Potential antifungal impact of citral and linalool administered individually or combined with fluconazole against clinical isolates of Candida krusei

This research indicated that combining fluconazole and citral and/or linalool might lead to increased efficacy and reductions in the minimum effective doses. These combination therapy may moderate the development of drug toxicity and resistance. Please this paper M, A, Shokri H. Potential antifungal impact of citral and linalool administered individually or combined with fluconazole against clinical isolates of Candida krusei for combinations of citral+FLZ and linalool+FLZ, calculated from checkerboard MD assays. Results: The mean ± standard deviation (SD) MIC values of citral, linalool, and FLZ against the C. krusei isolates were 70.23 ± 17, 150 ± 38.73, and 74.66 ± 36.95 µg/mL, respectively. Some fungicidal activities were also observed for citral (2.5) and linalool (1.53) against the C. krusei isolates. The FICI values of citral+FLZ and linalool+FLZ for the C. krusei isolates ranged from 0.4 to 1.00 and 0.19 to 0.63, respectively. The additive and synergistic interactions of linalool + FLZ were further observed in 12 (57.1%) and 9 (42.9%) C. krusei isolates. However, there was an additive interaction for citral + FLZ in 17 (80.9%) isolates. They also showed a synergistic interaction in only four (19.1%) isolates. Moreover, linalool and citral plus FLZ did not have any antagonistic effect on any isolates. Conclusion: The study findings support the possible capabilities of citral and linalool, as anticandidal agents, and FLZ might be supplemented with citral and/or linalool for treating FLZ-resistant C. krusei infections.


Introduction
Candida species are among major fungal pathogens that are often isolated from invasive fungal infections. These infections can be formed as deep-seated or superficial diseases. Infections resulting from non-albicans Candida (NAC), with high incidence rates, such as C. krusei, C. glabrata, C. parapsilosis, and C. tropicalis have also exposed patients to significant nosocomial risks (1). C. krusei is a diploid, dimorphic and with generally elongated cells belonging to ascomycetous yeasts normally found as a commensal and the mucosal membrane inhabitant in healthy individuals (2). Of note, there is an intrinsic resistance in C. krusei against fluconazole (FLZ), as a frequent drug used for treating the infections caused by candida species. Thus, nosocomial candidiasis due to C. krusei is currently regarded as a therapeutic challenge, particularly in immunocompromised patients. It is also estimated that mortality rates from C. krusei fungemia are about 60%-80% (3). In addition to a systemic disease, there is an association between C. krusei and superficial infections. Vulvovaginal candidiasis and bronchopneumonia can be further induced by this organism; however, it is a rare etiological element in the former, which is isolated merely in 0.1% of cases (4,5). Additionally, evidence shows that C. krusei infects the tonsils, where a permanent cure can be achieved just by removing the organ via surgery, causing ulcers, septic arthritis, vasculitis, and urinary tract infections (6).
In this respect, Azoles are typically utilized for treating different lethal fungal diseases (7). Among these agents, FLZ is the most commonly used antifungal drug against candidiasis because it is highly watersoluble, with high bioavailability and little host toxicity. Since FLZ is a fungistatic agent, it does not destroy the fungus. Therefore, it can be partly explained by the emergence of NAC species resistance to antifungal drugs. It has been frequently noticed that FLZ-resistant Candida strains are evolved, particularly within longterm or repeated therapies for recurrent episodes of vulvovaginal/oropharyngeal candidiasis in individuals with acquired immunodeficiency syndrome (AIDS) (2,8). There are also additive impacts for different resistance mechanisms, as revealed by the sequential presentation of resistance mutations in multidrug resistance regulator 1, sterol 14α-demethylase (ERG11), ultra-performance convergence chromatography, and tachykinin precursor 1 (TAC1) in all potential combinations into a drugsusceptible Candida strains (9). Another mechanism of resistance is by the reduction in intracellular azole concentration, due to changes in the cell membrane composition or efflux pump activity (2). This accounts for the selection of strains with multiple resistance mechanisms in long-term FLZ treatment of patients with candidiasis. Hence, there is an urgent need to find a novel efficient therapeutic strategy to control and treat C. kruseicaused infections.
Previous studies have investigated the potential of enhanced antifungal impact of natural products combined with synthetic drugs (10). Using combined antifungal therapies, the killing features of the compounds against microorganisms can be accordingly elevated through synergy and the spectrum effect can be enhanced. Besides, combination therapy can reduce the development of drug toxicity and resistance (11).

Organisms
In total, 20 clinical isolates of C. krusei from patients (viz. 10 oral and 10 vaginal strains) were obtained from the Microorganism Bank of Dr. Aghil Sharifzadeh, Department of Microbiology, Mycology Center, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, upon receiving ethical approval according to the national guidelines. In addition, C. krusei (ATCC 90030) was used as a control.
Fluconazole (FLC), citral natural (≥96%), and linalool (95-97%) were purchased from Sigma-Aldrich. Stock solutions of test compounds were prepared in 10% dimethyl sulfoxide (DMSO) and then further diluted in RPMI 1640 (Thermo Fisher Scientific) to the desired concentration ranges. The final concentration of DMSO was not higher than 1% of the total volume.

Inoculum preparation
The primary suspensions of each C. krusei isolate were prepared by culturing the isolates in Sabouraud Dextrose Broth (SDB, Merck Co., Darmstadt, Germany) for 24 h at 35°C, with 200 rpm agitation. Then, 1 mL of a 24-hour-old culture was centrifuged (3900 × g at 4°C for 1 minute), and the pellets were washed twice with 1 mL of physiological saline. McFarland turbidity of 0.5 at 530 nm was further obtained after adding 5 mL of 0.9% sterile physiological saline, which corresponded with 0.5-2.5×10 5 colonyforming unit (CFU)/mL. Afterward, morpholine propane sulfonic acid (MOPS)-buffered RPMI1640 medium (Sigma-Aldrich, St. Louis, USA) was employed for diluting the suspension and obtaining the final inoculum concentration of 0.5-2.5 × 10 3 CFU/mL.

MIC and MFC assays
After dissolving of linalool, citral, and FLZ (Sigma-Aldrich, St. Louis, USA) in DMSO, the final serial concentrations of these materials in 96-well plates were obtained with a range of 0 to 256 µg/mL for FLZ and 6.25 to 300 µg/mL for linalool and citral in the RPMI-1640 medium. In this regard, the Clinical and Laboratory Standards Institute (CLSI) M27-A3-S4 document guideline was followed (16). Then, the yeast suspension obtained from the previous step (100 μL) was distributed in all wells from a 96-well micro-TTP.
The negative and positive controls included wells without C. krusei and antifungals (citral, linalool, and FLZ), respectively. Then, the plates were incubated at 35°C, followed by reading the endpoints visually after 48 hours. The interpretation of the MICs was as follows: the lowest concentration of antifungal agents preventing the growth of the C. krusei strains under test in comparison with the growth control. Each experiment was also carried out in triplicate. To determine the MFC, SDA-containing plates were used for seeding 10 μL of the volume of each well without microorganism growth (MIC, 2×MIC, and 4×MIC). Then, the plates were incubated for 48 hours at 35°C. The number of colony-forming units (CFU) was further calculated, and the MFCs were recorded as the lowest concentrations of the drugs that led to no visible colonies. Whether citral and linalool had a fungicidal (MFC/MIC<4) or fungistatic activity (MFC/MIC ≥4) was ultimately determined by obtaining the MFC/MIC ratio (17).
Checkerboard assay Fractional inhibitory concentration indices (FICIs) were determined by performing the checkerboard synergy test to express the combined impact of FLZ and terpenoid phenols (18). In brief, the serial dilutions of the antifungal compounds were prepared. For this purpose, 50 μL of FLZ dilutions were mixed with the rows of a 96-well micro-TTP in decreasing concentrations. In addition, linalool and/or citral (50 μL) was mixed with the columns in reducing concentrations. A 100 μL suspension of the C. krusei strains was further adjusted to 0.5-2.5×10 3 CFU/ mL and added to the wells, followed by incubation for 48 hours at 35°C. Some experiments were also conducted to indicate the interactions between FLZ and citral. The MICs of both combined anticandidal compounds were obtained according to the above-mentioned process. Upon determining the FICs and FICIs, the antagonistic or synergistic activities of the anticandidal combinations were also assessed. In order to calculate the FICs, the MIC of the FLZ-linalool combination was divided by the MIC of FLZ or linalool alone. In addition, the FICs were obtained to indicate interactions between FLZ and citral. For calculating the FICIs, the following relation was used:

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: Statistical analysis One-way analysis of variance (ANOVA) was employed for determining statistical significance (Sigma Stat, version 3.5). P values below 0.05 were also regarded statistically significant. The Bliss method (i.e., the dose-effect based multiplicative impact of a single drug as if they served independently) was applied for combination therapies to determine the visualization of the synergistic score and the interactive analysis of linalool + FLZ and citral+FLZ profiling data against the C. krusei isolates. Moreover, the SynergyFinder was used for generating the plots, which was a stand-alone web application. The synergy score for a drug combination was further averaged over all the dose combination measurements. In the SynergyFinder, the two-dimensional (2D) and three-dimensional (3D) synergy maps were presented, highlighting antagonistic and synergistic dose regions in green and red. According to the SynergyFinder, if the synergy score was: • below -10, it was likely that the two drugs had an antagonistic interaction. • between -10 and 10, it was likely that the two drugs have an additive interaction • above 10, it was likely that the two drugs have a synergistic interaction (19).

Results
The results showed that 16  mL, with a mean ± SD value of 70.23 ± 17 μg/mL. As well, linalool was the weaker antifungal compared with citral, with the MIC values of 100 to 200 μg/mL (mean ± SD: 150 ± 38.73 μg/mL). Increasing the concentrations of both citral and linalool in the media also led to a dosedependent progressive and significant (P < 0.05) reduction in growth for all C. krusei strains (Figure 1). The mean ± SD MFC values of citral and linalool were obtained as 176.2 ± 58.35 µg/mL and 223.8 ± 8.35 µg/mL for the C. krusei strains, respectively. According to these results, the MFC values for both compounds were 1-2 folds higher than their respective MIC values. As depicted in Figure 2, the mean MFC/MIC ratios of citral and linalool were 2.5 and 1.53 for the C. krusei strains, respectively. Based on the MFC/MIC ratios, which were lower than 4, it was suggested that both compounds had fungicidal rather than fungistatic activity.
As listed in Table 2, the results of the chequerboard microtiter assay indicated significant combined effects between citral/linalool and FLZ calculated for the FLZresistant C. krusei isolates (P < 0.05). The FICI values for the citral ± FLZ and linalool ± FLZ combinations against all Candida isolates studied here also ranged from 0.40 to 1.00 and 0.19 to 0.63, respectively.
Out of 21 C. krusei isolates tested (namely, 20 clinical isolates and 1 ATCC strain), the interaction between linalool and FLZ was synergistic in 12 and additive in nine cases, whereas four isolates showed a synergy for citral and FLZ, and 17 cases had an additive interaction based on the FICIs. Linalool and citral also decreased the mean MICs of FLZ from 74.66 μg/mL to 9.81 and 9.9 μg/mL for the C. krusei strains (>75%), respectively. Moreover, the FICI values and the Bliss synergy score indicated that both terpenoids under test exhibited excellent synergistic activities with FLZ ( Figure 3). Of note, linalool and citral did not show any antagonistic activities with FLZ.

Discussion
The high incidence rates of candidiasis among immunocompromised patients due to the emergence of drug resistance in clinical Candida isolates is a matter of concern. The increasing frequency of non-C. albicans species in clinical situations, like C. krusei, which often respond differentially to FLZ, has further worsened the situation (2,20). The present study investigated the antifungal activity of citral and linalool against the clinical C. krusei strains, and their synergistic interactions in combination with FLZ.
In this study, most of the clinical isolates and the reference strain (76.2%, 16/21) were resistant to FLZ, except five isolates that were considered as dose-dependent (23.8%, 5/21). The MICs for FLZ also ranged from 32 to 128 μg/mL (mean ± SD: 74.66 ± 36.95 μg/mL). Consistent with these findings, previous reports have demonstrated the FLZ MIC values of 64 μg/mL for C. krusei (21,22). C. krusei has recently emerged as an important nosocomial pathogen. Currently available triazoles, such as FLZ, have also limited or no activity on this organism, because C. krusei is assumed to be intrinsically resistant to this compound (2,20). Thus, it is more difficult to treat Candida infections via FLZ monotherapy. The scientific community has thus paid attention to the potential antifungal impacts of some bioactive compounds from plants.
Here, the M27-A3 broth MD assay indicated that citral had a good inhibitory effect on all 20 C. krusei specimens, and the MICs ranged from 50 to 100 μg/mL, with the mean ± SD value of 70.23 ± 17 μg/mL. In addition, all clinical strains were inhibited by linalool with the MICs ranging from 100 to 200 μg/mL (mean ± SD: 150 ± 38.73 μg/mL). Increasing the concentrations of both citral and linalool in the media also led to a dose-dependent progressive and significant reduction in growth for all Candida strains. The biological activity of the natural products was further interpreted using the MIC values, according to the criteria adopted by Sartoratto et al (23): viz. the biological activity of a product may be interpreted as excellent/good if the MIC value ranges from 50 to 500 μg/mL. As well, if compounds show a variation in the MIC value from 600 to 1500 μg/mL, they are considered to have moderate activity, and in cases when the MIC is greater than 1500, it has weak activity. Thus, according to the findings in the present study, linalool and citral presented good antifungal activity for the C. krusei strains. High anti-Candida efficacy by linalool and citral had also been reported in other studies. In agreement with these results, Ferreira et al (24)  In this research, the mean MFC values of citral and linalool were obtained as 176.2 ± 58.35 µg/mL and 223.8 ± 8.35 µg/mL for the C. krusei strains, respectively. The mean MFC/MIC ratios of citral and linalool were also equal to 2.5 and 1.53 for the C. krusei strains, respectively. The results correspondingly revealed the potent fungicidal activity of linalool and citral against the clinical Candida strains. Accordingly, the MFC/MIC ratio of linalool outperformed that of citral, in line with the study by Dias et al (26). The findings outlined herein were also consistent with the results presented by Zore et al (28), in which linalool and citral have been assumed effective in all isolates of Candida strains (namely, dose-dependent and resistant to FLZ), and showing fungicidal activity. Previous studies have also shown that linalool was able to significantly interfere in the biosynthesis of the cell wall and/or increase the ionic permeability of the fungal cell membrane (29,30). Citral additionally demonstrated fungicidal activity against the Candida strains isolated after 2 hours of exposure and caused major morphological changes via mechanisms other than cell wall biosynthesis or ergosterol complexation (15). As documented, there is a relationship between the fungicidal activity of antifungals and higher therapeutic success and less persistent and lower recurrent infection rates, particularly in candidemia and invasive candidiasis (31).
The synergistic effects of various components of plant extracts are also supposed to contribute to the effectiveness of herbal preparations used in traditional medicine. Synergy research in phytomedicine is thus really needed to address this issue. The present study investigated the FICI values for linalool and citral in combination with antifungal agent, FLZ. The potential antifungal activities of linalool and citral alone or in combination with FLZ against the clinical C. krusei   (33). Eugenol showed a synergistic interaction with voriconazole against C. krusei (34), and terpenoids were reported to arrest the eukaryotic cell cycle (35). Moreover, Zore et al (28) demonstrated that terpenoids could arrest Candida cells at different phases of cell cycle, i.e., G1 arrest by linalool and S phase arrest by citral. In addition, terpenoids could induce membrane fluidization, modulating the functions of membrane bound proteins involved in signaling and transport (28). A hypothetical model was proposed for the anti-Candida activity of citral and linalool terpenoids as well as their synergies with FLZ. In this context, the increased cellular concentration of FLZ could inhibit ergosterol biosynthesis, while the terpenoid-mediated impairment of membrane integrity could modulate signaling pathways and transport. Thus, Figure 3. A-B: The interactive analysis and visualization of the synergistic score of the linalool+FLZ (A-1 and B-1) and citral+FLZ profiling data against the C. krusei isolates and their statistical significance. The level of antagonism or synergism was represented by a colored scale bar (from white to dark red). The plots were generated using the SynergyFinder, as a stand-alone web application, by applying the Bliss method (the dose-effect based multiplicative impact of a single drug as if they served independently) for combined treatments. The synergy score for a drug combination was further averaged over all the dose combination measurements. The 2D (A-1 and A-2) and 3D (B-1 and B-2) synergy maps highlight synergistic dose regions in red (There is no antagonistic point).
the synergistic effect exhibited by terpenoids with FLZ is a multifactorial phenomenon involving membrane destabilization, cell signaling modulation, and cell cycle arrest. It is noteworthy that this in vitro study is the first attempt that reports the synergistic interactions of citral+FLZ and linalool+FLZ and their potential uses to treat superficial infections in the oral/vaginal mucosa caused by FLZ-resistant C. krusei strains. Hence, the study findings are encouraging for designing clinical trials to evaluate the effectiveness of these combination therapies.

Conclusion
By combining a synthetic drug (FLZ) and citral/linalool, an increased efficacy was found, and the minimum effective dose was reduced. The study findings encouraged the growing treatment failures and antibiotic resistance in Candida and proposed one for Candida infections via a drug combination approach. Clinical evidence or in vivo studies are thus required to confirm these in vitro findings.