Short-Term Antifungal Treatments of Caprylic Acid with Carvacrol or Thymol Induce Synergistic 6-Log Reduction of Pathogenic Candida albicans by Cell Membrane Disruption and Efflux Pump Inhibition
Yoon Seol Bae Min Suk Rhee
Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea
Key Words
Candida albicans • Caprylic acid • Essential oil component • Synergistic antifungal activity • Membrane disruption • Efflux pump
Abstract
Background/Aims: Although naturally-derived antifungals have been investigated for their ability to inactivate Candida albicans, which is a major cause of candidiasis, they have shown a less than 3 log reduction in C. albicans or required treatment times of longer than 3 h. Thus, the naturally-derived antifungals used in previous studies could not substantially eradicate C. albicans within a short period of time. Methods: To improve the fungicidal effects of naturally-derived antifungals against C. albicans within short time periods, we developed composites showing antifungal synergism using caprylic acid (CA), carvacrol (CAR) and thymol (THM) for 1–10 min at 22/37°C. Using flow cytometry, we examined the mode of action for the synergism of these compounds on membrane integrity and efflux pump activity. Results: Whereas the maximum reduction by individual treatments was 0.6 log CFU/ml, CA + CAR/THM (all 1.5 mM) eliminated all pathogens (> 6.8 log reduction) after 1 min at 37°C and after 10 min at 22°C. The flow cytometry results showed that exposure to CA damaged the membranes in 15.7–36.5% of cells and inhibited efflux pumps in 15.4–31.3% of cells. Treatments with CAR/THM slightly affected cell membranes (in 1.8–6.9% of cells) but damaged efflux pumps in 14.4–29.6% of cells. However, the combined treatments clearly disrupted membranes (> 83.1% of cells) and pumps (> 95.0% of cells). The mechanism of this synergism may involve membrane damage by CA, which facilitates the entry of antifungals into the cytoplasm, and the inhibition of efflux pumps by CA, CAR or THM, causing their accumulation within cells and, leading to cell death. Conclusion: Antifungal composites (CA + CAR/THM) showing synergism (i.e., an additional 6 log reduction) within minutes at room/body temperature can be used to treat candidiasis and improve the microbiological safety of facilities contaminated with fungi as a novel alternative to synthetic antifungals.
Introduction
Fungal infections have emerged as a major public health issue worldwide, and there are growing numbers of immunocompromised patients (1). Of all fungal diseases, candidiasis is one of the most common opportunistic and nosocomial infections, which are most commonly caused by Candida albicans, accounting for 42.5% of cases of candidiasis (2). C. albicans is commonly found in the intestinal tract, mouth, throat, vagina or nails (3). More than 75% of women have vulvovaginal candidiasis once in their lifetime, and 30–50% of healthy individuals carry this pathogen as a part of their normal oral microflora (4). Furthermore, C. albicans is often disseminated from mucosal lesions, gastrointestinal colonization or unsterile medical instruments, such as catheters or dentures (5). The spread of C. albicans can lead to invasive candidiasis, which is the fourth most common bloodstream infection, afflicting 250,000 patients and causing more than 50,000 deaths worldwide every year (6). Even with antifungal therapy, the mortality of invasive candidiasis is greater than 40%. Thus, it is important to inactivate C. albicans (i.e., destroy the biological activities of C. albicans) before the pathogen spreads to other body areas.
A variety of intervention methods designed to inactivate C. albicans have been investigated. C. albicans is enveloped within a plasma membrane and cell wall, protecting it from harsh environments and playing important roles in pathogenesis (7, 8). Thus, the primary targets for controlling C. albicans are potentially related to the membrane and cell wall (7). Traditionally, antibiotics have been used in the prevention or treatment of candidiasis. Azoles are mostly used to inactivate C. albicans, and are drugs that work by inhibiting the biosynthetic pathway of ergosterol, which is a component of the cell membrane (9). However, the prolonged use of antibiotics results in drug-resistant C. albicans (10). Clinical resistance to antibiotics can cause gastrointestinal or endocrinologic disorders and oxidative drug metabolism in the liver (11, 12). A significant mechanism of antibiotics resistance is the overexpression of efflux pumps, which actively export drugs out of cells (13, 14). Thus, the inhibition of drug efflux pumps is considered to be an important method for inactivating fungi (10, 12, 15). Due to the undesirable side effects of antibiotic overuse, the development of new antifungal compounds as alternatives to antibiotics is necessary, with naturally-derived antimicrobials representing preferred sources for new pharmaceutical products (16).
However, the naturally-derived antimicrobial compounds used in previous studies cannot sufficiently eradicate C. albicans within a short time. For example, some studies showed slight inhibitory effects (< 3 log CFU/ml reduction) of naturally-derived antifungal agents (e.g., lauric acid, eugenol, thymol, and carvacrol) after treatments of more than 3 h (9, 17, 18). Thus, to increase the efficacy of naturally-derived antifungals, hurdle technologies using naturally-derived agents have been developed, and a number of studies have focused on the combined effects of antifungal compounds. The use of several antifungals in combination is expected to achieve a synergistic effect and require lower quantities of the antifungals (19). Our previous study demonstrated that combination technologies using various types of naturally-derived antimicrobial compounds (e.g., medium-chain fatty acids, essential oil compounds, organic acids, citrus fruit extracts, etc.) showed marked antibacterial effects against a variety of foodborne pathogenic bacteria (e.g., Escherichia coli O157:H7, Salmonella Typhimurium, Cronobacter spp., etc.) compared to treatments with individual compounds. Following these studies, we attempted to determine which naturally-derived agents optimally inactivate C. albicans. However, pathogenic fungi have greater resistance to environmental stresses than bacteria due to having nuclear membranes and greater cell sizes, making the elimination of fungi potentially more difficult than that for bacteria (20).
Among the many naturally-derived compounds that have been studied, caprylic acid (CA), carvacrol (CAR) and thymol (THM) were further investigated in this study based on their antifungal synergism within minutes, low required concentrations, pH value and nontoxicity toward humans. CA is a medium-chain fatty acid (MCFA) that is naturally present in coconut oil and mammalian breast milk and is used to improve microbiological safety in the food, health care and medical industries (9, 19). CAR and THM are essential oil components (EOs) extracted from various plant materials (seeds, flowers, buds, herbs, woods and roots) and have been used as naturally-derived antimicrobial compounds and preservatives (10, 21, 22). However, no studies have examined the fungicidal activity and mechanism of action of the combined treatment of CA, CAR and THM against C. albicans.
The aim of this study was to develop a novel complex of CA and EOs (CAR or THM) with synergistic fungicidal activity after short exposure times against C. albicans at room temperature (22oC) and body temperature (37oC). Moreover, to elucidate the mode of action underlying the antifungal effects of the combined treatment, the accumulation of antifungals in the fungal cytoplasm due to increased membrane permeability and the inhibition of efflux pump activity was analyzed.
Materials and Methods
Preparation of fungal cell suspensions
Three strains of C. albicans (ATCC 10231, 18804, and 11006) were obtained from the American Type Culture Collection and the Food Microbiology Culture Collection of Korea University (Seoul, Korea). Stock cultures were stored at -80°C in sabouraud dextrose broth (SDB; Difco, Becton Dickinson, Sparks, MD, USA) containing 20% glycerol and were sub-cultured on a monthly basis. Each C. albicans strain was separately cultured at 30°C for 24 h in SDB. To prepare the three-strains cocktail, all culture suspensions were centrifuged at 1,800 × g for 15 min (CentraCL2; International Equipment Company, Needham Heights, MA, USA). After discarding the supernatants, the three pellets were washed twice with 0.85% saline, resuspended in 0.85% sterile saline and combined in a sterile 50-ml centrifuge tube (Becton Dickinson, Franklin Lakes, NJ, USA) to yield the prepared cell suspension (approximately 9 log CFU/ml).
Antifungal efficacy tests of CA combined with CAR or THM
CA, CAR and THM were purchased from Sigma Chemical Co. (St. Louis, MO, USA). CA stock solutions (100 and 150 mM) and CAR or THM stock solutions (50, 100 and 150 mM) were prepared in 98% ethanol (EtOH) and used within a week after their preparation.
For the individual CA, CAR and THM treatments, a 0.1 ml aliquot of CA, CAR or THM stock solution was added to 9.8 ml of sterile 0.85% saline in a sterile glass tube (final CA concentrations: 1 or 1.5 mM; final CAR or THM concentrations: 0.5, 1, or 1.5 mM). For combination treatments of CA with CAR or THM, the two CA solutions (1 and 1.5 mM) were individually combined with each EO solution (CAR or THM; 0.5, 1 and 1.5 mM). Aliquots (0.1 ml) of the CA and EO stock solutions were added to 9.7 ml of sterile 0.85% saline (0.1 ml of CA + 0.1 ml of the EOs + 9.7 ml of saline, total volume 9.9 ml). The tubes containing each antimicrobial solution were then equilibrated to 22 or 37°C at 100 rpm in a shaking water bath (VS-1205SW1, Vision Scientific Co., Ltd., Daejeon, Korea) prior to the addition of 0.1 ml of C. albicans suspension to yield an initial cell density of approximately 7 log CFU/ml. All tested fungi were exposed to treatment solutions for 1, 5 or 10 min at 22 or 37°C in a shaking water bath (100 rpm). The fungal suspension was also added to 9.9 ml of 0.85% saline to estimate the initial concentration of the inoculum and to a 2% EtOH solution as a solvent control. All experiments were repeated six times.
Microbiological analysis
Candida samples (1 ml) that had been treated with the antimicrobial agents were diluted 10-fold in 0.85% sterile saline (9 ml), and 0.1 ml aliquots were spread onto duplicate sabouraud dextrose agar (SDA) plates. To lower the detection limit, 0.2 ml of undiluted sample were spread onto five plates (a total of 1 ml; the detection limit was 1 CFU/ml). The plates were then incubated at 35°C for 48 h to allow surviving C. albicans cells to form colonies, which were subsequently enumerated.
The recovery of injured cells was evaluated in samples treated with antifungal composites, which showed complete inactivation of the inoculated C. albicans. Treated samples (1 ml) were inoculated in 30 ml of SDB. After incubating at 30°C for 24 h, the enriched samples were streaked onto SDA plates. The recovery was reported as positive or negative according to the formation of colonies after the SDA plates were incubated at 35°C for 48 h. The experiments were repeated six times.
Stability test for antifungal efficacy of CA combined with CAR or THM
To examine the stability of the antifungal effects of CA combined with CAR or THM, long-term storage tests of the composites were performed. The antifungal effects of the CA + CAR or THM solutions stored for 14, 30, and 60 days at room temperature (22°C) were measured. Prepared C. albicans cell suspensions were added to each stored solution, and the microbiological analysis was performed as described in sections 2.2–2.3. In addition, the pH values of CA + CAR or THM stored for 0, 14, 30 and 60 days were measured using a pH/ion meter (S220 SevenCompact pH/Ion, Mettler-Toledo, Greifensee, Switzerland) at 22 or 37°C. The pH meter was calibrated with commercially available standard buffer solutions at pH 7.0, 4.0 and 2.0 (Mettler Toledo). The experiments were performed in triplicate.
Evaluation of pH effect on antifungal efficacy of combined treatments
To evaluate the net effect of the pH value on the synergistic antifungal activity of the antifungal composites, C. albicans cells were exposed to hydrochloric acid (HCl) solutions. For the same pH values of 1.5 mM CA (pH 4.0 at 22 and 37°C), 0.1 ml-aliquots of 0.18 mM HCl solution were added to 9.8 ml of sterile 0.85% saline in sterile glass tubes (0.0018 mM final HCl concentration, pH 4.0). Additionally, 0.1 ml of 0.18 mM HCl and 0.1 ml of CAR or THM stock solution were added to 9.7 ml of sterile 0.85% saline solution to prepare solutions with the same pH values as those of the 1.5 mM CA + 1.5 mM CAR or THM samples (pH 4.0 at 22 and 37°C). Individual and combined antifungal efficacy tests were performed as described above. The pH of each solution was determined as described in section 2.4. The experiments were performed in triplicate.
Analysis of membrane damage by flow cytometry via propidium iodide staining
Flow cytometry was performed to identify effects of CA, CAR or THM alone, or in combination (CA + CAR or THM), and of HCl solutions (pH 4.0) alone or in combination with 1.5 mM CAR or THM (pH 4.0), on the integrity of cell membranes. Propidium iodide (PI) molecular probe reagent (Invitrogen, Thermo Fisher Scientific Inc., Grand Island, NY, USA), a nucleic acid-binding fluorescent probe, was used to evaluate the effect of antifungals on cell membrane lesions. C. albicans suspensions (approximately 7 log CFU/ml) of untreated cells (living cells), treated cells, or dead cells (after treatment with 100% EtOH for 10 min) were prepared in sterile phosphate-buffered saline (PBS; pH 7.4) (23). Immediately after treatment, the cells were diluted 4-fold in sterile PBS to prevent further fungicidal activity. Residual chemicals were removed by three washes with centrifugation at 18,341 × g for 3 min at 4°C (Smart R17 Plus, Hanil Scientific Inc., Seoul, Korea). The diluted C. albicans cells were then reconcentrated (4-fold). All target cells were stained with 1.0 μg/ml of PI at 35°C for 30 min in the dark. Subsequently, the stained cells were washed three times with sterile PBS and used for flow cytometry analysis.
Flow cytometry was performed with a FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA) using a blue argon laser (15 mW) with an excitation wavelength of 488 nm. Data were analyzed using BD CellQuest Pro (Becton Dickinson Biosciences). Orange fluorescence emitted by PI was detected at ≤ 585 nm (FL2 channel). The signals were detected as logarithmic signals from a photodiode detector with a forward scatter voltage setting of E00, with data acquisition including 30,000 events at a flow rate of 12 μl/min. The results were depicted as histograms, where the M1 region was defined as live and dead cells, and the percentage of fungal cells in the M1 region was calculated using BD CellQuest Pro (Becton Dickinson, San Jose, CA). The experiments were performed in triplicate.
Analysis of efflux pump activity by flow cytometry with Nile red staining
The efflux pump activities of C. albicans cells treated with CA, CAR or THM alone; CA + CAR or THM; HCl solution (pH 4.0) alone; and HCl + CAR or THM (pH 4.0) were examined via flow cytometry by measuring the efflux of Nile red dye (Sigma-Aldrich, St. Louis, MO, USA) (24). Nile red is a specific substrate for C. albicans efflux pumps. Samples were prepared as described in section 2.6, except that PBS containing 2% glucose and 7 μM Nile red was used instead of PBS and 1.0 μg/ml PI. Flow cytometry was performed as described in section 2.6, except that the red fluorescence emitted by Nile red was detected at ≤ 670 nm (FL3 channel). The experiments were performed in triplicate.
Statistical analysis
Duplicate average plate counts from six repeated experiments were converted to log number of CFU/ml. All experimental fungal reduction data were examined by analysis of variance (ANOVA) using SAS version 9.4 (SAS Institute, Cary, NC, USA). Significant differences were determined using Tukey’s multiple comparisons tests (P < 0.05).
Results
Antifungal efficacy of CA combined with CAR or THM at 22°C
Fig. 1 shows the fungicidal effects of CA (1 and 1.5 mM) and the two EOs (0.5, 1, and 1.5 mM) alone or in combinations of CA with CAR or THM (1 + 0.5, 1 + 1, 1 + 1.5, 1.5 + 0.5, 1.5 + 1, and 1.5 + 1.5 mM) for 1, 5 and 10 min at 22°C. Control treatments (2% EtOH) showed no antifungal effects in any of the experiments (< 0.1 log reduction; P > 0.05). The maximum reduction by individual treatments was only 0.2 log CFU/ml with 1.5 mM CA for 1–10 min. For CA with 0.5 mM CAR or THM, fungicidal effects were not observed even when the treatment time and CA concentration were increased. Treatments of 1 mM CA combined with 1 mM CAR or THM also showed negligible reductions in C. albicans populations (< 0.6 log reduction). The antifungal effects of 1 mM CA + 1.5 mM CAR or THM and 1.5 mM CA + 1 mM CAR or THM were augmented by increasing treatment time. In these assays, a maximum reduction of 4.0 log CFU/ml was observed after treatment for 10 min, although the treatments for 1 min with the composites resulted in less than 1.2 log reductions. The combined treatments of 1.5 mM CA + 1.5 mM CAR or THM showed highly enhanced fungicidal effects, where after 10 min at 22°C, the composite treatments reduced the fungal population to undetectable levels (initial population: 6.8 log CFU/ml; detection limit: 1 CFU/ml). Indeed, the treatments resulted in more than 6.5 log CFU/ml reductions in C. albicans cells, although the sum of the antifungal effects achieved by individual treatments was only a 0.3 log reduction in the fungal population. The elimination of inoculated C. albicans cells was confirmed by enrichment tests.
Antifungal efficacy of CA combined with CAR or THM at 37°C
The antifungal effects of CA (1 and 1.5 mM), CAR or THM (0.5, 1, and 1.5 mM) alone and in combined treatments on target fungi are shown in Fig. 2. When fungal suspensions were treated with CA, CAR and THM alone for 10 min, negligible reductions in C. albicans suspension were observed (< 0.6 log reduction). However, the combined treatments with CA and CAR or THM showed synergistic antifungal effects. Antifungal synergism was enhanced by specific treatment times, temperatures, and concentrations of CA, CAR and THM. When C. albicans suspensions were exposed to composites of 1.5 mM CA + 0.5 mM CAR or THM and 1 mM CA + 1 mM CAR or THM at 37°C for 10 min, 2.4–3.3 log reductions in fungal population were observed, although the treatments at 22°C resulted in less than 0.6 log reduction. The combined treatments of 1 mM CA + 1.5 mM CAR or THM and 1.5 mM CA + 1 mM CAR or THM showed 3.0–3.7 log reductions in C. albicans cells after 1 min and 5.1 log reduction after 10 min. Indeed, the antifungal effects of the composites at 37°C were higher than those observed at 22°C. When 1.5 mM CA was used in combination with 1.5 mM CAR or THM at any time point, all fungal cells were entirely eradicated (> 6.9 log CFU/ml reduction, detection limit: 1 CFU/ml), and no cells were detected after further enrichment in SDB, whereas treatments of the composites at 22°C for 1–5 min did not reduce the C. albicans populations to undetectable levels. The 1.5 mM CA + 1.5 mM CAR or THM treatments showed additional 6.0–6.3 log reductions within 1–10 min, whereas the sums of log reductions by individual treatment ranged from 0.6–0.9 log reductions in C. albicans cells within 1–10 min.
Stability of the antifungal efficacy of CA combined with CAR or THM
Because the 1.5 mM CA + 1.5 mM CAR or THM treatments for 10 min at 22°C and for 1– 10 min at 37°C showed the greatest synergistic antifungal effects (Fig. 1 and 2), the composites were stored for 14, 30, and 60 days to determine the stability of their antifungal efficacy and assess any changes in pH. After storage for 0–60 days, the pH values were maintained at 4.0 (data not shown). The 1.5 mM CA and 1.5 mM CAR or THM composites stored for 14 days eradicated all fungal cells in an inoculum (> 6.9 log CFU/ml reduction) treated for 10 min at 22°C and for 1–10 min at 37°C (Table 1). When fungal suspensions were treated at 22°C for 10 min with the composites stored for 30 and 60 days, 5.1–5.2 log CFU/ml reductions of fungal populations were observed. Similarly, treatments with CA + CAR or THM stored for 60 days at 37°C for 1 min reduced the population of C. albicans by 5.7–5.9 log CFU/ml. Treatments for 10 min at 37°>C with the composites showed the highest fungicidal activity observed (> 6.8 log reduction).
Effects of CA, CAR or THM alone or in combination on cell membrane integrity
Based on the treatment conditions that showed the most synergistic effects at 22 or 37°C in the previous experiments, C. albicans was treated with 1.5 mM CA, 1.5 mM CAR or THM or a combination thereof (1.5 mM CA + 1.5 mM CAR or THM) at 22°C for 10 min and at 37°C for 1–10 min. The cells were subsequently stained with PI to analyze the membrane integrity of the treated cells by flow cytometry (Fig. 3). The proportions of damaged cells exposed to 1.5 mM CA alone were higher than those exposed to 1.5 mM CAR or THM under the same treatment times and temperatures. For example, the percentages of permeabilized fungal cells induced by 1.5 mM CA were 15.7% and 18.7%–36.5% after treatment for 10 min at 22°C and for 1–10 min at 37°C, respectively (Fig. 3a). However, Fig. 3b and 3c show that treatments with 1.5 mM CAR and THM alone had lower effects on cells when tested at each treatment condition (10 min at 22°C, 1.8–2.0%; 1–10 min at 37°C, 3.0–7.6%). Additionally, the percentages of permeabilized cells treated by each antifungal agent alone tended to increase with treatment temperature. When C. albicans cells were treated with the same compound for the same exposure duration (10 min), permeabilization was higher at 37°C [36.5% after 10 min treatment at 37°C with 1.5 mM CA (Fig. 3a); 6.9–7.6% after 10 min treatment at 37°C with 1.5 mM CAR or THM (Fig. 3b and 3c)] than that at 22°C [15.7% after 10 min treatment at 22°C with 1.5 mM CA (Fig. 3a); 1.8–2.0% after 10 min treatment at 22°C with 1.5 mM CAR or THM (Fig. 3b and 3c)]. The combined treatments resulted in 95.0–99.8% of fungal cells being permeabilized when treated at 22°C for 10 min and at 37°C for 1–10 min (Fig. 3d and 3e).
Effects of CA, CAR or THM alone or in combination on efflux pump integrity
According to the treatment conditions that resulted in the most marked fungicidal effects at 22 or 37°C, efflux pump activities after treatments with 1.5 mM CA, CAR or THM or their combinations thereof for 10 min at 22°C or for 1–10 min at 37°C were evaluated using flow cytometry by measuring the efflux of Nile red dye (Fig. 4). The results showed that individual treatments of 1.5 mM CA, CAR or THM considerably inhibited efflux pump activity [15.4–31.3% treated with 1.5 mM CA, (Fig. 4a); 14.4–29.6% of cells with 1.5 mM CAR or THM (Fig. 4b and 4c)]. Nile red accumulated at higher levels in C. albicans cells treated with CA, CAR or THM alone at 37°C for 10 min (25.8–31.3%) than those treated at 22°C (14.4–15.4%). Indeed, efflux pump function gradually decreased with increasing temperature. For the combined treatments, efflux pump activity was completely inhibited in 83.1–99.1% of cells (Fig. 4d and 4e).
Examination of the effect of pH on the antifungal efficacy of combined treatments
Because CA is a weak acid, the pH values of combined solutions were determined relative to CA (1.5 mM CA has a pH of 4.0). To measure the effects of pH on the synergistic fungicidal effects, fungal cells were exposed to 0.0018 mM HCl (pH 4.0) and 0.0018 mM HCl + 1.5 mM EOs (pH 4.0) for 10 min at 22 and 37°C (Table 2). Individual treatments of 0.0018 mM HCl at 22 and 37°C did not show any fungicidal effects (< 0.1 log CFU/ml reduction), while 1.5 mM CA alone caused a less than 0.4 log reduction in a C. albicans suspension. The combined treatments of 0.0018 mM HCl with 1.5 mM CAR or THM at 22 and 37°C resulted in less than 0.5 log reductions, whereas the combined treatments of 1.5 mM CA + 1.5 mM CAR or THM for 10 min at 22 and 37°C inactivated all fungal cells (> 6.7 log CFU/ml reductions).
Damages to C. albicans membranes and efflux pumps induced by treatments with HCl (pH 4.0) alone or in combination with 1.5 mM CAR or THM (pH 4.0) for 10 min at 22oC and 37°C were also examined (Fig. 5 and 6). Unlike the combined treatments with 1.5 mM CA + 1.5 mM CAR or THM (Fig. 3 and 4), exposure to HCl + CAR or THM showed negligibly damaged membranes and efflux pumps. The percentage of C. albicans cells with membranes that were damaged by HCl alone and by HCl + CAR or THM was only 2.1–4.2% (Fig. 5c and 5d) and 2.7–6.6% (Fig. 5e to h), respectively. The HCl treatments only slightly inhibited the efflux pump activity of C. albicans [1.4–3.7% of cells (Fig. 6c and 6d)], whereas the combined treatments of HCl and CAR or THM blocked transporter pump activity in 13.1–28.0% of cells (Fig. 6e to h).
Discussion
The results of the present study revealed that the optimized combinations of CA and CAR or THM at low concentrations had synergistic fungicidal effects within short durations against C. albicans. These combinations of naturally-derived agents may be promising alternatives to synthetic compounds used to control pathogenic fungi. We analyzed the changes in membrane integrity and efflux pump activity of treated C. albicans cells using flow cytometry. The synergistic mechanism may involve the increased entry of the antifungals into the cells due to the disruption of the fungal membrane, primarily caused by CA, and by inhibition of the efflux of antifungals by CA, CAR or THM. These novel findings can potentially lead to the development of a new antifungal approach to cope with human fungal infection and environmental fungal contamination.
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