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Article

Antimicrobial Activity and the Synergy Potential of Cinnamomum aromaticum Nees and Syzygium aromaticum (L.) Merr. et Perry Essential Oils with Antimicrobial Drugs

by
Soraia El Baz
1,*,
Bouchra Soulaimani
1,
Imane Abbad
1,
Zineb Azgaou
1,
El Mostapha Lotfi
2,
Mustapha Malha
2 and
Noureddine Mezrioui
1
1
Laboratory of Water Sciences, Microbial Biotechnologies, and Natural Resources Sustainability (AQUABIOTECH), Unit of Microbial Biotechnologies, Agrosciences, and Environment (BIOMAGE)-CNRST Labeled Research Unit N°4, Faculty of Sciences-Semlalia, University Cadi Ayyad, P.O. Box 2390, Marrakech 40000, Morocco
2
Laboratory of Spectroscopy, Molecular Modeling, Materials, Nanomaterials, Water and Environment, Environmental Materials Team, ENSAM, Mohammed V University, Rabat 10000, Morocco
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(3), 63; https://doi.org/10.3390/microbiolres16030063
Submission received: 22 January 2025 / Revised: 12 February 2025 / Accepted: 20 February 2025 / Published: 10 March 2025
Versions Notes

Abstract

:
Antimicrobial resistance is a growing global challenge, rendering many standard treatments ineffective. Essential oils (EOs) of cinnamon (Cinnamomum aromaticum Nees) and clove (Syzygium aromaticum (L.) Merr. et Perry) may offer an alternative solution due to their high antimicrobial properties and their abilities to fight resistant pathogens. This study evaluates the antimicrobial activity of these two EOs, and their synergistic potential when combined with two antibiotics (ciprofloxacin and vancomycin) and two antifungals (fluconazole and amphotericin B) against various bacterial and yeasts strains. The antimicrobial activities of each EO were evaluated by agar diffusion and broth microdilution assays, while the synergetic effects with antimicrobials were determined by calculating the fractional inhibitory concentration index (FICI) using the checkerboard method. The chemical composition of the EOs was analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). The identification of individual components in the EOs was achieved by comparing their mass spectra with the NIST MS Search database and by correlating their retention times with those of known standards. GC-MS analysis revealed that the main constituents of S. aromaticum EO were eugenol (71.49%) and β-caryophyllene (23.43%), while C. aromaticum EO were dominated by cinnamaldehyde (47,04%) and cinnamyl acetate (18.93%). Antimicrobial activity showed that cinnamon EO exhibits highest effectiveness against all tested strains, with inhibition zones (IZ) ranging from 16.99 mm to 53.16 mm, and minimum inhibitory concentrations (MIC) and minimum microbicidal concentrations (MMC) ranging from 0.039 mg/mL to 0.156 mg/mL. However, for clove EO, the IZ ranged from 9.31 mm to 29.91 mm, with MIC and MMC values from 0.313 mg/mL to 1.25 mg/mL. In combination with antibiotics (ciprofloxacin and vancomycin), the studied EOs showed promising synergistic effects with reduction up to 128-fold. As regards antifungals (amphotericin B, and fluconazole), the synergistic effects were recorded with MIC gains up to 32-fold. Our findings demonstrate that the EOs from C. aromaticum and S. aromaticum exhibit significant broad-spectrum antimicrobial activity against diverse yeast and bacterial strains. This highlights their potential as bases for the development of novel plant-based antimicrobial agents. Importantly, the observed synergistic effects of these EOs with conventional antibiotics support their integration into medical treatments as a strategy to address microbial resistance. Future research should aim to elucidate the mechanisms underlying these synergistic actions, optimize their application, and enhance their therapeutic efficacy.

1. Introduction

The discovery of antibiotics stands as one of humanity’s greatest discoveries of the 20th century. This discovery has significantly decreased the prevalence and mortality rate of infectious diseases [1]. However, the effectiveness of many antibiotics is currently under threat due to the emergence and development of antimicrobial resistance [2]. This is mainly a result of the overuse and sometimes irrational application of these antimicrobial drugs, which has allowed for certain microorganisms to become resistant [3]. Currently, antimicrobial resistance (AMR) is a persistent issue in global public health, with predictions estimating that it could cause up to 10 million deaths annually by 2050 [4]. The urgent need for new alternative antimicrobial agents has become a priority to address AMR issues [5]. Among these alternatives, essential oils (EOs) have long been recognized as natural products with interesting antimicrobial effectiveness against several microorganisms that are resistant to standard antimicrobials [6]. Besides the growing interest in developing plant-based antimicrobial compounds, another emerging strategy involve the use of EOs as natural agents to combat antimicrobial resistance, either by directly destroying pathogens or by resensitizing resistant microorganisms to antibiotic, offering a promising alternative to conventional antibiotics [7,8]. Numerous studies have reported that EOs can act on multiple target sites within bacterial cells, including disrupting cell membrane integrity, inhibiting protein synthesis, and altering nucleic acid synthesis. EOs have also been shown to interfere with the quorum sensing pathways that bacteria use for communication and coordination of gene expression [9]. These advantage of EOs support their use in synergistic combination with conventional antibiotics. This approach can improve the antimicrobial therapy and reinstate the use of outdated, inexpensive drugs that have become largely ineffective [10,11,12]. Generally, combinations of single EO or mixture of their major purified components, influence multiple biochemical processes in bacteria, producing a broad spectrum of antibacterial activities due to their capacity to interfere with various bacterial function [13]. Several studies have highlighted synergistic or additive effects between antibiotics and EOs. For example, oregano EO has demonstrated synergistic activity when combined with doxycycline, florfenicol, or sarafloxacin against E. coli [14]. Similarly, rosemary oil, when combined with ciprofloxacin, has shown synergistic effects against Klebsiella pneumoniae and Staphylococcus aureus [15]. Thyme EOs exhibited synergy with norfloxacin, gentamicin, pristinamycin, and ciprofloxacin against various bacterial strains [15,16,17]. Additionally, tea tree oil has been reported to enhance the efficacy of aminoglycoside antibiotics against E. coli, Yersinia enterocolitica, Serratia marcescens, and one strain of S. aureus [18].
In this context, clove (Syzygium aromaticum (L.)) and cinnamon (Cinnamomum aromaticum Nees) EOs have been explored as alternative therapeutic option to combat some resistant pathogens. C. aromaticum (synonym of Cinnamomum cassia), one of the oldest known spices, has been used for centuries for its distinct flavor and aroma [19]. Cinnamon has also been used in traditional medicine to treat various health issues including digestive disorders, inflammations, headaches, fever, nausea, anorexia, and colds [20]. Cinnamon EO exhibits remarkable antioxidant, and antimicrobial effects against a wide range of pathogens, including bacteria and fungi [21,22]. Similarly, Syzygium aromaticum EO, renowned for its diverse applications, is extensively used across pharmaceuticals, food, and fragrance industries [23]. Traditionally, clove EO has been used to treat asthma, digestive problems, toothaches, respiratory issues, headaches, and sore throats [24]. Moreover, this EO exhibits potent antibacterial, antifungal, antiviral, anti-inflammatory, cytotoxic, and anesthetic properties [23]. It has been found that EOs containing cinnamaldehyde or eugenol as their main components exhibit the strongest antibacterial activity against several bacteria, including Salmonella typhimurium, Listeria monocytogenes, and Bacillus cereus [25,26,27]. Cinnamaldehyde has been found to disrupts bacterial cell membrane by inhibiting ATPase activity [28] while eugenol also disrupts membranes by inhibiting ATPase activity and blocking efflux pumps [29]. The synergistic effects of clove and cinnamon EOs in combination with certain antibiotics have been well-documented in numerous studies, underlining their potential to restore the effectiveness of these conventional drugs and consequently to combat antimicrobial resistance. These findings present compelling opportunities to explore such synergies with more commonly used and currently less effective conventional drugs. Thus, the objective of the present study is to investigate the antimicrobial activity of EOs extracted from these two valuable medicinal and aromatic plants ‘C. aromaticum and S. aromaticum’, and to evaluate, for the first time, their synergistic potential with two antibiotics (ciprofloxacin and vancomycin) and two antifungals (fluconazole and amphotericin B) against a panel of both Gram-positive and Gram-negative bacteria, as well as some candida species.

2. Materials and Methods

2.1. Plant Materials and EOs Extraction

The plant materials of C. aromaticum and S. aromaticum used in this study were purchased from a local open-air market. The EOs were extracted from flower buds of S. aromaticum and from bark of C. aromaticum (Table 1). Dried plant material from each species was subjected separately to three steam distillations (3 × 200 g) for approximately 3 h using a Clevenger-type apparatus. The recovered oils were then dried with anhydrous sodium sulfate and stored in amber bottles at 4 °C until further use. The EO yield was calculated as % (v/w) based on the dry plant material using the following equation:
% Yield of the EO = (Volume of the extracted EO (mL)/Weight of the dried material (g)) × 100.

2.2. GC/MS Analysis

The GC-MS chemical analysis of the EO was performed using Thermo trace 1300 with a TG-5MS column (length: 30 m; internal diameter: 0.25 mm, film thickness: 0.25 µm) and coupled with mass spectrometer TSQ 8000 EVO (triple quadrupole). The initial temperature of the column was adjusted to 40 °C for 2 min, then increased to 180 °C at a rate of 4 °C/min. The temperature was further raised to 300 °C at a rate of 20 °C/min and held at this final temperature for 2 min. Helium was used as the carrier gas, with a flow rate of 1.5 mL/min, and the full scan mode was adopted. The injector temperature was maintained at 200 °C, and the transfer line temperature was set to 250 °C. The ionization source temperature was set at 200 °C with electron ionization (EI) at 70 eV. One microliter of EO, diluted in hexane at 1:10 (v:v) ratio, was injected manually in split mode. The components of the EOs were determined through comparison with the NIST MS Search database, and their retention times with those of main standards.

2.3. Antimicrobial Assay

2.3.1. Microorganism Strains

Antibacterial activity of the studied EOs was evaluated against a range of microorganisms, including five pathogenic bacteria, namely Listeria monocytogenes CECT 4032, Staphylococcus aureus CECT 976, Escherichia coli CECT 4783, Salmonella enterica ATCC 14028, and Klebsiella pneumoniae (clinical isolate). Additionally, four clinically isolated Candida species, provided by the Moroccan coordinated collection of microorganisms, were tested, namely Candida albicans CCMM-L4 and Candida glabrata CCMM-L7 from vaginal sampling, Candida krusei CCMM-L10 from human blood, and Candida parapsilosis CCMM-L18 from human skin.
The streak-plate technique was employed to subculture the pure cultures obtained to ensure the viability of the obtained yeast and bacterial cultures. A sterilized cotton swab was used to streak the inoculum onto nutrient agar plates for bacteria and Sabouraud Dextrose agar for yeast. After inoculation, the Petri dishes were carefully wrapped with parafilm to prevent contamination and ensure uniform growth of microorganisms. The strains were then incubated at a temperature of 37 °C for 24 h for bacteria and at 28 °C for 48h for yeasts, allowing for optimal growth and development of microorganisms. All tested strains were then maintained in glycerol 20% at −20 °C. Prior to antimicrobial susceptibility testing, each isolate was inoculated on Sabouraud dextrose agar (SDA) for yeasts and on nutrient agar for bacteria to ensure optimal growth characteristics and purity.

2.3.2. Disc Diffusion Assay

The antimicrobial activity of each EO was evaluated using the agar disc diffusion method, following the guideline provided by Clinical and Laboratory Standards Institute [30]. For this procedure, 0.1 mL of cell suspensions at 108 UFC/mL for bacteria and 105 UFC/mL for yeasts were spread onto the surface of Mueller Hinton (MHA) or Sabouraud Dextrose (SDA) agar plates, respectively. Sterile filter paper discs (6 mm diameter) were individually impregnated with 10 µL of each EO and placed on previously inoculated agar plates. All plates were kept at 4 °C for 2 h to allow the diffusion of EOs and then incubated at 37 °C for 18–24 h for bacteria and at 28° for 48 h for yeasts. After incubation, the inhibition zone diameters (IZ), including disc diameter, were measured in mm. Ciprofloxacin (5 µg/disc) and vancomycin (10 µg/disc) were used as standard antibacterial agents, while fluconazole (10 µg/disc) and amphotericin B (5 µg/disc) were used as standard antifungal agents. All assays were performed in triplicate.

2.3.3. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC)

The antimicrobial activity of the EOs were evaluated quantitatively using the broth microdilution method according to the CLSI guidelines: M07-A10 for bacteria [31] and M27-A3 for yeasts [32]. Briefly, 100 μL of two-fold serial dilutions of each EO, previously prepared in Mueller Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for yeast using 1% dimethylsulphoxide (DMSO), were added to microwells containing 100 µL of the cell suspensions at 106 UFC/mL for bacteria and 1 − 2 × 103 cells/mL for yeasts. The microplates were incubated for 24 h at 37 °C for bacteria and at 28 °C for 48 h for yeasts. The MIC was determined as the lowest EO concentration that inhibited macroscopic growth of the tested strains.
To determine the minimum microbicidal concentration (MMC), 0.1 mL was taken from all clear wells that showed no visible growth during MIC assays and spread onto MHA and incubated at 37 °C for 24 h for bacteria [33], or onto SDA and incubated at 28° for 48 h for yeasts [32]. The MMC was defined as the lowest concentration of the EO required to kill 99.99% of the microorganisms [31]. Ciprofloxacin and vancomycin were used as standard antibacterial agents, while fluconazole and amphotericin B were used as standard antifungal agents.

2.3.4. Synergistic Effect of EOs with Standard Antimicrobials

The checkerboard design, based on the microdilution method, was used to evaluate the synergistic interactions between the two studied EOs and conventional antimicrobial drugs [34,35]. The antimicrobial drugs used were two antifungals, namely fluconazole and amphotericin B, and two antibiotics, namely ciprofloxacin and vancomycin. For this, mixtures were prepared by adding 50 µL of the EO concentration to 50 µL of different concentrations of the antimicrobial drug. Each microwell was then inoculated with 100 µL of a cell suspension. The analysis of the different combinations of EO with antimicrobials was performed by calculating the MIC gain of antimicrobials, which expresses how many times the MIC has been reduced in the combined application, using the following formula: MIC gain = MIC of antimicrobial alone/MIC of antimicrobial in combination. Additionally, to determine the interaction types generated by different combinations, the fractional inhibitory concentration index (FICI) was calculated using the following formula: FICI = FIC (EO) + FIC (antimicrobial).
With FIC (EO) = MIC of EO in combination with antimicrobial/MIC of EO alone, and FIC (antimicrobial) = MIC of antimicrobial in combination with EO/MIC of antimicrobial alone, the FICI results were interpreted as follows: synergism was defined when FICI ≤ 0.5, additive effects when 0.5 < FICI ≤ 1, indifference when 1 < FICI ≤ 2, or antagonism when FICI ≥ 2 [36].

3. Results and Discussion

3.1. Yield and Chemical Composition of the EOs

The yields of the EOs of C. aromaticum and S. aromaticum are presented in Table 1. The results show an average yield of 0.74% for C. aromaticum. Other studies reported similar profiles with EO yield, ranging from 0.41 to 1.8% [37,38]. For S. aromaticum, the yield was 2.4%, which aligns well with value reported in previous study, where the yield was 2.52% [39]. However, other study reported lower EO yield ranging from 0.7 to 0.92% [40]. This variation in the EO yield may depend on several factors, including geographical location, extraction technique used, transport and storage conditions [41,42].
The composition of the volatile oils was determined by GC-MS, and the results, including the identified compounds, their relative contents, elution order, retention times (RT), and structural subclasses are presented in Table 2. In total, 16 constituents were identified in C. aromaticum EO, accounting for 98.14%, and 11 constituents were identified in S. aromaticum, accounting for 98.65% of the total volatile oil. The main constituents of S. aromaticum EO were eugenol (71.49%), β-caryophyllene (23.43%), and eugenol acetate (2%) (Table 2), which is consistent with previous studies [43,44,45,46]. However, other investigations reported similar profiles but with lower eugenol contents, ranging from 49% to 55% [47,48]. Regarding C. aromaticum EO, the major compounds identified were cinnamaldehyde (47.04%), cinnamyl acetate (18.93%), (Z)-cinnamyl alcohol (8.16%), and copaene (6.33%). This finding, in agreement with the literature, reports comparable profiles with some fluctuations in cinnamaldehyde contents (33–85%) [38,49,50]. This variation in the chemical composition of EOs depend on various intrinsic factors, such as the species, its phenological stage, the drying and distillation method, and the harvesting time. Additionally, extrinsic parameters related to climatic, edaphic and agro-ecological as well as storage conditions, can also influence the composition of EOs [41,42,51,52].

3.2. Antimicrobial Activity

The results of antimicrobial activities demonstrated that C. aromaticum and S. aromaticum EOs were active against all tested bacterial and yeast strains (Table 3 and Table 4). Generally, C. aromaticum EO has exhibited superior efficacy, with IZ ranging from 16.99 mm to 53.16 mm, MIC from 0.039 mg/mL to 0.078 mg/mL, and MMC from 0.039 mg/mL to 0.156 mg/mL. In contrast, S. aromaticum EO showed smaller IZ diameters, ranging from 9.31 mm to 29.91 mm, and higher MIC and MMC values, which ranged from 0.313 mg/mL to 1.25 mg/mL and from 0.625 mg/mL to 1.25 mg/mL, respectively.
For bacteria, the results obtained for C. aromaticum EO showed that all the bacterial strains tested were inhibited at MIC equal to 0.078 mg/mL, with the exception of S. aureus, which showed an increased sensitivity with MIC reduced to 0.039 mg/mL. Additionally, this EO demonstrated a bactericidal effect for all bacterial strains (Table 3). These results concur with previous studies, which have found that C. aromaticum EO is effective against various bacterial species, including E. coli, L. monocytogenes, Klebsiella sp., and S. aureus [53,54,55]. Furthermore, the phenylpropanoid cinnamaldehyde, which represents 71% of C. aromaticum EO, have been documented to inhibit certain microorganisms through different mechanisms such as cytoplasmic granulation, increased cytoplasmic acidity, disruption of cell wall structure, inhibition of enzyme synthesis, and depletion of intracellular ATP stores [56,57]. The antibacterial effect of S. aromaticum EO was relatively similar against the five strains tested, with IZ ranging from 9.31 ± 0.29 mm to 11.09 ± 0.33 mm. The MIC was 1.25 mg/mL for all bacterial strains, except E. coli and S. enterica, which appeared more sensitive with a reduced MIC of 0.625 mg/mL. This EO also demonstrated a bactericidal effect, as the MMC=MIC for all bacterial strains (Table 3). Similarly, previous studies have demonstrated the high antibacterial action of this EO against strains of K. pneumonia, E. coli and S. aureus [58,59,60]. Generally, S. aromaticum EO exhibits bactericidal activities that cause significant structural and functional damage to bacterial cell, such as holes in the envelope and cell deformities [61]. Moreover, S. aromaticum EO has been reported to inhibit critical bacterial enzymes and toxin production, which are crucial for the virulence of many pathogenic bacteria. For example, it inhibited the production of listeriolysin O toxin by L. monocytogenes [62]. Furthermore, another study demonstrated that eugenol, the main compound in this EO, exhibited potent antimicrobial efficacy on E. coli, L. monocytogenes, S. carnosus, and P. fluorescens [63]. Eugenol acts effectively by disrupting cell membranes and it inhibits essential bacterial functions [64].
Regarding Candida strains, C. aromaticum EO demonstrated IZ ranging from 35.69 ± 0.31 mm to 53.16 ± 0.43 mm, and all tested strains were inhibited at a concentration of 0.039 mg/mL. A pronounced fungicidal effect was observed against C. glabrata, C. krusei, and C. parapsilosis, with MIC values equal to the MMC (Table 4). These results show the fungicidal activity against the tested candida strains, highlighting the potential of this EO as an effective antifungal agent. In line with this finding, it has been reported that C. aromaticum EO possesses a remarkable antifungal activity, even at very low concentrations [49,65]. Moreover, cinnamaldehyde has been shown to be effective against a wide range of yeast strains such as Candida sp. [66,67]. It has been reported to be the responsible for the antifungal properties of cinnamon EO by damaging cell membranes, altering lipid profiles, inhibiting enzymatic activities, and disrupting the reproduction of various fungi [68,69]. As regards S. aromaticum EO, tested Candida strains showed varied responses toward this EO effect, with IZ ranging from 23.37 ± 0.26 mm to 29.91 ± 0.15 mm. The lowest MIC (0.313 mg/mL) was recorded for C. krusei, indicating increased sensitivity compared to other Candida strains, which were inhibited at the same MIC of 0.625 mg/mL (Table 4). These results suggest a potent anticandidal effect and significant variations in the sensitivity of Candida strains to S. aromaticum EO, aligning with the findings in the literature [24,70,71]. Pinto et al. [72] demonstrated that S. aromaticum EO and its main component eugenol possess antifungal properties against both standard and clinical yeast strains. Additionally, flow cytometric analyses and the inhibition of ergosterol synthesis revealed that their antifungal activities are due to extensive lesions in the cell membrane and a significant reduction in the amount of ergosterol [73,74]. The mechanism of action of antimicrobial agents varies depending on the target microorganism. Due to their hydrophobic nature, the chemical components of EOs tend to accumulate in the lipid-rich regions of cell membranes, leading to significant structural and functional damage [75].

3.3. Synergistic Effect of EOs with Standard Antibiotics

The synergistic effects of EOs with the two antibiotics (vancomycin and ciprofloxacin) are presented in Table 5 and Table 6. The combination of C. aromaticum EO with vancomycin demonstrated a notable synergistic effect against all strains, except for S. enterica, where indifference effect was observed (FICI = 1.25). The other bacterial strains showed FICIs ranging from 0.27 to 0.75, indicating synergy with 2- to 64-fold reductions in the vancomycin MIC. In combination with ciprofloxacin, C. aromaticum EO showed a total synergistic effect against S. aureus, E. coli, and S. enterica, with FICIs ranging from 0.26 to 0.50, corresponding to 4- to 128-fold reductions in the MIC of this antibiotic. Nevertheless, indifferent effect was observed against L. monocytogenes and K. pneumoniae (FICI = 1.25). Similarly, cinnamon EO showed synergistic effects with other standard antibiotics (e.g., ampicillin, chloramphenicol and piperacillin) against Staphlococcus sp., P. aeruginosa, and E. coli [76,77,78,79].
The synergistic effects of S. aromaticum EO with ciprofloxacin showed a total synergistic effect against all tested bacteria, except K. pneumoniae, where an additive effect was observed (FICI = 0.75), with a 2-fold reduction in the antibiotic MIC (Table 6). The FICI values obtained for the other bacterial strains ranged from 0.27 and 0.50, indicating synergistic interaction with a 4- to 64-fold reductions in the ciprofloxacin MIC.
Additionally, the combination of S. aromaticum EO with vancomycin demonstrated a synergistic effect against all bacterial strains, except S. enterica, where no effect was observed. The FICIs for this combination ranged from 0.31 to 0.75, leading to a 2- to 16-fold reductions in the MIC of vancomycin. In agreement with these findings, several studies have found that the combination of S. aromaticum EO and some antibiotics reduced their MICs against a number of bacteria, including E. coli, E. aerogenes, P. vulgaris, P. aeruginosa, and S. typhimurium [9,80,81,82]. Furthermore, the combination of Syzygium aromaticum EO with vancomycin has been shown to significantly enhance the antibiotic’s efficacy, reducing the MIC of vancomycin by up to 8- and 16-fold against Staphylococcus species [83]. Moreover, the main component of S. aromaticum EO, eugenol, has been demonstrated to interact synergistically with antibiotics such as gentamicin, ampicillin, and vancomycin [84]. A study investigating the combination of eugenol with ten antibiotics against Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Salmonella typhi-murium, and Proteus vulgaris demonstrated significant synergistic effect, reducing the MIC of the antibiotics by 5- to 1000-fold compared to their use alone [85]. Vancomycin disrupt bacterial cells by altering cell membrane permeability, inhibiting cell wall biosynthesis and inhibiting ribonucleic acid synthesis [86]. In contrast, clove increases the permeability of the cell membrane and interacts with cellular proteins due to its high eugenol content [85,87]. The combination of these agents target several cellular structures and functions, resulting in lethal effect on bacterial pathogens [83]. These synergistic effects suggest that the incorporation of S. aromaticum and C. aromaticum EOs in antibiotic therapy could reduce the concentration of drugs needed for treatment, enhance bacterial susceptibility, and prevent the harmful side effects of the medications [16].

3.4. Synergistic Effect of EOs with Standard Antifungals

The synergistic effects of EOs with the two antifungals (amphotericin B and fluconazole) showed a variable interaction types (Table 7 and Table 8). The combination between C. aromaticum EO and amphotericin B demonstrated a total synergy against C. albicans and C. glabrata (FICI = 0.50), with a 4-fold MIC reduction. However, this combination had an indifferent effect on C. krusei and C. parapsilosis (FICI = 1.25). On the other hand, the association of C. aromaticum EO with fluconazole had a synergistic effect only against C. glabrata (FICI = 0.50), resulting in an 8-fold MIC reduction, while no effect was observed against the remaining Candida species (Table 7). The combination of S. aromaticum EO with fluconazole or amphotericin B exhibited a total synergistic effect against C. albicans and C. glabrata, with FICIs ranging from 0.28 to 0.50, and MIC reduction from 4- to 32-fold (Table 8). However, no effect was observed against C. krusei and C. parapsilosis. Previous studies have similarly reported that some combinations of these two EOs and standard antifungals produce different interaction types against Candida species such as C. krusei, C. tropicalis, C. albicans, and C. dublinienses [7,88,89,90,91]. Several studies have demonstrated that eugenol and cinnamaldehyde interact synergistically with antifungal drugs such as fluconazole and amphotericin B against some Candida species [92,93,94]. Jafri et al. [91] demonstrated a synergistic effect of eugenol combined with fluconazole (FICI = 0.156) against C. albicans, and an additive interaction (FICI = 0.625) when it was combined with amphotericin B against the same strain. The addition of these EOs may offer a promising therapeutic option that can extend the spectrum of antimicrobial activity of antifungal drugs and reduce their overall side effects.
Despite these interesting results, the present study has several limitations. A major issue with this type of research is the vulnerability of EOs’ quality to natural conditions [95]. Previous studies have demonstrated that encapsulating EOs can enhance their stability and provide controlled release as well as protection against environmental factors such as oxygen, moisture, and light, which otherwise diminish their effectiveness [96,97]. Furthermore, this work is limited by the necessity to test the major components, eugenol and cinnamaldehyde, both individually and in combination with these antimicrobial drugs to better understand the mechanisms of action of these EOs.

4. Conclusions

From this study, it can be concluded that Cinnamomum aromaticum and Syzygium aromaticum EOs exhibit potent antimicrobial activities, with C. aromaticum being particularly effective against the tested strains. The synergistic effects of these EOs in combination with investigated antibiotics and antifungals resulted in significant reductions in MICs for a broad range of bacterial and fungal pathogens. Notably, the interactions with vancomycin and ciprofloxacin substantially enhanced the efficacy of these antibiotics against Staphylococcus aureus, Escherichia coli, and Candida glabrata. These findings support the integration of C. aromaticum and S. aromaticum EOs into conventional antimicrobial therapies, potentially reducing required drug dosages, enhancing microbial susceptibility, and mitigating adverse effects, thereby presenting a viable strategy to strengthen the antimicrobial arsenal against resistant pathogens. Further research is needed to elucidate the mechanisms underlying the synergistic interactions of these EOs with tested antimicrobial agents, evaluate their therapeutic implications, and optimize their use. Additionally, exploring the encapsulation of these EOs could address their volatility, improve their stability and solubility, and potentially extend their efficacy in clinical applications.

Author Contributions

Conceptualization: S.E.B., B.S., I.A. and N.M.; methodology: S.E.B., B.S., I.A. and N.M.; formal analysis: S.E.B. and B.S.; investigation: S.E.B., B.S., Z.A. and I.A.; composition analysis: S.E.B., B.S, E.M.L. and M.M.; writing—original draft: S.E.B., B.S. and I.A.; writing—Review and Editing: S.E.B. and B.S. supervision: N.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article.

Acknowledgments

The authors would like to the National Center for Scientific and Technical Research in Rabat, Morocco, (CNRST) for providing some analysis.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. The essential oil (EO) yields of C. aromaticum and S. aromaticum.
Table 1. The essential oil (EO) yields of C. aromaticum and S. aromaticum.
Plant SpeciesFamilyCommon NounLocal NamesPart of the Plant UsedOil Yield a (%)
C. aromaticumLauraceaeCinnamonKarfaBark0.74
S. aromaticumMyrtaceaeCloveKronfelFlower buds2.4
a yield of EOs determined based on their volume/weight of the sample used for distillation.
Table 2. Chemical composition of EOs extracted from the studied species.
Table 2. Chemical composition of EOs extracted from the studied species.
RT aCompounds bC. aromaticumS.aromaticum
8.11Nonane- c0.06
10.01Benzaldehyde1.37-
12.50α-Pinene-0.11
12.951.8-Cineole-0.05
15.01Linalool-0.06
17.212-Phenyl propanal1.98-
17.36endo-Borneol0.89-
18.34Methyl salicylate-0.35
20.09Carvone1.09-
21.30(Z)-Cinnamyl alcohol8.16-
22.21Cinnamaldehyde47.04-
22.30Isobornyl acetate2.58-
22.50Azulene1.76-
23.42Citral0.58-
24.09Eugenol3.0771.49
24.60Copaene6.33-
25.81Vanillin-0.04
26.212.6-Dimethyl-1.3.6-heptatriene-1.01
27.25Methyleugenol-0.05
27.68Benzene, 1-methyl-3.5-bis(1-methylethyl)-1.78-
29.13Cinnamyl acetate18.93-
29.58β-Caryophyllene-23.43
29.66Naphthalene, 2-methoxy-1.01-
32.631.5.9-Cyclododecatriene, (E.E.E)-1.13-
33.67Nerolidol0.44-
33.85Eugenol acetate-2
Oxygen-containing monoterpenes5.6371.6
Monoterpene hydrocarbons1.760.11
Oxygen-containing sesquiterpenes0.44-
Sesquiterpene hydrocarbons6.3323.43
Other83.983.51
TOTAL%98.1498.65
a RT retention times. b Compounds listed in order of elution. c Not detected.
Table 3. Inhibition zone diameters (IZ in mm), minimal inhibitory concentrations (MIC), and minimal microbiocidal concentrations (MMC) of C. aromaticum, S. aromaticum (mg/mL), and antibiotics (μg/mL).
Table 3. Inhibition zone diameters (IZ in mm), minimal inhibitory concentrations (MIC), and minimal microbiocidal concentrations (MMC) of C. aromaticum, S. aromaticum (mg/mL), and antibiotics (μg/mL).
Bacterial StrainsEssential OilAntibiotics
Cinnamomum aromaticumSyzygium aromaticumCipVanc
IZMICMMCIZMICMMCIZMICIZMIC
S. aureus21.63 ± 0.350.0390.15610.97 ± 0.451.251.2523.16 ± 0.030.3914.24 ± 0.211.56
L. monocytogenes26.35 ± 0.120.0780.15610.63 ± 0.351.251.258.68 ± 0.183.12514.88 ± 0.426.25
E. coli20.48 ± 0.150.0780.15610.97 ± 0.410.6250.62527.24 ± 0.140.396 ± 025
K. pneumoniae20.71 ± 0.060.0780.1569.31 ± 0.291.251.2524.82 ± 0.160.19513.87 ± 0.5350
S. enterica16.99 ± 0.380.0780.07811.09 ± 0.330.6250.62528.36 ± 0.420.786 ± 012.5
IZ: Inhibition-zone diameter (in mm); Cip: Ciprofloxacin (5 μg/disc); Vanc: Vancomycin (10 μg/disc).
Table 4. Inhibition zone diameters (IZ in mm), minimal inhibitory concentrations (MIC), and minimal microbiocidal concentrations (MMC) of C. aromaticum, S. aromaticum (mg/mL), and antifungals (μg/mL).
Table 4. Inhibition zone diameters (IZ in mm), minimal inhibitory concentrations (MIC), and minimal microbiocidal concentrations (MMC) of C. aromaticum, S. aromaticum (mg/mL), and antifungals (μg/mL).
Yeast StrainsEssential OilAntifungals
Cinnamomum aromaticumSyzygium aromaticumFlucAmph
IZMICMMCIZMICMMCIZMICIZMIC
C. albicans L441.65 ± 0.080.0390.07829.91± 0.150.6251.2528.98 ± 0.643.12512.55 ± 0.341.562
C. glabrata L735.69 ± 0.310.0390.03927.34 ± 0.150.6250.62526.64 ± 0.186.2514.47 ± 0.111.562
C. krusei L1053.16 ± 0.430.0390.03929.43 ± 0.290.3131.2519.33 ± 0.191.56214.97 ± 0.560.781
C. parapsilosis L1842.79 ± 0.230.0390.03923.37 ± 0.260.6251.2519.62 ± 0.406.258.12 ± 0.341.562
IZ: Inhibition-zone diameter (in mm); Fluc: Fluconazol (10 μg/disc); Amph: Amphotericin B (5 μg/disc).
Table 5. Fractional inhibitory concentrations indices (FICIs) and the gain of antibiotics combined with the C. aromaticum EO.
Table 5. Fractional inhibitory concentrations indices (FICIs) and the gain of antibiotics combined with the C. aromaticum EO.
Bacterial StrainsCiprofloxacin Vancomycin
MICMIC Cip + CaGainFICIEffectMICMIC Vanc + CaGainFICIEffect
S. aureus0.390.09740.50Synergism1.560.7820.75Additive effect
L. monocytogenes3.1253.12511.25Indifference6.250.195320.28Synergism
E. coli0.390.012320.28Synergism2512.520.75Additive effect
K. pneumoniae0.1950.19511.25Indifference500.87640.27Synergism
S. enterica0.780.0061280.26Synergism12.512.511.25Indifference
Cipro: Ciprofloxacine; Vanco: Vancomycine; Ca: Cinnamomum aromaticum EO.
Table 6. Fractional inhibitory concentrations indices (FICIs) and gain of antibiotics combined with the S. aromaticum EO.
Table 6. Fractional inhibitory concentrations indices (FICIs) and gain of antibiotics combined with the S. aromaticum EO.
Bacterial StrainsCiprofloxacin Vancomycin
MICMIC Cip + SaGainFICIEffectMICMIC Vanc + SaGainFICIEffect
S. aureus0.390.09740.50Synergism1.560.3940.50Synergism
L. monocytogenes3.1250.195160.31Synergism6.250.78180.37Synergism
E. coli0.390.024160.31Synergism2512.520.75Additive effect
K. pneumoniae0.1950.09720.75Additive effect503.13160.31Synergism
S. enterica0.780.012640.27Synergism 12.512.511.25Indifference
Cipro: Ciprofloxacine; Vanco: Vancomycine; Sa: Syzygium aromaticum EO.
Table 7. Fractional inhibitory concentrations indices (FICIs) and gain of antifungals combined with the C. aromaticum EO.
Table 7. Fractional inhibitory concentrations indices (FICIs) and gain of antifungals combined with the C. aromaticum EO.
Candida StrainsFluconazol Amphotericin B
MICMIC Fluc + CaGainFICIEffectMICMIC Amph + CaGainFICIEffect
C. albicans L43.1253.12511.25Indifference1.5620.3940.50Synergism
C. glabrata L76.250.7880.50Synergism1.5620.3940.50Synergism
C. krusei L101.5621.56211.25Indifference0.7810.78111.25Indifference
C. parapsilosis L186.256.2511.25Indifference1.5621.5621125Indifference
Fluco: Fluconazole; Ampho: Amphotericine B; Ca: Cinnamomum aromaticum EO.
Table 8. Fractional inhibitory concentrations indices (FICIs) and gain of antifungals combined with the S. aromaticum EO.
Table 8. Fractional inhibitory concentrations indices (FICIs) and gain of antifungals combined with the S. aromaticum EO.
Candida StrainsFluconazol Amphotericin B
MICMIC Fluc + SaGainFICIEffectMICMIC Amph + SaGainFICIEffect
C. albicans L43.1250.195160.31Synergism1.5620.39140.50Synergism
C. glabrata L76.250.195320.28Synergism1.5620.39140.50Synergism
C. krusei L101.5621.56211.25Indifference0.7810.78111.25Indifference
C. parapsilosis L186.256.2511.25Indifference1.5621.56211.25Indifference
Fluco: Fluconazole; Ampho: Amphotericine B; Sa: Syzygium aromaticum EO.
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El Baz, S.; Soulaimani, B.; Abbad, I.; Azgaou, Z.; Lotfi, E.M.; Malha, M.; Mezrioui, N. Antimicrobial Activity and the Synergy Potential of Cinnamomum aromaticum Nees and Syzygium aromaticum (L.) Merr. et Perry Essential Oils with Antimicrobial Drugs. Microbiol. Res. 2025, 16, 63. https://doi.org/10.3390/microbiolres16030063

AMA Style

El Baz S, Soulaimani B, Abbad I, Azgaou Z, Lotfi EM, Malha M, Mezrioui N. Antimicrobial Activity and the Synergy Potential of Cinnamomum aromaticum Nees and Syzygium aromaticum (L.) Merr. et Perry Essential Oils with Antimicrobial Drugs. Microbiology Research. 2025; 16(3):63. https://doi.org/10.3390/microbiolres16030063

Chicago/Turabian Style

El Baz, Soraia, Bouchra Soulaimani, Imane Abbad, Zineb Azgaou, El Mostapha Lotfi, Mustapha Malha, and Noureddine Mezrioui. 2025. "Antimicrobial Activity and the Synergy Potential of Cinnamomum aromaticum Nees and Syzygium aromaticum (L.) Merr. et Perry Essential Oils with Antimicrobial Drugs" Microbiology Research 16, no. 3: 63. https://doi.org/10.3390/microbiolres16030063

APA Style

El Baz, S., Soulaimani, B., Abbad, I., Azgaou, Z., Lotfi, E. M., Malha, M., & Mezrioui, N. (2025). Antimicrobial Activity and the Synergy Potential of Cinnamomum aromaticum Nees and Syzygium aromaticum (L.) Merr. et Perry Essential Oils with Antimicrobial Drugs. Microbiology Research, 16(3), 63. https://doi.org/10.3390/microbiolres16030063

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