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Erfaninia M, Alizadeh F. Killing Kinetics of Carvacrol against Fluconazole-Susceptible and -Resistant Isolates of Candida tropicalis. mljgoums 2023; 17 (1) :27-34
URL: http://mlj.goums.ac.ir/article-1-1381-en.html
URL: http://mlj.goums.ac.ir/article-1-1381-en.html
1- Department of Microbiology, Yasooj Branch, Islamic Azad University, Yasooj, Iran
2- Department of Microbiology, Yasooj Branch, Islamic Azad University, Yasooj, Iran ,mnalizadeh@yahoo.com
2- Department of Microbiology, Yasooj Branch, Islamic Azad University, Yasooj, Iran ,
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INTRODUCTION
Candida tropicalis has emerged as an important opportunistic fungal pathogen and the second most common cause of candidiasis. This Candida species has been also recognized as a potent biofilm producer, which is highly adherent to most human tissues and cells (1, 2). During biofilm formation, planktonic cells attach to a surface, resulting in the formation of a complex network of different layers of polymorphic cells, including hyphal cells, pseudohyphal cells, and round planktonic cells, which are enclosed in an extracellular polymeric matrix (3).
The transition from yeast to hyphal growth and involved signaling pathways are very well-established for Candida albicans. The environmental signals together with signaling pathway-specific transcription factors regulate the expression of target genes involved in filamentation. However, certain C. albicans morphological regulatory functions are evolutionarily conserved in C. tropicalis (4, 5).
Misuse and overuse of antifungals in the clinical and industrial sectors have led to the emergence of antifungal-resistant C. tropicalis strains, which could be life-threatening, particularly in vulnerable groups such as immunocompromised patients (1, 3, 6). Antifungal agents such as azoles, polyenes, and echinocandins are currently considered the last resort antibiotics for treating candidiasis (7). Resistance to these antifungals has been reported in C. tropicalis due to the increased reliance upon these lines of antibiotics (6). Azole-resistant Candida species produce efflux pumps, which can reduce the amount of intracellular drug content. Other antibiotic resistance mechanisms such as the alteration of the concentration or structure of antifungal target sites as well as alteration in the sterol composition of the fungal membrane are sufficient to confer azole resistance among Candida species (8, 9). Resistance to fluconazole has been commonly reported amongst clinical isolates of C. tropicalis (6). The point mutation Y132F has been reported in a single fluconazole-resistant C. tropicalis, which impacts susceptibility to fluconazole (11).
In order to mitigate issues caused by antifungal resistance, considerable effort has been directed toward the development of novel antifungal agents. Natural products have provided key scaffolds for drug development due to their relative diversity (12). Several studies have shown that natural products possess tremendous potential as an antifungal resource (13-16). A strong body of evidence suggests that essential oils can disrupt the structural integrity of the membrane and affect cell metabolism, eventually causing cell death (17-19). Despite the great potential of carvacrol-rich essential oils against C. tropicalis, no study has yet investigated the inhibitory effects of such essential oils on hyphal growth. Carvacrol, also known as cymophenol (2-methyl-5-propan-2-ylphenol), is a monoterpene phenol found in several aromatic plants. This compound has strong antimicrobial, acetylcholinesterase inhibitory, anti-tumor, anti-inflammatory, anti-mutagenic, anti-genotoxic, anti-elastase, anti-spasmodic, anti-hepatotoxic, anti-platelet, analgesic, angiogenic, insecticidal, and hepatoprotective activities (19, 20). The antifungal activity of carvacrol against C. tropicalis has been well demonstrated (13-16). Data obtained from time-kill kinetics have provided critical information regarding the effects of antimicrobials over time. Appiah et al. analyzed the time-kill kinetics of antifungal agents against C. albicans (21).
This study aimed to investigate the antifungal activity of carvacrol on planktonic and hyphal cells of clinical isolates of fluconazole-susceptible and -resistant C. tropicalis, with a focus on the time-kill kinetics.
MATERIALS AND METHODS
The study protocol was approved by the ethics committee of the Islamic Azad University of Yasuj, Iran (ethical code: IR. IAU.YASUJ.REC.1395.11). Fluconazole and carvacrol were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). A stock solution of carvacrol was made by dissolving carvacrol in dimethyl sulfoxide (DMSO). Ten clinical isolates of C. tropicalis SN-1, SN-2, IAU-1-8, and C. tropicalis ATCC 750 were used in this study. Clinical isolates of C. tropicalis SN-1 and SN-2 from the vagina of patients with recurrent vulvovaginal candidiasis were kindly provided by the Microbiology Laboratory of Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Iran. Clinical isolates of C. tropicalis IAU-1-8 were taken from diabetics and patients with liver disease. The isolates of C. tropicalis were identified based on the morphological, biochemical, and molecular characteristics such as germ tube production, colony appearance on sabouraud dextrose agar (SDA; Q-Lab, England), CHROMagar Candida (CHROMagar Company, France), urease test, polymerase chain reaction (PCR) using primers of ITS1 (5ʹ-TCC GTA GGT GAA CCT GCG G-3ʹ) and ITS4 (5ʹ-TCC TCC GCT TAT TGA TAT GC-3ʹ), and sequencing (22).
Fluconazole-resistant C. tropicalis isolates were identified by the disk diffusion (M44-A2) and broth microdilution (M27-A3 and M27-S4) methods according to the Clinical and Laboratory Standards Institute recommendations. The results were interpreted by the World Health Organization's WHONET software (23).
The minimum inhibitory concentration (MIC) of fluconazole and carvacrol against the isolates was determined by broth microdilution assay in accordance with CLSI M27-A3 as described previously (22). Two-fold dilutions were performed in each microplate well to a final volume of 50 μl of carvacrol and 50 μl of clinical isolates of fluconazole-susceptible and -resistant C. tropicalis suspension at approximately 0.5–5 × 103 CFU/ml. The MIC50 and MIC90 were defined as the lowest concentration of the drugs that inhibited 50% and 90% of the isolates, respectively. Wells with no antifungal agents and those containing medium alone were used as the positive and negative controls, respectively.
A standard inoculum of 1 × 106 CFU/ml was treated with the MIC90 of carvacrol in the time-kill assay through viable plate counting. Samples were incubated at 35 °C with shaking at 200 rpm. Immediately after the inoculation, viable counting was performed at different times (i.e., 0, 2, 4, 6, 8, 12, 24, and 48 hours). Next, 100 μl of the samples were subjected to 100-fold dilution with 0.85% (w/v) saline and then plated onto SDA and incubated at 35 °C for 24 hours. In the experimental groups, fluconazole-treated (positive control), untreated (negative control), and DMSO without antifungal agents were also included (16).
The inhibitory effect of carvacrol on hyphal growth was assessed according to the method described by Khodavandi et al. (24). In brief, a starting cell density of 1 × 106 CFU/ml was treated with carvacrol at concentrations equal to 1/4× MIC, 1/2× MIC, 1× MIC, and 2× MIC values. The samples were dispensed into the cell culture plates and incubated at 35 °C for 90 minutes. Subsequently, the mixture was incubated at 35 °C for 16 hours with shaking at 200 rpm. Finally, the samples were washed with phosphate buffer saline and observed via a light field microscope (Nikon, Japan).
All experiments were done in triplicate. The normality of data was assessed using the Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to compare the normally distributed data. The nonparametric tests including the Kruskal-Wallis test followed by Bonferroni’s post hoc test were used when appropriate. A p-value of ≤0.05 was considered statistically significant. Statistical analysis was performed in IBM SPSS Statistics (version 24, SPSS Inc., Chicago, IL, USA).
Table 1- Morphological, biochemical, and molecular characterization of C. tropicalis isolates
.PNG)
Table 2-The fluconazole-susceptibility results in clinical isolates of C. tropicalis using the disk diffusion and broth microdilution assays
R: resistant; S: sensitive; I: Intermediate; C.I.: Confidence Interval; Geom. Mean: Geometric Mean
RESULTS
Morphological, biochemical, and molecular characteristics of C. tropicalis isolates are shown in table 1. All clinical isolates of C. tropicalis produced metallic blue colonies on CHROMagar Candida, and other identification tests confirmed the isolates as C. tropicalis. The PCR process using the universal primer pairs generated a single PCR product of 524 bp. The nucleotide sequence of C. tropicalis isolates was analyzed using the Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequence displayed 100% identity with the respective gene sequence.
Based on the results, 50% of the clinical isolates of C. tropicalis were resistant to fluconazole (Table 2).
The MIC of carvacrol on planktonic cells was determined using the broth microdilution assay. According to the results, the MIC90 and MIC50 values of carvacrol against planktonic cells were 25.00-300.00 µg/ml and 12.50-100.00 µg/ml, respectively. However, carvacrol showed more potency in vitro with MIC90 and MIC50 values ranging from 25.00 to 150.00 μg/ml and 12.50 to 50.00 μg/ml, respectively against fluconazole-susceptible isolates (Table 3).
The killing kinetics of carvacrol was evaluated at a concentration equal to the MIC value by performing the time-kill analysis. A more complete killing profile of fluconazole-susceptible and -resistant C. tropicalis isolates was observed after 24 hours of treatment with carvacrol (Figure 1).
The results of one-way ANOVA showed significant differences amongst carvacrol-treated clinical isolates of C. tropicalis at different times. At 0-hour, there was no significant difference between the carvacrol- and fluconazole-treated C. tropicalis isolates and the control groups (Figure 1). After 2 to 48 hours, the number of fluconazole-susceptible isolates decreased significantly compared with the control groups (p<0.05). Of note, after 8 and 12 hours, a significant difference was observed between fluconazole-susceptible and -resistant C. tropicalis isolates (p<0.05) (Figures 1A, 1B). The number of fluconazole-resistant isolates decreased only in treatment with carvacrol after 2-48 hours (Figure 1C).
Both planktonic and hyphal cells of fluconazole-susceptible and -resistant C. tropicalis isolates reduced at different MICs after 16 hours (Figure 2).
.PNG)
Figure 1- Time-kill assay of (A) C. tropicalis ATCC 750, (B) clinical isolates of fluconazole-susceptible (SN-1), and (C) fluconazole-resistant (IAU-2) C. tropicalis isolates treated with MIC90 of carvacrol at different times (i.e., 0, 2, 4, 6, 8, 12, 24, and 48 hours). Positive control represents C. tropicalis treated with MIC of fluconazole. Data are shown as the mean and standard error of the mean of three independent experiments.
.PNG)
Figure 2- Light field microscopy of the inhibitory effect of different MICs of carvacrol on hyphal cells of clinical isolates of fluconazole-susceptible (SN-1) and -resistant (IAU-2) C. tropicalis after 16 hours. C. tropicalis ATCC 750 was used as a reference control. The images were taken under × 40 magnification. Bar = 50 µm.
Table 3- MIC90 and MIC50 of carvacrol and fluconazole against clinical isolates of C. tropicalis
DISCUSSION
The emergence and spread of antifungal resistance by opportunistic fungi have become a major challenge in healthcare settings, impeding treatment progress and causing substantial morbidity and mortality. This issue is further complicated by the emergence of azole-resistant fungal infections (25). Thus, finding plant-based, natural antifungals is of great importance for finding alternative solutions to the increasing resistance problem. The bioactivity of carvacrol, a natural phenolic monoterpenoid, has been documented in many studies (13-16, 19, 20). Thus, this study was carried out to investigate the killing kinetics of carvacrol against hyphal growth of C. tropicalis isolates. We first determined the bioactivity of carvacrol against planktonic cells of fluconazole-susceptible and -resistant C. tropicalis isolates and found that planktonic cells of the fluconazole-susceptible isolates were more sensitive to carvacrol. From the presented antifungal assay, small MIC values are noticed in clinical fluconazole-resistant isolates of C. tropicalis, indicating the antifungal potential of carvacrol. In a study conducted by Khodavandi et al. (24), carvacrol showed antifungal activity against multidrug-resistant C. albicans.
To further elucidate the overall effect of carvacrol on planktonic cells, the killing kinetics of both clinical isolates of fluconazole-susceptible and -resistant C. tropicalis isolates were determined via time-kill analysis. The time-kill profile showed a reduction in the number of planktonic cells, 2-48 hours after treatment with carvacrol, while a more complete killing profile was observed after 24 hours. This indicates the fungicidal potential of carvacrol against planktonic cells of fluconazole-susceptible and -resistant C. tropicalis isolates as previously proposed in other studies (24, 26).
Carvacrol exposure also caused a reduction in hyphae formation. Both clinical isolates of fluconazole-susceptible and -resistant shared a similar reduction of hyphae and planktonic cells. Studies by Manoharan et al. (27) and Raut et al. (28) also showed that essential oil components such as α-longipinene, linalool, and carvacrol can inhibit planktonic cells, hyphae, and biofilm of C. albicans. In line with our results, Tobaldini-Valerio et al. showed the antifungal and antibiofilm activities of propolis against Candida species (29). These authors also observed that the time-kill assay results were in accordance with the antifungal susceptibility results. Moreover, Lemos et al. (30) reported the reduction of planktonic and biofilm populations of Streptococcus mutans in different environmental conditions.
To the best of our knowledge, this study is the first to evaluate the killing kinetics of carvacrol on fluconazole-susceptible and -resistant isolates of C. tropicalis. The cellular and molecular mechanisms involved in the antifungal activity of carvacrol are not completely clear, but inhibition of ergosterol biosynthesis, the impairment of membrane integrity, endoplasmic reticulum stress, the unfolded protein response, and perturbing H+ and Ca2+ ion homeostasis have been proposed as possible mechanisms (26, 30). Niu et al. (31) found that carvacrol could trigger C. albicans apoptosis associated with Ca2+/calcineurin pathway.
The complex structure of the fungal cell is a limitation of the present study when assessing the antifungal activity of carvacrol.
CONCLUSION
This study investigated the antifungal effects of carvacrol against planktonic and hyphal cells of fluconazole-susceptible and -resistant C. tropicalis isolates. The findings revealed that carvacrol exhibits inhibitory effects on the planktonic and hyphal cells of fluconazole-susceptible and -resistant C. tropicalis isolates. Therefore, the antifungal potential of carvacrol as a natural antifungal could be further exploited for the treatment of resistant C. tropicalis infections.
ACKNOWLEDGEMENTS
The authors acknowledge Dr. Sadegh Nouripour-Sisakht from the Cellular and Molecular Research Center of Yasuj University of Medical Sciences for kindly providing the clinical isolates of C. tropicalis. The authors wish to thank the Islamic Azad University of Yasuj for providing the equipment to accomplish this investigation. The results presented in this study have been derived from a Master’s thesis approved by the Islamic Azad University of Yasuj, Iran.
DECLARATIONS
FUNDING
The authors received no financial support for the research, authorship, and/or publication of this article.
Ethics approvals and consent to participate
The study protocol was approved by the ethics committee of the Islamic Azad University of Yasuj, Iran (ethical code: IR. IAU.YASUJ.REC.1395.11).
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest regarding the publication of this article.
Candida tropicalis has emerged as an important opportunistic fungal pathogen and the second most common cause of candidiasis. This Candida species has been also recognized as a potent biofilm producer, which is highly adherent to most human tissues and cells (1, 2). During biofilm formation, planktonic cells attach to a surface, resulting in the formation of a complex network of different layers of polymorphic cells, including hyphal cells, pseudohyphal cells, and round planktonic cells, which are enclosed in an extracellular polymeric matrix (3).
The transition from yeast to hyphal growth and involved signaling pathways are very well-established for Candida albicans. The environmental signals together with signaling pathway-specific transcription factors regulate the expression of target genes involved in filamentation. However, certain C. albicans morphological regulatory functions are evolutionarily conserved in C. tropicalis (4, 5).
Misuse and overuse of antifungals in the clinical and industrial sectors have led to the emergence of antifungal-resistant C. tropicalis strains, which could be life-threatening, particularly in vulnerable groups such as immunocompromised patients (1, 3, 6). Antifungal agents such as azoles, polyenes, and echinocandins are currently considered the last resort antibiotics for treating candidiasis (7). Resistance to these antifungals has been reported in C. tropicalis due to the increased reliance upon these lines of antibiotics (6). Azole-resistant Candida species produce efflux pumps, which can reduce the amount of intracellular drug content. Other antibiotic resistance mechanisms such as the alteration of the concentration or structure of antifungal target sites as well as alteration in the sterol composition of the fungal membrane are sufficient to confer azole resistance among Candida species (8, 9). Resistance to fluconazole has been commonly reported amongst clinical isolates of C. tropicalis (6). The point mutation Y132F has been reported in a single fluconazole-resistant C. tropicalis, which impacts susceptibility to fluconazole (11).
In order to mitigate issues caused by antifungal resistance, considerable effort has been directed toward the development of novel antifungal agents. Natural products have provided key scaffolds for drug development due to their relative diversity (12). Several studies have shown that natural products possess tremendous potential as an antifungal resource (13-16). A strong body of evidence suggests that essential oils can disrupt the structural integrity of the membrane and affect cell metabolism, eventually causing cell death (17-19). Despite the great potential of carvacrol-rich essential oils against C. tropicalis, no study has yet investigated the inhibitory effects of such essential oils on hyphal growth. Carvacrol, also known as cymophenol (2-methyl-5-propan-2-ylphenol), is a monoterpene phenol found in several aromatic plants. This compound has strong antimicrobial, acetylcholinesterase inhibitory, anti-tumor, anti-inflammatory, anti-mutagenic, anti-genotoxic, anti-elastase, anti-spasmodic, anti-hepatotoxic, anti-platelet, analgesic, angiogenic, insecticidal, and hepatoprotective activities (19, 20). The antifungal activity of carvacrol against C. tropicalis has been well demonstrated (13-16). Data obtained from time-kill kinetics have provided critical information regarding the effects of antimicrobials over time. Appiah et al. analyzed the time-kill kinetics of antifungal agents against C. albicans (21).
This study aimed to investigate the antifungal activity of carvacrol on planktonic and hyphal cells of clinical isolates of fluconazole-susceptible and -resistant C. tropicalis, with a focus on the time-kill kinetics.
MATERIALS AND METHODS
The study protocol was approved by the ethics committee of the Islamic Azad University of Yasuj, Iran (ethical code: IR. IAU.YASUJ.REC.1395.11). Fluconazole and carvacrol were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). A stock solution of carvacrol was made by dissolving carvacrol in dimethyl sulfoxide (DMSO). Ten clinical isolates of C. tropicalis SN-1, SN-2, IAU-1-8, and C. tropicalis ATCC 750 were used in this study. Clinical isolates of C. tropicalis SN-1 and SN-2 from the vagina of patients with recurrent vulvovaginal candidiasis were kindly provided by the Microbiology Laboratory of Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Iran. Clinical isolates of C. tropicalis IAU-1-8 were taken from diabetics and patients with liver disease. The isolates of C. tropicalis were identified based on the morphological, biochemical, and molecular characteristics such as germ tube production, colony appearance on sabouraud dextrose agar (SDA; Q-Lab, England), CHROMagar Candida (CHROMagar Company, France), urease test, polymerase chain reaction (PCR) using primers of ITS1 (5ʹ-TCC GTA GGT GAA CCT GCG G-3ʹ) and ITS4 (5ʹ-TCC TCC GCT TAT TGA TAT GC-3ʹ), and sequencing (22).
Fluconazole-resistant C. tropicalis isolates were identified by the disk diffusion (M44-A2) and broth microdilution (M27-A3 and M27-S4) methods according to the Clinical and Laboratory Standards Institute recommendations. The results were interpreted by the World Health Organization's WHONET software (23).
The minimum inhibitory concentration (MIC) of fluconazole and carvacrol against the isolates was determined by broth microdilution assay in accordance with CLSI M27-A3 as described previously (22). Two-fold dilutions were performed in each microplate well to a final volume of 50 μl of carvacrol and 50 μl of clinical isolates of fluconazole-susceptible and -resistant C. tropicalis suspension at approximately 0.5–5 × 103 CFU/ml. The MIC50 and MIC90 were defined as the lowest concentration of the drugs that inhibited 50% and 90% of the isolates, respectively. Wells with no antifungal agents and those containing medium alone were used as the positive and negative controls, respectively.
A standard inoculum of 1 × 106 CFU/ml was treated with the MIC90 of carvacrol in the time-kill assay through viable plate counting. Samples were incubated at 35 °C with shaking at 200 rpm. Immediately after the inoculation, viable counting was performed at different times (i.e., 0, 2, 4, 6, 8, 12, 24, and 48 hours). Next, 100 μl of the samples were subjected to 100-fold dilution with 0.85% (w/v) saline and then plated onto SDA and incubated at 35 °C for 24 hours. In the experimental groups, fluconazole-treated (positive control), untreated (negative control), and DMSO without antifungal agents were also included (16).
The inhibitory effect of carvacrol on hyphal growth was assessed according to the method described by Khodavandi et al. (24). In brief, a starting cell density of 1 × 106 CFU/ml was treated with carvacrol at concentrations equal to 1/4× MIC, 1/2× MIC, 1× MIC, and 2× MIC values. The samples were dispensed into the cell culture plates and incubated at 35 °C for 90 minutes. Subsequently, the mixture was incubated at 35 °C for 16 hours with shaking at 200 rpm. Finally, the samples were washed with phosphate buffer saline and observed via a light field microscope (Nikon, Japan).
All experiments were done in triplicate. The normality of data was assessed using the Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to compare the normally distributed data. The nonparametric tests including the Kruskal-Wallis test followed by Bonferroni’s post hoc test were used when appropriate. A p-value of ≤0.05 was considered statistically significant. Statistical analysis was performed in IBM SPSS Statistics (version 24, SPSS Inc., Chicago, IL, USA).
Table 1- Morphological, biochemical, and molecular characterization of C. tropicalis isolates
Table 2-The fluconazole-susceptibility results in clinical isolates of C. tropicalis using the disk diffusion and broth microdilution assays
| CLSI disk diffusion assay | ||||||||||
| Antibiotic name | Antibiotic class | Code | Breakpoints | Number | %R | %I | %S | %R 95%C.I. | ||
| Fluconazole | Antifungals | FLU | 15 - 18 | 10 | 50 | 0 | 50 | 20.1-79.9 | ||
| CLSI broth microdilution assay | ||||||||||
| Antibiotic name | Antibiotic class | Code | Breakpoints | Number | %R | %I | %S | %R 95%C.I. | Geom. Mean | MIC Range |
| Fluconazole | Antifungals | FLU | S<=2 R>=8 | 10 | 50 | 0 | 50 | 20.1-79.9 | 9.849 | 2 - 64 |
RESULTS
Morphological, biochemical, and molecular characteristics of C. tropicalis isolates are shown in table 1. All clinical isolates of C. tropicalis produced metallic blue colonies on CHROMagar Candida, and other identification tests confirmed the isolates as C. tropicalis. The PCR process using the universal primer pairs generated a single PCR product of 524 bp. The nucleotide sequence of C. tropicalis isolates was analyzed using the Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequence displayed 100% identity with the respective gene sequence.
Based on the results, 50% of the clinical isolates of C. tropicalis were resistant to fluconazole (Table 2).
The MIC of carvacrol on planktonic cells was determined using the broth microdilution assay. According to the results, the MIC90 and MIC50 values of carvacrol against planktonic cells were 25.00-300.00 µg/ml and 12.50-100.00 µg/ml, respectively. However, carvacrol showed more potency in vitro with MIC90 and MIC50 values ranging from 25.00 to 150.00 μg/ml and 12.50 to 50.00 μg/ml, respectively against fluconazole-susceptible isolates (Table 3).
The killing kinetics of carvacrol was evaluated at a concentration equal to the MIC value by performing the time-kill analysis. A more complete killing profile of fluconazole-susceptible and -resistant C. tropicalis isolates was observed after 24 hours of treatment with carvacrol (Figure 1).
The results of one-way ANOVA showed significant differences amongst carvacrol-treated clinical isolates of C. tropicalis at different times. At 0-hour, there was no significant difference between the carvacrol- and fluconazole-treated C. tropicalis isolates and the control groups (Figure 1). After 2 to 48 hours, the number of fluconazole-susceptible isolates decreased significantly compared with the control groups (p<0.05). Of note, after 8 and 12 hours, a significant difference was observed between fluconazole-susceptible and -resistant C. tropicalis isolates (p<0.05) (Figures 1A, 1B). The number of fluconazole-resistant isolates decreased only in treatment with carvacrol after 2-48 hours (Figure 1C).
Both planktonic and hyphal cells of fluconazole-susceptible and -resistant C. tropicalis isolates reduced at different MICs after 16 hours (Figure 2).
Figure 1- Time-kill assay of (A) C. tropicalis ATCC 750, (B) clinical isolates of fluconazole-susceptible (SN-1), and (C) fluconazole-resistant (IAU-2) C. tropicalis isolates treated with MIC90 of carvacrol at different times (i.e., 0, 2, 4, 6, 8, 12, 24, and 48 hours). Positive control represents C. tropicalis treated with MIC of fluconazole. Data are shown as the mean and standard error of the mean of three independent experiments.
Figure 2- Light field microscopy of the inhibitory effect of different MICs of carvacrol on hyphal cells of clinical isolates of fluconazole-susceptible (SN-1) and -resistant (IAU-2) C. tropicalis after 16 hours. C. tropicalis ATCC 750 was used as a reference control. The images were taken under × 40 magnification. Bar = 50 µm.
Table 3- MIC90 and MIC50 of carvacrol and fluconazole against clinical isolates of C. tropicalis
| Isolates /Antifungal | Carvacrol | Fluconazole | |||||||||||||
| MIC90 rang (µg/ml) | Geom. Mean | 95%C.I. | MIC50 rang (µg/ml) | Geom. Mean | 95%C.I. | MIC90 rang (µg/ml) | Geom. Mean | 95%C.I. | MIC50 rang (µg/ml) | Geom. Mean | 95%C.I. | ||||
| C. tropicalis ATCC 750 | 50-75 | 57.23 | 58.3 ± 16.3 (42 to 74.6) |
25 | 25 | 25 ± 0 (25 to 25) |
2 | 2 | 2 ± 0 (2 to 2) |
0.25 | 0.25 | 0.25 ± 0 (0.25 to 0.25) |
|||
| IAU-1 | 100-125 | 107.72 | 108 ± 16.3 (91.7 to 124) |
25-50 | 31.5 | 33.3 ± 16.3 (17 to 49.6) |
2 | 2 | 2 ± 0 (2 to 2) |
0.25 | 0.25 | 0.25 ± 0 (0.25 to 0.25) |
|||
| IAU-2 | 150 | 150 | 150 ± 0 (150 to 150) |
50 | 50 | 50 ± 0 (50 to 50) |
64 | 64 | 64 ± 0 (64 to 64) |
16 | 20.16 | 21.3 ± 10.5 (10.8 to 31.8) |
|||
| IAU-3 | 25 | 25 | 25 ± 0 (25 to 25) |
12.5-25 | 15.75 | 16.7 ± 8.17 (8.53 to 24.9) |
2 | 2 | 2 ± 0 (2 to 2) |
0.25 | 0.25 | 0.25 ± 0 (0.25 to 0.25) |
|||
| IAU-4 | 100-125 | 107.72 | 108 ± 16.3 (91.7 to 124) |
25 | 25 | 25 ± 0 (25 to 25) |
32 | 32 | 32 ± 0 (32 to 32) |
16 | 16 | 16 ± 0 (16 to 16) |
|||
| IAU-5 | 50 | 50 | 50 ± 0 (50 to 50) |
12.5 | 12.5 | 12.5 ± 0 (12.5 to 12.5) |
2 | 2 | 2 ± 0 (2 to 2) |
0.25 | 0.25 | 0.25 ± 0 (0.25 to 0.25) |
|||
| IAU-6 | 150 | 150 | 150 ± 0 (150 to 150) |
25 | 25 | 25 ± 0 (25 to 25) |
32 | 32 | 32 ± 0 (32 to 32) |
8 | 10.08 | 10.7 ± 5.23 (5.47 to 15.9) |
|||
| IAU-7 | 300-325 | 316.44 | 308 ± 16.3 (292 to 324) |
100 | 100 | 100 ± 0 (100 to 100) |
64 | 64 | 64 ± 0 (64 to 64) |
16 | 20.16 | 21.3 ± 10.5 (10.8 to 31.8) |
|||
| IAU-8 | 300-325 | 316.44 | 308 ± 16.3 (292 to 324) |
50 | 50 | 50 ± 0 (50 to 50) |
64 | 64 | 64 ± 0 (64 to 64) |
32 | 32 | 32 ± 0 (32 to 32) |
|||
| SN-1 | 100 | 100 | 100 ± 0 (100 to 100) |
25-50 | 31.5 | 33.3 ± 16.3 (17 to 49.6) |
2 | 2 | 2 ± 0 (2 to 2) |
0.25 | 0.32 | 0.333 ± 0.163 (0.17 to 0.496) |
|||
| SN-2 | 150 | 150 | 150 ± 0 (150 to 150) |
50 | 50 | 50 ± 0 (50 to 50) |
2 | 2 | 2 ± 0 (2 to 2) |
0.25 | 0.25 | 0.25 ± 0 (0.25 to 0.25) |
|||
DISCUSSION
The emergence and spread of antifungal resistance by opportunistic fungi have become a major challenge in healthcare settings, impeding treatment progress and causing substantial morbidity and mortality. This issue is further complicated by the emergence of azole-resistant fungal infections (25). Thus, finding plant-based, natural antifungals is of great importance for finding alternative solutions to the increasing resistance problem. The bioactivity of carvacrol, a natural phenolic monoterpenoid, has been documented in many studies (13-16, 19, 20). Thus, this study was carried out to investigate the killing kinetics of carvacrol against hyphal growth of C. tropicalis isolates. We first determined the bioactivity of carvacrol against planktonic cells of fluconazole-susceptible and -resistant C. tropicalis isolates and found that planktonic cells of the fluconazole-susceptible isolates were more sensitive to carvacrol. From the presented antifungal assay, small MIC values are noticed in clinical fluconazole-resistant isolates of C. tropicalis, indicating the antifungal potential of carvacrol. In a study conducted by Khodavandi et al. (24), carvacrol showed antifungal activity against multidrug-resistant C. albicans.
To further elucidate the overall effect of carvacrol on planktonic cells, the killing kinetics of both clinical isolates of fluconazole-susceptible and -resistant C. tropicalis isolates were determined via time-kill analysis. The time-kill profile showed a reduction in the number of planktonic cells, 2-48 hours after treatment with carvacrol, while a more complete killing profile was observed after 24 hours. This indicates the fungicidal potential of carvacrol against planktonic cells of fluconazole-susceptible and -resistant C. tropicalis isolates as previously proposed in other studies (24, 26).
Carvacrol exposure also caused a reduction in hyphae formation. Both clinical isolates of fluconazole-susceptible and -resistant shared a similar reduction of hyphae and planktonic cells. Studies by Manoharan et al. (27) and Raut et al. (28) also showed that essential oil components such as α-longipinene, linalool, and carvacrol can inhibit planktonic cells, hyphae, and biofilm of C. albicans. In line with our results, Tobaldini-Valerio et al. showed the antifungal and antibiofilm activities of propolis against Candida species (29). These authors also observed that the time-kill assay results were in accordance with the antifungal susceptibility results. Moreover, Lemos et al. (30) reported the reduction of planktonic and biofilm populations of Streptococcus mutans in different environmental conditions.
To the best of our knowledge, this study is the first to evaluate the killing kinetics of carvacrol on fluconazole-susceptible and -resistant isolates of C. tropicalis. The cellular and molecular mechanisms involved in the antifungal activity of carvacrol are not completely clear, but inhibition of ergosterol biosynthesis, the impairment of membrane integrity, endoplasmic reticulum stress, the unfolded protein response, and perturbing H+ and Ca2+ ion homeostasis have been proposed as possible mechanisms (26, 30). Niu et al. (31) found that carvacrol could trigger C. albicans apoptosis associated with Ca2+/calcineurin pathway.
The complex structure of the fungal cell is a limitation of the present study when assessing the antifungal activity of carvacrol.
CONCLUSION
This study investigated the antifungal effects of carvacrol against planktonic and hyphal cells of fluconazole-susceptible and -resistant C. tropicalis isolates. The findings revealed that carvacrol exhibits inhibitory effects on the planktonic and hyphal cells of fluconazole-susceptible and -resistant C. tropicalis isolates. Therefore, the antifungal potential of carvacrol as a natural antifungal could be further exploited for the treatment of resistant C. tropicalis infections.
ACKNOWLEDGEMENTS
The authors acknowledge Dr. Sadegh Nouripour-Sisakht from the Cellular and Molecular Research Center of Yasuj University of Medical Sciences for kindly providing the clinical isolates of C. tropicalis. The authors wish to thank the Islamic Azad University of Yasuj for providing the equipment to accomplish this investigation. The results presented in this study have been derived from a Master’s thesis approved by the Islamic Azad University of Yasuj, Iran.
DECLARATIONS
FUNDING
The authors received no financial support for the research, authorship, and/or publication of this article.
Ethics approvals and consent to participate
The study protocol was approved by the ethics committee of the Islamic Azad University of Yasuj, Iran (ethical code: IR. IAU.YASUJ.REC.1395.11).
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest regarding the publication of this article.
Research Article: Research Article |
Subject:
Microbiology
Received: 2021/04/14 | Accepted: 2021/07/16 | Published: 2023/01/20 | ePublished: 2023/01/20
Received: 2021/04/14 | Accepted: 2021/07/16 | Published: 2023/01/20 | ePublished: 2023/01/20
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