If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Listeria monocytogenes has gained increasing attention as a pathogen of public health importance owing to large numbers of food-borne outbreaks of listeriosis. Because of negative consumer perception of chemical preservatives, attention is shifting towards natural alternatives. Particular interest has been focused on the potential application of plant essential oils. The objective of the present study was to determine ultrastructural changes brought about by essential oils from two types of thyme, Thymus eriocalyx and Thymus x-porlock, on Listeria monocytogenes.
Materials and methods
Minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations and bactericidal kinetics of the oils were determined. Listeria monocytogenes were treated with essential oils from two thyme species and observed under a transmission electron microscope.
Results
The oils from the above plants were found to be strongly antimicrobial. Analysis of the oils by gas chromatography and gas chromatography/mass spectrometry lead to the identification of 18 and 19 components in T. eriocalyx and T. x-porlock oils, respectively. Listeria monocytogenes treated with essential oils from the two thyme species exhibited a thickened or disrupted cell wall with increased roughness and lack of cytoplasm.
Conclusion
The antilisterial effects of thyme oil are stronger than the action of electric shocks in combination with nisin reported in the literature. It is concluded that essential oils such as thyme oil, which inhibited the growth of L. monocytogenes at low concentrations, could be considered as preservative materials for some kinds of foods; they could find an application as additives to foodstuffs in storage to protect them from listerial contamination.
Finding healing power in plants is a traditional and ancient concept. However, since the advent of potent synthetic antibiotics in the 1950s, the use of plant derivatives as antimicrobials has become almost nonexistent. In recent years the essential oils and extracts of many plant species have become popular, and attempts to characterize their bioactive principles have gained momentum in many pharmaceutical and food-processing applications.
The antimicrobial activities of essential oils isolated from many plants have been recognized, albeit empirically, for centuries; only recently have such properties been confirmed.
The essential oils produced by different plant species are in many cases biologically active.
Thyme is stated to possess carminative, antispasmodic, antitussive, expectorant, secretomotor, bactericidal, anthelmintic and astringent properties. At present, the essential oils of many Thymus species are widely used as flavoring agents in food processing and many pharmacological preparations, and thyme oil is still among the world's top 10 most used essential oils.
Previous studies on the antimicrobial activity of the essential oils of some Thymus spp, most of them possessing large quantities of phenolic monoterpenes, have shown activity against viruses,
Listeria monocytogenes is a Gram-positive non-spore forming bacterium with a short rod shape, ranging in length from 500 to 2000 nm and in width from 400 to 500 nm. It is a psychrotolerant food-borne pathogen that can be present in milk from infected cows and is known to cause listeriosis in humans.
making post-process contamination a significant concern for ready-to-eat meat produce. Cross contamination from plant workers is a further possible cause of Listeria spp getting into food products after heat treatment.
Most of these studies have been conducted using essential oils in microbiological media and there are no published studies involving the investigation of morphological changes in Listeria spp as a result of essential oil treatment. Consequently, little is understood about their effectiveness at the ultrastructural level. The inactivation of Listeria with the aim of food preservation is of the utmost importance to the food industry. The objective of this study was, therefore, to investigate under a transmission electron microscope (TEM) the morphological changes in Listeria monocytogenes as a result of exposure to the essential oils from Thymus eriocalyx and Thymus x-porlock.
Material and methods
Microbial strain and growth media
L. monocytogenes (PTCC 1298) obtained from the Iranian (Persian) Type Culture Collection was maintained on blood agar. A single colony from blood agar plate was inoculated into a brain heart infusion (BHI) broth tube. The tube was incubated at 37 °C on a shaker to propagate the cells. The contents of the BHI broth tube were transferred to a 100-ml flask containing sterile BHI broth at log phase (OD600 = 0.4–0.5) to scale up the bacterial suspension volume. An initial bacterial suspension containing 108 CFU/ml was made from the flask broth culture. Subsequent dilutions were made from the above suspension, which were then used in the tests.
Oil isolation
The plants, Thymus eriocalyx and Thymus x-porlock, were collected from the National Botanical Garden of Iran during May and June 2003. The fresh leaves were hydrodistilled for 90 min in full glass apparatus. The oils were isolated using a Clevenger-type apparatus. The extraction was carried out for 2 h after a 4-h maceration in 500 ml of water. The oils were stored in dark glass bottles in a refrigerator until use.
Oil analysis
The essential oils were analyzed by gas chromatography (9-A-Shimadzu) and gas chromatography/mass spectrometry (Varian-3400) column: DB-1 (dimethyl polysiloxane), 60 m × 0.25 mm fused silica capillary column, film thickness 0.25 μm, using a temperature program of 50–250 °C at a rate of 4 °C/min, injector temperature 250 °C, carrier gas helium (99.99%), inlet pressure 3 kg/cm2. The constituents were identified by comparison of their mass spectra with those in the computer library and with authentic compounds. The identifications were confirmed by comparison of their retention indices with those of authentic compounds or with literature data.
Oil dilution solvent
Of all the solvents tested, such as ethanol, methanol, acetone, butanol and diethyl ether, methanol was selected as the diluting agent for the oils as it did not exhibit antilisterial activity. Oil dilutions of 1/2, 1/4, 1/8 and 1/16 were made with methanol. These dilutions were used in antilisterial analysis. Undiluted oil was taken as dilution 1. This solvent also served as control.
Antilisterial analysis
The fresh oils were tested for their antilisterial activities. The disc diffusion method
was used for antilisterial screening as follows: sterile Mueller–Hinton agar medium (Merck) was prepared and distributed into Petri plates of 90 mm diameter. This medium was used for antibiogram assays. The disc size used was 6 mm (Whatman No. 1) paper. Different dilutions of the oils were made with methanol. The listerial suspension at 108 CFU/ml was streaked over the surface of the Mueller–Hinton agar using a sterile cotton swab in order to get a uniform microbial growth on both control and test plates. Under aseptic conditions, the discs were placed on the agar plates and then 10 μl from each of the oil dilutions were put on the discs. Dilution solvent (10 μl methanol) was added to the discs on the control plates. The plates were then incubated at 37 °C for 24 h in order to get reliable microbial growth. Diameters of microbial inhibition zones were measured using vernier calipers.
The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were assessed according to our modified procedure.
MBC was determined by a broth dilution method in test tubes as follows: 50 μl from each of the various dilutions of the oils were added to 5 ml BHI broth tubes containing 107 cells/ml. The tubes were then incubated at 37 °C for 24 h on an incubator shaker so as to evenly disperse the oil throughout the broth in the tubes. The highest dilution (lowest concentration) showing no visible growth was regarded as the MIC. From the tubes showing no growth, 0.1 ml of the cells were spread on BHI agar plates in triplicate to determine if the inhibition was reversible or permanent. MBC was determined as the highest dilution (lowest concentration) at which no growth occurred on the plates. Mean values of three such assays were recorded as the MBC.
Bactericidal kinetics of the oils
Each oil (50 μl), at the dilution determined by MBC, was added to 5 ml BHI broth tubes containing 1/10, 1/100 and 1/1000 dilutions of initial bacterial suspension of 108 CFU/ml to obtain 107, 106 and 105 CFU/ml, respectively, and were then incubated at 37 °C for 60 min in an incubator shaker. Samples (0.1 ml) were taken after 5, 10, 15, 20, 25, 30, 45 and 60 min. The samples were immediately washed with sterile phosphate buffer pH 7.0, centrifuged at 10 000 rpm for 1 minute, resuspended in the buffer and were then spread cultured on BHI agar for 24 h at 37 °C. Phosphate buffer was used as diluent when needed. Bactericidal experiments were performed three times. Microbial colonies were counted from triplicates after incubation period and the mean total number of viable cells per ml was calculated. The mean total number was converted to log10 viable cells using routine mathematical formulae.
Transmission electron microscopy
Five ml of 107 CFU/ml in BHI were exposed to 50 μl of each oil at the dilution determined by MIC and were then incubated at 37 °C for 5 min in an incubator shaker. The control cells were treated with solvent only. The bacterial suspensions were then centrifuged in sterile plastic centrifuge tubes at 3000 rpm for 10 min at 10 °C. The supernatant was discarded and the Listeria resuspended in sterile 0.1 M cacodylate buffer (1.4 g cacodylate powder, 50 ml distilled water, 2.7 ml 1 N HCl, pH 7.2) for 2 h at 4 °C and was then centrifuged for 10 min at 3000 rpm at 10 °C. The supernatant was discarded and the cells suspended in 2 ml of glutaraldehyde solution (2.5%) in 0.1 M cacodylate buffer (pH 7.2), and transferred to a microcentrifuge tube, and allowed to fix for 2 h at 4 °C. The fixing solution was washed with six consecutive (5 min) washes with cacodylate buffer. The cells were post fixed with 2% osmium tetroxide in 0.1 M cacodylate buffer. The cells were washed three times with cacodylate buffer, and dehydrated in ethyl alcohol in a series of 50%, 60%, 70%, 80%, 90%, and 95% dilutions for 10 min each. A final dehydration step was carried out for 3 h in 100% ethyl alcohol with changes every 30 min. The cells were then suspended in propylene oxide for 30 min with a change at the 15th min. They were then suspended in propylene oxide:araldite (1:1) for 1 h and overnight in propylene oxide:araldite (1:3) and were then transferred to fresh araldite (10 ml Araldite CY 212 (Ciba), 10 ml Hardner 964 B (Ciba), 0.22 ml dibutyl phthalate, 0.3 ml dimethyl phthalate (DMP) for 4 h. The polymerization of the resin to form specimen blocks was accomplished in an oven at 45 °C for 24 h and then at 70 °C for 48 h. The specimen blocks were hand trimmed with a razor blade and sectioned with an ultramicrotome with 1-micron thickness (sections appearing blue in color under ultratome) for light microscopic observations and 0.1-micron thickness (sections appearing blue in color under ultratome) for transmission electron microscopic observations. The ultrathin sections were placed on 200 mesh copper grids. The sections were stained with 12.5% alcoholic uranyl acetate (UO2(OH3COO)2·H2O) in methanol for 20 min, then with lead citrate (25 mg lead citrate Pb3(C6H5O7)2·3H2O dissolved in 1 ml of 1 N sodium hydroxide and the final volume was made to 10 ml by adding 9 ml double-distilled water) and were then washed with double-distilled water for 1 min, dried under a reading lamp for 30 min and viewed with a JEOL 100 (Japan) transmission electron microscope (TEM) operating at 80 kV.
Results
Essential oils were extracted from Thymus eriocalyx and Thymus x-porlock yielding 1.2% and 1.0% w/w oil, respectively. Chemical analysis of the components of the oils led to identification of 18 and 19 components in T. eriocalyx and T. x-porlock oils, respectively. The profile of the oil components from T. eriocalyx was similar to that of our previous report
with slight changes in concentrations. The major components of T. eriocalyx and T. x-porlock oils were thymol (63.8, 31.7%), α-phellandrene (13.30, 38.7%), cis-sabinene hydroxide (8.1, 9.6%), 1,8-cineole (2, 1.7%), and α-pinene (1.31, 2%).
Preliminary experiments were carried out in vitro using the disc diffusion and tube dilution methods to investigate antimicrobial action of the essential oils. Various concentrations of essential oils from T. eriocalyx and T. x-porlock, tested on the relevant agar plates and broth tubes, showed very strong antimicrobial properties (Table 1). The oils from T. eriocalyx and T. x-porlock were bactericidal at the initial oil dilution of 1/8 (250 ppm) with growth inhibition zones of 27 and 23 mm respectively (Table 1). A study of the bactericidal kinetics of the essential oils revealed complete elimination of various microbial loads of L. monocytogenes within the first 20 min of exposure (Figure 1, Figure 2). No viable bacterium was detected after 20 min. Listeria monocytogenes treated with essential oils from the two thyme species exhibited thickened or disrupted cell walls with increased roughness and lack of cytoplasm (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7). This pattern of abnormalities was evident in almost all the cells scanned under TEM.
Table 1Antimicrobial effect of various concentrations of thyme essential oils on the basis of growth inhibition zone (mm) with corresponding inhibitory or lethal properties
Thymus eriocalyx
Thymus x-porlock
Oil dilution
1
1/2
1/4
1/8
1/16
1
1/2
1/4
1/8
1/16
Oil concentration (ppm)
2000
1000
500
250
125
2000
1000
500
250
125
Zone of inhibition (mm) of L. monocytogenes
38
42
44
27
19
37
40
30
23
19
MIC
+
+
+
+
+
+
+
+
+
+
MBC
+
+
+
+
−
+
+
+
+
−
+ Indicates inhibitory or lethal effectiveness of the oils.
− Indicates no inhibitory or lethal effectiveness of the oils.
Figure 4EM graph (×80 000) of Listeria monocytogenes exposed to 1/16 dilution of essential oil from Thymus x-porlock exhibiting decreased cell size (probably for survival). The cell wall (CW) is undergoing degenerative changes. The cells are getting closer to each other (arrows).
Figure 5EM graph (×30 000) of Listeria monocytogenes exposed to 1/8 dilution of essential oil from Thymus x-porlock exhibiting cell wall and organelle damage. Cytoplasm has lost its even distribution showing clumping of cytoplasmic material (white arrows). The cells are getting closer to each other (smaller black arrows).
Figure 6EM graph (×80 000) of Listeria monocytogenes exposed to 1/8 dilution of essential oil from Thymus x-porlock exhibiting cell wall and organelle damage. Cytoplasm has lost its even distribution showing clumping of cytoplasmic material (white arrow). The cells are getting closer to each other (black arrow).
Figure 7EM graph (×80 000) of Listeria monocytogenes exposed to 1/8 dilution of essential oil from Thymus eriocalyx. The cell shows severe damage to the cell wall and organelles.
The results show that Listeria monocytogenes was completely eliminated within 20 min of exposure to the essential oils in culture broth at 250 ppm (Figure 1, Figure 2). Such a delay in, or inhibition of, microbial growth is particularly useful in terms of food safety. This indicates higher efficacy of thyme oils as compared to those of clove, which have been recommended for usage in short-term storage of products.
This difference is attributable to the chemical composition of the essential oils. The two varieties of thyme used in the present study showed similar but varying levels of oil components and this influenced the antibacterial properties of the oils. Thymus x-porlock oil was a stronger bactericidal agent than Thymus eriocalyx oil (Figure 1, Figure 2).
A transmission electron microscopic study of untreated cells of L. monocytogenes showed a continuous thin smooth cell wall (CW), cell membrane (CM) and nuclear material (Figure 3). Cells of L. monocytogenes exposed to the MIC dilution (125 ppm) of essential oil from T. x-porlock exhibited decreased size and the cells were found to be closer together (arrows in Figure 4) probably in an attempt to manage their survival. The cell wall exhibited budding scars and underwent degenerative changes showing splitting of the wall layers. This budding scar manifestation has been reported by Harrison et al.
in yeast cells as a result of pulse electric fields (PEF). The thickened appearance of the cell wall was more pronounced at the polar regions. Maisner-Patin and Richard
reported that exposure of Listeria innocua to nisin concentrations of 500 and 4000 IU/ml induced cell wall thickening as well as irregularities. As the oil concentration increased, the cell wall of L. monocytogenes lost smoothness and uniformity (Figure 5, Figure 6). Calderon-Miranda et al.
reported that the surface roughness of the cell walls of L. innocua exposed to 32 pulses of electric field intensity of 30 kV/cm increased slightly. Our observations indicate more severe effects of thyme essential oils on the listerial cell wall, leading to cell wall rupture (Figure 7), than in those treated with nisin or an electric field intensity above 40 kV/cm
It seems that the essential oils used in this study have similar effects to those of nisin.
Cell membrane disruption and lack of cytoplasm was evident at an early stage of treatment of the cells with the lower concentration of thyme oil (Figure 4). Cytoplasm lost its even distribution and showed clumping of intracellular materials. The cells also exhibited lack of cytoplasm as a result of the decrease of the cell membrane functionality as a barrier (Figure 4, Figure 5, Figure 6). Similar observations have been reported by Pothakamury et al.
for Staphylococcus aureus treated with an electric field intensity of 20 kV/cm and 64 pulses. A report on the action of tea tree oil shows loss of cytoplasmic contents of S. aureus.
Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time–kill lysis leakage and salt tolerance assays and electron microscopy.
The findings suggest that thyme oils have good potential as antilisterial substances in food preservation as they may be more acceptable to consumers and the regulatory agencies in comparison to synthetic chemical compounds. Although high concentrations of essential oils may adversely affect the organoleptic properties of food,
lower concentrations may be sufficient for food safety in situations where bacterial load is low, in addition to the pleasant flavor of thyme being imparted to the food. These results on the mechanism of action of thyme oils on the inactivation of L. monocytogenes will help in the development or modification of processing conditions, or the implementation of a new preservation factor to complement those already employed in food preservation and safety.
Acknowledgements
The authors wish to thank Shahed University (Tehran, Iran) for the sanction of research grants to conduct the present study. Special thanks to Mr Mohammad Habibi for his assistance in our microbiology laboratory.
Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time–kill lysis leakage and salt tolerance assays and electron microscopy.
00177-3&id=gr1.jpg){kind=link}
00177-3&id=gr2.jpg){kind=link}
00177-3&id=gr3.jpg){kind=link}
00177-3&id=gr4.jpg){kind=link}
00177-3&id=gr5.jpg){kind=link}
00177-3&id=gr6.jpg){kind=link}
00177-3&id=gr7.jpg){kind=link}