Skip to main content
Download PDF

Evaluation of antifungal and apoptotic effects of linalool, citral, and carvacrol separately and in combination with nystatin against clinical isolates of Pichia kudriavzevii

BMC Microbiology volume 24, Article number: 333 (2024) Cite this article

Abstract

Pichia kudriavzevii (formerly Candida krusei) poses a significant threat to immunocompromised patients due to its inherent resistance to various antifungal drugs. This study explored the anticandidal potential of citral, linalool, and carvacrol in combination with nystatin against P. kudriavzevii strains.

Using the microdilution method following CLSI guidelines, Minimum Inhibitory Concentrations (MICs) and fungicidal concentrations (MFCs) were determined. Citral exhibited MIC values ranging from 50 to 100 µg/ml, averaging 70.24 ± 16.99 µg/ml, while carvacrol had MIC values of 50 to 100 µg/ml, averaging 86.90 ± 16.99 µg/ml. Linalool demonstrated weaker antifungal activity, with MIC values between 100 and 200 µg/ml, averaging 150 ± 38.73 µg/ml. The study assessed the synergistic effectsof these phenols with nystatin through fractional inhibitory concentration indices (FICIS). In addition, flow cytometry was employed to assess apoptosis induction in P. kudriavzevii cells.

Carvacrol displayed a remarkable synergistic effect in combination with nystatin against all 21 isolates tested. Conversely, linalool showed synergy in 17 isolates, while citral exhibited synergy in only 2 isolates. These findings highlight distinct patterns of synergy between the different compounds and nystatin against P. kudriavzevii. Also, Carvacrol emerged as the most potent inducer of apoptosis across all P. kudriavzevii strains, followed by citral and linalool. This suggests that carvacrol not only possesses a stronger antifungal effect but also has a more pronounced ability to trigger programmed cell death in P. kudriavzevii. In conclusion, the study supports the potential of carvacrol, citral and linalool, as anticandidal agents, suggesting their supplementation with nystatin for treating P. kudriavzevii infections.

Peer Review reports

Introduction

Candida species are among the most significant factors in fungal infections in humans and animals giving rise to a variety of invasive diseases ranging from potentially life-threatening bloodstream candidiasis to non-life-threatening mucocutaneous candidiasis such as genitourinary candidiasis, vulvovaginal candidiasis, and oropharyngeal candidiasis [1]. These infections are more common in individuals with underlying risk factors such as cancer and leukemia, diabetes mellitus, prolonged antibiotic and corticosteroid treatments, AIDS, pregnancy, burns, transplant recipients, and especially in cases involving some form of leukopenia or other white blood cell deficiencies [2]. P. kudriavzevii, a diploid, dimorphic fungus belonging to the Non albicans Candida (NAC group), can cause a variable spectrum of infections, ranging from mucosal colonization to life-threatening invasive diseases. Cutaneous and mucosal candidiasis are the most prevalent forms [2, 3]. P. kudriavzevii is currently recognized as one of the challenges in the fields of human and veterinary medicine worldwide due to its rapid adaptation to antifungal drugs, and reduced susceptibility to azoles and polyenes facilitated by its drug resistance [2]. P. kudriavzevii, unlike some other Candida strains, demonstrates more resistance to common antifungal drugs such as fluconazole (FLZ) and nystatin [4].Nystatin, an antifungal medication, is primarily employed to treat fungal infections, particularly those caused by the Candida genus. Administered topically in the form of creams or gels, it targets infections on the skin, mouth, and genital tract [5]. By disrupting the fungal cell membrane, particularly by binding to ergosterol and creating pores, nystatin brings about a reduction and destruction of fungal cells [6]. However, factors like prolonged drug use and genetic changes have contributed to the emergence of resistance to nystatin in various Candida species, including P. kudriavzevii [7]. The rising prevalence of drug-resistant candidal infections, particularly among high-risk patients, underscores the critical need for alternative treatment options [8]. These alternatives should ideally address three key challenges: combating resistance, minimizing side effects associated with synthetic drugs, and preventing recurrent infections. Combination therapies hold promise in potentially improving treatment effectiveness and reducing toxicity [9].

For millennia, humans have relied on medicinal plants, deeply intertwining their use with the evolution of civilization. In the pre-19th century era, herbal remedies were the predominant treatment for various ailments. Despite the subsequent dominance of chemical compounds in modern medicine, scientists are rekindling interest in traditional medicine and medicinal plants. This resurgence stems from a desire to explore natural remedies, mitigate the side effects associated with synthetic drugs, and seek alternative treatments, among other considerations [10].

Phenolic terpenoids, particularly carvacrol, are potent antifungal agents found in plant essential oils. They exhibit a broad spectrum of activity against various fungal pathogens, including Candida species [11]. Citral is one of the primary components of citrus peel oil, particularly found in orange peel. It exhibits antimicrobial, antifungal, and antiparasitic properties, which have led to citral being recognized as a natural preservative for the pharmaceutical, food, perfume, and cosmetics industries as well [12]. Linalool, which is naturally found in many fragrant flowers and plants, is a monoterpene alcohol and associated with various commercial applications, including cosmetics, personal care, pest control, and medicine, most of which are based on its pleasant scent and has also been tested as bactericide and fungicide [13].

Studies have demonstrated anticancer, anti-inflammatory, antimicrobial, antihyperlipidemic, antinoceptive, analgesic, anxiolytic, antidepressive, neuroprotective, and antifungal activities against yeasts and molds in citral and linalool [14].

Furthermore, some phenolic compounds have no adverse effects on natural cells [15]. Combination therapy has become an important topic in formulating new therapeutic strategies against fungal infections. They can enhance efficacy, overcome drug resistance, and reduce the toxicity of antifungal drugs [16]. Studies have shown that combining antifungal drugs with phenolic compounds can lead to lower MIC values compared to using them alone. This combination is also expected to reduce virulence factors of Candida species, such as adhesion, hydrophobicity, and biofilm formation [16]. The objective of this study is to determine the MIC (Minimum Inhibitory Concentration) of plant phenols, including linalool, citral, and carvacrol, and investigate their antifungal and apoptotic effects separately and in combination with nystatin against P. kudriavzevii fungi.

Materials and methods

Organisms

In this study, 20 clinical isolates of P. kudriavzevii were isolated from patients (10 oral isolates and 10 vaginal isolates) and were kept in the microbial bank of the mycology center of the Faculty of Veterinary Medicine, University of Tehran. The effects of nystatin (≥ 98%), natural citral (≥ 96%), linalool (95–97%) and carvacrol (≥ 96%) on the growth of P. kudriavzevii was investigated. P. kudriavzevii ATCC 90,030 was used as the control strain. The tested compounds were purchased from Sigma Aldrich and diluted in RPMI 1640 medium to the final concentration. The final concentration of DMSO did not exceed 1% of the total volume.

Antifungal susceptibility testing

Inoculum preparation

Candida yeast was cultured on Sabouraud Dextrose Agar (SDA; Merck, Germany) at 37 °C for 16 to 18 h. In order to prepare the standard yeast suspension, spectrophotometry and slide hemocytometer methods were used, then the optical absorption of suspension was evaluated at a wavelength of 625 nm so that in this case the number of yeast is 0.5–2.5 × 105 CFU/ml. We considered the above solution as a stock solution. RPMI1640 medium buffered with MOPS (Sigma-Aldrich, St. Louis, USA) was then used to dilute the suspension to obtain a final inoculum concentration of 0.5–2.5 × 103 CFU/ml.

MIC and MFC determination assays

The study aimed to determine the MIC of nystatin, linalool, carvacrol, and citral on candidate cells, following the M27-A3 method outlined by the Clinical and Laboratory Standards Institute (CLSI) guideline. Standard solutions of nystatin, citral, linalool, and carvacrol from Sigma-Aldrich were dissolved in DMSO, resulting in serial concentrations of 1–256 µg/ml for nystatin and 6.25–300 µg/ml for citral, linalool, and carvacrol. Each well of a 96-well plate received 100 µl of the compound solution and 100 µlof yeast suspension (0.5–2.5 × 103 CFU/ml). After incubating the plates for 24–48 h at 35 °C, the MIC was defined as the lowest concentration of the treated wells at which no visible growth was detected.The MFC was determined by culturing 10 µl from MIC, double MIC, and four times MIC wells on Saburo dextrose agar, with the absence of colonies considered as MFC. It should be noted that each assay was repeated three times. Fungicidal or inhibitory activity was assessed using the MFC/MIC ratio. A ratio less than 4 indicated fungicidal activity, while a ratio equal to or greater than 4 signified fungus growth inhibition.

Checkerboard assay

The study assessed synergistic effects of plant phenols and antifungal drugs using the checkerboard microdilution method [17]. Then 50 µlof each of the phenolic compounds, combined with antifungal drugs in a 1:1 volume ratio, were added to the first row of a 96-well plate and serially diluted. Each well received 100 µlof culture with inoculated material (0.5–2.5 × 103 CFU/ml). Following 48-hour incubation at 35 °C, MIC values were recorded. To validate results, the test was conducted in triplicate. The combined interactions were evaluated based on the Loewe additivity zero-interaction theory, utilizing FICI according to the following formula.

Drug A FIC = Drug A MIC in a combined manner/ Drug A MIC individually; Drug B FIC = Drug B MIC in a combined manner/Drug B MIC individually.

The FICI is given as the sum of the FIC of drug A and drug B, and its interpretation is as follows:

Additive impact, 0.5 < FICI < 1.0; indifference, synergy, FICI ≤ 0.5, 1.0 < FICI ≤ 4.0, antagonism, FICI > 4.0.

Apoptosis and necrosis analysis

To ascertain the mode of cell death induced by phenolic compounds, apoptosis in P. kudriavzevii cells was examined using apoptosis markers and flow cytometry. The assay was performed in triplicate for each treatment group and control group. Briefly, P. kudriavzevii cells were incubated in Saborud Dextrose Broth (SDB) with phenolic compounds (half, equal, and double MIC concentrations) to achieve logarithmic phase growth for 18 h at 30 °C. Subsequently, cells were centrifuged for 3 h at 200 rpm at 30 °C, washed in 0.1 M potassium phosphate buffer, and maintained in PBS solution for 15 min at 25 °C. After centrifugation for 5 min at 1500 rpm, the cells were treated with 5 mg of annexin V-FITC and 5 mg of Propidium Iodide (PI) following the staining kit protocol. The suspension was incubated in the dark for 15 min at 25 °C. Annexin V-binding buffer was added, and the samples were analyzed using a BD Accuri C6 flow cytometer to assess early apoptosis, late apoptosis, necrosis, and the number of live cells.

Statistical analysis

Statistical analysis utilized GraphPad Prism software (version 5), SPSS software (version 24), and one-way variance ANOVA (Sigma Stat version 3.5), with a significance threshold set at P ≥ 0.05. The Bliss model, assuming independent drug effects, calculated the expected combined effect based on the probability of independent events. Synergy Finder web software facilitated the creation of interaction diagrams, curves, and matrix diagrams for dose-response effects. The software, employing Bliss mathematical models and the cNMF algorithm, analyzed the matrix of yeast cell viability percentages resulting from spectrophotometer readings for antifungal drugs and phenolic compounds combinations (e.g., linalool with nystatin).

Two-dimensional and three-dimensional diagrams depicted areas of antagonistic and synergistic effects, marked in green and red, respectively. The combined effect coefficient below − 10 indicated antagonism, between − 10 and − 10 denoted additivity, while a score above 10 signified synergy. Results were interpreted based on these criteria in the context of the analyzed data by the software.

Results

MIC and MFC values

The results showed that 13 isolates of the studied organisms (61.9%) with MICs in the range of 16 to 32 µg/ml had dose-dependent sensitivity to nystatin, and 6 isolates of P. kudriavzevii (28.57%) with MIC equal to 64 µg/ml were resistant to nystatin, while only 2 isolates (9.53%) were sensitive to nystatin with MIC equal to 8 µg/ml. The mean MIC of nystatin was equal to 35.05 ± 20.47 µg/ml (Table 1).

Table 1 In vitro susceptibilities of clinical and standard strains of P. Kudriavzevii to linalool, citral, carvacrol and nystatin
Full size table

Based on the data obtained from this study and determination of MIC, MFC and MFC/MIC ratio related to linalool, carvacol and citral, it was determined that the tested phenolic compounds were active against all P. kudriavzevii isolates studied (Table 1). Thus, citral had MIC values ​​between 50 and 100 µg/ml, with an average of 70.24 ± 16.99 µg/ml. Also, the MIC values ​​for carvacrol plant phenol were in the range of 50 to 100 µg/ml, with an average of 86.90 ± 16.99 µg/ml. linalool had a weaker antifungal activity than citral and carvacrol, so that the MIC values ​​between 100 and 200 µg/ml with an average of 150.00 ± 38.73 µg/ml were determined for this phenol.

Based on the analysis of the data obtained from the study by the web-based Synergy Finder software, increasing the concentration of linalool, carvacol and citral in the culture medium led to a progressive and significant dose-dependent decrease in the growth of all P. kudriavzevii strains. Also, the average MIC values ​​in the combination of linalool and nystatin decreased from 150 to 35.05 µg/ml to 44.05 and 6.19 µg/ml respectively, in the case of citral and nystatin from 70.24 to 35.05 µg/ml to 31.55 and 13.33 µg/ml respectively, and in the case of carvacrol and nystatin, it decreased from 86.90 to 35.05 µg/ml to 23.21 and 2.67 µg/ml, respectively (P ≥ 0.05) (Fig. 1).

Fig. 1
figure 1

Synergy Finder software analyzed dose-response matrix curves and plots depicting growth inhibition percentages. The images illustrate the impact of increasing concentration of individual phenolic compounds (A: Citral, B: Linalool, C: Carvacrol, D: Nystatin) against the standard strain of P. kudriavzevii Additionally, matrix diagrams (E-G) demonstrate dose-response interactions between phenolic compounds and nystatin by F algorithm for combined treatments against P. kudriavzevii isolates (E: Citral and Nystatin; F: Linalool with Nystatin; G: Carvacrol and Nystatin)

Full size image

Checkerboard assay

Based on the statistical analysis of the checkerboard microdilution test results according to CLSI-M27-A3 standard instructions, the values ​​obtained from which are listed in Table 2, FICI values ​​in the combination of citral plus nystatin in 21 isolates of P. kudriavzevii were calculated in the range of 0.46 to 1.25 with an average of 0.82, linalool plus nystatin equal to 0.38 to 0.83 and an average of 0.49, and carvacrol plus nystatin in the range of 0.22 to 0.50 with an average of 0.36.

Table 2 The results of the checkerboard microdilution test
Full size table

The combination of linalool and nystatin exhibited a synergistic state in 17 P. kudriavzevii isolates and an additive state in 4 isolates. Citral and nystatin interaction resulted in an additive state in 17 isolates, a synergistic state in 2, and an ineffective state in 2. Notably, the combination of carvacrol with nystatin consistently yielded a synergistic state in all studied isolates, as indicated by the FICI calculations (Table 2).

The concordance between FIC index values and Bliss model synergy scores, assessed through Synergy Finder web software, highlighted the prevalent synergistic activity of the triphenoids carvacrol and linalool when combined with nystatin. For citral, the predominant mode was additive when combined with antifungal drugs. Notably, antagonistic activity was absent in the combinations of citral, linalool, and carvacrol with nystatin (Fig. 2).

Fig. 2
figure 2

Synergistic score and statistical analysis of the data obtained from the interaction of the combination of citral, linalool and carvacrol with nystatin against P. kudriavzevii isolates studied. The two-dimensional (2D) and three-dimensional (3D) diagrams have marked the synergistic areas in red color. The areas with the highest bioenergy score are marked inside the square box. A-1) 2D diagram of citral-nystatin synergistic score. A-2) 3D diagram of citral-nystatin synergistic score. B-1) 2D diagram of linalool-nystatin synergistic score. B-2) 3D diagram of linalool-nystatin synergistic score. C-1) 2D diagram of carvacrol-nystatin synergistic score. C-2) 3D diagram of carvacrol-nystatin synergistic score

Full size image

Apoptosis and necrosis

Cells exposed to varying concentrations of citral, carvacrol, and linalool underwent dual staining with annexin V-FITC and propidium iodide (PI). Annexin V-FITC identified apoptotic cells marked by phosphatidylserine (PS) release, while PI staining identified necrotic cells. Flow cytometry analysis graphs in Fig. 3, and 4 showcased the impact of three concentrations of each compound on treated P. kudriavzevii strains and the control sample. Additionally, Fig. 3, derived from flow cytometry analysis, illustrated the percentage effect of these concentrations on the treated strains alongside the control sample. Among the tested compounds, carvacrol demonstrated the highest efficacy in inducing apoptosis against all P. kudriavzevii strains, with citral and linalool following in activity. So that in the concentrations of 0.5×MIC, 1×MIC and 2×MIC respectively, carvacrol decreased the initial apoptosis rate of 2.6%, 0.25% and 0%, delayed apoptosis 6.42%, 33.3% 0.042% and necrosis 5.23%, 66.4% and 99.6%, and for citral concentrations were 2.01%, 0.032% and 0% for early apoptosis, delayed apoptosis 1.13%, 28% 0.469% and for cell necrosis equal to 20.6%, 70.6% and 98.6%. Also, in the case of linalool phenol, the initial apoptosis rate was 1.44%, 0.102% and 0.092%, delayed apoptosis was 0.664%, 15% and 16.2% and the rate of cell necrosis against P. kudriavzevii isolates studied at concentrations of 0.5×MIC, 1×MIC and 2×MIC were equal to 10.2%, 76.4% and 75.8%.

Fig. 3
figure 3

cell apoptosis by flow cytometry using double staining with annexin V-FITC and propidium iodide (PI). P. Kudriavzevii cells were exposed to concentrations of 0.5×MIC, 1×MIC and 2×MIC of carvacrol. Untreated cells were considered as control. In each quadrant of the graph, the density in Q1 represents necrotic cells (annexin − PI+); Q2 indicates delayed apoptotic cells (annexin + PI+); Q3 indicates early apoptotic cells (annexin + PI−) and Q4 indicates live cells (annexin- PI-)

Full size image
Fig. 4
figure 4

The percentage of cells that showed necrosis, early apoptosis and delayed apoptosis after being exposed to different concentrations of carvacrol, citral and linalool

Full size image

The most significant necrosis effect occurs with double the MIC concentration of carvacrol, followed by twice the MIC of citral, and then the MIC concentration of linalool. The highest delayed apoptosis effect is associated with the MIC concentration of carvacrol, followed by citral, and then double the MIC concentration of linalool. The total percentage of dead cells (necrosis and apoptosis) in all three phenols at 1× MIC and 2× MIC concentrations was more than 90% (Fig. 4), among the phenols tested, carvacrol at MIC concentration with a total of 99.725% (Total percentage of necrosis, delayed apoptosis and early apoptosis) had the greatest effect among the phenols tested.

Discussion

Candida species are the most important causes of fungal infections in humans and animals. P. kudriavzevii, which mainly affects immunocompromised patients, is recognized as an emerging opportunistic pathogen worldwide with its multidrug resistance, ability to rapidly adapt to antifungal therapies, and complex susceptibility profile [18]. Since 1960, P. kudriavzevii infections have increased alarmingly, and are increasing drug resistance [2]. The present study investigated the antifungal activity of carvacrol, citral and linalool against clinical P. Kudriavzevii strains and their synergistic interactions in combination with nystatin. In this study, using the data obtained from the broth microdilution method and obtaining the MIC values, it was determined that 61.9% of the studied organisms had dose-dependent sensitivity and 28.57% were resistant to nystatin. This is while only 2 isolates (9.53%) were sensitive to nystatin drug. Although nystatin has inhibitory effects, it can also show fungicidal activity in high concentrations. The primary activity of nystatin both in vitro and in clinical applications is mainly against yeasts. The spectrum of activity of C. albicans and other Candida spp. is significantly inhibited by nystatin [19]. With the increasing prevalence of non-albicans candida infections and the occurrence of acquired resistance, finding alternative treatments to eliminate resistance, prevent repeated relapses and side effects of chemical and synthetic drugs seems necessary. In this regard, one of the treatment methods that researchers are interested in is the use of plant compounds with antimicrobial properties such as phenolic compounds and terpenoids [20, 21]. Based on the data obtained from the present study, the average MIC values were determined ​​for linalool (150 µg/ml), carvacol (86.90 µg/ml) and citral (70.24 µg/ml) and MFC 223.8, 103.09 and 176.2 µg/ml, respectively and MFC/MIC ratio of these three compounds equal to 1.53, 1.2 and 2.5, respectively. It also showed that the tested phenolic compounds were active against all studied P. kudriavzevii isolates. So that all three compounds in the culture medium led to a progressive and significant decrease in dose-dependent growth of all P. kudriavzevii strains and had inhibitory and lethal effects. While this study confirms the antifungal activity of carvacrol, citral, and linalool against P. kudriavzevii, it is important to note that these effects were observed at concentrations significantly higher than those typically used in clinical settings for Candida infections. This highlights the need for further research to optimize the delivery methods or explore synergistic combinations with existing antifungal drugs to achieve therapeutic efficacy at lower concentrations.

By examining the effects of carvacrol and cinnamaldehyde, Cadena et al. stated that all tested plant compounds had a lethal effect on planktonic cells in 35 isolates of different Candida spp. including 3 isolates of P. kudriavzevii [22]. Leite et al.. measured the MIC and MFC values ​​of citral against C. albicans 64 µg/ml and 256 µg/ml, respectively [23]. Wani et al.. investigated the effects of citral derivatives by broth microdilution method against C. albicans, and according to the ratio of MFC/MIC (less than 4), they confirmed the fungicidal activity of this compound, and this study was consistent with our results [24]. The results of the study Dias et al., showed that the best antifungal activity of linalool on Candida tropicalis with MIC = 500 mg/ml and then for C. albicans with MIC = 1.00 mg/ml and finally with MIC = 2.00 mg/ml is for P. kudriavzevii, also the MFC of linalool against P. kudriavzevii was determined to be 2.00 mg/ml. Based on the conditions of the study and based on the obtained results, it can be concluded that the Candida strains tested are sensitive to linalool [25].

Comparing the results of Bliss model synergy scoring and FIC values ​​in this study showed that the synergistic activity of carvacrol and linalool in combination with nystatin is dominant. Also, these data about citral showed the predominance of the additive mode in the combination of citral and nystatin. Finally, no antagonistic activity was observed in any of the studied tripinoids in combination with nystatin. The average calculated values ​​of FICI for the combination citral-nystatin equal to 0.82, linalool-nystatin equal to 0.49 and carvacrol-nystatin equal to 0.36 were calculated.

Rosato et al.. investigated the combined effect of nystatin with Origanum Vulgare and Pelargonium graveolens essential oils against Candida spp. including P. kudriavzevii and found that Pelargonium graveolens containing linalool in combination with nystatin has synergistic and additive effects (FICI ≤ 0.5 to FICI = 1) [26]. In other study, Shaaban et al.. investigated the improvement of the effectiveness of antifungal drugs in combination with monoterpenes against Candida auris, and among the phenols tested, carvacrol with an average MIC of 125 µg/ml was the most active phenol and in combination with nystatin by reducing the MIC of the mentioned antifungal drugs, it has left an additive effect in 96% of the tested strains respectively [27]. In other study, De Paiva et al.. investigated the relationship between the use of nystatin and citral in Cymbopogon citratus, against Candida spp. isolated from the oral cavity and this combination decreased the MIC of nystatin and showed additive (78.9%), ineffective (17.8%) and synergistic (3.4%) effects, and they have not observed an antagonistic effect in it [28].

In the current study, based on the flow cytometry results, citral, carvacrol and linalool phenols induced apoptosis and necrosis in P. kudriavzevii cells. Among the compounds tested, carvacrol was the most active compound against all P. kudriavzevii strains to induce apoptosis, followed by citral and linalool. The highest necrosis effect is related to twice the MIC concentration of carvacrol, then twice the MIC of citral, and then the MIC concentration of linalool. The highest delayed apoptosis effect was related to the MIC concentration of carvacrol, then citral, and then twice the MIC concentration of linalool.

Chao Niu et al., investigated the mechanisms of apoptosis induction by carvacrol in C. albicans and reducing its survival rate, showed that the treatment of cells with this phenol causes cell membrane permeability and depolarization. Also, the relationship between DNA fragmentation and inhibition of metacaspase activation was confirmed with cell death and apoptosis in exposure to carvacrol. In addition, the total and mitochondrial level of Reactive Oxygen Species (ROS) increased and caused disruption in cell function. In addition, carvacrol increases cytosolic and mitochondrial calcium levels [29]. Liu et al.. confirmed the effect of citral in inducing apoptosis in Malassezia furfur by confirming the removal of phosphatidylserine, DNA fragmentation and interactions involved in metacaspase activation [30]. By investigating the effects of citral, Wani et al. showed cell cycle arrest and induction of apoptosis signaling pathways in C. albicans by this compound. Also, DNA fragmentation, phosphatidylserine release from the cell, mitochondrial breakdown, and cell cycle arrest were also confirmed in this study, and it was concluded that cell death of the apoptosis type is induced in C .albicans [24]. Using phosphatidylserine (PS) and annexin V-FITC, Khan et al.. investigated the apoptosis process under the influence of different concentrations of linalool in C. albicans. So that by being placed in MIC1/4, it led to the occurrence of morphological changes related to apoptosis, While necrosis was shown at higher concentrations [31].

The effect of citral on P. kudriavzevii isolates has not been investigated in other studies. The reported differences in the effective concentrations of carvacrol, citral and linalool in different studies can be caused by the difference in the source of Candida spp. Therefore, the findings of this study can be used to design clinical trials to evaluate the effectiveness of these combined treatments.

Conclusions

This study demonstrates that carvacrol, citral, and linalool possess antifungal activity against P. kudriavzevii strains. Notably, carvacrol exhibited the strongest antifungal effect, and both carvacrol and linalool displayed synergistic activity with nystatin. Furthermore, all three phenolics induced cell death (apoptosis) in P. kudriavzevii. These findings suggest the potential of these plant phenolics, particularly carvacrol, as alternative or adjunctive therapy to combat drug-resistant P. kudriavzevii infections.

Data availability

The data presented in this study are available from the corresponding author upon request.

References

  1. Zaoutis TE, Argon J, Chu J, Berlin JA, Walsh TJ, Feudtner C. The epidemiology and attributable outcomes of candidemia in adults and children hospitalized in the United States: a propensity analysis. Clin Infect Dis. 2005;41(9):1232–9.

    Article  PubMed  Google Scholar 

  2. Jamiu AT, Albertyn J, Sebolai OM, Pohl CH. Update on Candida krusei, a potential multidrug-resistant pathogen. Med Mycol. 2021;59(1):14–30.

    Article  PubMed  CAS  Google Scholar 

  3. Yadav JS, Bezawada J, Yan S, Tyagi RD, Surampalli RY. Candida krusei: biotechnological potentials and concerns about its safety. Can J Microbiol. 2012;58(8):937–52.

    Article  PubMed  CAS  Google Scholar 

  4. Nenoff P, Krüger C, Neumeister C, Schwantes U, Koch D. In vitro susceptibility testing of yeasts to nystatin–low minimum inhibitory concentrations suggest no indication of in vitro resistance of Candida albicans, Candida species or non-candida yeast species to nystatin. Clin Med Investig. 2016;22(3):1.

    Google Scholar 

  5. Sousa F, Nascimento C, Ferreira D, Reis S, Costa P. Reviving the interest in the versatile drug nystatin: a multitude of strategies to increase its potential as an effective and safe antifungal agent. Adv Drug Deliv Rev. 2023;199:114969.

    Article  PubMed  CAS  Google Scholar 

  6. Ellepola AN, Samaranayake LP. The in vitro post-antifungal effect of nystatin on Candida species of oral origin. J Oral Pathol Med. 1999;28(3):112–6.

    Article  PubMed  CAS  Google Scholar 

  7. An L, Tan J, Wang Y, Liu S, Li Y, Yang L. Synergistic effect of the combination of Deferoxamine and Fluconazole in Vitro and in vivo against Fluconazole-Resistant Candida Spp. Antimicrob Agents Chemother. 2022;66(11):e00725–22.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Katiraee F, Afshar SA, Pirmahalleh SR, Shokri H. In vitro antifungal activity of essential oils extracted from plants against fluconazole-susceptible and-resistant Candida albicans. Curr Med Mycol. 2017;3(2):1.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Perrine-Walker F. Caspofungin resistance in Candida albicans: genetic factors and synergistic compounds for combination therapies. Braz J Microbiol. 2022;53(3):1101–13.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Soliman SS, Semreen MH, El-Keblawy AA, Abdullah A, Uppuluri P, Ibrahim AS. Assessment of herbal drugs for promising anti-candida activity. BMC Complement Altern Med. 2017;17:1–9.

    Article  CAS  Google Scholar 

  11. Lima IO, Pereira FD, Oliveira WA, Lima ED, Menezes EA, Cunha FA, Diniz MD. Antifungal activity and mode of action of carvacrol against Candida albicans strains. J Essent Oil Res. 2013;25(2):138–42.

    Article  CAS  Google Scholar 

  12. Silva CD, Guterres SS, Weisheimer V, Schapoval EE. Antifungal activity of the lemongrass oil and citral against Candida Spp. Braz J Infect Dis. 2008;12:63–6.

    Article  PubMed  Google Scholar 

  13. Dias IJ, Trajano ER, Castro RD, Ferreira GL, Medeiros HC, Gomes DQ. Antifungal activity of linalool in cases of Candida spp. isolated from individuals with oral candidiasis. Brazilian J Biology. 2017;78:368–74.

    Article  Google Scholar 

  14. Leite MC, Bezerra AP, Sousa JP, Guerra FQ, Lima ED. Evaluation of antifungal activity and mechanism of action of citral against Candida albicans. Evid Based Complement Altern Med. 2014;2014(1):378280.

    Article  Google Scholar 

  15. Lin D, Xiao M, Zhao J, Li Z, Xing B, Li X, Kong M, Li L, Zhang Q, Liu Y, Chen H. An overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes. Molecules. 2016;21(10):1374.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Baddley JW, Poppas PG. Antifungal combination therapy: clinical potential. Drugs. 2005;65:1461–80.

    Article  PubMed  CAS  Google Scholar 

  17. Asadi S, Nayeri-Fasaei B, Zahraei-Salehi T, Yahya-Rayat R, Shams N, Sharifi A. Antibacterial and anti-biofilm properties of carvacrol alone and in combination with cefixime against Escherichia coli. BMC Microbiol. 2023;23(1):55.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Abbas J, Bodey GP, Hanna HA, Mardani M, Girgawy E, Abi-Said D, Whimbey E, Hachem R, Raad I. Candida krusei fungemia: an escalating serious infection in immunocompromised patients. Arch Int Med. 2000;160(17):2659–64.

    Article  CAS  Google Scholar 

  19. Pakshir K, Akbarzadeh MA, Bonyadpour B, Mohagheghzadeh AA. In vitro activity and comparison of clotrimazol, fluconazol and nystatin against Candida Vaginitis isolates in Shiraz, 2008. J Rafsanjan Univ Med Sci. 2010;9(3):210–20.

    Google Scholar 

  20. Fichtenbaum CJ, Koletar S, Yiannoutsos C, Holland F, Pottage J, Cohn SE, Walawander A, Frame P, Feinberg J, Saag M, Van der Horst C. Refractory mucosal candidiasis in advanced human immunodeficiency virus infection. Clin Infect Dis. 2000;30(5):749–56.

    Article  PubMed  CAS  Google Scholar 

  21. Guo N, Liu J, Wu X, Bi X, Meng R, Wang X, Xiang H, Deng X, Yu L. Antifungal activity of thymol against clinical isolates of fluconazole-sensitive and-resistant Candida albicans. J Med Microbiol. 2009;58(8):1074–9.

    Article  PubMed  CAS  Google Scholar 

  22. Miranda-Cadena K, Marcos-Arias C, Mateo E, Aguirre-Urizar JM, Quindós G, Eraso E. In vitro activities of carvacrol, cinnamaldehyde and thymol against Candida biofilms. Biomed Pharmacother. 202;143:112218.

  23. Leite MC, Bezerra AP, Sousa JP, Guerra FQ, Lima ED. Evaluation of antifungal activity and mechanism of action of citral against Candida albicans. Evid Based Complement Alternat Med. 2014;2014(1):378280.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Wani MY, Ahmad A, Aqlan FM, Al-Bogami AS. Citral derivative activates cell cycle arrest and apoptosis signaling pathways in Candida albicans by generating oxidative stress. Bioorg Chem. 2021;115:105260.

    Article  PubMed  CAS  Google Scholar 

  25. Dias IJ, Trajano ER, Castro RD, Ferreira GL, Medeiros HC, Gomes DQ. Antifungal activity of linalool in cases of Candida spp. isolated from individuals with oral candidiasis. Braz J Biol. 2017;28:78:368–74.

    Article  Google Scholar 

  26. Rosato A, Vitali C, Piarulli M, Mazzotta M, Argentieri MP, Mallamaci R. In vitro synergic efficacy of the combination of nystatin with the essential oils of Origanum vulgare and Pelargonium graveolens against some Candida species. Phytomedicine. 2009;16(10):972–5.

    Article  PubMed  CAS  Google Scholar 

  27. Shaban S, Patel M, Ahmad A. Improved efficacy of antifungal drugs in combination with monoterpene phenols against Candida Auris. Sci Rep. 2020;10(1):1162.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. De Paiva LF, Teixeira-Loyola AB, Schnaider AB, De Souza AC, Lima LM, Dias DR. Association of the essential oil of Cymbopogon citratus (DC) Stapf with nystatin against oral cavity yeasts. Acad Bras Cienc. 2022.

  29. Niu C, Wang C, Yang Y, Chen R, Zhang J, Chen H, Zhuge Y, Li J, Cheng J, Xu K, Chu M. Carvacrol induces Candida albicans apoptosis associated with Ca2+/calcineurin pathway. Front Cell Infect Microbiol. 2020;10:192.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Liu YT, Lee MH, Lin YS, Lai WL. The inhibitory activity of Citral against Malassezia furfur. Processes. 2022;10(5):802.

    Article  CAS  Google Scholar 

  31. Khan A, Ahmad A, Khan LA, Manzoor N. Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans. Res Microbiol. 2014;165(6):411–9.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We appreciate the University of Tehran for financial support. We are also grateful to Dr. Alireza Khosravi and Dr. Iradj Ashrafi Tamai for friendly providing the necessary materials for this study.

Funding

This work was supported by the University of Tehran.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

    Aghil Sharifzadeh, Bahar Nayeri Fasaei, Sepideh Asadi & Mahdi Houshmandzad

  2. DVM, Student of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran

    Narges Fatemi & Mohammad Hosein Ghaffari

Authors
  1. Aghil Sharifzadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bahar Nayeri Fasaei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sepideh Asadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Narges Fatemi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mahdi Houshmandzad
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mohammad Hosein Ghaffari
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization: (A) Sharifzadeh and (B) Nayeri fasaei; Methodology: M. Houshmandzad, S. Asadi and A.Sharifzadeh; Investigation: A.Sharifzadeh, S.Asadi and M. Houshmandzad; data curation: M. Ghaffari, A.Sharifzadeh and N. Fatemi; writing-original draft preparation: (A) Sharifzadeh, S.Asadi, M. Houshmandzad, M. Ghaffari and N.Fatemi; writing-review and editing: A.Sharifzadeh and (B) Nayeri; supervision: A.Sharifzadeh.

Corresponding author

Correspondence to Aghil Sharifzadeh.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Institutional Review Board (IRB) of the Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, under the ethical code 6/7/300511. For any aspects of the study that involved human subjects, written informed consent was obtained from all participants or their legal guardians, as applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sharifzadeh, A., Fasaei, B.N., Asadi, S. et al. Evaluation of antifungal and apoptotic effects of linalool, citral, and carvacrol separately and in combination with nystatin against clinical isolates of Pichia kudriavzevii. BMC Microbiol 24, 333 (2024). https://doi.org/10.1186/s12866-024-03487-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12866-024-03487-y

Keywords

BMC Microbiology

ISSN: 1471-2180

Contact us