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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

This study examined the antibacterial mode of action of Taxus cuspidata leaf essential oil (TCEO) against foodborne pathogenic bacteria. Gas chromatography–mass spectrometry analysis of microwave-extracted TCEO resulted in examination of 34 different compounds, representing 81.63% of the total oil. The TCEO (1,000 μg per disc) showed antibacterial effect against Bacillus cereus ATCC 13061, Staphylococcus aureus ATCC 12600, Listeria monocytogenes ATCC 7644, Salmonella typhimurium ATCC 43174 and Escherichia coli ATCC 43889 as diameters of inhibition zones (22.0 ± 0.4 to 34.0 ± 1.2 mm). The minimum inhibitory concentration and minimum bactericidal concentration values of TCEO against the tested pathogens were found in the range of 250–1,000 μg/mL. Also, the TCEO had potential inhibitory effect on the viable counts of test pathogens. The TCEO revealed its mode of action on membrane integrity as confirmed by marked release of extracellular adenosine triphosphate, 260 nm absorbing materials and potassium ion efflux. The scanning electron microscopy analysis using TCEO further confirmed severe morphological alterations on the cell membrane of test pathogens.

Practical Applications

Because of frequent foodborne outbreaks and rising concern of consumers on the use of chemical preservatives in foods, the food industry is emphasizing the use of natural antibacterial agents from plant origin as safe food preservatives. Some selected plant materials are used as natural antimicrobials in food systems to prevent the growth of foodborne pathogens, resulting in the extension of shelf life of processed foods. Taxus cuspidata leaf essential oil (TCEO) is shown by this study to elicit its antibacterial action against foodborne pathogens through membrane permeabilization and can be used to control the growth of several foodborne pathogens. Hence, it is anticipated that TCEO may have greater potential for its practical application as an affective food preservative.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

Foodborne pathogens are a major public health concern in both developed and developing countries, and account for considerably high cases of human and animal diseases (Tayel and El-Tras 2010). The World Health Organization characterizes this issue as “one of the most prevalent health problems and a major cause of the reduction in economic output” (WHO 2008). Also, the increasing international trade in commodities and food products has raised the risk of spreading pathogenic bacteria from production sites to distant places. Furthermore, contamination with foodborne pathogens is a major problem in livestock production (Sittiwet and Puangpronpitag 2009). This has resulted on extensive use of chemical preservatives to prevent growth of foodborne pathogens in food industry (Natta et al. 2008). However, there has been an increasing consumer demand for foods free from toxic effects of added synthetic preservatives because some chemical preservatives have been associated with gastrointestinal disorders, hypersensitivity, allergic reaction, immunity suppression, and carcinogenic and teratogenic attributes (Pundir et al. 2010). In addition, bacterial resistance to currently used antibiotics has also led to the development of new and safer antimicrobial agents from plant origin to combat against various infectious diseases (Cock 2008).

Moreover, greater consumer awareness regarding the use of synthetic preservatives can lead to negative health consequences, which in turn has encouraged food processors to look for natural, effective and nontoxic food additives for using in food system with a broad spectrum of antimicrobial activity (Burt 2004). Food safety is an essential concern of both consumers and the food industry, especially as the number of reported cases of foodborne infections continues to increase (Alzoreky and Nakahara 2003). Plant-based essential oils are gaining vital importance as potential food preservatives and are considered generally recognized as safe by the United States Food and Drug Administration. Moreover plant-based essential oils showed a wide acceptance from consumers (Burt 2004). The antimicrobial components are commonly found in the essential oil fractions and it is well established that many have a wide spectrum of antimicrobial activity, with the potential for control of foodborne pathogens within food systems (Burt 2004).

Essential oils are natural volatile organic compounds that can be obtained by distillation, enfleurage, expression or solvent extraction, but the method of microwave-assisted distillation is the most commonly used for commercial production (Bousbia et al. 2009). Essential oils have been shown to possess antibacterial, antiviral, antifungal, insecticidal, repellant, anti-inflammatory, spasmolytic and antioxidant properties (Burt 2004; Cakir et al. 2004). Moreover, use of essential oils in consumer goods is expected to increase in the future because of the risk of “green consumerism,” which stimulates the use and development of plant-derived products (De Silva 1996; Burt 2004), as both consumers and regulatory agencies are more comfortable with the use of natural antimicrobials.

Taxus cuspidata Sieb et Zucc. (Japanese yew or spreading yew) is a member of the genus Taxus, native to Japan, Korea, northeast China and Russia, which is a broadly columnar, evergreen shrub with linear, spiny-tipped, dark green leaves. T. cuspidata has been used in traditional medicine in the treatment of inflammation, renal disorders, cancer and diabetes (Wang et al. 2007). Recently, T. cuspidata has been found to possess antidiabetic activity in streptozotocin-induced diabetic mice (Zhang et al. 2012). Previously, an anticancer compound, paclitaxel, isolated from T. cuspidata stem has also confirmed its role on microtubule prevention from disintegration (Schiff et al. 1979; Wang et al. 2007). However, various potentially toxic chemicals containing cardiotoxic taxine alkaloids present in Taxus species are of great concern (Wilson et al. 2001), and more than 150 taxanes and other compounds have been isolated and characterized from T. cuspidata (Wang et al. 2010).

Although antimicrobial efficacy of various essential oils has been reviewed previously, to the best of our knowledge, no systematic research on the chemical composition and mode of antimicrobial action of microwave-assisted extracted T. cuspidata leaf essential oil (TCEO) had been conducted so far against a wide range of foodborne microorganisms. Therefore, this study was undertaken in order to investigate the effectiveness of TCEO on survival and growth of selected foodborne pathogens using in vitro models. Furthermore, antibacterial mechanism of action was investigated by determining the release of extracellular adenosine triphosphate (ATP), potassium ions and cellular materials, and morphological alterations were investigated by scanning electron microscopy (SEM).

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

Chemicals and Instruments

The standard antibacterial compound, tetracycline, was purchased from Sigma-Aldrich (St. Louis, MO). Spectrophotometric measurements were performed using a 96-well microplate enzyme-linked immunosorbent assay (ELISA) reader (Infinite M200, Tecan, Männedorf, Switzerland) and a luminometer (Synergy HT multimode microplate reader, Biotek, Winooski, VT).

Plant Material and Oil Extraction

The leaves of T. cuspidata were collected from the campus of Yeungnam University and identified by the help of a botanist, and a voucher specimen number (YU-TCS 0072) has been deposited in the library of School of Biotechnology, Yeungnam University, Korea. The leaves were dried under shade at room temperature. The dried material (200 g) was immersed in water (2 L) and subjected to hydrodistillation using a microwave-assisted extraction apparatus for 2 h. The microwave extraction apparatus was purchased from KMD Engineering, Yangju-si, Korea. The commercial microwave apparatus was fitted with automated thermo controller system having oven power capacity of 40 W, operating at a frequency of 15 GHz. The distillate was collected and mixed with dichloromethane, shaken and kept in a separating funnel. The lower layer of dichloromethane containing the essential oil was collected and evaporated using rotary evaporator at room temperature. Finally, the oil was dried over anhydrous sodium sulfate and preserved in a sealed vial at 4C until tested and analyzed.

Gas Chromatography–Mass Spectrometry Analysis

A detailed chemical composition analysis of TCEO was performed using a gas chromatography–mass spectrometry (GC-MS) system (Jeol JMS 700 mass spectrometer [JEOL Ltd., Tokyo, Japan]) equipped with an Agilent 6890N GC DB-5 MS-fused silica capillary column (30 × 0.25 m i.d., film thickness 0.25 μm; Agilent Technologies, Inc., Santa Clara, CA). For GC-MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as the carrier gas at a constant flow rate of 0.06 L/h. Injector and MS transfer line temperature were set at 280 and 250C, respectively. The initial oven temperature of 50C was maintained for 2 min, and then increased to 250C at a rate of 10C/min followed by holding at 250C for 10 min. Diluted samples (1/100, v/v, in methanol) of 1.0 μL were injected manually in the split-less mode. The relative percentage of the oil constituents was expressed as percentages by peak area normalization. Identification of the TCEO components was based on GC retention time on a DB-5 capillary column relative to computer matching of electron ionization mass spectra using Wiley and NIST libraries for the GC-MS system (Adam 2001).

Test Microbial Strains

The foodborne pathogenic bacteria used in this study included Bacillus cereus ATCC 13061, Escherichia coli ATCC 43889, Listeria monocytogenes ATCC 7644, Staphylococcus aureus ATCC 12600 and Salmonella typhimurium ATCC 43174. The bacterial pathogens were obtained from the Korea Food and Drug Administration, which were maintained on nutrient agar medium at 4C.

Determination of Antibacterial Activity by Agar Disc Diffusion Assay

Standard agar diffusion method was used for the determination of antibacterial efficacy of TCEO (Bajpai et al. 2009). Petri plates were prepared by pouring 20 mL of nutrient agar medium and allowed to solidify. Plates were dried, and 1 mL of standardized inoculum containing 107 cfu/mL of bacterial suspension was poured and uniformly spread, and the inoculum was allowed to dry for 5 min. A Whatman no. 1 sterile filter paper disc (6 mm in diameter; Whatman plc, Maidstone, UK) was impregnated with 10 μL (1,000 μg per disc) of TCEO. The air-dried discs were placed on nutrient agar plates and incubated at 37C for 24 h. The TCEO was dissolved in 5% dimethyl sulfoxide (DMSO). Negative controls were prepared using only DMSO. Standard reference antibiotic, tetracycline (20 μg per disc, from Sigma-Aldrich), was used as the positive control against the tested foodborne pathogenic bacteria. Diameters of zone of clearance surrounding the discs (including the disc) were measured in millimeter using a Vernier calliper (Sciencetown, Daejeon, Korea). Each assay in this experiment was performed in triplicate.

Determination of Minimum Inhibitory and Minimum Bactericidal Concentrations

The minimum inhibitory concentration (MIC) of the TCEO was tested by twofold serial dilution method (Bajpai et al. 2009). The TCEO was first dissolved in DMSO and incorporated into nutrient broth (NB) medium for bacterial pathogens to obtain a concentration of 2,000 μg/mL, and serially diluted to achieve 1,000, 500, 250, 125, 62.5, 31.25, 15.62 and 7.81 μg/mL, respectively. Overnight broth cultures (10 μL) of tested pathogens (approximately 107 cfu/mL) were transferred to each tube. The control tubes containing only bacterial suspensions were incubated at 37C for 24 h. The lowest concentration of TCEO, which did not show any visible turbidity of test organisms after macroscopic evaluation, was determined as MIC, which was expressed in μg/mL. Further, the concentrations showing complete inhibition of visual growth of bacterial pathogens were identified, and 50 μL of each culture broth was transferred on the agar plates and incubated for specified time and temperature as mentioned earlier. The complete absence of growth of bacterial colonies on the agar surface is the lowest concentration of sample and was defined as the minimum bactericidal concentration (MBC). The determinations were performed in triplicate.

Effect of TCEO on Viable Counts of Bacterial Pathogens

Active cultures for viable count assay were prepared in NB medium, grown at 37C for 24 h. Simultaneously, stock bacterial suspensions for a number of viable counts were prepared by microbial spread plate count method so that the final suspension contained approximately 107 cfu/mL. For each strain, 1 mL of active stock solution (approximately 107 cfu/mL) was transferred to 2 mL of Eppendorf tube (DASLAB, Barcelona, Spain). The cultures were then centrifuged at 10,000 rpm for 10 min. The pellets were retained and resuspended with 1 mL of phosphate-buffered saline (PBS). For viable counts, each of the tubes containing resuspended bacterial suspension (approximately 107 cfu/mL) of B. cereus ATCC 13061 and E. coli ATCC 43889 was inoculated with 100 μL of TCEO at MIC in 900 μL NB, and kept at 37C. Samples for viable cell counts were taken out at 0-, 40-, 80-, 120-, 160- and 200-min time intervals. The viable plate counts were monitored as follow: after incubation, 100 μL of the resuspended culture was diluted in 900 μL PBS, thereby diluting it 10-fold. One hundred microliters of sample of each treatment was diluted and spread on the surface of NB agar. The colonies were counted after 24 h of incubation at 37C (Bajpai et al. 2009). The controls were inoculated without TCEO for each bacterial strain with same experimental condition as mentioned earlier. Each assay in this experiment was replicated three times.

SEM Analysis

To determine the effect of TCEO, at MIC, on the morphology of B. cereus ATCC 13061 and E. coli ATCC 43889, a SEM study was performed. Control samples were prepared without TCEO. Further, to observe the morphological changes, the SEM analysis was modified from the previous method (Kockro et al. 2000; Bajpai et al. 2009). The bacterial samples were washed gently with 50 mM phosphate-buffered solution (pH 7.2), fixed with 100 mL glutaraldehyde (2.5%) and 100 mL osmic acid solution (1%). The specimens were dehydrated using sequential exposure per ethanol concentrations ranging from 50 to 100%. The ethanol was replaced by tertiary butyl alcohol. After dehydration, the specimens were dried with carbon dioxide. Finally, the specimens were sputter coated with gold in an ion coater for 2 min, followed by microscopic examinations using a scanning electron microscope (S-4300; Hitachi, Hitachi City, Japan).

Measurement of Extracellular ATP Concentration

To evaluate the detrimental the effect of TCEO on membrane integrity, the extracellular ATP concentrations were determined (Lee et al. 2002). The working cultures of B. cereus ATCC 13061 and E. coli ATCC 43889 containing approximately 107 cfu/mL were centrifuged for 10 min at 1,000 × g and the supernatants were discarded. The cell pellets were washed thrice using sodium phosphate buffer (0.1 mol/L; pH 7.0), and cells were collected by centrifugation under the same experimental conditions. A cell suspension (107 cfu/mL) was prepared with 9 mL of sodium phosphate buffer (0.1 M; pH 7.0), and 0.5 mL of cell solution was taken into the Eppendorf tube for the treatment of TCEO. Then, the different concentrations (MIC) of TCEO were added to the cell solution. Samples were maintained at room temperature for 30 min, centrifuged for 5 min at 2,000 × g and incubated on ice immediately to prevent ATP loss until measurement. The extracellular (upper layer) ATP concentrations were measured using an ATP bioluminescent assay kit (Sigma-Aldrich), which comprised ATP assay mix containing luciferase, luciferin, MgSO4, dithiothreitol, ethylenediaminetetraacetic acid, bovine serum albumin and tricine buffer salts. After an addition of 100 μL of ATP assay mix to 100 μL of supernatant, the extracellular ATP concentration of the supernatants was determined using a luminometer (Synergy HT multimode microplate reader, Biotek). The excitation and the emission wavelengths were 420 and 520 nm, respectively; excitation band pass and emission band pass were 1 and 2 nm, respectively.

Assay of Potassium Ions Efflux

A previously described method (De Souza et al. 2010) was used to determine the amount of the released potassium ion (K+). The free K+ ion concentration in bacterial suspensions of B. cereus ATCC 13061 and E. coli ATCC 43889 was measured after the exposure of bacterial cells to TCEO at the MIC in sterile peptone water (0.1 g/100 mL) for 0, 30, 60 and 120 min. The extracellular K+ ion concentrations were measured photometrically using a kalium/potassium kit (Quantofix, GmbH, Wiesbaden, Germany) at each preestablished interval. Controls were tested similarly without TCEO, and the results were expressed as amount of extracellular free potassium (mmol/L) in the culture media in each interval of incubation with respect to time.

Measurement of Release of 260 nm Absorbing Cellular Materials

The release of 260 nm absorbing materials such as DNA and RNA from B. cereus ATCC 13061 and E. coli ATCC 43889 cells was measured according to the method described by De Souza et al. (2010). Aliquots of 2 mL of the bacterial inocula in sterile peptone water (0.1 g/100 mL) were incubated at 37C in the presence of TCEO at final concentrations equivalent to their MICs. At the time intervals of 0-, 30- and 60-min treatment, cells were centrifuged at 3,500 × g and the absorbance of the collected supernatant was determined by 96-well plate ELISA reader at 260 nm. Controls were tested similarly without TCEO. The concentration of the cell constituents released was expressed in terms of optical density (OD260) in each interval with respect to the last time.

Statistical Analysis

All experiments were performed in triplicate. The data were recorded as mean ± standard deviation by measuring three independent replicates. One-way analysis of variance followed by Duncan's test was performed to test the significance of differences between means obtained among the treatments at 5% level of significance using a SAS software (version 9.2; SAS Institute, Inc., Cary, NC). Differences were considered significant at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

Chemical Composition of TCEO

The GC-MS analysis of the TCEO led to the identification of 34 different components, representing 81.63% of the total oil. The identified compounds are listed in Table 1 as a relative peak area of each constituent according to their elution order on DB-5 MS-fused silica capillary column. The microwave-assisted hydrodistillation of T. cuspidata leaves gave light yellowish oil with the major proportion of the oil containing oxygenated mono- and sesquiterpenes, diterpenes, aliphatic hydrocarbons, aliphatic and aromatic acids, phenolics, alkaloids, and esters as well as imidazole, quinoline and isoquinoline derivatives along with some other essential phytoconstituents. Previously we found chemical composition analysis and antioxidant efficacy of T. cuspidata stem essential oil (data not shown). When compared, it was found that T. cuspidata stem essential oil contained similar proportions of monoterpenes, sesquiterpenes, diterpenes, phenolics, hydroxy derivatives, fatty acids and their esters, steroids and alkaloids. However, the major individual components in T. cuspidata stem essential oil were noted to be as ethyl linoleolate (9.0%), longiborneol (7.9%), 13-diepoxy-14,15-bisnorlabdane (7.0%), ambrettolide (4.5%), allylhydroquinone (2.7%), hexahydrofarnesol (2.6%), 2,2-dimethyl-3-hexanol (2.3%), α-3-ethyl-lumiflavin (2.3%), chavicol (2.2%), 2 (3H)-dihydromethyl furanone (2.0%) and 3-β-hydroxyandrostan-5,7-diene (2.0%).

Table 1. GC-MS Chemical Composition Analysis of the Essential Oil Obtained by Microwave-Assisted Hydrodistillation from Taxus cuspidata Leaves
No.SIaRTb (min)CompoundcCompositiond (%)Methode
  1. a

    Library search purity value.

  2. b

    Retention time.

  3. c

    Compounds listed in order of elution from a DB-5 capillary column.

  4. d

    Percentage based on gas chromatography–mass spectrometry (GC-MS) peak normalization.

  5. e

    Identification based on computer matching of electron ionization mass spectra using Wiley and NIST libraries for the GC-MS system.

  6. EI-MS, electron impact-mass spectroscopy; RT, retention time; SI, Search Index (library search purity value).

 16622.78Crotonic acid1.06EI-MS
 26923.93n-Hexyl vinylalcohol3.54EI-MS
 37714.73Benzyl alcohol0.69EI-MS
 47135.23Octilin1.37EI-MS
 58065.70Linalool1.23EI-MS
 63946.63Nor-antipyrine methylated1.71EI-MS
 78847.13α-Terpineol2.45EI-MS
 87637.23Myrtenol0.94EI-MS
 96937.512,3-Dihydrobenzofuran0.93EI-MS
106317.61Geraniol0.81EI-MS
116417.663-Phenylpropanol0.47EI-MS
125247.80Benzene acetic acid0.49EI-MS
135567.98Nerol1.36EI-MS
148288.49Endobornyl acetate1.82EI-MS
155328.54p-Cymen-α-ol0.34EI-MS
166569.693,4-Xylenol2.83EI-MS
1751810.33E-procainamide4.59EI-MS
1846410.73β-Elemenone1.61EI-MS
1971212.44Ethyl phthalate28.15EI-MS
2076712.49(–) Caryophyllene oxide2.17EI-MS
2150915.17Isophytol0.88EI-MS
2243116.003-Methyl-4,4-diphenyl-2-cyclohexen-1-one4.20EI-MS
2365916.39Butyl phthalate1.08EI-MS
2445416.422-Quinoline carboxylic acid0.90EI-MS
2518516.52Norhydrofluorocurine1.20EI-MS
2648916.592-Acetyl-1,2-dihydro-1-isoquinoline-carbonitrile1.26EI-MS
2742116.923-Eicosyne0.94EI-MS
2854017.89Neophytadiene1.29EI-MS
2967118.00Methyl stearate0.97EI-MS
3023218.071,2-Benzenedicarboxylic acid0.85EI-MS
3127821.352,3-Dimethyl-3H-phenanthrol-[3,4-D]imidazol-10-ol1.29EI-MS
3235921.52Dihydro-5-tetradecyl-2-(3H)-furanone1.31EI-MS
3328524.65Didesethylflurazepam dehydration product2.99EI-MS
3457226.23Aspidofractinine-3-methanol2.65EI-MS

Antibacterial Activity

Preliminary screening of in vitro antibacterial activity of TCEO against the tested foodborne pathogenic bacteria was qualitatively and quantitatively determined by the presence or absence of inhibition zones. The data obtained from the disc diffusion method indicated that the TCEO at 1,000 μg per disc displayed a variable degree of antimicrobial activity against the tested foodborne pathogenic bacteria, including both gram-positive and gram-negative bacteria (Table 2). In this assay, B. cereus ATCC 13061, L. monocytogenes ATCC 7644 and St. aureus ATCC 12600 were found to be the most inhibited and susceptible bacterial pathogens by the TCEO with their respective diameters of inhibition zones of 34.0 ± 1.2, 27.0 ± 0.3 and 34.0 ± 0.8 mm, whereas Sa. typhimurium ATCC 43174 and E. coli ATCC 43889 were inhibited moderately with diameters of inhibition zones of 22.0 ± 0.4 and 24.0 ± 0.6 mm, respectively (Table 2). Moreover, TCEO exhibited significant and greater antibacterial efficacy as did by the standard compound tetracycline at the used concentration. The diameters of inhibition zones of TCEO against gram-positive bacteria were found to be higher than gram-negative bacteria. The DMSO as a negative control had no inhibitory effect at the used concentration.

Table 2. Antibacterial Activity of Taxus cuspidata Leaf Essential Oil against Foodborne Pathogens
Bacterial pathogenDiameter of inhibition zone (mm)TCEO
TCEO*TetracyclineMICMBC§
  1. *Diameter of inhibition zones of T. cuspidata leaf essential oil (TCEO), tested volume 10 μL, corresponding to 1,000 μg per disc.

  2. †Standard antibiotic tetracycline (20 μg per disc).

  3. ‡Minimum inhibitory concentration (MIC; values in μg/mL).

  4. §Minimum bactericidal concentration (MBC; values in μg/mL).

  5. Values in the same column with different superscripts are significantly different (P < 0.05). Data are expressed as mean ± standard deviation (n = 3).

Bacillus cereus ATCC 1306134.0 ± 1.2a25.5 ± 0.1b250500
Listeria monocytogenes ATCC 764427.0 ± 0.3b27.0 ± 0.7a5001,000
Staphylococcus aureus ATCC 1260034.0 ± 0.8a22.0 ± 0.2c250500
Salmonella typhimurium ATCC 4317422.0 ± 0.4d12.5 ± 0.2e5001,000
Escherichia coli ATCC 4388924.0 ± 0.6c14.4 ± 0.7d5001,000

MIC and MBC

In this assay, the TCEO showed potent inhibitory effect as MIC and MBC values against all the investigated foodborne pathogens. As presented in Table 2, the MIC and MBC values of TCEO against the tested gram-positive bacteria, B. cereus ATCC 13061, L. monocytogenes ATCC 7644 and St. aureus ATCC 12600 were found in the range of 250–500 μg/mL, whereas for gram-negative bacteria such as Sa. typhimurium ATCC 43174 and E. coli ATCC 43889, the MIC and MBC values were ranged from 500 to 1,000 μg/mL. Among the bacteria, B. cereus and St. aureus were found to be the most susceptible pathogens to the TCEO, with MIC and MBC values of 250 and 500 μg/mL, respectively. Although both gram-positive and gram-negative bacteria were found susceptible to TCEO in this study, Sa. typhimurium ATCC 43174 and E. coli ATCC 43889 showed less susceptibility to TCEO.

Cell Viability

Based on the susceptibility of the investigated foodborne pathogens, one gram-positive (B. cereus ATCC 1306) and a gram-negative (E. coli ATCC 43889) bacteria were selected as the model organisms for further studies to confirm the mechanism of antibacterial action of TCEO. In this regard, further study was carried out to evaluate the effect of TCEO on the viable counts of the selected bacteria such as B. cereus ATCC 13061 and E. coli ATCC 43889. The effect of TCEO on the growth of investigated bacterial pathogens demonstrated reduced cell viability at the MIC (Fig. 1). The effect of 0- to 80-min exposure of TCEO did not cause severe decline on the inhibition of cell viability of the tested pathogens; however, considerable amount of inhibitory effect was observed on the inhibition of the cell viability of the tested bacteria of E. coli ATCC 43889 and B. cereus ATCC 13061 at the exposure time of 120 min. Interestingly, the exposure of the TCEO for 160 min revealed complete inhibition of cfu numbers against B. cereus ATCC 13061 (Fig. 1a) and E. coli ATCC 43889, and no cfu formation was observed against the tested pathogens (Fig. 1b).

figure

Figure 1. Effect of Taxus cuspidata Leaf Essential on the Viability of the Tested Foodborne Pathogenic Bacteria, Bacillus cereus ATCC 13061 (a) and E. coli ATCC 43889 (b); CT: Control without Treatment

Data are expressed as mean ± standard deviation (n = 3).

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SEM Analysis

The electron microphotographs from SEM reveal various morphological damages because of the treatment with appropriate antimicrobial agents; hence, SEM analyses were carried out to further visualize the effect of TCEO on the surface morphology of B. cereus ATCC 13061 and E. coli ATCC 43889 cells as compared with the controls (Fig. 2). Untreated cells (control) presented no changes in cell surface morphology of the investigated foodborne pathogens and showed a regular, complete and smooth surface (Fig. 2a,d). On the contrary, B. cereus ATCC 13061 and E. coli ATCC 43889 cells treated with TCEO at MIC (250 and 500 μg/mL, respectively) revealed severe detrimental effect on the cell morphology of the investigated pathogens, showing disruption of cell membrane and swelling of the cells (Fig. 2b,e). Moreover, initial exposure of TCEO to the tested pathogens revealed large surface collapse and abnormal cell breaking, as well as complete lysis or dead cell formation (Fig. 2c,f).

figure

Figure 2. Scanning Electron Microscopy of Bacillus cereus ATCC 13061 (a–c) and Escherichia coli ATCC 43889 (d–f) Treated with Taxus cuspidata Leaf Essential Oil

(a and d) Controls showing a regular and smooth surface. (b and e) Disruption and swelling of the cells. (c and f) Surface collapse or lysed cell formation.

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Extracellular ATP Concentration

The effect of TCEO on the determination of extracellular ATP concentrations in B. cereus ATCC 13061 and E. coli ATCC 43889 cells is presented in Fig. 3. The extracellular ATP concentration in the untreated cells (control) of B. cereus ATCC 13061 and E. coli ATCC 43889 was 0.94 and 0.20 pg/mL, respectively (Fig. 3). B. cereus ATCC 13061 and E. coli ATCC 43889 cells treated with TCEO at the MIC showed significant (P < 0.05) increase in the release of extracellular ATP concentration, indicating that the TCEO was able to cause pore formation, which allowed the passage of ATP molecules from the treated cells. In this assay, the extracellular ATP concentrations for B. cereus ATCC 13061 and E. coli ATCC 43889 cells were measured to be 1.82 (Fig. 3a) and 1.51 pg/mL (Fig. 3b), respectively. This phenomenon could be due to a consequence of the membrane damage induced by the TCEO, resulting in the increased extracellular ATP concentrations from the treated cells.

figure

Figure 3. Effect of Taxus cuspidata Leaf Essential Oil (TCEO) on Extracellular Adenosine Triphosphate (ATP) Concentration of Bacillus cereus ATCC 13061 (a) and E. coli ATCC 43889 (b)

Data are expressed as mean ± standard deviation (n = 3). Values with different superscripts are significantly different (P < 0.05).

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Potassium Ions Efflux

Further mechanism of antibacterial action of TCEO against the tested foodborne pathogens was confirmed using the assay for efflux of K+ ions from the treated cells of B. cereus ATCC 13061 and E. coli ATCC 43889 at the MIC (250 and 500 μg/mL, respectively) (Fig. 4). In this assay, the leakage of K+ from the bacterial cells occurred immediately after the addition of TCEO following a steady loss along the evaluated intervals (Fig. 4a,b). However, no leakage of K+ ions was observed when B. cereus ATCC 13061 and E. coli ATCC 43889 grew in media without TCEO (control). K+ ion efflux of gram-positive bacterium was 1.2 times higher than gram-negative bacterium.

figure

Figure 4. Effect of Taxus cuspidata Leaf Essential Oil on the Leakage of Potassium Ions from the Tested Foodborne Pathogenic Bacteria Bacillus cereus ATCC 13061 (a) and E. coli ATCC 43889 (b), CT: Control without Treatment

Data are expressed as mean ± standard deviation (n = 3).

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Release of 260 nm Absorbing Materials

An additional approach for determining the mechanism was performed to confirm the antibacterial action of TCEO against gram-positive and gram-negative bacteria of foodborne origin on the basis of release of cell constituent such as DNA and RNA (260 nm materials) from the treated cells of B. cereus ATCC 13061 and E. coli ATCC 43889. The optical density at 260 nm (OD260) of the culture filtrates of B. cereus ATCC 13061 and E. coli ATCC 43889 cells exposed to TCEO at the MIC revealed an increasing release of 260 nm absorbing materials with respect to exposure time (Fig. 5). However, no changes in the OD260 of untreated cells (control) of B. cereus ATCC 13061 and E. coli ATCC 43889 were observed during the study. After 60 min of treatment, approximately more than twofold increase was observed in the optical density of the bacterial cell culture filtrates treated with TCEO (Fig. 5a,b). A significant loss (P < 0.05) of cytoplasmic constituents indicated an important and irreversible damage to the plasma membrane from the bacterial cells treated with TCEO.

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Figure 5. Effect of Taxus cuspidata Leaf Essential Oil (TCEO) on the Release Rate of 260 nm Absorbing Material from Bacillus cereus ATCC 13061 (a) and E. coli ATCC 43889 (b)

Data are expressed as mean ± standard deviation (n = 3). Values with different superscripts are significantly different (P < 0.05).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

In this study, the results of the antibacterial screening including the results from disc diffusion assay and MIC and MBC values illustrated that TCEO had strong and consistent inhibitory effect against some representative foodborne pathogens as confirmed by its inhibitory effect showing different susceptibility rate against the tested foodborne pathogens. In recent years, several researchers have reported that monoterpene or sesquiterpene hydrocarbons and their oxygenated derivatives, which are the major components of essential oils, exhibit potential antimicrobial activity (Burt 2004; Cakir et al. 2004; Bajpai et al. 2008). These findings strongly support the outcomes of this study as the TCEO was also found to contain oxygenated sesquiterpenes and their respective hydrocarbons, confirming its efficacy as natural antimicrobial agent.

In addition, the results from cell viability assay revealed that exposure of TCEO had a rigorous effect on the cell viability of the investigated foodborne pathogens. The TCEO exerted its maximum bactericidal activity as evident by the significant reduction in microbial counts and complete inhibition of B. cereus ATCC 13061 and E. coli ATCC 43889 cells at the exposure of 160 min for both the tested pathogens. Previously, we have confirmed the inhibitory effects of various plant-based essential oils on the cell viability of various foodborne and food spoilage pathogens (Bajpai et al. 2008, 2009, 2012).

The SEM generated photomicrographs of the investigated pathogens showed changes in cell morphology and topography. The distortion of the cell physical structure may cause the expansion and destabilization of the membrane, hence can increase membrane fluidity, which in turn can increase passive permeability and manifest itself as a leakage of various vital intracellular constituents, such as ions, ATP, nucleic acids and amino acids (Helander et al. 1998; Cox et al. 2001). Changes in membrane fluidity usually occur due to alterations in membrane lipid composition (Sikkema et al. 1995) and are considered to be a compensatory mechanism to counter the lipid disordering effects of the treatment agent. These morphological alterations in bacterial cells could be associated with damage in the cell wall and cell membrane of the investigated foodborne pathogens treated with TCEO, leading to disruption and formation of lysed cell. Previously, such morphological alterations have been reported for foodborne pathogens treated with plant-based essential oils (Bajpai et al. 2009).

The production of ATP in prokaryotes occurs both in the cell wall and in the cytosol by glycolysis (Helander et al. 1998). Hence, it is expected that alterations on intracellular and external ATP balance can be affected due to the action of the essential oils on the cell membrane. Conversely, ATP being a principal energy carrier is used for many cell functions including transport work moving substances across cell membranes, which might be a potential target parameter to understand the mechanism of action of antimicrobial agents. The results of our study showed an increasing rate of extracellular ATP concentrations after B. cereus ATCC 13061 and E. coli ATCC 43889 cells exposed to TCEO at MIC. This might occur due to significant impairment in membrane permeability of the tested bacteria by TCEO, which caused the intracellular ATP leakage through defective cell membrane. Furthermore, reduction in intracellular ATP may also occur due to decreased rate of ATP synthesis and increased rate of ATP hydrolysis inside the cells. Previously, similar findings on this phenomenon have also been reported for various antibacterial agents (Herranz et al. 2001). Burt (2004) also reported that exposure of B. cereus cells to oxygenated monoterpenes resulted in decreased level of intracellular ATP while disproportionately increased the level of extracellular ATP. Similarly, Helander et al. (1998) found that B. subtilis ATCC 6633 cells treated with essential oil components resulted in decreased level of intracellular ATP pool and increased levels of extracellular ATP pool.

Another apparent mechanism of antimicrobial action of TCEO was visualized by the confirmation on the release of 260 nm absorbing materials when the investigated foodborne pathogens were exposed to TCEO at the MIC. The macromolecules of a bacterial cell including DNA and RNA (nucleic acids), which reside throughout the interior of the cell, in the cytosol, are the key structural components. The transfer of cellular information through the processes of translation, transcription and DNA replication occur within the same compartment and can interact with other cytosolic structures. Measurement of specific cell leakage markers such as 260 nm absorbing materials is an indicative of membrane sensitivity to specific antimicrobial agent in relationship to unexposed cells. In this study, exposure of B. cereus ATCC 13061 and E. coli ATCC 43889 to TCEO caused rapid loss of 260 nm absorbing materials from the treated bacterial cells. Previously, similar findings on the leakage of 260 nm absorbing materials have been reported against foodborne pathogens treated with other plant-based essential oil (De Souza et al. 2010).

The plasma membrane is the target of many antimicrobial agents including plant-based essential oils (Bajpai et al. 2012). When bacterial membranes become compromised, small molecules are left out. Potassium ions are the major intracellular cations in bacteria that act as cytosolic-signaling molecules, activating and/or inducing enzymes, and transport systems that allow the cell to adapt to elevated osmolarity. Furthermore, the mechanism of antimicrobial action of TCEO was confirmed on the basis of K+ ions efflux from B. cereus ATCC 13061 and E. coli ATCC 43889 cells when exposed to TCEO at MIC. The bacterial cytosolic membrane provides a permeability barrier to the passage of small ions such as K+ ions and allows cells to control the entry and exit of different compounds. This impermeability to small ions is maintained and even regulated by the structural and chemical composition of the membrane itself. Increases in the efflux of K+ will indicate a disruption of this permeability barrier. Maintaining ion homeostasis is integral to the maintenance of the energy status of the cell in addition to other membrane-coupled energy-transducing processes, such as solute transport, regulation of metabolism, control of turgor pressure, motility and maintains proper enzyme activity (Cox et al. 2001). Therefore, even relatively slight changes to the structural integrity of cell membranes can detrimentally affect cell metabolism and lead to cell death (Cox et al. 2001). This suggests that, in the case of B. cereus ATCC 13061 and E. coli ATCC 43889 cells, monitoring K+ efflux and release of 260 nm absorbing materials may be more sensitive indicators of membrane damage (Cox et al. 2001).

These results suggest that storage of the TCEO components in the plasma membrane causes instant loss of their integrity and become increasingly more permeable to essential cell constituents and ions that might be responsible for setting up an antibacterial activity. Severe leakage of cytosolic materials is used as an indication of gross and irreversible damage to the cell membrane (Cox et al. 1998). From the results that the amount ratio of loss of 260 nm absorbing materials was as extensive as the leakage of K+ ions, this may indicate that the membrane structural damage sustained by B. cereus ATCC 13061 and E. coli ATCC 43889 cells resulted in release of macromolecular cytosolic constituents (Cox et al. 2001). Similarly, the effect of essential oil components such as carvacrol on proton motive force of bacteria has strongly been correlated to leakage of various substances such as ions, ATP, nucleic acids and amino acids (Helander et al. 1998).

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

This study showed that T. cuspidata leaf essential oil (TCEO) possessed significant in vitro antibacterial properties against foodborne pathogens including both gram-positive and gram-negative bacteria. We conclude that TCEO exerts its antibacterial effect through permeabilization of the cell membrane associated with generalized membrane-disrupting effects, and this corresponds to a simultaneous reduction in cell viability, loss of 260 nm absorbing materials and potassium ion efflux with decreased pool of extracellular ATP being indicative of loss of cell membrane integrity. Moreover, the SEM observations also support the aforementioned hypothesis, and strongly indicate the membrane disrupting activity of TCEO. This should lead to effective industrial application of the TCEO in food industry as a natural antimicrobial agent to inhibit the growth of foodborne pathogens. The heterogeneous composition of TCEO and the antimicrobial activities of many of its components are an indication that many essential oil components exert antibacterial action via several mechanisms. Further research is required to fully understand the mechanisms involved in order to justify the real applications of TCEO in food practices as a natural antibacterial agent.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

This work was supported by a Grant No. PJ00950603 from Systems and Synthetic Agrobiotech Center through Next-Generation BioGreen 21 Program, RDA, Korea.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References
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