Abstract
Antibiotic resistance is a major public health problem. The search for new therapeutic alternatives is becoming urgent. Essential oils are a promising alternative. This study aimed to evaluate the antibacterial activities of essential oils from selected plants on multidrug-resistant zoonotic strains isolated from dogs. Essential oils from dried Thymus vulgaris leaves, Cinnamomum verum bark and Cuminum cyminum seeds were extracted and tested on five multidrug-resistant Escherichia coli and four Staphylococcus aureus isolated from dogs in southern Benin. The study showed that T. vulgaris essential oil was bacteriostatic, with an MIC equal to 2.5 µl ml−1 and a minimum bactericidal concentration (MBC) of 17 µl ml−1 for E. coli strains and 11.25 µl ml−1 for S. aureus strains. Regarding C. verum essential oil, its bacteriostatic power was characterized by an MIC of 1.25 µl ml−1 for the isolates tested and an average MBC of 11.50 µl ml−1 for E. coli and 12.19 µl ml−1 for S. aureus. On the other hand, C. cyminum essential oil was ineffective on the strains investigated. Additionally, T. vulgaris essential oil contained predominantly thymol (36.57%), p-cymene (30.51%) and carvacrol (7.62%), whilst C. verum essential oil contained cinnamaldehyde (88.76%). This study reveals the antibacterial activity of T. vulgaris dry leaf and C. verum bark essential oils on multi-resistant E. coli and S. aureus isolated from dogs. These two essential oils may be alternative candidates for combating antibiotic-resistant E. coli and S. aureus infections.
Keywords: Benin, Cinnamomum verum, Escherichia coli, essential oils, Staphylococcus aureus, Thymus vulgaris
Data Summary
The authors confirm all supporting data, code and protocols have been provided within the article.
Introduction
Antibiotic resistance is a major threat to human and animal health. Indeed, the misuse of antibiotics in humans and animals has been identified as the main cause of the development of antibiotic-resistant pathogens [1]. This misuse includes the use of antibiotics (administration, dispensing or prescribing) for reasons other than treatment, non-completion of prescribed treatments and/or incorrect doses of antibiotics (under- or overdosing). The study conducted by O'Neill [2] showed that resistance to anti-infectives could be responsible for more than 10 million deaths a year, making it the leading cause by 2050, with an economic cost of US$100 billion if precautions are not taken.
Furthermore, close contact between pets and humans (petting, licking or physical injury), as well as the domestic environment, facilitates the transmission of antibiotic-resistant bacteria and genes [3]. Studies have reported antibiotic resistance in strains of Staphylococcus aureus, Escherichia coli, Salmonella spp., Pseudomonas aeruginosa, Streptococcus pyogenes, coagulase-negative Staphylococcus and Staphylococcus pseudintermedius isolated from dogs [4].
In Benin, multi-resistant strains of E. coli and S. aureus have been isolated from both free-ranging and caged dogs, posing a health threat to all those who come into contact with dogs harbouring these micro-organisms.
Nowadays, faced with the increase in drug-resistant bacteria and the limited availability of new, effective antibacterial agents, researchers are prompted to look for new strategies to replace or assist synthetic antibiotics [5]. Essential oils are known to possess a broad spectrum of bioactivities, including antimicrobial, anti-inflammatory, antioxidant, antiviral and antiproliferative properties [6,8]. The antimicrobial properties of essential oils have been known since antiquity and represent the most exploited to date. They can act as both bacteriostatic and bactericidal agents, being capable of inhibiting bacterial growth, thus blocking bacteria’s ability to reproduce and killing bacterial cells [5].
Given the consequences of infections caused by antimicrobial-resistant agents, there is an urgent need to look for alternatives to combat multi-resistant bacteria infections, which are emerging in dogs in our country. The present study aimed to evaluate the antibacterial activity of Thymus vulgaris, Cinnamomum verum and Cuminum cyminum essential oils on multi-resistant E. coli and S. aureus isolated from dogs in southern Benin.
Methods
This study was approved by the Ethical Committee of Research Unit on Communicable Diseases (URMAT in French) of the Polytechnic School of Abomey-Calavi of the University of Abomey-Calavi (N°004/EPAC/LARBA/URMAT/CE/R).
Plant samples used
C. verum bark (YH 1014/HNB), C. cyminum seeds (YH 1015/HNB) and dry leaves of T. vulgaris (YH 1016/HNB) purchased at the market and identified at the National Herbarium of Benin were used in this study.
Essential oil extraction
Essential oils were extracted from dried T. vulgaris leaves, C. verum bark and C. cyminum seeds purchased at the market, at the ‘Laboratoire d’Etude et de Recherche en Chimie Appliquée’. The hydrodistillation method described by Kpatinvoh et al. [9] was used. One hundred grams of each plant material were introduced into 1 l of distilled water, and the mixture was distilled for 3 h in a Clevenger-type hydrodistillation apparatus. The essential oil obtained was separated from the aqueous phase, then dried with sodium sulphate (Na2SO4) and kept cool at 4 °C in a refrigerator for later use [8].
Determination of inhibition diameters of essential oils
The antibacterial activities of essential oils from dry T. vulgaris leaves, C. verum bark and C. cyminum seeds were evaluated on five multi-resistant strains of E. coli (Ec01, Ec02, Ec03, Ec04 and Ec05) and four multi-resistant S. aureus strains (Sa01, Sa02, Sa03 and Sa04) isolated from dogs in southern Benin (Table 1). The disc diffusion method described by Sessou et al. [7] and slightly modified was used. A bacterial suspension was prepared by placing one colony in 5 ml of Mueller–Hinton broth (MHB) (Oxoid, Basingstoke, UK) and adjusted to 108 c.f.u. ml−1. The inoculum thus prepared was inoculated onto Mueller–Hinton agar (Oxoid, Basingstoke, UK). Sterile Whatman paper discs (6 mm in diameter) coated with 10 µl of the essential oil were placed on the previously seeded agar. A negative control was prepared in the same way as the experimental test, and sterile distilled water was added in place of the essential oil. The experimental plates were incubated at 37 °C for 24 h. After incubation, the diameters of the zone of inhibition around the discs were measured. Essential oils with a diameter greater than 14 mm at 10 µl were selected for determination of MIC, MBC and antibiotic potency [7].
Table 1. Antibiotic susceptibility profile of multi-resistant E. coli and S. aureus strains studied.
| Bacteria Antibiotics |
E. coli | S. aureus | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Ec01 | Ec02 | Ec03 | Ec04 | Ec05 | Sa01 | Sa02 | Sa03 | Sa04 | |
| Penicillin G | R | R | R | R | R | R | R | R | R |
| Amoxicillin-clavulanic acid | S | S | S | S | I | S | S | S | S |
| Tetracycline | R | R | R | R | R | R | R | R | R |
| Gentamicin | S | S | S | S | S | S | S | S | S |
| Streptomycin | R | R | R | R | R | R | R | R | R |
| Erythromycin | – | – | – | – | – | R | S | R | S |
| Chloramphenicol | S | S | S | S | S | R | S | S | S |
| Ceftazidime | S | S | S | S | S | I | S | S | I |
| Cefotaxime | S | S | S | S | S | S | S | S | S |
| Cotrimoxazole | R | R | R | R | R | R | R | R | S |
I, intermediate resistance; R, resistant to the antibiotic; S, susceptible to the antibiotic.
Determination of the MIC and MBC of effective essential oils
The MIC and MBC of the essential oils effective on the bacteria tested were determined following the method described by Sessou et al. [7].
Concerning MIC, a stock solution of each essential oil was prepared from 2 ml of MHB, 40 µl of the essential oil and one drop of Tween 80. Thus, 100 µl of MHB was dispensed into each well of a 96-well microplate. One hundred microlitres of the oil stock solution were added to the first well, and successive dilutions of reason 2, well by well, were made up to the eleventh well in each row. All wells except those in the eleventh row were inoculated with 100 µl of bacterial suspension at 106 c.f.u. ml−1, equal density on the McFarland scale. The microplate was then covered with parafilm and incubated at 37 °C for 18–24 h. The eleventh row represents the negative control, whilst the twelfth represents the positive control. The MIC is the lowest concentration for which no visible growth was noted. The MIC was considered to be the concentration of the well in which no visible bacterial growth was observed.
The MBC was determined successively to the MIC. Briefly, the contents of each well, ranging from the MIC value to the highest concentrations, were streaked onto the surface of Mueller–Hinton agar poured into Petri dishes. The wells of the positive and negative control rows were also streaked on the same agar to ensure the absence of bacterial growth in the wells of the negative control row and the presence of bacterial strains in the wells of the positive row. Inoculated agar plates were incubated at 37 °C for 24 h. On reading, MBC was the lowest extract concentration for which there were 0.01% surviving bacteria for the wells [7].
Determining the antibiotic potency of essential oils
The antibiotic potency of the strain is determined by calculating the MBC/MIC ratio. When this ratio is less than or equal to 4, the essential oil is said to be bactericidal, and when the ratio is greater than 4, the essential oil is said to be bacteriostatic [10].
Analysis of the constituents of essential oils effective against multi-resistant bacteria
GC/MS
T. vulgaris leaves and C. verum bark essential oils were analysed on a Hewlett–Packard gas chromatograph model 7890, coupled to a Hewlett–Packard MS model 5875, equipped with a DB5 MS column (30 m×0.25 mm; 0.25 µm), programmed from 50 °C (5 min) to 300 °C at 5 °C min−1, with a 5 min hold. Helium was used as a carrier gas (1.0 ml min−1) with split mode injection (1 : 30); injector and detector temperatures were 250 °C and 280 °C, respectively. The mass spectrometer was operated in electron impact mode at 70 eV, with an electron multiplier (2500 eV) and an ion source temperature (180 °C); mass spectra data were acquired in scan mode in the m/z 33–450 range.
GC/flame ionization detector
T. vulgaris leaves and C. verum bark essential oils were analysed on a Hewlett–Packard gas chromatograph model 6890, equipped with a DB5 MS column (30 m×0.25 mm; 0.25 µm), programmed from 50 °C (5 min) to 300 °C at 5 °C min−1, with a 5 min hold. Hydrogen was used as a carrier gas (1.0 ml min−1), with split mode injection (1 : 60); injector and detector temperatures were 280 °C and 300 °C, respectively. The essential oil was diluted in hexane: 1 out of 30. Compounds determined by GC in the various essential oils were identified by comparing their retention indices with those of reference compounds in the literature and confirmed by GC-MS by comparing their mass spectra with those of reference substances.
Statistical analysis
Data from the analyses were entered into an Excel spreadsheet and analysed using R 4.3.1 software. Inhibition diameters were expressed as mean±sd. A one-factor ANOVA and an unpaired Student’s t-test were performed to compare the means of inhibition diameters. For a P-value<0.05, the difference between the means compared was statistically significant, and for a P-value>0.05, it was statistically non-significant.
Results
Antibacterial activity of the essential oils tested
Analysis of Table 2 shows that E. coli and S. aureus strains are sensitive to T. vulgaris and C. verum essential oils but are resistant to C. cyminum essential oil. The average diameter of the zone of inhibition of T. vulgaris was 31 and 36.5 mm, respectively, for E. coli and S. aureus strains, whilst that of C. verum was 33.8 and 35.25 mm, respectively, for the same strains.
Table 2. Average diameters of the inhibition zones of the essential oils of the plants studied.
| Strains | Code | T. vulgaris | C. verum | C. cyminum |
|---|---|---|---|---|
| Mean | Mean | Mean | ||
| E. coli | Ec01 | 32 | 35 | 0 |
| Ec02 | 34 | 32 | 0 | |
| Ec03 | 30 | 35 | 0 | |
| Ec04 | 29 | 34 | 0 | |
| Ec05 | 30 | 33 | 0 | |
| Mean±sd | 31±2.00ba | 33.8±1.30ca | 0.00aa | |
| S. aureus | Sa01 | 27 | 35 | 10 |
| Sa02 | 44 | 35 | 13 | |
| Sa03 | 32 | 37 | 8 | |
| Sa04 | 43 | 34 | 13 | |
| Mean±sd | 36.5±8.35ba | 35.25±1.26ba | 11±2.44ab |
Means followed by different letters are statistically different at the 5% threshold.
The first letters indicate comparisons of averages within the same bacterium, and the second letters indicate comparisons of means of the diameters of each oil between the two bacterial species studied.
MIC and MBC of essential oils effective on multi-resistant strains
The MIC of T. vulgaris essential oil was the same for E. coli and S. aureus strains (2.5±0.00 µl ml−1), whilst the average MBC was 17 µl ml−1 for E. coli strains and 11.25 µl ml−1 for S. aureus strains. For C. verum essential oil, an MIC equal to 1.25 µl ml−1 was obtained for E. coli and S. aureus strains, whilst a mean MBC equal to 11.50 µl ml−1 was recorded for E. coli strains and 12.19 µl ml−1 for S. aureus strains (Table 3).
Table 3. MIC and MBC of T. vulgaris and C. verum essential oils.
| Strains | Code | T. vulgaris | C. verum | ||
|---|---|---|---|---|---|
| MIC (μl ml−1) | MBC (μl ml−1) | MIC (μl ml−1) | MBC (μl ml−1) | ||
| Mean±sd | Mean±sd | Mean±sd | Mean±sd | ||
| E. coli | Ec01 | 2.5±00 | 20±00 | 1.25±00 | 10±00 |
| Ec02 | 2.5±00 | 5±00 | 1.25±00 | 2.5±00 | |
| Ec03 | 2.5±00 | 20±00 | 1.25±00 | 15±00 | |
| Ec04 | 2.5±00 | 20±00 | 1.25±00 | 15±00 | |
| Ec05 | 2.5±00 | 20±00 | 1.25±00 | 15±00 | |
| Mean±sd | 2.5±00a | 17±6.71b | 1.25±00a | 11.50±5.48a | |
| S. aureus | Sa01 | 2.5±00 | 20±00 | 1.25±00 | 15±00 |
| Sa02 | 2.5±00 | 15±00 | 1.25±00 | 15±00 | |
| Sa03 | 2.5±00 | 5±00 | 1.25±00 | 15±00 | |
| Sa04 | 2.5±00 | 5±00 | 1.25±00 | 3.75±00 | |
| Mean±sd | 2.5±00a | 11.25±7.5a | 1.25±00a | 12.19±5.62a | |
Means followed by different letters are statistically different at the 5% threshold.
Antibiotic potency of T. vulgaris and C. verum essential oils effective on multi-resistant strains
Analysis of Table 4 shows that T. vulgaris and C. verum essential oils were bacteriostatic against the multi-resistant E. coli and S. aureus strains tested.
Table 4. Antibiotic power of T. vulgaris and C. verum essential oils.
| T. vulgaris | C. verum | |||
|---|---|---|---|---|
| Strains | MBC/CMI | Antibiotic potency | MBC/CMI | Antibiotic potency |
| E. coli | 6.80 | Bacteriostatic | 9.20 | Bacteriostatic |
| S. aureus | 4.50 | Bacteriostatic | 9.75 | Bacteriostatic |
Chemical composition of T. vulgaris and C. verum essential oils
Analysis of the essential oils revealed thymol (36.57%), p-cymene (30.51%) and carvacrol (7.62%) as the major compounds in T. vulgaris essential oil (Table 5). In C. verum essential oil, cinnamaldehyde was found to be the major component with a percentage of 88.76% (Table 6).
Table 5. Chemical composition of T. vulgaris essential oil.
| No. | Components | RT | RI | Percentage (%) |
|---|---|---|---|---|
| 1 | Camphene | 3.5094 | 1074 | 0.72 |
| 2 | d-Limonene | 5.6248 | 1194 | 0.44 |
| 3 | Eucalyptol | 5.7768 | 1202 | 1.42 |
| 4 | Gamma-Terpinene | 6.6085 | 1240 | 0.43 |
| 5 | p-Cymene | 7.2039 | 1267 | 30.51 |
| 6 | 1-Octen-3-ol | 11.5015 | 1444 | 1.42 |
| 7 | (+)−2-Bornanone | 12.8575 | 1498 | 0.48 |
| 8 | Linalool | 13.9004 | 1540 | 2.45 |
| 9 | 2-Isopropyl-5-methyl-anisole | 14.9475 | 1582 | 3.08 |
| 10 | Terpinen-4-ol | 15.1333 | 1589 | 1.10 |
| 11 | 2-Isopropyl-1-methoxy-4-methylbenzene | 15.205 | 1592 | 1.07 |
| 12 | Estragole | 16.7715 | 1656 | 0.82 |
| 13 | Endo-borneol | 17.4639 | 1685 | 2.18 |
| 14 | γ-Cadinene | 18.8446 | 1739 | 0.62 |
| 15 | Anethole | 20.6982 | 1810 | 1.65 |
| 16 | p-Cymen-8-ol | 21.3231 | 1833 | 0.51 |
| 17 | Caryophyllene oxide | 24.7769 | 1955 | 1.68 |
| 18 | 2-Propanone, 1-(4-methoxyphenyl)- | 29.9196 | 2129 | 0.39 |
| 19 | 3-Allyl-6-methoxyphenol | 30.2869 | 2142 | 2.52 |
| 20 | Thymol | 31.0891 | 2169 | 36.57 |
| 21 | 3-Methyl-4-isopropylphenol | 31.5029 | 2183 | 1.15 |
| 22 | Carvacrol | 31.8027 | 2193 | 7.62 |
| 23 | Eugenol acetate | 32.9976 | 2234 | 1.16 |
RI, retention index; RT, retention time.
Table 6. Chemical composition of C. verum essential oil.
| No. | Components | RT | RI | Percentage (%) |
|---|---|---|---|---|
| 1 | Anethole | 20.6984 | 1810 | 1.79 |
| 2 | 2-Propenal, 3-phenyl- | 22.4084 | 1872 | 2.47 |
| 3 | Cinnamaldehyde, trans | 26.7362 | 2022 | 88.76 |
| 4 | Cinnamyl acetate | 29.8438 | 2127 | 3.20 |
| 5 | Cinnamaldehyde, o-methoxy | 37.914 | 2403 | 3.78 |
RI, retention index; RT, retention time.
Discussion
This study assessed the antibacterial activity of T. vulgaris, C. cyminum and C. verum oils on multidrug-resistant E. coli and S. aureus isolated from dogs in southern Benin and determined the chemical composition of the effective essential oils.
The results of this study revealed the bacteriostatic power of T. vulgaris dry leaf and C. verum bark essential oils on multi-resistant E. coli and S. aureus strains.
The antimicrobial activity of T. vulgaris essential oil depends on the percentage composition of its main components [11]. In this study, thymol presented the highest percentage in the T. vulgaris essential oil tested (36.57%). This percentage may explain the bacteriostatic power of this oil. Indeed, thymol, chemically known as 2-isopropyl-5-methylphenol, is an edible monoterpene phenol found in abundance in certain plants such as T. vulgaris [12,13]. Studies investigating the mechanism of thymol’s antibacterial activity have indicated that its ability to integrate into the lipid layer of the cell membrane increases surface curvature. The hydrophilic part of the thymol interacts with the polar part of the membrane, whilst the hydrophobic benzene ring and aliphatic side chains sink into the inner part of the biological membrane [11]. This interaction leads to major changes in membrane structure, with destabilization of the lipid layer, reduced elasticity and increased fluidity. The change leads to increased permeability to potassium and hydrogen ions and also affects the activity of internal membrane proteins such as enzymes and receptors. After incorporation into the cell membrane, thymol interacts with the proteins embedded in it via a variety of non-specific mechanisms, leading to changes in the conformation and activity of internal and membrane proteins. As a result, cell membrane tension and destabilization can be induced by the presence of thymol [11].
The bacteriostatic power of T. vulgaris essential oil may also be linked to the presence of p-cymene in the oil tested. p-Cymene, known as p-cymol or p-isopropyltoluene, is an alkyl-substituted aromatic compound naturally present in the essential oils of various aromatic plants, including Artemisia, Protium, Origanum and Thymus. It is related to the terpene family, in particular the monocyclic monoterpenes [14]. Studies on the biological activities of this molecule have shown that it has synergistic antibacterial activity with other molecules such as carvacrol [15,16].
Carvacrol is a natural monoterpene phenol that is particularly abundant in the essential oils of Origanum vulgare, T. vulgaris, Lepidium flavum, Citrus aurantium bergamia and other plants [17,18]. Carvacrol interacts with the cell membrane via hydrogen bonding, making membranes and mitochondria more permeable and disintegrating the outer cell membrane [19]. Its antibacterial activity on E. coli [20] and methicillin-resistant S. aureus (ATCC-33591) strains has been demonstrated [21]. The bacteriostatic power of T. vulgaris essential oil can also be attributed to the synergistic activity of thymol, p-cymene and carvacrol [6,22, 23].
C. verum essential oil’s bacteriostatic power against E. coli and S. aureus strains can be linked to its high cinnamaldehyde content. Cinnamaldehyde is a chemical compound that occurs naturally as a trans-stereoisomer, namely, (2E)-3-phenylprop-2-enal or trans-cinnamaldehyde, which is particularly abundant in the essential oils of plants in the Cinnamomum genus [5]. Trans-cinnamaldehyde has been shown to possess substantial antimicrobial activity, as well as a range of medicinal properties [24,26]. The antibacterial activities of this molecule extend to a range of Gram-positive and negative bacteria, such as E. coli, Bacillus subtilis, Staphylococcus spp., Listeria spp., Salmonella spp., Lactobacillus sakei, Campylobacter jejuni, Vibrio spp., Pseudomonas spp., Porphyromonas gingivalis, S. pyogenes and Cronobacter sakazakii [27].
In this study, the essential oil of C. cyminum seeds proved ineffective on the multidrug-resistant E. coli and S. aureus strains investigated. However, in the study by Sharifi et al. [28] C. cyminum seed oil showed bacteriostatic and bactericidal activities on multidrug-resistant S. aureus isolated from patients and from the milk of cattle suffering from mastitis. This difference in results is linked to the different sources of bacteria studied, the resistance mechanisms developed by these strains and the chemical composition of the oils tested.
Conclusion
This study demonstrated the antibacterial activity of T. vulgaris dry leaf and C. verum bark essential oils on multi-resistant E. coli and S. aureus strains. Both oils are bacteriostatic on the zoonotic agents studied. The study also revealed the components responsible for antibacterial activity. Thymol, p-cymene and carvacrol were the main components of T. vulgaris essential oil, whilst cinnamaldehyde was the main component of C. verum essential oil. The two oils investigated in this study may be alternative candidates for combating infections with resistant bacterial agents, such as E. coli and S. aureus.
Acknowledgements
The authors thank the Ministry of Science and Technology (DST), the Ministry of External Affairs (MEA) of the Government of India (GoI) and the Federation of Indian Chambers of Commerce and Industry (FICCI), who, through the C.V. Raman International Fellowship for African Researchers, funded the work on antibacterial activity of Thymus vulgaris and Cinnamomum verum essential oils on the strains.
Abbreviations
- MBC
minimum bactericidal concentration
- MHB
Mueller–Hinton broth
Footnotes
Funding: This work received funding from the Ministry of Science and Technology (DST), the Ministry of External Affairs (MEA) of the Government of India (GoI) and the Federation of Indian Chambers of Commerce and Industry (FICCI) through the C.V. Raman International Fellowship for African Researchers to A.B.Y.
Author contributions: A.B.Y.: conceptualization, statistical analysis, writing original draft, review and editing; A.D. and R.N.B.: methodology; F.M. and M.-L.F.: essential oil analysis; P.A., L.B.-M., S.F., K.A. and P.S.: supervision, review and editing.
Ethical statement: The study was approved by the Ethical Committee of Research Unit on Communicable Diseases (URMAT in French) of the Polytechnic School of Abomey-Calavi of the University of Abomey-Calavi (N°004/EPAC/LARBA/URMAT/CE/R).
Contributor Information
Ayaovi Bruno Yaovi, Email: brunoyaovi@gmail.com.
Arpita Das, Email: a_das@bt.iitr.ac.in.
Rama N. Behera, Email: rama_b@bt.iitr.ac.in.
Paulin Azokpota, Email: azokpotap@yahoo.fr.
Souaïbou Farougou, Email: farougou@gmail.com.
Lamine Baba-Moussa, Email: laminesaid@yahoo.fr.
Franck Michels, Email: fmichels@uliege.be.
Marie-Laure Fauconnier, Email: marie-laure.fauconnier@uliege.be.
Kiran Ambatipudi, Email: kiran.ambatipudi@bt.iitr.ac.in.
Philippe Sessou, Email: sessouphilippe@yahoo.fr;philippe.sessou@uac.bj.
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