Antimicrobial Evaluation of Melaleuca alternifolia and Melaleuca citrina Essential Oils Against Listeria monocytogenes and Escherichia coli Applied in Disinfection

MATERIALS AND METHODS

Study area: This research was conducted over the period of 6 months, from May to October, 2022.

Plant materials: The Melaleuca alternifolia (Maiden and Betche) Cheel. and Melaleuca citrina (Curtis) Dum. Cours. plant materials were collected from Long An and Hau Giang Province, Vietnam, respectively. The taxonomic identification of these plant samples was verified by a plant taxonomist, Phung T.H., MSc of the Botanical Laboratory, Department of Biology, School of Education, Can Tho University, Vietnam. Voucher specimens [Ma15092022-LA0001 and Mc20092022-HG0018] were deposited at Can Tho University, Vietnam.

Extraction of essential oil using steam distillation: The oil extraction used the steam distillation method, which was performed at the Laboratory of Organic Chemistry,

Department of Chemistry, School of Education, Can Tho University, Vietnam. Fresh leaves and twigs (850 g each) of Melaleuca alternifolia and Melaleuca citrina samples were used as ingredients combined with 3 L of distilled water to conduct the oil extraction experiment. The ingredients were contained in a round-bottom flask of distillation apparatus which was fairly and tightly packed to fit completely. The steam was allowed to move through the raw materials due to the leaving of several spaces. There is a flow of water drained into the condenser, allowing the oil to flow into the tube. When the steam distillation process was initiated, the water was heated by heating the mantle resulting in the vapor passing through the samples. During the essential oil extraction, the temperature needed to be reached the boiling point of TTO which is 165°C and stabilized until the end of this process. After the oil distillation was finished, the essential oil was collected, followed by performing the “liquid-liquid extraction” technique. The obtained TTO was contained in a sealed amber glass sample tube and preserved at 4°C within the refrigerator^26,27.

The essential oil collected from the steam distillation process was separated by liquid-liquid extraction technique. The distilled solution contained three layers after adding diethyl ether, including diethyl ether, TTO and from top to bottom, respectively. This is because the density of each substance is not similar, which were 0.713 g mL^–1 (diethyl ether), 0.885-0.906 g mL^–1 (essential oil) and 1.000 g mL^–1 (water)²⁷. After extracting with diethyl ether, there was a small amount of water in this solution and anhydrous sodium sulfate was added to eliminate the water.

The yield of TTO extraction (% v/w) was calculated according to the following formula²⁸:

Gas Chromatography-Mass Spectrometry (GC-MS) analysis: The GC-MS analysis of the oils was performed on a GC/MS Thermo Machine, Thermo Xcalibur software, NIST MS Search 2.0 Mass Spectral Library with a DB-5 capillary column (0.32 mm×30 m×0.25) and the carrier gas was used in this system which was Helium. The temperature of the sample injection on the column was 240°C. The temperature program commenced at 50°C and was maintained within 1 min. In the first stage, the temperature increased at the rate of 2°C/min to 70°C, subsequently, it was adjusted to rise to 150°C, with a speed of 5 min. In the third stage, the temperature rose 10 min and reached a peak of 230°C²⁹.

Antibacterial assay: Two bacterial species were used to investigate the antibacterial activity of tea tree oil which are Gram-positive bacteria: Listeria monocytogenes obtained from the Department of Microbiology, VNU HCMC-University of Science and Gram-negative bacteria: Escherichia coli (ATCC 25922) collected from Department of Molecular Biology, Biotechnology Research and Development Institute, Can Tho University. In this study, the antimicrobial ability of both types of Melaleuca oils was evaluated by several following methods including agar well diffusion associated with the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the microdilution method.

Agar well diffusion: According to the research of Valgas et al.³⁰, the agar disc diffusion method was used to assess the antimicrobial activity of TTO with a modification related to the nutrient broth medium. The bacteria were cultured in tubes containing 20 mL of Luria-Bertani (LB) broth and incubated overnight in the warm condition of 37°C. The bacterial inoculum measured the optical density at the wavelength of 600 nm (OD₆₀₀) and standardized with sodium chloride 0.85% solution to obtain turbidity which was comparable to the figure of McFarland 0.5 standard (1.0×10⁸) CFU mL^–1. The microbial suspension was consistently spread with sterile cotton swabs on the surface of the Petri dish containing LB agar. Two series of essential oil serial dilutions were prepared including a series that yielded concentrations of 10, 20, 30% (v/v) and the other performed at 25, 50 and 75% (v/v) which were diluted by Dimethyl Sulfoxide (DMSO) 15% solution. Amoxicillin (5 mg mL^–1) and DMSO 15% solution was used as positive and negative control, respectively. There were 5 wells with a diameter of 7 mm cut in the agar surface of each 90 mm Petri dish with 20 mm between each other. About 50 μL of natural extracts at different concentrations, positive and negative control were added in each well. Subsequently, the plates were incubated at 37°C for 24 hrs and the diameter of the inhibitory zone was measured in mm. These experiments were triplicated.

Determination of MICs: The MIC is defined as the lowest concentration of antimicrobial agent that prevents the visible growth of microorganisms³¹. In other words, the testing samples with certain concentrations prepared in tubes, among which, the concentration would be determined as MIC if its turbidity was visualized by unaided eyes compared to the turbidity of control samples after incubation for 24 hrs³². The microdilution assay using the 96-well microplates method combined with OD measurement was applied to evaluate the antibacterial activity and determine the MIC of investigated samples against 2 strains of bacteria (L. monocytogenes and E. coli)³³. The bacterial cultures were similarly prepared and adjusted the turbidity according to the McFarland 0.5 standard as the description above. The bacterial inoculum was diluted at the ratio of 1:1 of each inoculum in fresh LB broth to utilize in microdilution experiments. The oil extract solution was prepared in a series of densely ascending concentrations from 0.1 to 10% (v/v) and applied to the microplate, followed by the addition of bacterial culture. Eventually, each well had 200 μL as the total volume. The microplates were measured at the OD value at the wavelength of 600 nm by using a spectrophotometer (Thermo Scientific Multiskan GO) and incubated at 37°C for 24 hrs. Afterward, the OD value was measured again to assess bacterial growth. To indicate only the bacterial turbidity, the total OD value did not contain the OD of the background (LB fresh). After 24 hrs of incubation, if the difference between this OD value and the figure measured at 0 hr was lower than the change in OD value recorded in wells containing only bacterial culture without oil extracts, it was determined that the bacterial growth was prevented.

Determination of MBCs: The MBC is determined as the least concentration of antimicrobial substances required to kill microorganisms³⁰. The experiments determining the MBCs were continuously conducted after the MICs of tested samples had been detected. Many tested samples having concentrations which were the same and higher than the MICs, were selected to perform experiments related to MBCs. The determination of MBCs was tested by sub-culturing the broth dilution on agar plates without a test antimicrobial agent. The sterile LB agar was prepared in Petri dishes and 50 μL of each distinct concentration sample was spread on the surface of the nutrient agar by using a glass spreading rod. Additionally, the experiment could be conducted by drop plate methods of counting viable bacteria³⁴. The Petri plates were incubated at the relevant condition of 37°C for 24 hrs. Subsequently, the bacterial growth in the Petri dish was observed via the appearance of colonies and the MBCs were determined as the lowest concentration of the series without the colony formation.

Statistical analysis: The data were expressed as Mean±Standard deviation. One-way ANOVA Test was used to analyze the difference between groups of data. The p<0.05 was considered statistically significant. All statistical tests of variation were performed with Minitab Software Version 18 (Pennsylvania, USA).

RESULTS AND DISCUSSION

Productivity of essential oil extraction: The productivity of both kinds of Melaleuca oils extraction by steam distillation was evaluated in several different stages. Generally, the yield of M. alternifolia oil extraction was highly greater than the figure of M. citrina. As shown in Table 1, the M. alternifolia oil volume rose as the extraction time increased and reached the highest value at 120 min. However, the figures related to the yield of oil extraction at two different times, 100 and 120 min, were not significant. Thus, the time was examined as relevant for the extraction process, which was 100 min, which could avoid the time and energy consumption in case of conducting distillation within 120 min. The average productivity of M. alternifolia extraction was 2.84±0.12% which was higher than that the yield had been recorded in the previous study, in the range of 1 to 2% of the weight of fresh plant ingredients²¹. There were some reasons that could influence the yield of essential oil extraction, typically the moisture content of the raw ingredients (leaves and twigs) and the temperature that the mixture of ingredients and steam were heated²⁷. In addition, the extraction time could be a factor affecting productivity. If the distillation process was conducted in a short time which was not long enough to vaporize most of the oil content in raw material, the yield of extraction would be low and the utilization of material would not be effective. This is the reason why our research performs the experiment to evaluate the yield of essential oil extraction according to distinct times to select the relevant time which could optimize the extraction process.

Table 2 demonstrated the yield of M. citrina distillation and 100 min were selected as the suitable extraction time which had been clarified above. The extraction productivity of M. citrina oil was 0.57±0.03%, quite low in general, which was nearly similar to the findings of previous investigation related to the percentage yields of M. citrina, approximately 0.7% (leaves) and 0.5% (stems)³⁵.

GC-MS analysis: The GC-MS analysis result of Melaleuca oils illustrated that there were 16 peaks in the sample of M. alternifolia oil while, the M. citrina oil had 12 peaks recorded (Fig. 1 and 2). The number of peaks were identified equal to the quantities of constituents contained in the oil. As shown in Table 3, there were two main components with high percentages including eucalyptol (51.88%) and 1-terpene-4-ol (22.71%) obtained in M. alternifolia oil. The 1-terpene-4-ol and α-Terpineol were isomers of terpene-4-ol³⁶, therefore, the percentage of terpene-4-ol calculated in M. alternifolia oil was 30.08% which achieved the required standard of ISO 4730:2004. This result was similar to the figure recorded in the previous research of Chi et al.²⁸. The terpene-4-ol was assessed as the key component which greatly contributed to the antibacterial activity of the essential oil²³. Besides, the other compounds obtained in the oil such as α-Terpineol and Globulol were also accepted with a certain amount following the ISO 4730:2004 standard.

The components of M. citrina oil were different from M. alternifolia. The main compounds of M. citrina oil were eucalyptol (76.55%) and α-pinene (7.78%), which were higher than the figure recognized in M. alternifolia oil. However, the important component greatly attributed to the antimicrobial ability was terpene-4-ol, accounting for the small percent of 4.93%. This might generally influence the effectiveness of M. citrina oil in resisting microbials. The distinction related to the extraction productivity and components of oils might be involved in the different genetics and geographical conditions in particular places where the tea tree had been cultivated²⁶.

Fig. 1:

Chromatogram of M. alternifolia oil from GC-MS analysis

Fig. 2:

Chromatogram of M. citrina oil from GC-MS analysis

Antibacterial activity by agar well diffusion: The experimental figures reported in Table 4 proved that both types of Melaleuca oils expressed the antibacterial activity against L. monocytogenes (Gram-positive bacteria) and E. coli (Gram-negative bacteria) via the diameter of inhibition zone observed in the Petri dishes (Fig. 3). The obtained results detected that the antimicrobial ability of essential oil was proportional to its concentration meaning that the inhibitory zone expanded according to the increase in oil concentration. It was apparently seen that L. monocytogenes had a higher susceptibility to the oil than E. coli with a larger inhibition zone in different concentrations. Among them, the highest diameters of inhibitory zone recorded in L. monocytogenes at the concentration of 75% were 16.00±1.00 mm and 13.67±0.58 mm for M. alternifolia and M. citrina, respectively while the figure evaluated in E. coli were 12.67±1.16 mm and 12.33±1.16 mm.

Fig. 3:

Inhibition zones observed in the Petri dishes, (a) 10, 20 and 30% and (b) Petri dish performed the antibacterial activity at the concentration of 25, 50 and 75%

Fig. 4:

Turbidity of testing samples observed in L. monocytogenes before and after incubation of 24 hrs

The value of the inhibitory zone measured at 50% and 75% of M. alternifolia oil against two strains of bacteria were not significant to the data observed at positive control (Amoxicillin (5mg mL^–1)) (15.67±0.58 mm and 11.67±0.58 mm for L. monocytogenes and E. coli, respectively). It was convinced that the M. alternifolia oil at the concentration of 50 and 75% had an antibacterial activity which was similar to positive control. In the case of M. citrina oil, the diameter of inhibitory zones was mostly smaller than the figure of M. alternifolia oil at many concentrations (Table 3) and insignificant in comparison with the inhibitory zone of positive control at the level of 75%. Thus, the antimicrobial activity of M. alternifolia oil was dominant compared to M. citrina oil due to the difference related to the constituents obtained in each kind of essential oil. Furthermore, the essential oil was extracted from different organs of the same plant which could indicate the distinct levels of antimicrobial activity. The Melaleuca oils in this research mainly extracted from leaves and twigs showed differences in antibacterial expression, which was lower than the data recognized in the oils distilled from flowers³⁷.

This could be examined that both Melaleuca oils indicated the broad spectrum of antibacterial activity against both Gram-positive and the Gram-negative bacteria³⁸. Particularly, the experimental data of Melaleuca oils (Table 3) delineated that the inhibitory effect on Gram-positive bacteria was higher than on Gram-negative bacteria, which agreed with the results of some previously published investigations³⁹.

Determination of MICs and MBCs: The MIC of M. alternifolia oil against L. monocytogenes was 0.2%, which was comparable to the study of Oussalah et al.⁴⁰ and Liu et al.⁴¹, lower than the MICs recorded in E. coli, was 0.3%, insignificant to the baseline MICs (0.25-0.5%) revealed in previous research⁴². The M. alternifolia oil had MICs <4% (v/v) in resisting both bacteria above and was assessed as the high antibacterial activity⁴⁰. The MICs recorded in experiments with M. citrina oil were greater than the data in M. alternifolia oil, which were 3.0 and 1.25% for E. coli and L. monocytogenes, respectively. The turbidity of testing samples in different concentrations was shown in Fig. 4 and 5. It was evidently seen that the turbidity of MICs could be observed by unaided eyes compared to the control. The experimental results related to MICs in this investigation proved that the tea tree oil enabled to inhibition a wide range of pathogenic microorganisms at low levels of concentration²³.

Current research detected the MBCs of M. alternifolia oil against both E. coli and L. monocytogenes whereas, these figures on M. citrina have not been recorded yet. The M. alternifolia oil nearly killed the viability of L. monocytogenes at 12.5% (v/v) mean while, the MBCs in E. coli was higher, 47.5% with the modes of action described in the article of Carson et al.²³.

Fig. 5:

Turbidity of testing samples observed in E. coli before and after incubation of 24 hrs

This result demonstrated that the Gram-negative bacteria (E. coli as an example) was difficult to destroy its cells in comparison with Gram-positive bacteria, which was explained by the study of Jones⁴³, grounded on the difference in membrane structure between Gram-positive and Gram-negative bacteria. To be specific, the membrane of Gram-positive bacteria was structured by a single outer membrane outside covering a cell wall while, Gram-negative bacteria had three layers containing an inner membrane, a cell wall and an outer membrane, posing an additional barrier to molecule entry.

CONCLUSION

The investigation evaluated the extraction of both M. alternifolia and M. citrina oil to optimize the distillation time. It was easily seen that the Melaleuca oils richly included antibacterial components which enabled to prevent the growth of both Gram-positive and Gram-negative bacteria, such as L. monocytogenes and E. coli. Due to the difference in the compositions of each kind of essential oil, the bioactivity is also distinct between M. alternifolia and M. citrina oil, typically the percentages of some major compounds indicating the high antibacterial ability as terpene-4-ol, α-Terpineol and eucalyptol. Furthermore, the findings of this study expressed the potential of applying essential oils as natural antibacterial agent’s in vitro conditions which might limit some side effects and could be an alternative to antibiotics. It is necessary to restrict the antibiotics-resistance or multi-drug resistance which has increasingly occurred in microorganisms, particularly bacteria.

SIGNIFICANCE STATEMENT

This study convinced that the essential oils extracted from both kinds of Melaleuca species, especially Melaleuca alternifolia, have effective antimicrobial activity even with some dangerous-diseased bacteria as L. monocytogenes and E. coli, which could be applied to disinfect in scope of laboratories and processed factories to restrict the risk of bacterial contamination. Furthermore, the Melaleuca essential oils are examined as promising alternatives for antibiotics in usage of antibacterial as well as prevent side effects for consumers. Besides, utilization of the essential oils as well as natural plant extracts, also contributes to the environmental protection due to their friendly effects which would be beneficial for our lives.

ACKNOWLEDGMENT

This research was supported in finance by Can Tho University, code: TSV2022-156. We highly appreciate the advice from Ms. Phung T.H., MSc of the Botanical Laboratory, the support in experimental equipment of laboratories from College of Natural Science and School of Education, Can Tho University.