Abstract
Objective
The increasing emergence of antibiotic- and biocide-resistant microorganisms has reduced the effectiveness of conventional antimicrobial strategies, highlighting the need for alternative approaches, particularly in clinical and high-risk environments. This study evaluated the antimicrobial activity and mode of action of fifteen essential oils against pathogenic microorganisms in the vapor phase. Pathogens were selected based on clinical relevance, transmission potential via direct or indirect contact, and their ability to cause severe infections, particularly in immunocompromised patients.
Methodology
The chemical composition of essential oils was determined by gas chromatography–mass spectrometry (GC/MS). Antimicrobial activity was assessed against five bacterial strains and two Candida species using the vapor-phase diffusion method. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) were applied to visualize structural alterations in microbial cells following exposure to the most active essential oils.
Results
Coriandrum sativum fruit essential oil exhibited strong efficacy against Staphylococcus aureus and Pseudomonas aeruginosa. Ocimum basilicum was particularly effective against Escherichia coli. Moreover O. basilicum and Coriandrum sativum EO significantly suppressed pigment production in S. aureus. The most efficient oils against Candida albicans were Syzygium aromaticum, Mentha x piperita and C. sativum. SEM analyses revealed structural deformations in C. albicans cells, including surface shrinkage, irregular contours, partial collapse, surface rupture, and pore formation. TEM images showed disrupted cell walls, increased vacuolization, and compromised intracellular integrity following exposure to essential oil vapors.
Conclusion
This study contributes to understanding the anticandidal potential and morphological effects of vapor-phase essential oils, highlighting their relevance beyond antibacterial activity. The application of essential oils in their vapor phase presents notable advantages, including the avoidance of direct contact and the feasibility of use in high-risk environments, such as hospitals and communal sanitation facilities, where microbial contamination is a significant concern.
Introduction
Down the ages, medicinal and aromatic plants have garnered considerable attention due to their wide range of applications. These plants and their essential oils (EOs) have been traditionally used in the treatment of several illnesses, sanitation, food flavouring and preservation, as well as in the fragrance industry, aromatherapy, and other therapeutic applications. 1 Owing to their unique chemical composition which is rich in bioactive compounds, they continue to be regarded as promising natural therapeutic agents. They are environmentally favorable due to their volatility and biodegradability, while also exhibiting specific biological activities.2,3
EO are derived from the naturally found volatile compounds within aromatic plants. Their complex composition, primarily consisting of terpenes such as monoterpenes and sesquiterpenes together with their oxygenated derivatives, phenolic, and aliphatic compounds, contributes to their diverse biological activities.4,5 EOs play an important role in nature due to the wide variety of compounds in their composition. 6 They protect plants against harmful animals, bacteria, fungi, and some allelopathic effects, and attract certain insects and bees for pollination because of their strong odor and volatile nature. 7
Antibiotic resistance which has been increasingly developed by pathogenic microorganisms has limited the use of existing antibiotics. Therefore, research into antimicrobials of natural origin is increasing consistently. In particular, essential oils have attracted noteworthy attention for a long time due to their significant antibacterial, antifungal, antiviral, antiprotozoal, insecticidal, and antioxidant properties. For this reason, they may be considered as generally safe and promising alternatives to biocides and antibiotics.
Table 1 summarizes the pharmacological properties of several
List of Essential Oils and Their Properties.
After the Covid-19 pandemic, which affects the whole world has become a risk factor in our lives, airborne and nosocomial pathogens have been continuing to cause concern. Especially in hospitals and washrooms of the transmission of pathogens by inhalation and surface contact can be caused very serious health problems. Microbial colonization and persistence in surface can be reduced with constantly use of sanitizers. However, they have the toxic effects of these chemicals used on human and environment also permanence of the disinfecting effects after their application is limited.26,27
Among various application methods, the use of essential oils in their vapor phase represents the most affordable and simple approach to combat pathogenic bacteria and fungi, especially in hospitals, washrooms, and other public areas prone to contamination, helping to eliminate or at least slow down pathogen growth. Several studies have shown that essential oils in their vapor phase can display stronger and faster antimicrobial effects than their liquid counterparts. This vapor-based approach provides contact-free disinfection, lower toxicity, and practical applicability for controlling airborne and surface pathogens in hospital and public environments. 28
This study aimed to screening of fifteen selected essential oils in controlling the growth of five common bacterial pathogens and two Candida species, particularly those associated with nosocomial infections, using the vapor-phase diffusion assay. Most potent EOs were analysed by TEM and SEM techniques to better understand their mode of action at the cellular level.
Materials and Methods
This study was conducted between July 2024 and August 2025 at the Pharmacognosy Laboratory, Anadolu University, Eskişehir, Turkey.
Essential Oils Samples
Certified essential oils were supplied by commercial sources (Tabimer, Eskişehir, Türkiye, Defne Essencia, Antalya, Türkiye). The authentic samples of carvacrol (Frutarom), eugenol (Sigma-Aldrich), linalool (Sigma-Aldrich), terpinen-4-ol (Sigma-Aldrich) were used as standards in bioactivity assay. Carvacrol, a well-known broad-spectrum antimicrobial volatile compound was employed as a natural positive control agent in this study.29,30
Gas Chromatography/Mass Spectrometry (GC/MS)
Analysis of the EOs were performed by SHIMADZU GC-2010 Plus Gas Chromatograph system and equipped with mass detector (MS). A 25 m 0.25 mm 0.25 mm CP-Sil 5 CB column was used. The injection port temperature was 250 °C.
The column temperature was held at 60 °C for 10 min and then increased to 270 °C at a rate of 4 °C/min and held at 270 °C for 10 min. Helium was used as the carrier gas at a constant flow rate of 1.2 mL/min in split mode 50:1. The injected sample included 1.5 mL of diluted EO in hexane (1/100, v/v). For GC/MS detection, an electron ionization system with ionization energy of 70 eV was used over a scan range of 35–450 m/z. The ion source temperature was 200 °C. The components of commercial EOs were analysed with the standard mass spectra provided by Wiley and Adams Library. The retention indices were obtained by applying an oil sample with a C9-C40 linear hydrocarbons mixture.31,32
Antimicrobial Assays
Bacterial and Fungal Strains
Antimicrobial activity was evaluated against five pathogenic bacterial strains and two fungal strains. EOs were tested against two Gram-positive bacteria (Staphylococcus aureus ATCC 6538 and Staphylococcus epidermidis ATCC 12228) and three Gram-negative bacteria (Escherichia coli NRRL B-3008, Pseudomonas aeruginosa ATCC 9027, and Serratia marcescens NRRL B-2544). Anticandidal activity was assessed using two strains of Candida species (Candida albicans ATCC 90028 and Candida parapsilosis ATCC 22019). Culture stocks stored at −85 °C were inoculated onto Mueller-Hinton Agar (MHA) and incubated at 35 ± 2 °C for 24 h, while fungal strains were inoculated onto Potato Dextrose Agar (PDA) and incubated at 35 ± 2 °C for both 24 and 48 h to evaluate the time-dependent effects of essential oils.
Vapor Phase Test (VPT)
Antibacterial Assay
Antibacterial activity was investigated using a vapor diffusion technique. Bacterial suspensions were prepared in 0.85% saline and adjusted to a 0.5 McFarland standard (approximately 1.5 × 108 CFU/mL). Tryptic Soy Agar (TSA) plates were inoculated with 80 µL of the suspension. The pure essential oils (10 µL) were impregnated on each sterile 6-mm-diameter absorbent blank filter paper disc that was placed in the centre of the lid of the agar plates. Control plates were prepared by placing carvacrol disks for positive controls. The experiment was performed in three replicates. Petri dishes were sealed with parafilm and incubated for 24 h at 37 °C. Diameters of the inhibition zones were measured at the end of the incubation.33,34
Anticandidal Assay
Candida suspensions were also adjusted to a 0.5 McFarland standard and inoculated onto Potato Dextrose Agar (PDA) plates. After impregnation with essential oils, the plates were sealed with parafilm and incubated at 37 °C for 24 and 48 h plates. Colony diameters were measured at the end of each incubation period. Essential oils that completely inhibited Candida growth after 24 h of incubation were retested at a lower concentration (5 µL). Experiments with higher doses (20 µL) were carried out to determine the minimum effective concentrations of oils that exhibited no effect and those with documented antifungal activity in the literature.34–36
Scanning Electron Microscopy (SEM)
Samples of each test microorganism treated with the most effective essential oils were subjected to SEM and TEM analyses to elucidate the mode of action exerted by the oils. Samples were prepared according to partly modified methods of Ramirez-Camacho et al 2024. 37 Agar media from the edge of the inhibition zone were cut into 1 × 1 cm squares aseptically, then fixed and dehydrated for SEM analysis. The agar pieces were fixed in 2.5% glutaraldehyde prepared in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature. Following fixation process, the samples were rinsed with 0.1 M sodium phosphate buffer and dehydrated through a graded ethanol series of 50%, 70%, 90%, 96%, and 99%. Following dehydration, the samples were placed on metal stubs and allowed to dry in a desiccator for overnight. Finally, the specimens were sputter-coated with gold-palladium for 1 min at 30 mA and imaged using a Zeiss Ultra Plus SEM at an accelerating voltage of 5 kV.
Transmission Electron Microscopy (TEM)
Cells from the edge of the inhibition zones on assay plate were collected and aseptically transferred into Eppendorf tubes. Cells was fixed in 0.1 M phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde. After primary fixation, the cells were washed with sodium phosphate buffer and post-fixed in 2% osmium tetroxide (OsO₄; EMS, Hatfield, PA, USA) for 2 h at 4 °C. Dehydration was performed using a graded ethanol series (70%, 90%, 96% and 99%). Dehydrated samples were embedded in EPON 812 epoxy resin and ultrathin sections (up to 100 nm thick) were obtained. Sections were stained with uranyl acetate and lead citrate (EMS, Hatfield, PA, USA). Structural and morphological analyses of Candida cells were performed using a Hitachi HT7800 transmission electron microscope (Eskişehir Osmangazi University, Arum, Türkiye). 38
Statistical Analyses
All experiments were conducted in triplicate. The results are expressed as the mean ± standard deviation.
Results
GC-MS Analysis
The essential oils were analysed by GC/MS. Table 2 shows the major compounds of the tested oils.
Inhibition Zone Diameters (mm) of Essential Oils Against Tested Bacterial Strains.
* Literature data
Vapor Diffusion Assay
According to the results of the vapor diffusion assays, tested EOs and selected major constituents exhibited inhibitory effects at varying degrees at 10 µL. C. sativum EOs has demonstrated significant antibacterial activity. Furthermore, both O. basilicum and C. sativum essential oils have been shown to exert a marked inhibitory effect on pigment production in S. aureus ATCC 6538.
In addition, M. piperita, O. basilicum, and C. camphora oils have exhibited notable antimicrobial properties. However, it has been shown that the vapor phase of EOs possesses significant inhibitory effects, particularly against Candida species. Vapor diffusion experiments have also performed with the major components of most effective essential oils having a zone above 15 mm diameter. Inhibition zone diameters (mm) of the essential oils and carvacrol against five bacteria and two Candida strains are shown in Table 3. Carvacrol, the major constituent of Origanum essential oils, is a well-documented compound for its antibacterial and anticandidal properties. Due to its broad-spectrum antimicrobial efficacy, it was employed as a natural and volatile positive control in the present study.
Inhibition zone diameters (mm) of essential oils against tested bacterial strains.
–: No inhibition zone observed in any replicate; ± Standard deviations were calculated from two independent experiments performed in triplicate.
Considering the different susceptibility properties of the tested Candida strains to the essential oils, 5 μL and 10 μL concentrations were tested. For example, O. basilicum oil were not produced any inhibition zone at initial concentration of 5 μL, whereas when applied 10 μL, it exhibited remarkable inhibitory zone 39 mm in diameter. In certain cases, complete inhibition occurred at 10 μL without the formation of a visible inhibition zone. Accordingly, all experiments were replicated using two distinct concentrations. C. albicans was completely eradicated after applying 10 µL of clove, coriander, and peppermint essential oils at both 24- and 48-h incubation periods. Consequently, trials were repeated with a reduced concentration of 5 µL, and the inhibition zone diameters were measured (Table 4).
Inhibition Zone Diameters (mm) and Effective Doses of Essential Oils Against Candida strains After 24- and 48-h of Incubation.
+: The essential oil completely killed all cells; -: No inhibition zone observed in any replicate; ± Standard deviations were calculated from two independent experiments performed in triplicate.
Morphological Alteration
For SEM and TEM analyses, microbial cells were collected from the inhibition zones formed around the vaporized from essential oil discs, as illustrated in Figure 1.
Sample collection zones for SEM and TEM analysis. Agar blocks were excised from the marked inhibition zones on Petri dishes after 24-h exposure to EOs vapors.
SEM Analysis Results
Scanning electron microscopy revealed significant ultrastructural alterations in both E. coli and S. aureus cells after 24-h exposure to essential oil vapors. In E. coli cells treated with O. basilicum EOs (10 µL), distinct morphological changes were observed, including surface irregularities, cell swelling, elongation and membrane disruption (Figure 2b). Aggregation of bacterial cells further indicated increased membrane permeability and loss of cellular integrity.
SEM images of E. coli and S. aureus cells after 24-h treatment: (a) Untreated E. coli cells displaying typical rod-shaped morphology with smooth, intact surfaces; (b) E. coli cells exposed to O. basilicum essential oil showing membrane disruption, surface irregularities, cell elongation and partial collapse, (c) Control S. aureus cells with characteristic spherical shape and intact cell walls. (d) S. aureus cells treated with C. sativum essential oil exhibiting aggregation, cell size reduction, surface deformation, and loss of structural integrity.
In S. aureus cells treated with C. sativum EOs (10 µL), SEM images displayed severe deformation and collapse of cell surfaces. The treated cells showed clustering, irregular contours, and ruptured membranes (Figure 2d). These findings suggest that the vapor-phase essential oils interfere with bacterial cell envelope integrity, ultimately leading to cell death.
Following exposure to essential oil vapors, C. albicans cells showed notable morphological alterations, according to SEM analysis
SEM images of C. albicans cells after 24-h treatment: (a) Control cells exhibiting intact, smooth morphology; (b) C. albicans exposed to C. sativum EOs, showing severe structural damage, cell collapsed, and pore formation. (c) C. albicans treated with M. × piperita EOs, showing severe cell wall deformation, irregular shapes, and surface shrinkage (d) S. aromaticum EOs vapor-treated cells displaying surface irregularities and formation of deep pits on the cell surface.
TEM Analysis Results
According to TEM analysis, E. coli, S. aureus and C. albicans cells exhibited distinct ultrastructural alterations following treatment with various essential oils. Control E. coli cells (Figure 4a) exhibited typical rod-shaped morphology with smooth, continuous cell membranes and well-organized intracellular content. Similarly, untreated S. aureus cells (Figure 4c) showed intact, spherical cell structures with well-defined and smooth cell walls. In contrast, EO-treated cells displayed marked structural damage. E. coli cells exposed to EOs (Figure 4b) exhibited disrupted and irregular cell membranes, with evident leakage of intracytoplasmic contents, suggesting loss of membrane integrity. Likewise, S. aureus cells treated with EOs (Figure 4d) showed pronounced alterations, including compromised cell walls, discontinuities in the membrane structure, and intracellular disorganization. These morphological deformations indicate that the essential oils exert their antimicrobial activity by disrupting membrane integrity and inducing cellular leakage, ultimately leading to cell death.
The control cells of E. coli (4a) and S. aureus (4c), examined by TEM, exhibited intact cell membranes and well-defined, smooth cell walls, typical of healthy bacterial morphology. In contrast, the treated cells (4b (E. coli) and 4d (S. aureus) showed disrupted cell membranes, irregular cell wall structures, and leakage of cytoplasmic contents, indicating severe structural damage induced by EOs exposure.
In the Candida cell control group (Figure 5a), cells displayed typical morphological features, including an intact and well-defined cell wall, distinct mitochondria, a clearly visible nucleus, and small, regularly shaped vacuoles. In contrast, cells exposed to the EOs exhibited substantial structural alterations. A significant increase in vacuolization was observed in cells exposed to clove essential oil, potentially indicating disruption of cellular homeostasis or activation of autophagic processes. In contrast, the extrusion of cellular contents observed after treatment with coriander EO suggests a loss of membrane integrity, leading to cytoplasmic leakage. Furthermore, cells treated with peppermint EO showed pronounced morphological alterations, including irregular cell shapes and potential deformation of the cell membrane. These findings indicate that each essential oil exerts its antifungal activity through distinct mechanisms that disrupt cellular ultrastructure and ultimately induce cell death.
(a) control cell presents a regularly outlined cell wall, mitochondria, nucleus and small vacuoles. (b) The cell exposed to C. sativum EOs vapor cell exhibiting extrusion of cellular contents and membrane disruption. (c) Cells exposed to M. x piperita EOs vapor, displaying irregular shapes and membrane deformation. (d) Cells treated with S. aromaticum EOs vapor, showing increased vacuolization.
Discussion
In several studies, vapor-phase essential oils (VP-EOs) have been shown to be more potent antimicrobials than the corresponding liquid forms. This has been demonstrated for thyme, citrus, eucalyptus, tea tree, and lemongrass oils.41,42
In the present study, we evaluated the effects of 15 widely used essential oils in vapor phase on five bacterial species and two pathogenic Candida strains. The most effective essential oils identified in this study were C. sativum, O. basilicum, M. × piperita, and S. aromaticum, demonstrating strong antibacterial and antifungal properties. In contrast, several oils, including C. bergamia, B. serrata, M. fragrans, L. x intermedia, M. viridiflora, C.atlantica, I. verum, and Z. officinale, did not exhibit any observable antibacterial activity against the tested strains. The most potent EOs were subjected to detailed SEM and TEM analysis to elucidate their mode of action.
E. coli treated with O. basilicum SEM revealed membrane disruption, surface irregularities, cell elongation, and partial collapse. TEM analysis revealed disrupted and irregular cell membranes with cytoplasmic leakage, indicative of compromised membrane integrity. Comparable ultrastructural alterations have been documented in previous similar studies. 43
According to Tyagi and Malik, lemongrass oil vapor (LGOV) exerted a more detrimental effect on E. coli compared to its liquid form. TEM analysis revealed membrane rupture accompanied by cytoplasmic leakage, SEM demonstrated loss of turgidity and surface roughening, and atomic force microscopy (AFM) indicated marked reductions in cell height.44,45 These findings support the idea that VP-EOs largely destroy bacteria by disrupting their membranes, and they are consistent with our SEM/TEM data for O. basilicum vapor.
Previous studies using disk-diffusion methods have demonstrated that C. sativum EOs vapor induces morphological disturbances in S. aureus, such as cell swelling, irregular cell walls, and clustering. 46 In our study, exposure to coriander EO vapor produced similar morphological deformation in SEM images, including cell clustering, irregular contours, and membrane rupture. TEM corroborated these findings, showing compromised cell walls, membrane discontinuities, and intracellular disorganization—consistent with vapor-based structural damage leading to cell death. These results indicate that the volatile constituents of coriander oil can induce similar ultrastructural damage without direct liquid contact, thereby supporting the potential of vapor-phase application as an effective contact-free antibacterial strategy.
In addition to structural damage, our observations revealed a visible reduction in the characteristic golden pigment (staphyloxanthin) in S. aureus cells exposed to C. sativum and O. basilicum EOs in vapor phase. This aligns with previous studies showing that Geranium essential oil, can inhibit staphyloxanthin biosynthesis by interfering with the CrtM enzyme pathway, a key component in pigment production. Since staphyloxanthin is associated with oxidative stress resistance and virulence, its suppression suggests that VP-EOs may not only be bactericidal but also reduce S. aureus pathogenicity by downregulating its virulence factors. 47
Against Candida spp., M.× piperita EOs vapor exhibited strong and consistent inhibitory activity against both C. albicans and C. parapsilosis at a volume of 5 µL. The previous study by Tyagi and Malik clearly states that M. x piperita EOs is effective against both S. aureus and fungal C. albicans in both liquid and vapor phases. Notably, the rapid killing effect of the vapor phase on C. albicans (100% viability reduction in 8 h) was highlighted. 48 O. basilicum, C. sativum and S. aromaticum EOs vapor also displayed potent antifungal activity. SEM analyses mirrored prior vapor-phase results, such as those by Tyagi & Malik, which showed severe structural damage, cell collapse, pore formation, surface shrinkage, and irregular shapes. TEM further revealed profound membrane damage and cell wall disruption in treated C. albicans cells, reinforcing that vapor-phase oils impart profound ultrastructural harm comparable to or exceeding prior observations. 44
While Candida species are often harmless in healthy individuals, this study addresses two critical aspects: protecting immunocompromised patients from opportunistic infections and exploring the potential of essential oil vapors for disinfecting contamination-prone areas and surfaces in public or healthcare settings to prevent fungal spread. Candida spp. are generally responsible for mucosal, dermal, wound-associated, and medical device-related infections. Vapor-phase compounds can reach these surfaces without direct liquid contact, which may be advantageous in clinical scenarios where surface-level fungal control is needed. 49
In particular, this is the first comprehensive investigation to assess the effects of extensively utilized vapor-phase essential oils on pathogenic bacteria and fungi at the cellular level. The primary advancement in this work is the use of SEM and TEM to visualize the morphological effects caused by vapor-phase essential oils and to demonstrate their anticandidal potential. These results offer novel insights on the antifungal effect of vapor-phase treatments and broaden our understanding of them beyond their antibacterial activity.
Limitations
This study has several limitations that should be acknowledged. First, microscopy provided mainly qualitative insights into anticandidal and antibacterial activity, as no mechanistic assays (eg, membrane potential, leakage analysis, ROS quantification, lipid peroxidation, or proteomics) were performed. Second, all experiments were conducted in vitro, which does not fully replicate in vivo complexity. Moreover, the study focused specifically on evaluating essential oils as surface-active or vapor-phase antimicrobial agents, limiting clinical extrapolation. Future studies should include in vivo assessments, toxicity analyses, and pharmacokinetic and stability evaluations to support practical application.
Conclusion
This study demonstrates that vapor-phase essential oils (VP-EOs) exhibit moderate bactericidal and strong fungicidal activity in the vapor phase. SEM and TEM analyses revealed that the underlying mechanism involves significant microbial membrane disruption and structural collapse. These findings highlight VP-EOs as promising natural, vaporized biocides, particularly effective against Candida species, offering significant advantages for applications in healthcare, food safety, and air decontamination without direct contact.
Footnotes
Acknowledgment
We thank Dr. Nesil Ertorun, Department of Biology, Eskişehir Technical University (ESTU), and Central Research Laboratory Application and Research Center, Eskişehir Osmangazi University (ESOGU), Eskişehir, Türkiye, for technical support and providing the facilities for SEM and TEM analyses.
Ethical Approval Statement
This study did not involve human participants, human data, or animal subjects. Ethical approval is not applicable for this article.
Consent to Participate
Not applicable. This study did not involve human participants.
Author Contribution
B.N.M.: Investigation, Data Collection, Formal Analysis, Writing–Original Draft; A.İ.: SEM/TEM Analysis, Validation, Formal Analysis; S.H.: Formal Analysis, Data Curation, Support in Investigation; G.İ.: Supervision, Conceptualization, Methodology, Project Administration, Writing – Review & Editing.
Funding
The authors received financial support for the research of this article: Participation in the 11th International Mediterranean Symposium on Medicinal and Aromatic Plants (MESMAP-11) was supported by the Scientific and Technological Research Council of Türkiye (TÜBİTAK) through the 2224-A Grant Program for Participation in International Scientific Events Abroad (2025/first Term). This study was also supported by Anadolu University Scientific Research Projects Coordination Unit under grant number YTT-2024-2568.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Data Availability Statement
The data presented in this study are available on request from the corresponding author.