Unveiling novel insights: geraniol’s enhanced anti-candida efficacy and mechanistic innovations against multidrug-resistant candida strains

Abstract

Objectives

This study addressed the need for new treatments for severe Candida infections, especially resistant strains. It evaluated the antifungal potential of geraniol alone and with fluconazole against various Candida spp., including resistant strains, and investigated geraniol’s mechanism of action using flow cytometry.

Methods

The research assessed the inhibitory effects of geraniol on the growth of various Candida species at concentrations ranging from 110 to 883 µg/ml. The study also explored the potential synergistic effects when geraniol was combined with fluconazole. The mechanism of action was investigated through flow cytometry, with a particular emphasis on key enzymes associated with plasma membrane synthesis, membrane permeability changes, mitochondrial membrane depolarization, reactive oxygen species (ROS) induction, and genotoxicity.

Results

Geraniol demonstrated significant antifungal activity against different Candida species, inhibiting growth at concentrations within the range of 110 to 883 µg/ml. The mechanism of action appeared to be multifactorial. Geraniol was associated with the inhibition of crucial enzymes involved in plasma membrane synthesis, increased membrane permeability, induction of mitochondrial membrane depolarization, elevated ROS levels, and the presence of genotoxicity. These effects collectively contributed to cell apoptosis.

Conclusions

Geraniol, alone and in combination with fluconazole, shows promise as a potential therapeutic option for Candida spp. infections. Its diverse mechanism of action, impacting crucial cellular processes, highlights its potential as an effective antifungal agent. Further research into geraniol’s therapeutic applications may aid in developing innovative strategies to address Candida infections, especially those resistant to current therapies.

Key messages

Research on geraniol’s anticandidal activity and mechanisms against clinical Candida strains is vital due to increasing antifungal resistance. Candida spp. cause severe infections, and geraniol provides a promising natural alternative. Understanding its action enhances fungal knowledge and supports innovative therapies for clinically relevant infections, positively impacting clinical practice.

Introduction

Candida species infections pose significant healthcare challenges, contributing to increased mortality rates and substantial economic burdens. These infections, contributing to over 1.5 million deaths annually [1, 2], represent a growing global health burden. These alarming statistics emphasize the urgent need to understand and actively combat fungal pathogens. In the United States, the impact of fungal infections extends far beyond public health, significantly affecting the economy. In 2018, the U.S. recorded an astounding total of 666,235 diagnosed fungal infections, resulting in a substantial economic impact of $6.7 billion. This underscores the critical need for effective strategies to address this public health issue [3]. Among these cases, candidiasis has emerged as a prevalent and economically burdensome infection, with 457,080 documented cases. Its invasive form has contributed to the second-highest mortality rate of 17%. Complications arise when this yeast develops resistance to antifungal drugs or spreads to other regions of the human body, a condition known as invasive candidiasis. These highlights need for effective therapeutic strategies [4]. Within pathogenic fungi, infections caused by drug-resistant Candida species continue to pose a relevant challenge to public health [5]. In this circumstance of COVID-19, immunocompromised patients and those admitted to Intensive Care Units (ICUs) are at heightened risk for new infections, including Candida. The appearance of COVID-19 associated candidiasis (CAC) further complicates this challenge. This situation emphasizes the urgent need for comprehensive research and effective therapeutic strategies [6, 7].

Candida species, integral components of the human microbiota, naturally inhabit various parts of the body, such as the skin, oral cavity, and gastrointestinal and urogenital tracts. Under normal circumstances, these microorganisms coexist harmoniously with the host. However, disturbances in the microbiota or the host’s immune system can pave the way for these microorganisms to transition from commensals to harmful pathogens [8]. While C. albicans is traditionally recognized as the most common species associated with humans, recent decades have witnessed a concerning increase in infections caused by non-albicans Candida species that are becoming more resistant to treatment, adding another layer of complexity to the management of these infections [9, 10]. C. auris, in particular, has gained notoriety as an emerging multi-resistant yeast, associated with high mortality rates and significant transmissibility [11].

Antifungal resistance exacerbates the challenge posed by these fungal infections. These pathogens not only exhibit intrinsic resistance, but the use of antifungals for prophylactic or empirical treatment has led to the emergence of multi-drug resistant fungal strains [12]. The development of adaptive resistance and tolerance mechanisms to antifungals is well-documented, including alterations or overexpression of drug target proteins, activation of cellular stress responses, and upregulation of drug transporters [13]. To avert a global crisis stemming from our limitations in controlling fungal infections and to prevent critical failures in the realms of medicine and food security [14, 15], there is substantial concern, and immediate measures must be taken to discover and develop new antifungal agents [16, 17].

Recognizing the severity of the circumstances, the World Health Organization (WHO) [18] has classified certain species, including Candida auris, Candida albicans, Candida glabrata, Candida tropicalis, and Candida parapsilosis, as critical or high-priority targets. Candida species represent a significant cause of severe nosocomial infections [19–21]. Given the scarcity of therapeutic options available for Candida infections, the search for new treatment strategies becomes a fundamental public health concern [22]. Natural products and combination therapy emerge as promising pathways to discover new molecular targets, improve treatment efficacy, overcome resistance, and reduce toxicity associated with traditional antifungal agents [23, 24].

Within this context, geraniol (C10H18O), an aliphatic monoterpene present in over 250 essential oils, stands out as a promising candidate for future explorations. Although commonly used as a fragrant compound in commercial products, geraniol exhibits several notable biological activities and pharmacological properties [25, 26]. Its antimicrobial properties are well-documented, particularly its efficacy against fungi and both Gram-positive and Gram-negative bacteria, with significant impact on the Candida and Staphylococcus genera [25]. Given the pressing need for innovative therapeutic options against Candida spp., it is imperative to investigate the precise mechanisms by which geraniol exerts its antimicrobial effects against these fungal species.

Considering the increasing incidence of infections and the diminishing efficacy of currently available drugs, the importance of this research is paramount [27]. Understanding these mechanisms is vital for the innovation of new therapeutic strategies and for the implementation of effective measures to control the spread of drug-resistant fungal infections in clinical settings. By elucidating the mechanisms of action and synergy with conventional antifungals [1, 28], as the threat of fungal infections continues to rise, the importance of this research cannot be underestimated, offering renewed hope in the battle against these lethal pathogens [29]. This research promises to pave the way for the development of more effective and targeted treatments. Particularly when considering the diverse spectrum of Candida infections and the various challenges they present in clinical settings, the comprehensive understanding provided by this investigation is of utmost importance.

Therefore, this study aims to evaluate the antifungal activity of geraniol, both alone and in combination with fluconazole, against a variety of Candida spp. Furthermore, the study seeks to unravel the possible underlying mechanisms of geraniol’s antifungal action, employing flow cytometry and scanning electron microscopy (SEM) (Fig. 1). By utilizing SEM the research aims to provide a detailed morphological analysis, offering insights into structural changes in Candida cells exposed to geraniol. This multifaceted approach that combines flow cytometry and SEM enhances our understanding of the antifungal efficacy of geraniol and elucidates its impact at both the cellular and structural level.

Fig. 1.

Study procedures and tests overview. Exploring the anticandidal activity of geraniol and possible mechanisms of action against strains of Candida spp

Materials and methods

Microorganisms

In this study, 14 clinical isolates of Candida spp. susceptible and resistant to fluconazole (6 C. albicans, 4 C. glabrata, 2 C. parapsilosis and 1 C. tropicalis) were used, along with C. parapsilosis ATCC 22,019 and C. krusei ATCC 6258 as controls. In addition, C. auris 01256P derived from CDC B11903 were also used. All strains belong to the collection of the Laboratory for Bioprospection of Antimicrobial Molecules, affiliated with the Faculty of Pharmacy of Federal University of Ceará (LABIMAN/FF/UFC).

Drugs

Geraniol, fluconazole, and amphotericin B were purchased commercially from Sigma-Aldrich Co. (MO, USA). For the assays, geraniol, and amphotericin B were dissolved in dimethylsulfoxide (DMSO) at a concentration ≤ 2.5% for immediate use, while fluconazole was dissolved in sterile distilled water.

Evaluation of antifungal activity

Determination of minimum inhibitory concentration (MIC)

The broth microdilution technique was performed according to the M27-A3 protocol [30] to determine the MIC, which for amphotericin B is defined as the lowest drug concentration that prevents any discernible growth, and for fluconazole, as the lowest concentration capable of inhibiting 50% of fungal growth. Geraniol was tested in a concentration range from 2 to 1024 µg/mL and fluconazole between 0.125 and 64 µg/mL. The isolates were classified as susceptible (S), susceptible dose-dependent (SDD), or resistant ^® to fluconazole according to document M27-S4 [31].

Evaluation of the interaction between geraniol and fluconazole by the checkerboard technique

The checkerboard test was performed according to [32] based on the MIC values of geraniol and fluconazole. We prepared solutions containing varying concentrations of the drugs in combination. The specific concentrations of geraniol and fluconazole used, based on the sensitivity profile of each isolate [33], are detailed in Table 1.

Table 1.

Antifungal effect of geraniol alone and association with fluconazole against Candida spp. strains

Straina	MICb (µg/mL) Alone		MICc (µg/mL) Association		FICId	Interpretatione
	FLC	GER	FLC	GER
C. parapsilosis ATCC 22,019	1	110	0.125	13.78	0.25	Synergistic
C. krusei ATCC 6258	32	220	16	110	1	Additive
C. auris 01256P	2	110	2	110	2	Indifferent
C. albicans 1*	16	110	16	110	2	Indifferent
C. albicans 2	32	441	32	441	2	Indifferent
C. albicans 3	32	441	32	441	2	Indifferent
C. albicans 4	32	110	32	110	2	Indifferent
C. albicans 5	1	220	0.25	55.125	0.5	Synergistic
C. albicans 6	64	220	64	220	2	Indifferent
C. albicans 7	0.25	220	0.25	220	2	Indifferent
C. glabrata 1*	64	220	16	55.125	0.5	Synergistic
C. glabrata 2	8	110	8	110	2	Indifferent
C. glabrata 3	8	220	8	220	2	Indifferent
C. glabrata 4	4	220	4	220	2	Indifferent
C. parapsilosis 1	8	110	8	110	2	Indifferent
C. parapsilosis 2	8	883	8	883	2	Indifferent
C. tropicalis 1*	32	117	32	117	2	Indifferent

GER: Geraniol, FLC: fluconazole

^aCandida spp. strains belonging to the arsenal of strains of the Laboratory of Bioprospection of Antimicrobial Molecules (LABIMAN)

^b MIC of fluconazole and geraniol isolated

^c MIC post association, obtained for fluconazole and geraniol

^d Fractional Inhibitory Concentration Index (FICI)

^e Interpretation of the associated activity based on the FICI in synergistic, additive, indifferent or antagonistic

* Strains used in flow cytometry assays

From this, we calculated the Fractional Inhibitory Concentration Index (FICI), where

These values were interpreted in accordance with [27], whereby a FICI ≤ 0.5 is considered synergistic (SYN), 0.5 < FICI ≤ 1 is additive (ADI), 1 < FICI ≤ 4 is indifferent (IND), and FICI > 4 is antagonistic (ANT).”

Assays performed by flow cytometry

Preparation of yeast cells for treatment with geraniol

The cells were collected during exponential growth to prepare the suspensions, using representative isolates of C. albicans, C. glabrata, and C. tropicalis. The protocol was performed according to [28, 33], in which the cells were centrifuged (2500 rpm for 5 min) and washed three times with 0.85% saline solution. Thereafter, the cell concentrate was resuspended in RPMI 1640 (pH 7.0) buffered with 0.165 M MOPS (Sigma-Chemical, MO, USA) at a concentration of approximately 10⁴ cells/ml. Then the cells were treated with geraniol (MIC/2 and MIC), fluconazole (32 µg/ml) and AMB (4 µg/ml) and incubated for 24 h at 35 ^oC. All flow cytometry tests were performed using the same FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).

Evaluation of cell viability

For the evaluation of cell density, cells were treated with propidium iodide (PI) at a concentration of 2 mg/l 24 h after drug exposure. A total of 10,000 events were analyzed per experiment, and cell debris was omitted from the analysis [28].

Determination of mitochondrial transmembrane potential (∆ψm)

After washing the cells with PBS, they were incubated with rhodamine 123 (Rho) at 5 mg/l and 37 ^oC for 30 min in the absence of light. Rho retention was used to determine ∆ψm. A total of 10,000 events were investigated per experiment, with cell debris omitted from the analysis [28, 33].

Analysis of the production of reactive oxygen species (ROS)

After drug exposure, cells were incubated with 20 µM of CM-H2DCFDA [5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate acetyl ester] for 30 min at 35 °C in the absence of light. Intracellular esterases hydrolyze CM-H2DCFDA into the non-fluorescent compound 2′,7′-dichlorodihydrofluorescein (DCFH), which is rapidly oxidized to the highly fluorescent DCF. The production of ROS is proportional to the intensity of fluorescence generated by DCF [33].

Investigation of phosphatidylserine externalization

Cells were digested with 2 mg/l of 20T zymolyase (Seikagaku Corp., Abingdon, United Kingdom) in potassium phosphate buffer (1 M sorbitol, pH 6.0) for 2 h at 30 °C. Afterward, the protoplasts were stained with Annexin V labeled with FITC and PI, using the FITC-Annexin V apoptosis detection kit (Nexin Kit, Guava Technologies). Thus, cells were washed with potassium phosphate buffer and incubated in annexin binding buffer containing FITC-Annexin V and PI for 20 min, followed by analysis. A total of 10,000 events were analyzed per experiment and cell debris was omitted from the analysis [33].

Statistical analysis

Assays were performed in triplicate on different days, using the geometric mean for MIC analysis. For assays performed by flow cytometry, data were submitted to two-way analysis of variance (ANOVA) followed by the Newman-Keuls test (p < 0.05) using the Prism program, version 5.01 (GraphPad Software, CA, USA).

Scanning electron microscopy (SEM)

For SEM, a representative isolate was used, which corresponded to C. albicans 1. Initially, an inoculum containing 0.5–2.5 × 10³ CFU/mL in RPMI buffered with 0.165 M MOPS was prepared. This was treated with geraniol at MIC and 2xMIC and incubated for 24 h at 35^oC. Then, on glass coverslips previously treated with 2.5% silane, the cells were added and fixed with 2.5% glutaraldehyde solution (Sigma-Aldrich, MO, USA) in 0.15 M cacodylate buffer (Electron Microscopy Sciences, PA, USA) and 0.1% alcian blue (Sigma-Aldrich, São Paulo, Brazil) overnight at 4^oC. Next, the cells were washed twice with cacodylate buffer solution, and alcoholic dehydration was performed at concentrations corresponding to 30, 50, 70, 80, 90, 95, 100% ethanol for 10 min each, followed by drying for 20 min at 35 ^oC. After this period, 150 µL of hexamethyldisilazane (Sigma-Aldrich) was added to the cover slips, which were left at rest until the reagent was completely evaporated. Finally, the coverslips were coated with 10 nm of gold (Emitech, East Sussex, UK; Q150T) and observed under a Quanta 450 FEG microscope [34, 35].

Results

Geraniol has antifungal activity against Candida spp. isolates

Geraniol showed antifungal activity with MIC50% ranging from 110 to 883 µg/ml against Candida spp. Isolates susceptible and resistant to fluconazole were used, with MIC50% between 0.25 and 64 µg/ml. About the association between geraniol and fluconazole, 77.8% were indifferent, 16.7% synergistic, and 5.5% additive (Table 1).

Geraniol causes a reduction in the number of viable cells of Candida spp.

The treatment with geraniol at MIC/2 generated a significant reduction in cell viability compared to the control against C. albicans and C. glabrata, also occurring at the MIC against the three tested isolates. There was no statistically significant reduction in viability in comparison with the control in the C. tropicalis strain at MIC/2. Compared to fluconazole, the three species under analysis showed decreases (p < 0.05) in viability in the treatment with geraniol (MIC/2 and MIC). Treatment with amphotericin B led to a reduction (p < 0.05) in viability compared to the control and fluconazole in all isolates (Fig. 2). Exposure of cells to fluconazole did not reduce cell viability compared to the control.

Fig. 2.

Cell damage caused by geraniol. Cell viability analysis of C. albicans, C. glabrata and C. tropicalis after treatment with FLC (fluconazole) (32 µg/mL), GER (geraniol) (MIC/2 and MIC). RPMI and AMB (amphotericin B) (4 µg/mL) were used as negative and death controls, respectively. * p < 0.05 compared with the control, ** p < 0.05 compared with FLC

The exposure of Candida spp. to geraniol causes changes in the ∆ψm

Exposure of Candida cells to geraniol (MIC/2 and MIC) and amphotericin B significantly increased mitochondrial depolarization in C. albicans, C. glabrata, and C. tropicalis compared to the control and treatment with fluconazole (p < 0.05) (Fig. 3). The treatment of cells with fluconazole did not generate a significant difference in relation to the control.

Fig. 3.

Evaluation of the mitochondrial transmembrane potential (ΔΨm) on strains of Candida spp. The treatments were FLC (fluconazole) (32 µg/mL), GER (geraniol) (MIC/2 and MIC). RPMI and AMB (amphotericin B) (4 µg/mL) were used as negative and death controls, respectively. * p < 0.05 compared with the control, ** p < 0.05 compared with FLC

The production of ROS is associated with the action of geraniol on Candida spp.

Geraniol (MIC/2 and MIC) and amphotericin B induced the production of ROS in cells of C. albicans, C. glabrata, and C. tropicalis in comparison with the control and treatment with fluconazole (p < 0.05). Exposure of Candida spp. to fluconazole did not generate significant production of ROS (Fig. 4).

Fig. 4.

Reactive oxygen species in Candida spp. strains. After treatment for 24 h with FLC (fluconazole) (32 µg/mL), GER (geraniol) (MIC/2 and MIC). RPMI and AMB (amphotericin B) (4 µg/mL) were used as negative and death controls, respectively. * p < 0.05 compared with the control, ** p < 0.05 compared with FLC

Phosphatidylserine externalization after geraniol treatment in Candida spp. cells

Geraniol (MIC/2 and MIC) and amphotericin B generated phosphatidylserine externalization in all analyzed isolates in comparison with untreated cells and treatment with fluconazole (p < 0.05). Fluconazole did not show this pattern in cells exposed to this drug (Fig. 5).

Fig. 5.

Phosphatidylserine externalization in Candida spp. strains. After treatment for 24 h with FLC (fluconazole) (32 µg/mL), GER (geraniol) (MIC/2 and MIC). RPMI and AMB (amphotericin B) (4 µg/mL) were used as negative and death controls, respectively. * p < 0.05 compared with the control, ** p < 0.05 compared with FLC

Geraniol causes morphological changes in C. albicans cells

The SEM images revealed that geraniol caused alterations in the planktonic cells of C. albicans. Figure 6A shows intact fungal cells. In turn, Fig. 6B shows the treatment of the cells with geraniol at MIC, and the presence of shrinkage in the yeast can be observed, indicated by an arrow. Figure 6C shows that exposure to geraniol at 2xMIC caused intense damage to the morphology of the microorganism, with a large amount of cellular debris.

Fig. 6.

Scanning electron microscopy of C. albicans cells. (a) Untreated control. (b) Geraniol at MIC. (c) Geraniol at 2x MIC. Magnification: 10,000x; bar: 10 μm

Discussion

Natural products have been garnering attention from researchers for the discovery of antifungal compounds, based on the potential existence of new mechanisms of action and reduction of resistance emergence. Thus, there has been significant progress in this area recently, with about 200 anti-Candida natural products isolated from different sources in the last decade [36].

Our results demonstrated that geraniol has antifungal activity against susceptible and fluconazole-resistant Candida spp. strains. This finding echoes the results of study [37], which showed that geraniol inhibited 50% of the growth of C. albicans and C. glabrata at concentrations of 130 µg/mL, and 80 µg/mL for C. tropicalis. Furthermore, our findings are in tune with the study of [38], which also explored the efficacy of geraniol. They observed that the ability of geraniol to inhibit the growth of Candida spp. was 256 µg/mL. On the other hand, study [39] observed the same effect of the compound at concentrations of 16 µg/mL for 90% of the tested C. albicans strains, results considerably lower than ours. However, it is important to note that, although the sources of geraniol are the same in both studies and in the present work, methodological differences can influence the values of Minimum Inhibitory Concentration (MIC) found. Therefore, direct comparisons of MICs may not be appropriate without considering these variables.

Therefore, any inference about the effect of geraniol being influenced by genetic variability among strains should be made with caution, Although MICs themselves do not influence genetic variability, they are used to measure the effects of genetic variability on the susceptibility of fungal strains to antifungal drugs. Genetic variability can lead to differences in MICs, indicating different levels of drug resistance [40].

Regarding the pharmacological interaction with fluconazole, the indifferent results found in most strains (77.8%) reveal that, when used concomitantly, there is no negative interference in the activity of the drugs, possibly enabling their joint use [41] reported only synergistic results of the association of the same drugs, with lower inhibitory concentrations against the tested C. albicans strains, but smaller sample quantities were used than in the present study, which also found synergism counterpart of the tested strains (16.7%). Such data reveal that the concomitant use of geraniol and fluconazole is safe, as there was no decrease in the antifungal activity of both compounds. In addition, in a smaller portion of the tested strains, a reduction in the effective concentration of both was observed, which can contribute to reducing the risk of toxicity and improving the efficacy of treatment. It is important to note that although fluconazole is widely used, it is not first-line therapy for all Candida spp. infections, as in the case of invasive candidiasis, where echinocandins are generally preferred.

The effect of geraniol was also confirmed by the significant reduction in cell viability (p < 0.05) of the strains exposed to MIC/2 and MIC concentrations. These results were more promising than those observed for fluconazole, a drug used for the treatment of Candida spp. infections.

Studies in the literature suggest that the antifungal effect of geraniol may be related to the microbial membrane that alters cell permeability by penetrating between the acyl chains that make up the lipid bilayers of the membrane [25, 42, 43]. The consequences of the inhibition of ergosterol synthesis could be observed by SEM, showing the shrinkage of the fungal cell with the treatment with geraniol. These results corroborate the other results described so far in the present work and those found in other parts of the literature for the antifungal mechanism of geraniol [43–46]. reported that the mechanism of action of geraniol is not related to direct binding to ergosterol, as occurs with amphotericin B. Other studies have shown that terpenoids, the group to which geraniol belongs, interfere with the cell cycle of C. albicans [37, 46, 47].

Complementing the evaluation of the possible mechanism of action, the results obtained by flow cytometry demonstrated that the exposure of the microorganisms to geraniol causes an increase in fungal mitochondrial depolarization, an effect that is linked to the increase in intracellular levels of ROS, also observed in the test. Large amounts of these substances have toxic potential in cells, causing damage to DNA and the plasma membrane, for example, potentiating the effects previously described on these structures, leading to cell death [44, 45, 48], possibly by apoptosis, identified by the externalization of phosphatidylserine. The study conducted [46] demonstrated that in C.glabrata geraniol does not attack only one part of the fungus; it targets several pathways. Among them, weaken the cell walls, interfere with the internal processes of the fungus, and even trigger a mechanism of apoptosis corroborating as our findings.

Studies related the lipophilic character of geraniol to its antimicrobial mechanism [25, 49], causing it to interact and bind to the lipid and enzymatic components of the microbial plasma membrane, increasing permeability and facilitating the entry of the drug into the cell, possibly allowing greater binding to other intracellular sites, and causing the effects already demonstrated in the present work.

The results of the study consolidate the benefit of geraniol as an antifungal agent that can be safely used in topical formulations, as it was approved the Generally Recognized as Safe (GRAS) status by the USA indicating that it is a safe substance which is a significant advantage when considering the development of new antifungal treatments [50–52].

However, there is a need for a careful evaluation of its cytotoxic and genotoxic effects to ensure safe use in humans [53]. In the literature, there are already studies evaluating whether geraniol has a cytotoxic effect on cancer cells [42, 50, 51]. Regarding genotoxicity, studies suggest that geraniol does not present genotoxic effects in the tested human cell lines under the conditions that were studied [53, 54]. Still in terms of cytotoxicity, the results of the study [55] are positive in relation to geraniol that did not present cytotoxic effects in human lymphocytes at the tested concentrations up to 5,000 µg/ml, and the cut-off point for cytotoxicity was considered at 70% cell viability. Study [56] performed pharmacokinetics, bioavailability of geraniol the results suggest that geraniol is absorbed and distributed efficiently in the body, reaching the central nervous system, and potentially exerting its therapeutic effects showing a rapid and significant presence of geraniol in the bloodstream, with the oral route reaching a bioavailability of 92%.

In summary, the antifungal effect of geraniol against Candida spp. possibly occurs in a multifactorial manner, through interference in the synthesis of ergosterol, probably not by direct binding to the molecule. The resulting increase in microbial membrane permeability facilitates the entry of the compound, allowing it to bind to other intracellular sites, such as mitochondria and enzymes related to DNA, leading to cell death by apoptosis. Geraniol has antifungal activity against different species of Candida, without loss of efficacy when associated with fluconazole. The mechanism of action is likely related to interference in the synthesis of ergosterol, with a consequent increase in the permeability of the microbial plasma membrane and subsequent binding to different intracellular sites, causing mitochondrial depolarization and formation of ROS and genotoxicity, leading to cell apoptosis and death. This study contributes to ongoing efforts to better understand the mechanism of action of geraniol and thereby develop new and effective treatments for fungal infections, meeting a critical need in modern healthcare.

Funding

This study was supported by grants and fellowships from the research support agencies CNPq, CAPES and FUNCAP/Ceará.

Data availability

All data generated or analysed during this study are included in this published article: “Unveiling novel insights: geraniol’s enhanced anti-candida efficacy and mechanistic innovations against multidrug-resistant candida strains”.

Declarations

Competing interest

The authors declare no conflicts of interest concerning this article.