the same time had antagonistic effects. CitationFarah et al. (2001, Citation2002) also confirmed the lack of antimicrobial activity of 1,8-cineole against Staphylococcus aureus, Escherichia coli, and Aspergillius niger.
In this report, we are concerned with the general effect of the oil upon the growth parameters of a S. aureus suspension. The detailed study of each of the principal components of Eucalyptus essential oil will be the object of a separate study.
The adaptation and exponential phase results
For Eucalyptus globulus oil concentrations in the range 0–1600 ppm, there is a CPR during the adaptation phase, with a specific reduction rate μAdapt(c) proportional to the oil concentration. During the exponential growth phase, the observed specific growth rate is the difference between the specific growth rate in pure W.I.B. and μAdapt(c). Thus, the reduction in the specific growth rate during the exponential phase is the same as the CPR during the adaptation phase. This suggests that the action of the Eucalyptus globulus oil does not depend upon the cell states during the adaptation and exponential phases.
The adaptation time of S. aureus to the W.I.B. + oil growth medium over the concentration range is practically constant and corresponds with the adaptation time of S. aureus precultured in Müller-Hinton broth and inoculated into W.I.B. This suggests again that the presence of the Eucalyptus oil in the growth medium does not affect in a substantial way the internal metabolic cell transformations involved in the adaptation process.
The stationary phase results
During the adaptation and exponential phases, Eucalyptus globulus produces a CPR, with a specific reduction rate μ (c) that depends upon the oil concentration c. If this specific reduction rate is less than the cell division rate, then it is to be expected that, once the stationary phase is reached, CFUMax(c) = CFUMax(0). In fact, we do observe that there is no effect upon CFUMax (). Whatever the initial concentration in cells or concentration in Eucalyptus, the final cell concentration in the medium is always the same. The only factor that varies is the time necessary to reach this maximum cell concentration.
During the stationary phase there is no CPR. This is slightly surprising. The hypothesis that there is some component of the oil that has a nonspecific destructive effect upon the integrity of the cell membrane is frequently evoked, often with little or no convincing experimental evidence. If there is some such agent in Eucalyptus oil, then one would also expect it to continue acting during the stationary phase, i.e., one should observe a CPR, and this is not the case.
A possible explanation is that this hypothetical nonspecific inactivating agent is depleted as the suspension grows. If this is the case, then once the inactivating agent has been reduced to a concentration below a threshold level, the instantaneous specific growth rate μ (t,c) in the medium containing a concentration c of Eucalyptus should become equal to μ (t,0) at some point before the stationary phase is reached. The time at which this occurs corresponds with the moment at which all the “inactivating factor” in the Eucalyptus oil has been reduced to below its threshold level. The instantaneous specific growth rates as a function of time and as a function of instantaneous cell suspension concentration are shown in and . We observe that μ (t,c) does not converge to μ (t,0) at some time before the beginning of the stationary phase and that in , μ (t,c) is equal to μ(t,0) for all Eucalyptus oil concentrations only when the cell suspension concentration is close to the stationary phase cell concentration. This indicates that the lack of CPR during the stationary phase is not due to some inhibiting factor in the Eucalyptus that has been depleted.
Figure 6. Instantaneous specific growth rate, as function of time, for different oil concentrations.
Figure 7. Instantaneous specific growth rate as function of the maximum cell concentration percentage for various oil concentrations.
This leaves us with the possibility that it is the bacteria themselves that have changed in some way so as to be resistant to the Eucalyptus oil in the medium. The second representation of the specific growth rates () shows that this adaptation is achieved, for all the Eucalyptus oil concentrations used, when the cell concentration has reached about 70% of its final value. This is very near to the final cell concentration and the microbes are either in, or close to, a stationary phase physiologic state.
The resistance of the stationary phase S. aureus to the Eucalyptus oil suggests the following phenomenological scenario:
Suppose that S. aureus cells possess a “target” via which some component of the Eucalyptus oil inactivates the cell.
When the target is “accessible,” the cell is inactivated. If, due to physiologic transformations within the cell, the target becomes “inaccessible,” then the oil does not inactivate the cell.
When such cells are inoculated into a medium containing Eucalyptus, there is a CPR during the initial adaptation phase and during the exponential growth phase, so the target is accessible.
As the stationary phase is reached, internal physiologic transformations make the target inaccessible and so the cells are not inactivated by the Eucalyptus.
The results indicate that there is no residual effect of the acquired resistance to inactivation of the stationary phase cells by Eucalyptus oil when these cells are re-inoculated into the growth medium containing Eucalyptus oils and that successive growth cycles of cells in a medium containing Eucalyptus oils do not modify the acquired resistance of the stationary phase cells. These observations suggest that the resistance to the Eucalyptus is not due to some mutation effect but that the accessibility-inaccessibility of the Eucalyptus target is the result of reversible physico-biochemical transformations within the cells as they enter the stationary state.
Conclusions
We have applied techniques that enable us to measure quantitatively the action of an antimicrobial agent upon the five characteristic growth parameters of a microbial culture. The methods were used to study the action of Eucalyptus globulus essential oil upon a suspension of Staphylococcus aureus in a standard growth medium. We showed that the Eucalyptus oil produced a cultivable population reduction during both the adaptation phase and the exponential growth phase with the same specific reduction rate. This reduction rate was determined as an explicit function of the oil concentration. The oil does not affect the adaptation time. Our results suggest that the oil inactivates the bacteria via a “target” that is accessible to the oil while the internal cell transformation prepares the cell for its exponential growth period, but later, as the cells transform themselves into the stationary phase, this target ceases to be “accessible” to the oil. This process is reversible; the change from “accessible” to “inaccessible” occurs when the bacteria are transforming into the stationary phase.
The next step in the elucidation of the action of Eucalyptus oil against S. aureus would be to test individually, and in combination, different fractional components of the oil in order to determine those that do have an effect upon the bacteria.
Acknowledgement
We would like to thank the ‘Région d'Aquitaine, France’ for partial financial support in the acquisition of the conductance device and Spiral Plater apparatus.
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