A newly developed assay to study the minimum inhibitory concentration of Satureja spinosa essential oil

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1 Journal of Applied Microbiology ISSN ORIGINAL ARTICLE A newly developed assay to study the minimum inhibitory concentration of Satureja spinosa essential oil N.G. Chorianopoulos 1, R.J.W. Lambert 2, P.N. Skandamis 1, E.T. Evergetis 3, S.A. Haroutounian 3 and G.-J.E. Nychas 1 1 Department of Food Science and Technology, Laboratory of Microbiology and Biotechnology of Foods, Agricultural University of Athens, Athens, Greece 2 Nestle Research Centre Lausanne Vers-Chez-Les-Blanc, Lausanne, Switzerland 3 Department of Science, Laboratory of General Chemistry,Agricultural University of Athens, Athens, Greece Keywords MIC, Satureja, conductance, absorbance, impedance, natural antimicrobials, inhibition. Correspondence P.N. Skandamis, Department of Food Science and Technology, Laboratory of Microbiology and Biotechnology of Foods, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece. pskan@aua.gr 2005/0501: received 9 May 2005, revised 4 August 2005 and accepted 8 September 2005 doi: /j x Abstract Aims: The minimum inhibitory concentration (MIC) of Satureja spinosa essential oil against Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, Salmonella serovar Enteritidis PT4 and Bacillus cereus was comparatively assessed with an established optical density method as well as a novel impedimetric method. Methods and Results: The impedimetric analysis takes into account information of microbial growth, such as detection time, maximum conductance, and slope of the conductance curve. For each pathogen two levels of inoculation were studied, a high (10 5 CFU ml )1 ) and a low level (10 2 CFU ml )1 ). Non-linear regression analysis was used to fit the data using a modification of a previously published model, from which a more exact value can be obtained for the MIC. Both methods gave similar MICs as shown by t-test statistical analysis. Salm. Enteritidis seems to be the least sensitive to the action of S. spinosa essential oil followed by L. monocytogenes, E. coli, B. cereus and Staph. aureus. The MICs of low inoculum were lower than that of high inoculum. Conclusions: The new impedimetric assay of MIC of essential oils can be considered a reliable rapid method for screening antimicrobial effectiveness of natural additives. Significance and Impact of the Study: Determination of the minimum inhibitory concentration of an essential oil with the simple conductance technique and further study of the mode of action of its components is a good combination for obtaining additional knowledge for industrial application of such natural additives. Introduction The antimicrobial action of essential oils in food systems is well known in the literature (Nychas et al. 2003). Their use can be considered an additional intrinsic determinant to increase the safety and shelf life of foods (Tassou et al. 1995; Koutsoumanis et al. 1998; Skandamis and Nychas 2000). However, despite the antimicrobial action of essential oils, their practical application is hampered by some limitations: the strong smell of these substances when used at effective doses and the decrease in their effectiveness when they are added to complicated food ecosystems (Davidson 1997). Therefore, knowledge of the minimum inhibitory concentration (MIC) is important in order to apply the minimum essential concentration capable of preventing microbial growth (Lambert and Pearson 2000). Among the suggested methods for MIC determination of antimicrobials is the innovative-automated technique 778 ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006)

2 N.G. Chorianopoulos et al. A new assay for determining the MIC of Lambert and Pearson (2000), which is a combination of absorbance measurements with the common dilution method (Mann and Markham 1998; Lambert et al. 2001). However, absorbance measurements also have some limitations (Nychas et al. 2003). Specifically, turbidometry detects only the upper part of microbial growth curves, and requires calibration in order to correlate the results with viable counts obtained on agar media (Koch 1981; Bloomfield 1991; Daalgard and Koutsoumanis 2001; Skandamis et al. 2001). In addition, the physiological state of the cells (injured or healthy), the state of oxidation of the essential oil, as well as inadequate dissolution of the compound may also affect absorbance measurements in growth media (Nychas et al. 2003). An impedance technique, on the other hand, is able to monitor microbial metabolism in real time (Nychas et al. 2003) and therefore has an advantage over that of absorbance. Furthermore, impedance measurements are independent of the active state of cells (including their shape and size) and the possible oxidation of the essential oil. With regards to the latter, impedance technique has been successfully used to quantify microbial populations in turbid liquid foods, such as milk (Felice et al. 1999), where optical density measurements are not feasible. In addition, the inhibitory effect of mint essential oil (Tassou et al. 1995) and that of antimicrobials of animal origin, such as immunoglobulins and lactoferrin (Lindmark-Månsson et al. 2000) have also been evaluated with conductance technique in broths and milk, respectively. Due to these reasons, the impedimetric method is recognized as an alternative rapid method not only for screening the biocide activity of novel antimicrobial agents, but also for estimation of growth kinetics in mathematical modeling (Ayres et al. 1993, 1998; Johansen et al. 1995; Tassou and Nychas 1995; Tassou et al. 1995, 1997; MacRae et al. 1997; Koutsoumanis et al. 1998; Lachowicz et al. 1998). The aim of the present study was to develop an alternative rapid method to absorbance methods, based on impedimetry, for the estimation of the MIC of Satureja spinosa essential oil against five common pathogenic bacteria (Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella serovar Enteritidis PT4 and Bacillus cereus) at two different initial inoculum levels and to compare the results with an absorbance technique. Materials and methods Plant material Samples of S. spinosa were collected during the period of flowering on the island of Crete and the essential oil was purified by steam distillation according to Chorianopoulos et al. (2004). Bacterial cultures Staphylococcus aureus (ATCC 6538), E. coli O157:H7 (NCTC 12900), L. monocytogenes Scott A (kindly provided by Dr. Eddy Smid ATO-DLO), Salm. enterica serovar Enteritidis PT4 (kindly provided from University of Surrey) and B. cereus FSS 134 were grown overnight in flasks containing BH broth (Merck cat. No ), with shaking, at the appropriate temperature for each bacteria. The cells were harvested by centrifugation at 3000 g for 15 min and 4 C (ALC 4239R). Subsequently, they were washed twice and resuspended in Ringer solution (LabM) to provide bacterial concentration between CFU ml )1. Antibacterial assays The antimicrobial activity of the tested essential oil was monitored using the following two methods: (a) Conductance method. Bacterial growth was monitored by conductance measurements using the Malthus system (Radiometer International, Copenhagen, Denmark). A typical Malthus conductivity cell contains platinum electrodes that allow the detection of the conductance changes as a response to the bacterial metabolism in the growth medium. Thus, it is feasible to monitor the conductance changes at defined intervals and record the corresponding data. The detected changes are expressed in microsiemens (ls), which are recorded graphically as conductance curves. Three stock solutions of the essential oil (0Æ5, 0Æ4 and 0Æ3% v/v) were prepared by mixing the essential oil directly with BH broth. From the three stock solutions, seven half-fold serial dilutions were made using the growth medium (BH broth), covering the range from 0Æ5 to 0Æ00453% v/v essential oil. Preliminary experiments in our lab with various essential oils have repeatedly showed that mixing essential oils at concentrations up to 0Æ6 0Æ8% with broths (including BH broth) resulted in homogeneous solutions. This is likely associated with the emulsifying properties of the broth ingredients. Therefore, in the current study all the examined concentrations of essential oil, including those of the stock solutions were in the range of essential oil solubility in BH broth. Moreover, direct mixing of essential oil with broth has also been employed in previous reports involving the evaluation of the inhibitory effect of essential oil using impedance (Tassou et al. 2000) and optical density method (Lambert et al. 2001). Specifically, concentrations of mint essential oil (0Æ4 1Æ2%) added directly into nutrient broth (Tassou et al. 2000; NB), as well as oregano essential oil concentrations up to 0Æ1% added directly in tryptone soy broth (Lambert et al. 2001; TSB) were evenly distributed ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006)

3 A new assay for determining the MIC N.G. Chorianopoulos et al. in both laboratory media (i.e. NB and TSB respectively) since they managed to exert the expected inhibitory effect against the tested organisms. The aforementioned stock solutions of essential oil in BH broth were vortexed well (1 min) to ensure sufficient mixing with the growth medium, and the first series of half-fold dilutions (i.e. 0Æ25, 0Æ20 and 0Æ15%) from each stock solution (i.e. 0Æ5, 0Æ4 and 0Æ3%) was prepared immediately to prevent any possible separation of essential oil from broth. The half-fold serial dilutions were then dispensed to 2Æ0 ml final volume into the sterile glass reaction cells of the Malthus. The reaction cells were incubated at 37 C for 30 min and subsequently inoculated with 0Æ2 ml of an 18 h culture of each pathogen grown at optimum temperature in the same growth media. To examine the possible effect of inoculum size on the MIC, two levels of inoculation (10 5 and 10 2 CFU ml )1 ) were used. Each experiment was carried out in triplicate. The reaction cells were incubated for 48 h at 37 C for E. coli and Salm. serovar Enteritidis, 35 C for B. cereus and 30 C for L. monocytogenes and Staph. aureus. Agitation only of those Malthus cells that contained 0Æ4% and 0Æ5% essential oil (i.e. concentrations close to the limits of essential oil solubility) was performed every 6 h, between two successive readings of conductance (6 min interval) to enhance the dissolution of essential oil in the growth medium. b) Optical density (OD) method. Bacterial growth in BH was also monitored through changes in optical density of bacterial suspensions in the presence of multiple concentrations of Satureja essential oil as follows. The same stock solutions and half-fold dilutions of essential oil as those used for conductance measurements were prepared in BH (i.e. the growth medium) as described above. Aliquots (0Æ225 ml) of growth medium mixed with essential oil were transferred to the wells of a 96-well microplate. The bacterial suspensions were diluted tenfold in Ringer s solution (Skandamis et al. 2001; LabM). A 0Æ025 ml portion from the appropriate dilution was added to the wells containing the growth medium (final volume 0Æ25 ml) in order to give a population of approximately 10 5 CFU ml )1 and 10 2 CFU ml )1. Microplates were incubated in an identical manner to the Malthus method, and OD measurements were taken at 600 nm in a microplate reader set to perform 2 s agitations before each OD reading. Each experiment was carried out in triplicate. Analysis The basis of the data analyses is the comparison of the area under the OD/time curve of the control with the areas of the tests. Similarly, the analyses of the Malthus data used the comparison of the area under the conductance/time curve. In both techniques the effect on the growth is manifested by a reduction in the area under the OD/time or conductance/time curve relative to a control well at any specified time. By calculating the area using the trapezoidal rule, the relative amount of growth can be obtained using the ratio of the test area to that of the control, termed the fractional area, fa (Lambert and Pearson 2000). Measurement of MIC and NIC Lambert and Pearson (2000) published an investigation into the dose responses of Staph. aureus and Pseudomonas aeruginosa against several inhibitors. In that publication a Gompertz function was used to fit the observations and produce a dose response profile. The Lambert Pearson model (LPM) of 2000 was updated by Lambert and Lambert (2003), Eqn 1). " fa ¼ exp x # P2 ð1þ P 1 where fa is the fractional area, x is the inhibitor concentration (mg l )1 ), P 1 is the concentration at maximum slope (of a log x vs fa plot) and P 2 is a slope parameter. The MIC for a single substance was defined as the intercept of the concentration axis to the tangent at the maximum gradient of the fa/log concentration curve. From the LPM this can be formulated as MIC ¼ P 1 exp 1 ð2þ P 2 The non-inhibitory concentration is the concentration below which normal visible growth was observed, this was defined as the intercept of the tangent at the maximum gradient of the fa/log concentration curve to the fa ¼ 1 contour. From the LPM this can be formulated as NIC ¼ P 1 exp 1 e ð3þ P 2 where e is the value of the exponential of 1 (approx. 2Æ718). Data were fitted to the LPM using non-linear least squares regression analysis assuming equal variance. A more rigorous approach has been developed by Lambert (2001) but in this work the discrepancy between the assumption made and the use of a weighting scheme to correct for variance had little impact on the parameters found. 780 ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006)

4 N.G. Chorianopoulos et al. A new assay for determining the MIC Results A typical example of the inhibition caused by the essential oil as recorded by the two methods is presented in Fig. 1. The inhibition due to the increase in essential oil concentration is more evident with the conductance curves (a) Absorbance (b) Conductance ( S) Time (min) (Fig. 1b) than with the optical density curves (Fig. 1a). Specifically, the essential oil affects both the detection time and the slope of the conductance curves, whereas in the respective curves of optical density method, concentrations of essential oil lower than the MIC affect only the maximum absorbance. Plotting the inhibitor concentration on a logarithmic scale with the fractional area gives a characteristic sigmoid-shaped curve (Figs 2 and 3). Terms have been assigned to two specific concentrations; the non-inhibitory concentration, NIC, i.e. the concentration above which the inhibitor begins to have a negative effect on growth, and the minimum inhibitory concentration, MIC, which marks the concentration above which no growth is observed relative to the control. The experimental data were modelled using eqn (1). The MIC and NIC values based on optical density and conductance technique, for each pathogen and level of inoculation, were calculated from eqns (2) and (3) respectively (Table 1). It needs to be noted, that the estimated MICs from both techniques were similar (Table 1, Fig. 4). For example, equal MICs were calculated for the high inoculum level (10 5 CFU ml )1 ) of Salm. serovar Enteritidis by both optical density and conductance technique (Table 1). These MICs (0Æ068%) were also the highest observed inhibitory concentrations (Table 1). The MICs for the low inoculum level (10 2 CFU ml )1 ) of the same pathogen were 0Æ063% v/v based on optical density technique and 0Æ064% v/v according to conductance. Similarly, in cases of Staph. aureus, B. cereus, L. monocytogenes and E. coli the calculated MIC was lower at the low level of inoculation (10 2 CFU ml )1 ) for each pathogen (Table 1). From Fig.. 2 and 3 it is evident that lower essential oil concentrations are required to inhibit the lower populations of pathogen. In Fig. 4 plots of the MICs and NICs from the optical density technique against the MICs and NICs from the conductance technique are compared. The results show that there is good agreement between the two methodologies Time (min) Figure 1 Typical graphs for Salm. serovar Enteritidis at 10 5 CFU ml )1 level of inoculation as observed by absorbance measurements (a) and conductance measurements (b) at increasing concentrations: ( ) 0%, ( )0Æ0062%, (s) 0Æ0181%, (n) 0Æ0363%, (d) 0Æ05%, ( )0Æ0625%, ( ) 0Æ0725%, ()) 0Æ1%, (d) 0Æ15% and (e) 0Æ5% (v/v) of S. spinosa essential oil in BH broth. Discussion The potential use of essential oils as natural antimicrobial agents is less exploited than their uses as flavoring and antioxidant compounds. To our knowledge, in contrast to the extended documentation for the majority of essential oils, there is limited, if any, information relevant to the use of S. spinosa essential oil, even as a flavouring agent or as a preservative. However, this plant has been empirically used as an ornamental, pharmaceutical and bee plant in the Cretan diet, and emphasizes the contribution of the Mediterranean diet to the prevention of cardiovas- ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006)

5 A new assay for determining the MIC N.G. Chorianopoulos et al. (a) 1 2 (b) Fractional area 0 4 Fractional area Concentration % v/v Concentration % v/v Figure 2 The inhibition profile of S. spinosa essential oil against Listeria monocytogenes with optical density technique (a) and conductance technique (b); solid symbols for the high level of inoculation (10 5 CFU ml )1 ) and open symbols for the low level of inoculation (10 2 CFU ml )1 ); (d) observed Fractional Area, ( ) predicted Fractional Area. cular disease (Renaud et al. 1995). With regards to the antimicrobial properties of the essential oils of Satureja species, the available information is limited to S. thymbra (Muller-Riebau et al. 1995; Chorianopoulos et al. 2004), S. cuneifolia (Baydar et al. 2004), S. parnassica (Tzakou and Skaltsa 2003; Chorianopoulos et al. 2004), S. hortensis (Gulluce et al. 2003) and S. spinosa (Chorianopoulos et al. 2004). Measurement of the MIC is a basic requirement in studying the antimicrobial activity of preservative agents and biocides (Lambert and Pearson 2000). The MIC of several essential oils against food pathogens has been determined. The most commonly tested essential oils are oregano and thyme (Smith-Palmer et al. 1998; Hammer et al. 1999; Nychas et al. 2003; Burt 2004). Others include sage, clove, lemongrass, tea bush, rosemary and turmeric (Burt 2004). In the recent review by Burt (2004) the reported ranges of MIC for these essential oils are of the magnitude; 0Æ05 1, 0Æ02 1, 0Æ04 4 and 0Æ02 0Æ05% v/v for E. coli, B. cereus, Staph. aureus and L. monocytogenes, respectively. According to our results the MICs of S. spinosa essential oil were closest to that of oregano for the same pathogens. Moreover, in a recent report, we showed that the essential oil of S. spinosa has a bacteriostatic and bactericidal activity on pathogens in very small amounts similarly to oregano essential oil (Chorianopoulos et al. 2004). The two major components of S. spinosa essential oil are carvacrol and thymol that constitute 60% of the total essential oil (Chorianopoulos et al. 2004). These major components are also found at such levels in the composition of oregano essential oil (Kokkini 1994; Kokkini et al. 1997; Muller-Riebau et al. 1997; Aligiannis et al. 2001; Baydar et al. 2004), which is the most commonly studied essential oil for antibacterial use (Sivropoulou et al. 1996; Skandamis and Nychas 2000; Lambert et al. 2001; Skandamis and Nychas 2001). 782 ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006)

6 N.G. Chorianopoulos et al. A new assay for determining the MIC (a) 1 2 (b) Fractional area 0 4 Fractional area Concentration % v/v Concentration % v/v 0 2 Figure 3 The inhibition profile of S. spinosa essential oil against E. coli 0157:H7 with optical density technique (a) and conductance technique (b); solid symbols for the high level of inoculation (10 5 CFU ml )1 ) and open symbols for the low level of inoculation (10 2 CFU ml )1 ); (d) observed Fractional Area, ( ) predicted Fractional Area. Table 1 MIC and NIC values and their standard errors from optical density technique and conductance technique (% v/v concentration of S. spinosa essential oil in BH broth) Microorganism Inoculum level* Optical Density Conductance MIC NIC MIC NIC Staphylococcus aureus (ATCC 6538) High 0Æ042 ± 0Æ001 0Æ025 ± 0Æ003 0Æ043 ± 0Æ002 0Æ027 ± 0Æ001 Low 0Æ035 ± 0Æ003 0Æ015 ± 0Æ002 0Æ036 ± 0Æ002 0Æ020 ± 0Æ002 E. coli O157:H7 (NCTC 12900) High 0Æ066 ± 0Æ002 0Æ039 ± 0Æ001 0Æ064 ± 0Æ001 0Æ041 ± 0Æ002 Low 0Æ054 ± 0Æ001 0Æ025 ± 0Æ004 0Æ056 ± 0Æ001 0Æ030 ± 0Æ002 Listeria monocytogenes Scott A High 0Æ067 ± 0Æ002 0Æ042 ± 0Æ003 0Æ067 ± 0Æ002 0Æ044 ± 0Æ001 Low 0Æ061 ± 0Æ001 0Æ021 ± 0Æ001 0Æ062 ± 0Æ001 0Æ023 ± 0Æ002 Salm. serovar Enteritidis PT4 High 0Æ068 ± 0Æ002 0Æ044 ± 0Æ003 0Æ068 ± 0Æ001 0Æ049 ± 0Æ001 Low 0Æ063 ± 0Æ002 0Æ042 ± 0Æ004 0Æ064 ± 0Æ001 0Æ045 ± 0Æ002 B. cereus FSS 134 High 0Æ057 ± 0Æ003 0Æ040 ± 0Æ002 0Æ058 ± 0Æ002 0Æ043 ± 0Æ003 Low 0Æ049 ± 0Æ001 0Æ022 ± 0Æ001 0Æ050 ± 0Æ002 0Æ023 ± 0Æ002 *Two inoculation levels were used; a high level (10 5 CFU ml )1 ) and a low level (10 2 CFU ml )1 ). ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006)

7 A new assay for determining the MIC N.G. Chorianopoulos et al. (a) MIC optical density MIC conductance (b) NIC optical density NIC conductance Figure 4 Comparison of MIC (a) and NIC (b) values between optical density technique and conductance technique for all pathogens and inoculum levels; MIC OD ¼ 0Æ952 MIC cond +0Æ0033, r 2 ¼ 0Æ992; NIC OD ¼ 0Æ997 NIC cond +0Æ031, r 2 ¼ 0Æ981 ( ) Staphylococcus aureus, (d) E. coli O157:H7, ( ) Listeria monocytogenes, (n) Salm. serovar Enteritidis and ( ) B. cereus; solid symbols for the high level of inoculation (10 5 CFU ml )1 ) and open symbols for the low level of inoculation (10 2 CFU ml )1 ). Lambert et al. (2001) suggested that the inhibitory effect due to the combination of thymol and carvacrol was enough to account for the inhibition observed with the oregano essential oil itself. Moreover, the strong inhibitory effect of essential oil on bacterial membranes (Davidson 1997; Ultee et al. 1999) is highly associated with the presence of these two compounds, which are known to cause structural and functional damage to plasma membranes (Sikkema et al. 1995). In broth system studies a number of different techniques exists for determining the MIC (Burt 2004). The most commonly used methods are the enumeration of populations by viable counts and the optical density measurements. The first method is time-consuming and labor-intensive. Although OD measurements can be automated, it is not a perfect method as does not take into account information such as the possible oxidation of essential oil and the physiological state of cells (Nychas et al. 2003) that may interfere with measurements. Lambert and Pearson (2000) made an advance in the methodology and analysis of data from OD measurements for the determination of MICs. In our case, a further step to this method was to replace the optical density measurements with conductance measurements to overcome some of the problems inherent in the OD technique. Indeed, our results pointed at better curve fittings using impedance measurements than those obtained by optical density measurements, especially, at concentrations near to the MIC. It is possible that, this was due to inactivated cells, which induced changes in absorbance, but not in conductance. As a matter of fact, use of impedance measurements for determining the MIC has the advantage of being more sensitive near to the growth boundaries, where bacterial metabolism is slow. Furthermore, the results related to the MICs of two different initial levels of inoculation, reinforce the practical use of the conductance technique and agreed with the results of Lambert (2000) according to which, inhibition was dependent on the inoculum size. In conclusion, the conductance technique may be used as an alternative rapid method for determining the MIC of novel antimicrobial agents. However, further research is needed in order to evaluate the effectiveness of the MICs of S. spinosa essential oil in food ecosystems and to establish whether this natural compound has a role as an antimicrobial agent in food safety. Moreover, additional knowledge on the mode of action of such components in combination with data on their MICs would allow for effective industrial application of such natural additives. References Aligiannis, A., Kalpoutzakis, E., Mitaku, S. and Chinou, I.B. (2001) Composition and antimicrobial activity of the essential oils of two Origanum species. J Agric Food Chem 49, Ayres, H.M., Furr, J.R. and Russell, A.D. (1993) A rapid method of evaluating permeabilizing activity against Pseudomonas aeruginosa. Lett Appl Microbiol 17, Ayres, H.M., Payne, D.N., Furr, J.R. and Russell, A.D. (1998) Use of the Malthus-AT system to assess the efficacy of permeabilizing agents on the activity of antibacterial agents 784 ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006)

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