1. Background
Due to bacterial resistance to common antibiotics, surreptitious use of antimicrobial drugs by the general population, and high rates of allergies and side effects in chemical treatments, it is essential to find antimicrobial compounds with minimum side effects. Use of herbs for their medicinal properties dates back to long ago. Researchers are now seeking alternative antimicrobial agents with plant origins (1-3). Herbal medicine as a traditional health approach is popular among 80% of the world’s population in Latin America, Asia, and Africa, and it has been shown to have fewer side effects (3, 4). According to previous research, there are various compounds and substances in different plants, including essential oils (EO), peptides, water, ethanol, phenol, methanol, soluble butanol compounds, and chloroform (4, 5).
It has been shown that EOs have insecticidal, antifungal, antiviral, antibacterial, and antioxidant properties. Moreover, some EOs have been used in cancer treatment (6-8), while others are used in food preservation, fragrance, and aromatherapy industries. EOs are valuable sources of biologically active compounds. Accordingly, there has been a growing interest in the antimicrobial effects of extracts from aromatic plants, particularly EOs (9).
Eucalyptus species are commonly used in traditional medicine. Eucalyptus is a large native genus from Australia, which belongs to the Myrtaceae family and includes nearly 900 species and subspecies (10). Eucalyptus species are well-known for their rapid growth. In fact, some species have exceptional growth and are among the tallest trees in the world (20 - 50 m) (11). Eucalyptus species are important sources of gum, tannins, polyphenols, terpinenes, proteins, and dyes.
EO is the most important product, easily distilled from Eucalyptus leaves. The EO of several Eucalyptus species, such as Eucalyptus maidenii, includes 83.59% 1,8-cineole (eucalyptol) (12, 13). Eucalyptus EO is commonly used in deodorizing and cleaning products, as well as cough suppressants and decongestants (14). It has been used for the treatment of many diseases, such as influenza, dysentery, and skin disorders (1). Today, it is a common over-the-counter drug for cold treatment and has been long used to treat pneumonia, common colds, bronchitis, sore throat, and headache (15).
2. Objectives
The purpose of this study was to evaluate the antibacterial activity of EOs from the leaves of Eucalyptus camaldulensis, the major Eucalyptus species cultivated in Khuzestan, South of Iran, against the growth of drug-resistant bacteria.
3. Methods
3.1. Plant Collection and Identification
Eucalyptus was collected from botanical gardens, and leaf samples were identified at the herbarium of the faculty of pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran in September 2014.
3.2. EO Preparation
The identified plant leaves were weighed accurately and washed with distilled water. Then, the plant leaves were dried in shade at room temperature for 3 days. The dried leaves were chopped into small pieces, and EO was extracted by a Clevenger device. Extraction was performed by mixing 150 g of Eucalyptus leaves after 4 hours of maceration in 500 mL of distilled water. EOs were kept in dark glass bottles at -12°C until further use. The EO yield was 1% (16, 17).
3.3. Bacterial Strains
The microorganisms used in the present study were Escherichia coli, Acinetobacter baumannii, Proteus vulgaris, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Salmonella para typhi, Salmonella infantis, Salmonella enteritidis (Gram-negative), and Staphylococcus aureus (Gram-positive). The resistant bacteria were obtained from the department of biology, faculty of basic sciences, University of Shahed, Bu-Ali hospital, Tehran, Iran.
3.4. Gas chromatography-Mass Spectrometry (GC-MS)
The most potent antibacterial EO from E. camaldulensis was analyzed by GC-MS. GC analysis was performed in a Shimadzu-9A gas chromatograph with a flame ionization detector. Quantitative analysis was performed with EuroChrom 2000 (Knauer), using the area normalization method, regardless of response factors. The analysis was performed by a DB-5 fused-silica column (30 m × 0.25 mm; film thickness, 0.25 μm; J&W Scientific Inc., Rancho Cordova, CA, USA).
The operating conditions were as follows: detector temperature, 250°C; injector temperature, 265°C (helium as the carrier gas). The oven temperature was set at 40 - 250°C, changing at a rate of 4°C per minute. The GC-MS unit was a gas chromatograph (Varian Model 3400), coupled with a Saturn-II ion trap detector. The column was the same as that of GC, and the GC conditions were the same as described above. The MS conditions were as follows: ionization potential, 70 EV; electron multiplier energy, 2000 V. The characteristics of EO components were derived from GC retention indices related to C7-C25 n-alkanes.
3.5. Determination of Antimicrobial Activity of EO
3.5.1. Inoculum Preparation
All bacterial strains were prepared freshly. The plates were cultured on a nutrient agar medium (NA, Que Lab, Canada) in several directions and parallel lines. Then, the plates were incubated for 24 hours at 37°C. For each test, to evaluate the antimicrobial effects, fresh bacteria were used, and fresh medium was prepared.
3.5.2. Preparation of Microbial Suspension
Preparation of microbial suspension requires a 24-hour culture from each microorganism. Accordingly, 24 hours before the experiments, the microorganisms were inoculated from stock medium to nutrient agar slant. In each sterile tube, 5 mL of Mueller-Hinton Broth (MHB; Que Lab, Canada) was poured and incubated with different strains of bacteria. The tubes were placed in a shaker-incubator at 37°C for 24 hours at 200 rpm (IKA KS 4000I Control, Germany). Afterwards, 100 μL of each suspension was added to sterile tubes, containing 4 mL of MHB and incubated again. Finally, absorbance of the samples was determined by a spectrophotometer (UNIC-UV-2100, USA) at 620 nm. The time needed to achieve the target concentration in the range of 0.08 - 0.13 nm was considered as the optimal interval and varied for each microbial strain (18-20).
3.5.3. Determination of Inhibition Zone
In vitro antibacterial activities were tested against 8 bacterial isolates with agar well diffusion assay. The antimicrobials in the plant EOs diffused into the medium and interacted with organisms which were seeded freshly in the plate. The test was performed in sterile petri dishes (diameter, 80 mm), containing MHB agar medium (25 mL; pH, 7). Four sterile blank Whatman discs (diameter, 6 mm; Teb Padtan, Iran) were also placed in each plate.
Under aseptic conditions, different quantities of the extracted EO (5, 10, and 20 μL) were placed on paper discs. Blank discs were used as the positive controls. All the plates were incubated for 24 hours at 37°C, and diameters of microbial inhibition zones were measured with a ruler. Ultimately, antibacterial activity was evaluated as the average inhibition zone diameter in millimeters from triplicate samples. The final mean diameter was recorded, and the total inhibition zone diameter was scaled, including discs with 6 mm-diameter filters (16, 21, 22).
3.6. Measurement of MIC
The susceptibility of resistant bacteria to E. camaldulensis EO was assessed using the macrodilution method. Briefly, before preparing the microbial suspension, 20 sterile tubes, each containing MHB, were prepared. Different concentrations of E. camaldulensis EO (0.5 - 18 μL) were placed in the tubes, while in the 20th tube, as the negative culture, no amount of EO was used. Then, 100 μL of fresh bacterial suspension was added to each tube and incubated in a shaker-incubator for 24 hours at 37°C. The tubes were assayed for microorganism growth, and MIC was specified. MIC was defined as the lowest concentration of EO, which inhibited the visible growth of microorganisms. The concentration related to the first clear culture tube was considered as the MIC (16).
3.7. Measurement of MBC
MBC was described as the highest dilution (the lowest concentration) with no growth on the plates. On MHB medium plates, 100 μL of clear tubes was cultured using the spreader method, and all the plates were incubated for 24 hours at 37°C. If there was no growth in the MHB medium plate, MBC was determined. To ensure the test results, each episode was repeated 3 times (16).
4. Results
The output of the extraction process was estimated at 1.33%. The results of GC-MS analysis of E. camaldulensis EO are presented in Table 1. The GC-MS analysis indicated 17 compounds, constituting 99.96% of total EO. The major component was 1,8-cineole (55.2%). The other compounds included β-selinene (6.88%), hexadecanoic acid (5.5%), allo-aromadendrene (4.62%), 3-carene-δ (4.04%), γ-terpinene (3.94%), 8-octadecenoic acid (3.8%), β-gurjunene (3.3%), 9,10-dehydro-isolongifolene (3.1%), limonene (2.31%), α–pinene (2.07%), valencene (1.46%), aromadendrene (1.32%), β-elemene (1.02%), longifolene (0.5%), and ledene (0.5%). The results of antibacterial activity assay of E. camaldulensis EO against 8 resistant bacteria are presented in Table 2.
Table 1.The Chemical Composition of the Essential Oil (EO) of Eucalyptus camaldulensis
| No. | Oil Compounds | % | RI |
|---|---|---|---|
| 1 | Α-pinene | 2.07 | 941 |
| 2 | 3-Carene-δ | 4.04 | 1008 |
| 3 | Limonene | 2.31 | 1025 |
| 4 | 1,8-Cineole | 55.2 | 1030 |
| 5 | γ-Terpinene | 3.94 | 1062 |
| 6 | Clovene | 0.4 | 1359 |
| 7 | β-Elemene | 1.02 | 1388 |
| 8 | β-Gurjunene | 3.3 | 1430 |
| 9 | allo-aromadendrene | 4.62 | 1439 |
| 10 | Longifolene | 0.5 | 1404 |
| 11 | Aromadendrene | 1.32 | 1448 |
| 12 | Valencene | 1.46 | 1482 |
| 13 | β-selinene | 6.88 | 1484 |
| 14 | Ledene | 0.5 | 1485 |
| 15 | 9,10-Dehydro-isolongifolene | 3.1 | 1913 |
| 16 | Hexadecanoic acid | 5.5 | 1921 |
| 17 | 8-Octadecenoic acid | 3.8 | 1939 |
| Total compounds | 99.96 |
Abbreviation: RI, retention indices.
Table 2.The Antibacterial Activitya and MIC and MBCb of Eucalyptus camaldulensis EO Against Resistant Bacteriaa
| No. | Bacteria | 5 μL | 10 μL | 20 μL | MIC | MBC |
|---|---|---|---|---|---|---|
| Gram-Positive | ||||||
| 1 | Staphylococcus aureus 2 | 11 | 15 | 22 | 1500 | 2500 |
| 2 | Staphylococcus aureus D2 | 10 | 19 | 28 | 1000 | 2000 |
| 3 | Staphylococcus aureus D3 | 15 | 17 | 28 | 1000 | 2000 |
| 4 | Staphylococcus aureus D5 | 11 | 16 | 30 | 1000 | 2000 |
| 5 | Staphylococcus aureus D7 | 12 | 17 | 29 | 1000 | 2000 |
| 6 | Staphylococcus aureus D8 | 13 | 17 | 30 | 1000 | 2000 |
| Gram-Negative | ||||||
| 7 | Escherichia coli E1 | 10 | 19 | 24 | 1500 | 2500 |
| 8 | Escherichia coli E2 | 7 | 18 | 25 | 1500 | 2500 |
| 9 | Escherichia coli F3 | 8 | 19 | 26 | 1500 | 2500 |
| 10 | Acinetobacter baumannii A2 | 14 | 22 | 30 | 1000 | 2000 |
| 11 | Acinetobacter baumannii A5 | 15 | 21 | 30 | 1000 | 2000 |
| 12 | Acinetobacter baumannii A7 | 14 | 20 | 30 | 1000 | 2000 |
| 13 | Proteus vulgaris | 10 | 15 | 20 | 2500 | 3500 |
| 14 | Proteus vulgaris 1 | 14 | 16 | 20 | 2500 | 3500 |
| 15 | Proteus vulgaris 2 | 13 | 15 | 19 | 2500 | 3500 |
| 16 | Shigella sonnei 34 | 12 | 15 | 18 | 3000 | 4500 |
| 17 | Shigella sonnei 3 | 8 | 12 | 15 | 3500 | 4500 |
| 18 | Shigella sonnei 5 | 8 | 11 | 14 | 3500 | 4500 |
| 19 | Pseudomonas aeruginosa P2 | 8 | 13 | 20 | 2500 | 4000 |
| 20 | Pseudomonas aeruginosa G2 | 9 | 12 | 20 | 2500 | 4000 |
| 21 | Klebsiella pneumoniae I1 | 18 | 27 | 35 | 500 | 1500 |
| 22 | Klebsiella pneumoniae K1 | 17 | 26 | 33 | 500 | 1500 |
| 23 | Salmonella typhi A1 | 8 | 10 | 13 | 4000 | 6000 |
| 24 | Salmonella typhi A2 | 10 | 10 | 13 | 4000 | 6000 |
| 25 | Salmonella para typhi B | 8 | 14 | 17 | 3500 | 4500 |
| 26 | Salmonella typhi 146 | 10 | 11 | 14 | 4000 | 6000 |
| 27 | Salmonella infantis | - | 9 | 11 | 6000 | 8000 |
| 28 | Salmonella enteritidis 119 | - | 8 | 11 | 6000 | 8000 |
Abbreviations: MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration.
aDisc diameter, 6.2 mm.
The results showed that E. camaldulensis EO was effective against the tested organisms. The highest antibacterial activity of E. camaldulensis EO was reported against K. pneumoniae I1, which showed significant susceptibility to EO at concentrations of 5, 10, and 20 μL per disc per petri plate, based on the large growth inhibition diameters (18, 27, and 35 mm, respectively). Following K. pneumoniae strains, the largest inhibition zone was observed for A. baumannii, ranging from 14 to 30 mm.
The S. aureus strains showed minor difference in the inhibition zone diameter, compared to A. baumannii strains and susceptible bacteria. The activity of 5, 10, and 20 μL of Eucalyptus EO against E. coli F3 was significant (8, 19, and 26 mm, respectively). E. camaldulensis EO also showed a relatively good activity against P. vulgaris 1 (14, 16, and 20 mm). Moreover, it showed relatively moderate inhibition against P. aeruginosa P2 (8, 13, and 20 mm).
The antibacterial activity of 5, 10, and 20 μL of Eucalyptus EO against S. sonnei 3 and 5 strains indicated inhibition zone diameters of 8, 12, and 15 mm and 8, 11, and 14 mm, respectively. Eucalyptus EO exhibited relatively poor activity against S. typhi at concentrations of 20 and 30 μL with inhibition zone diameters of 10 and 13 mm, respectively. The lowest activity at 20 μL (inhibition zone diameter, nearly 11 mm) was demonstrated against S. infantis and S. enteritidis 119. At an EO concentration of 5 μL per disc per petri plate, no activity was found against S. infantis and S. enteritidis. The MIC and MBC of E. camaldulensis EO for 8 resistant bacteria are presented in Table 2.
The lowest MIC and MBC of EO were reported against K. pneumoniae (500 and 1500 ppm, respectively), and they were considered as the most sensitive bacteria. Moreover, the MIC and MBC for A. baumannii and S. aureus strains were 1000 and 2000 ppm, respectively. It was revealed that the MICs for E. coli strains (1500 ppm) were lower than those of P. vulgaris and P. aeruginosa strains (2500 ppm). The MBCs against P. vulgaris and P. aeruginosa were 3500 and 4000 ppm, respectively.
The MIC of EO against S. sonnei was 3500 ppm. Although EO was effective against most tested pathogenic strains, its effectiveness against S. infantis and S. enteritidis 119 was significantly lower (6000 and 8000 ppm, respectively). Therefore, higher levels of antibacterial activity are required in the treatment of infections caused by S. infantis and S. enteritidis 119 if they are not toxic to the tissues.
5. Discussion
Medicinal plants have been used for the treatment of infectious diseases. With respect to ecophysiological differences among plants grown in different geographical areas, research is necessary to discover their pharmaceutical efficacy (23). Recent emergence of drug-resistant bacteria highlights the importance of antimicrobial activity (11, 24). In this study, hydrodistillation of E. camaldulensis leaves yielded 1.33% EO (considering the fresh weight of young leaves) with a spicy aromatic odor. These results are in line with reports from the literature, indicating yields of 1.3 - 1.8% (considering the fresh weight of immature E. globulus leaves) in Buenos Aires (25) and 1.8% (considering the fresh weight of immature E. globulus leaves) in Montenegro (26). Despite limited consistent evidence in the literature, the yield was estimated at 1.9 - 2.7% (considering the fresh weight of immature E. globulus leaves) in Morocco (27), 2.68% in Argentina (28), and 3.91% in Brazil (considering the fresh weight of young E. cinerea leaves) (29).
Antibacterial activity of EO has been attributed to the presence of some active components. Earlier research has shown that the antibacterial activity of EOs is because of their major components (30). The analysis of E. camaldulensis EO indicated 1,8-cineole as the main component. Because of the high content of 1,8-cineole (73.07%), EO is categorized as a medicinal or eucalyptol type (29). Overall, cineole is monoterpenoid cyclic ether, which can affect the cytoplasmic membrane of target bacteria (11). The 1,8-cineole content in E. globulus has revealed larvicidal and ovicidal activities against Haemonchus contortus (31).
In a study by Damjanovic-Vratnica et al. the main component, 1,8-cineole (85%), was active against S. aureus, E. coli, and A. baumannii in most E. globulus EOs (26). In another study, 1,8-cineole (72.71%) was the most abundant component in E. globulus EO, which was active against Lactococcus garvieae (32). Identification of these compounds with great biological activities is vital, as it helps determine chemical compositions, which can be helpful in designing novel medications with remedial activities against human pathogens.
It is very difficult to compare the obtained data with the literature, as several parameters can affect the results, such as different chemical structures because of environmental factors (eg, day length, nutrients, temperature, and geography) (33). According to the results, EO of native E. camaldulensis leaf grown in Khuzestan is a significant antibacterial agent against both Gram-negative and Gram-positive drug-resistant pathogenic bacteria. The tested bacteria in our study were sensitive to EO, although the extent of antibacterial effect varied, depending on the type of microorganisms. The maximum effect was observed against K. pneumoniae, while the lowest effect was reported against S. infantis and S. enteritidis.
In a study by Cimanga et al. 5 μL of E. urophylla and E. globulus EO showed an inhibition zone diameter of 18 mm against K. pneumoniae strains, which is similar to the results of the present study (34). According to our results, E. camaldulensis EO at a concentration of 20 μL displayed major activity against A. baumannii with an inhibition zone diameter of 30 mm, while in another study, Damjanovic-Vratnica et al. showed an inhibition zone diameter of 36 mm for E. globulus in Montenegro (26).
Inhibition of S. aureus is of great importance, as resistant strains from this species appear each year. Treatment can be a major problem in near future, especially in cases with hospital-acquired infections, which are resistant to methicillin and vancomycin to some extent (11). It has been reported that Gram-negative bacteria have lower sensitivity to volatile EOs of Eucalyptus, compared to Gram-positive bacteria. This can be due to differences in the cell structure of these bacteria, as Gram-positive bacteria have more mucopeptides in their cell wall structure, while Gram-negative bacteria only have a thin layer of mucopeptides; also, lipoprotein and lipopolysaccharides comprise most of the cell structure; therefore, Gram-negative bacteria are more resistant (1, 35, 36).
Borumand et al. determined the antibacterial activity of C. sativum EO against S. aureus and reported MIC and MBC of 1000 ppm. Similar inhibitory effects and better bactericidal properties were reported against Eucalyptus (37). Moreover, Gandomi Nasrabadi et al. reported the MIC of Artemisia absinthium EO against S. aureus to be 3000 ppm, which is less effective than Eucalyptus in our study (38). Damjanovic-Vratnica et al. also showed the significant antimicrobial activity of E. globulus leaf EO against S. aureus bacteria (26).
Ghalem and Mohamed showed that the effects of E. camaldulensis leaf EO against S. aureus bacteria were similar to the present study (35). Furthermore, in a study by Ghaderi et al. Anethum graveolens EO with 312.5 ppm, Coriandrum sativum EO with 625 ppm, and Achillea millefolium EO with 10,000 ppm were effective against the growth of Escherichia coli. However, Achillea millefolium had weaker effects than Eucalyptus in our study (39).
Cimanga et al. showed similar results about the antibacterial activity of EOs from E. citriodora and Monodora myristica (14 mm) against P. vulgaris (14 mm) (34). Since Pseudomonas species can metabolize a wide range of organic compositions (accordingly, it is applied widely in bioremediation), their high level of resistance can be explained. In our experiment, the MIC of E. camaldulensis EO against P. aeruginosa was 2500 ppm, while in the study by Ghaderi et al. Coriandrum sativum and Anethum graveolens EO showed MICs of 5000 and 1250 ppm, respectively (39). In the study by Borumand et al. the MBCs for Coriandrum sativum and Anethum graveolens EO against Salmonella typhimurium were found to be more than 4000 ppm (37).
6. Conclusions
The EO of E. camaldulensis (Myrtaceae family) grown in Iran exhibited major activities against different pathogenic microorganisms. Treatment can be difficult considering the emergence of strains showing resistance to a wide range of antibiotics. The obtained results confirm the potential use of E. camaldulensis EO as an alternative antibacterial agent and a natural drug for the treatment of various infectious diseases.
