Antifungal susceptibility and biofilm production of Candida species– causative agents of female genital tract infections

Abstract

Background

Recurrent vulvovaginal candidosis (RVVC) is a chronic infection affecting 8-10% of women worldwide. Biofilm production of the infecting species and reduced sensitivity to antimycotics could contribute to the recurrence of this infection. This study aimed to examine the biofilm production ability and antifungal susceptibility of genital yeast isolates to determine their virulence potential.

Methods

Matrix-assisted laser desorption in ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to identify 300 Candida species. Using crystal violet method, strains were categorized into non-producers, weak, moderate, and strong biofilm producers (BFP). Antifungal susceptibility testing was performed using commercial Integral System YEASTS Plus test (ISYPT) and broth microdilution method (BMM).

Results

MALDI-TOF MS identified 150 Candida albicans, 124 non-albicans Candida (NAC), and 26 Saccharomyces cerevisiae strains. Within 138 (46.0%) BFP, 23 (16.7%) were strong, 44 (31.9%) moderate, and 71 (51.4%) weak. BMM was done for 43 BFP selected isolates with nystatin MIC ˃1.25 μl, fluconazole MIC ˃64 μl, and clotrimazole MIC ˃1.0 μl determined by ISYPT. Compared to all examined isolates, BMM confirmed that: i) C. albicans and NAC BFP showed low sensitivity to fluconazole (12% and 4%, respectively); ii) all BFP showed low sensitivity to nystatin (12.7% C. albicans, 14.5% NAC, and 23.1% S. cerevisiae); iii) clotrimazole in vitro was the most efficient regarding C. albicans and S. cerevisiae strains, but in 4.0% NAC BFP for this antimycotic higher MIC was established.

Conclusion

Novel antimycotics or possible combinations of antifungal agents and natural products could be a new treatment option for RVVC.

Introduction

Yeasts of the genus Candida colonize the vulvovaginal mucosa of 20–30% of women without any clinical symptoms or signs of infection. Moreover, based on recent molecular research, this percentage is significantly higher (64%) [1]. As long as the yeasts remain in balance with the host environment, with other representatives of the microbiota and immune system, Candida spp. are saprophytic, commensal organisms [2]. However, many risk factors can cause changes in the environment, leading to dysbiosis, which predisposes to multiplication and overgrowth of the present Candida spp. that affects host inflammatory processes followed by the onset of symptomatic infection, vulvovaginal candidosis (VVC).

A disturbed balance between yeasts-colonizers and the host may occur due to many predisposing risk factors such as immunosuppression, diabetes, pregnancy, hormonal imbalance, use of broad-spectrum antibiotics and glucocorticoids, allergies [3–6], mechanical and oral contraceptives, estrogen therapy, as well as individual characteristics of affected women [7]. All predisposing factors mentioned are well recognized as enabling yeasts to switch from a harmless commensal to a pathogenic role and influence the development of symptomatic infection characterized by vulvovaginal itching, irritation, burning, soreness, fissuring, redness, vaginal discharge, and dyspareunia. Because of the multiplicity of risk factors and the very high percentage of women with Candida colonization, it is understandable that 75% of women will undergo at least one episode of VVC, and 50% have had this experience two or more times in their lifetime. More important is the fact that 8–10% of all women develop recurrent vulvovaginal candidosis (RVVC), defined as at least 3–4 episodes of VVC per year [8, 9].

One of the assumptions is that recurrence might result from a higher pathogenic potential of infecting species and low sensitivity or resistance to treatments used. It has been shown that Candida spp. are capable of forming biofilms, which are defined as a formation consisting of a community of microorganisms protected from the immune system, showing decreased susceptibility to antimicrobials and enhancing the spread of antimicrobial resistance [10, 11]. In order to investigate a possible higher pathogenic potential of the strains in this study, we investigated the biofilm production of yeast isolates originating from the female genital tract, as well as their antifungal susceptibility to antimycotics.

Materials and methods

Study design

This study examined biofilm production and antifungal susceptibility for 300 collected vulvovaginal yeast isolates (150 C. albicans and 150 non-albicans Candida species (NAC) and non-Candida yeasts). Before enrolment, all participants gave prior approval. The study design was approved by the Ethics Committees of the Public Health Institute Niš and Medical Faculty, University of Niš, Serbia.

Isolation and identification of Candida species

In order to collect 300 isolates (150 C. albicans and 150 NAC and non-Candida yeast), the vaginal swabs of 4569 women with signs and symptoms of infection were observed. The microbiological examination was carried out on each patient in accordance with standard protocols that included bacteriological and parasitological testing in addition to mycological analysis. Isolation of yeasts was performed by inoculating patient material on Sabouraud dextrose agar (SDA) and Candida-Chromogenic medium (Liofilchem​​/Bacteriology products, Italy) and incubating at 37 °C for 3–5 days. The primary differentiation of C. albicans was based on the pigmentation of colonies on the Chromogenic medium and by the germ tube test. However, all isolates were typed using protein fingerprint analysis obtained by matrix-assisted laser desorption in ionization-time of flight mass spectrometry (MALDI-TOF MS) according to the Bruker protocol (Bruker Daltonics, Bremen, Germany). Briefly, all strains were subcultured on SDA for 24 h at 37 °C. After incubation, thin colony layers were made on a polished steel target plate, and the spots were covered with 1 µL of 70% formic acid and alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix. After drying at room temperature, the plate was placed into the MALDI-TOF MS Biotyper Sirius one IVD System (Bruker Daltonics, Bremen, Germany), and strains were identified using MBT Compass software, ver. 4.1.100 in automatic runs operated by flexControl, ver. 3.4.207.20 (Bruker Daltonics, Bremen, Germany). The database used to compare generated mass spectra in this study was MBT Compass Library, Revision H, 3893 species/entries. Identification was accepted if the log score values were greater than 1.7.

Biofilm production assay

Biofilm production assays were performed by a crystal violet (CV) method under static conditions in 96-well microtiter plates [12, 13]. After preparing fungal suspensions, RPMI-1640 medium supplemented with 0.8% glucose was added to each well and then inoculated so that the final concentration was 5 × 10⁵. Testing was done in triplicates, and after an incubation period of 48 h at 35 °C, well contents were gently aspirated, washed twice with sterile phosphate-buffered saline (PBS, pH = 7.4), dried and stained with 0.5% CV for 20 min.

Such prepared microtiter plates were washed, and wells were filled with 250 µl of 96% (v/v) ethanol. After 45 min of destaining, 150 µl of the obtained solution was transferred into a sterile microtiter plate, and the absorbance was measured at 595 nm using an ELISA reader (Multiskan™ FC Microplate Photometer, Thermo Scientific™). The strains were categorized according to their biofilm producing ability into the following groups: non-producers, weak, moderate, and strong biofilm producers [12].

Antifungal susceptibility testing

Integral system YEASTS plus test

Antifungal susceptibility testing for all strains was performed using the Integral System YEASTS Plus (ISYP) commercial test (Liofilchem S.r.l. Roseto D.A. Teramo, Italy). This antifungal susceptibility kit testing allows the selection of yeasts with nystatin (NY) MIC ˃1.25 µg /L, clotrimazole (CLO) MIC ˃1 µg /L, and fluconazole (FLU) MIC ˃64 µg /L. The test was performed and interpreted according to the manufacturer’s instructions. Referent strains, C. albicans ATCC 24,433 and C. krusei ATCC 6258, were used as control strains in all assays.

Broth microdilution method

For 43 strains with biofilm production ability, which showed low sensitivity in the ISYP test and resistance to commonly used antifungals (NY, FLU, and CLO) in treatment for VVC, susceptibility testing was performed using the Broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [14].

The tested antimycotics (NY, FLU, and CLO obtained from Sigma-Aldrich Company, Steinheim, Germany) were dissolved in 100% dimethyl sulfoxide (DMSO) at concentrations of 640 µg/mL for FLU and 160 µg/mL for NY and CLO. After providing double dilutions of antimycotics in 96-well plastic microtiter plates, such solutions were transferred to another 96-well microtiter plates previously filled with RPMI-1640 Medium (Sigma-Aldrich, Darmstadt, Germany) with L-glutamine without bicarbonate. The prepared suspensions of selected yeast strains with a density of 0.5 McFarland standard (obtained from colonies cultivated on SDA for 24 h at 35 °C) were added to the wells with antimycotics and RPMI and to growth control (RPMI without antimycotics). The density of the fungal suspensions applied to the microtiter plate was in the range of 0.5–2.5 × 10³ CFU/mL. The concentrations of FLU ranged from 0.031 to 64 µg/mL and of both NY and CLO from 0.008 to 16 µg/mL. The microdilution test was done in triplicates for every strain. Inoculated microplates were incubated for 24 h at 37 °C. The minimum inhibitory concentration (MIC) endpoint was defined as 100% of growth inhibition for NY and 50% of growth inhibition for the other drugs. The quality control strains were C. krusei ATCC 6258 and C. parapsilosis ATCC 22,019.

Statistical analysis

Standard methods of descriptive statistics were used in the statistical processing of the data. The numerical features were compared with the t-test or the Mann-Whitney test, depending on the data distribution. Categorical characteristics were compared using the Chi-square test or Fisher’s test. Statistical data processing was performed in the software package R and RStudio (ver 2021.09.0 + 351, 2021-09-20 for Windows).

Results

Microbial identification

The research included 300 yeast isolates of the female genital tract material. Based on the colonies’ color change on the Chromogenic medium and the germ tube test, the isolates were classified into 150 C. albicans and 150, both NAC and non-Candida species. All strains were identified using MALDI-TOF MS, and the distribution of the species is shown in Fig. 1. Mass spectrometry confirmed the 150 C. albicans strains, and the other half of the isolates were identified as 124 NAC species (C. glabrata, C. krusei, C. kefyr, C. parapsilosis, C. lusitaniae, and C. norvengensis) as well as 26 strains of S. cerevisiae.

Fig. 1.

Distribution of the examined yeast species

Biofilm production

The CV method used for biofilm quantification revealed that out of 300 examined yeast strains, 138 (46.0%) showed biofilm production ability. Among biofilm producers, 23 (16.7%) were classified as strong, 44 (31.9%) as moderate, and 71 (51.4%) as weak producers. A statistically significant difference in the prevalence of producers between species was recorded (p < 0.001).

While the most producers were observed among C. albicans strains (63.3%), compared to these isolates, NAC species (29.0%) and S. cerevisiae (26.9%) showed significantly weaker biofilm production capacity (p < 0.001 and p = 0.001, respectively). The group of 23 strong biofilm producers included 18 strains of C. albicans, representing a significant majority compared to other species (two strains of C. glabrata and C. kefyr each and one strain of C. lusitaniae). The distribution of species based on biofilm production ability is shown in Fig. 2.

Fig. 2.

Biofilm production ability of examined species. *NAC- Non-albicans Candida species

Antifungal susceptibility testing to nystatin, clotrimazole and fluconazole among biofilm producing strains

In order to summarize the virulence potential of examined yeasts, we analyzed the effectiveness of recommended and commonly used antimycotics in treating VVC (NY, CLO, FLU) by observing the results of ISYP testing and biofilm production. For this purpose, broth microdilution assay was done on planktonic cells of selected 43 isolates with higher biofilm production ability (previously determined by the CV method) and resistance rates (determined by the ISYP test). The susceptibility of chosen strains (19 C. albicans, 15 C. glabrata, two C. krusei, six S. cerevisiae, and one C. kefyr) was summarized in Table 1. Results revealed that 94,7% of selected C. albicans, 50% of C. krusei strains, and one tested strain of C. kefyr were resistant to FLU (MIC ≥ 64 µg/mL) according to the susceptibility and resistance classifications commonly used for both systemic and mucosal infections [14]. These proposed standards classify the isolates of Candida species with MICs of < 8 µg/mL for FLU as susceptible (S); 16–32 µg/mL susceptible in the dose-dependent (SDD) category, and ≥ 64 µg/mL as resistant (R). On the other hand, only four strains of C. glabrata were resistant, while all of the S. cerevisiae isolates were sensitive to FLU.

Table 1.

Broth microdilution for 43 selected strains

	Cumulative % of isolates with MIC (µg/mL) of:
Antifungal agent (µg/mL)	< 0.008	0.016	0.031	0.063	0.125	0.25	0.5	1	2	4	8	16	32	64	> 64
C. albicans
Flu	-	-	-	-	-	-	-	-	-	-	-	-	5.3	21.1	100.0
Clo	-	-	-	5.3	-	15.8	73.7	100.0	-	-	-	-	-	-	-
Ny	-	-	-	-	-	-	-	-	5.3	26.3	94.7	100.0	-	-	-
C. glabrata
Flu	-	-	-	-	-	-	-	-	6.7	26.7	46.7	66.7	80.0	100.0	-
Clo	-	-	-	6.7	33.3	46.7	53.3	66.7	86.7	100.0	-	-	-	-	-
Ny	-	-	-	-	-	-	-	-	-	53.3	100.0	-	-	-	-
C. krusei
Flu	-	-	-	-	-	-	-	-	-	-	-	-	50.0	100.0	-
Clo	-	-	-	-	-	100.0	-	-	-	-	-	-	-	-	-
Ny	-	-	-	-	-	-	-	-	-	-	100.0	-	-	-	-
C. kefyr
Flu	-	-	-	-	-	-	-	-	-	-	-	-	-	-	100.0
Clo	-	-	-	100.0	-	-	-	-	-	-	-	-	-	-	-
Ny	-	-	-	-	-	-	-	-	100	-	-	-	-	-	-
S. cerevisiae
Flu	-	-	-	-	-	-	-	-	33.3	66.7	100.0	-	-	-	-
Clo	-	-	-	-	66.7	83.3	100.0	-	-	-	-	-	-	-	-
Ny	-	-	-	-	-	-	-	-	-	66.7	100.0	-	-	-	-

* Flu-Fluconazole, Clo - Clotrimazole, Ny– Nystatin

Analyzing the effectiveness of CLO, all of the tested C. albicans, C. krusei, C. kefyr, and S. cerevisiae strains were susceptible to this antimycotic. Nevertheless, a third of the C. glabrata isolates showed resistance to CLO with MICs of > 1 µg/mL, which was the breakpoint adopted by Richter et al. [15].

Although breakpoints for NY have not been defined by CLSI and EUCAST, some authors suggested that topical NY MICs of ≤ 1 µg/mL can be considered susceptible [15, 16]. Accordingly, all selected strains (biofilm producers that showed lower sensitivity in the ISYP test) were resistant to this antifungal agent.

Evaluation of all results (Fig. 3; Table 2) regarding biofilm producers and the number of determined species having high MIC values of examined antimycotics by broth microdilution assay, it can be pointed out that among 150 C. albicans strains 12.0% and 12.7% of them, besides the ability to produce biofilm, have low sensitivity to FLU and NY respectively. On the contrary, in all biofilm producers, CLO demonstrated satisfactory efficiency in vitro. Among 124 NAC biofilm producer species, 4.0%, 14.5%, and 4.0% had low sensitivity to FLU, NY, and CLO, respectively. Although for six S. cerevisiae biofilm producers, lower MIC values ​​were recorded for FLU and CLO, all of those strains showed high MIC values for NY, which implies that 23.1% of all investigated S. cerevisiae strains had higher virulence potential. If all isolates are taken into account, it can be highlighted that the prevalence of virulent strains which implies production of biofilm and lower susceptibility to NY, FLU, and CLO, as well as to the combinations of these antifungals FLU + NY, CLO + NY, FLU + CLO, and to all three antimycotics, was 7.7%, 14.3%, 1.7%, 7.7%, 1.7%, 1%, and 1% respectively.

Fig. 3.

Algorithm of study design. NAC– non-albicans Candida, BFP– biofilm producers, FLU– fluconazole, NY– nystatin, CLO– clotrimazole

Table 2.

Biofilm producers of different yeast species with high MIC values

Antimycotics	Number of BFP with high MIC values
	C. albicans		NAC species		S. cerevisiae		Total		p
	N	% of total C. albicans strains	N	% of total NAC strains	N	% of total S. cerevisiae strains	N	%
FLU	18a	12.0	5	4.0	0	0.0	23	7.7*	0.015
NY	19	12.7	18	14.5	6	23.1	43	14.3*	0.375
CLO	0	0.0	5b	4.0	0	0.0	5	1.7*	0.027
FLU + NY	18c	12.0	5	4.0	0	0.0	23	7.7	0.015
CLO + NY	0	0.0	5d	4.0	0	0.0	5	1.7	0.027
FLU + CLO	0	0.0	3	2.4	0	0.0	3	1.0	0.116
FLU + NY + CLO	0	0.0	3	2.4	0	0.0	3	1.0	0.116

BFP– biofilm producers; NAC– non-albicans Candida; FLU– fluconazole; NY– nystatin; CLO– clotrimazole; High MIC values for FLU ≥ 64 µg/mL, for NY > 1 µg/mL, for CLO > 1 µg/mL; a– significantly more C. albicans than NAC strains (p = 0.015), b– significantly more NAC than C. albicans strains (p = 0.027), c– significantly more C. albicans than NAC strains (p = 0.015), d– significantly more NAC than C. albicans strains (p = 0.027); *–significant difference between susceptibility of all strains to FLU (7,7%), NY (14,3%) and CLO (1,7%)(p = 0,006), FLU vs. NY (p = 0,009), FLU vs. CLO (p˂0,001), NY vs. CLO (p˂0,001)

Discussion

With the recent adoption of molecular identification of fungi, it has been established that the genus Candida implies a taxonomically unclear group comprising some very distant species. Through new taxonomic studies, several medically important species that were previously members of this genus, such as C. glabrata, C. krusei, C. kefyr, C. lusitaniae, and C. norvegensis, were now regrouped as Nakaseomyces glabrata, Pichia kudriavzevii, Kluyveromyces marxianus, Clavispora lusitaniae, and Pichia norvegensis, respectively. In this study yeasts are stated according to the previous classification until a general consensus is reached. Many authors believe that old terms can still be used because the new taxonomy could confuse clinicians in interpreting the results of mycological analyses. Besides, recently reported data on the species Saccharomyces (S.) cerevisiae as the causative agent of VVC [17] support the consideration of changing diagnosis with proposed names, which could be vulvovaginal fungal infection or vulvovaginal yeast infection.

Vulvovaginal candidosis is a highly prevalent mucosal infection of the female genital tract. While most symptomatic disease outbreaks emerge as sporadic acute VVC episodes, 8–10% of women experience at least 3–4 symptomatic episodes per year, defined as recurrent VVC (RVVC). This complex condition occurs due to an unbalanced vaginal microbiome, host risk, and specificity of Candida strains [18]. It is presumed that the ability of biofilm production is a significant virulence factor of Candida species in the pathogenesis of infection [19] due to their role in facilitating the development of persister cells, which are mostly responsible for the resistance of yeasts to antifungal medications, consequently leading to the development of RVVC [20, 21].

Nevertheless, biofilm production by Candida spp. on the vaginal mucosa in vivo and its role in the VVC is still controversial. Although some reports have questioned this fact due to the lack of histological evidence [22, 23], emphasizing that most of the experiments were conducted in vitro [24, 25], numerous studies have suggested that the biofilm formation process, especially by C. albicans, is very important in the pathogenesis of VVC [26, 27]. The study conducted by Wu et al. provided evidence to support this theory and demonstrated that biofilm growth of C. albicans on the vaginal epithelium is associated with histological damage to mucosal epithelial cells and local inflammation [28]. However, diverse results can be seen in the literature regarding the biofilm production of C. albicans compared to NAC or non-Candida (NC) strains. The utilization of scanning electron microscopy to observe biofilms of various Candida spp. revealed distinct structural variations that differ for each species [29]. For example, C. albicans forms a basal blastospore layer with a thick, exopolysaccharide and hyphal matrix-based overlaying layer. On the contrary, biofilms of excluded yeasts from the Candida genus as C. glabrata are considerably less extensive [19, 30, 31]. Candida krusei forms dense and complex biofilms composed of pseudohyphal structures entrenched within a polymer matrix [29]. Additionally, it is known that significant differences in positive biofilm findings were observed between species isolated from different sites [29, 31].

In this study, 46% of the examined strains had biofilm production ability. Half of the strains were isolates of C. albicans and were 18 strong (12%), 28 moderate (18,7%), and 49 weak biofilm producers (32,7%). In a group of NAC species, from a total of 124 examined, 36 (29%) had the biofilm production ability with only five strong (4%), 12 moderate (18,7%), and 19 weak producers (15,3%). Our results showed that C. glabrata had the biofilm production ability in 33,9% of isolates [two strong (3,3%), nine moderate (14,6%) and 10 weak producers (16,1%)]. Moreover, all isolates of C. lusitanie, 47,6% of C. kefyr, and only 6,1% of C. krusei were able to produce biofilm. Additionally, the biofilm production ability is detected for 7 out of 26 strains (26.9%) of non-Candida species, the recently established causative agent of VVC, yeast S. cerevisiae (four moderate, three weak producers). Our results demonstrate that a significantly high percentage of both C. albicans and NAC species, as well as S. cerevisiae, showed the ability to produce biofilm, i.e., indicate a higher virulence of the causative agents, which may represent one of the predispositions for unsuccessful therapy and recurrence of infection [18].

Moreover, a characteristic of yeast that can influence the unsuccessful treatment besides biofilm production capacity is antifungal resistance. Since the management of VVC is frequently determined empirically, without laboratory evidence, a choice of therapy is prescribed following official protocols and recommendations, which include using antifungals for initial therapy (sporadic cases), as well as the recommended treatment for recurrent forms to maintain remission (antifungal drug, dose, and duration of maintenance therapy). All organizations recommend using CLO and NY for local application or systemic treatment with FLU or ITR. To maintain remission in recurrent forms, FLU is recommended as the first choice [2]. In our study, a commercial ISYP antimycogram test was used as screening testing to assess the sensitivity of yeasts to highly recommended antifungals (NY, CLO, and FLU). To thoroughly evaluate the examined yeasts with the ability for biofilm production and which showed low sensitivity (NY-MIC ˃1.25 mg/L, CLO-MIC ˃1 mg/L, and FLU-MIC ˃64 mg/L), we tested the efficacy of these antimycotics using broth microdilution assay.

The results of our study revealed that in addition to the ability of strains to produce biofilms, isolates also exhibited significant resistance to the tested antimycotics. It was determined that compared to all examined isolates, C. albicans and NAC biofilm producers showed resistance to FLU (MIC ≥ 64 µg/mL) in 12% and 4%, respectively. All biofilm producers showed low sensitivity to NY (MIC ˃1 µg/mL), which implies that 12.7% C. albicans, 14.5% NAC, and 23.1% S. cerevisiae had higher virulence potential. On the other hand, CLO in vitro was the most efficient regarding C. albicans and S. cerevisiae strains. However, 4% of NAC species, more precisely C. glabrata, besides the ability to form biofilm, showed low sensitivity to this antimycotic (MIC ˃1 µg/mL). The evaluation of all results also demonstrated that, considering biofilm producers, a high percentage of low sensitivity was established to NY (14.3%) and FLU (7.7%), while CLO was the most efficient in vitro, whereby only 1.7% of isolates were biofilm producers and showed lower sensitivity to this antimycotic.

Prior to the start of the current century, only a few case reports of vulvovaginitis caused by C. albicans strains resistant to FLU (MIC ≥ 64 µg/mL) were documented [32]. However, some of the study’s results in the following years showed a significant increase of resistant C. albicans to FLU [33], besides highlighting the low effectiveness of NY [15, 16, 34]. Many studies also determined the higher FLU MIC values (128 µg/mL) for NAC species C. glabrata and C. krusei. Besides C. albicans, low effectiveness of NY was detected for C. krusei, C. tropicalis, and C. parapsilosis, as well as for S. cerevisiae strains [15, 35]. As for susceptibility to CLO, in the study of Frej-Madrzak et al., similar results as ours were observed, where the high sensitivity of C. albicans and wide range of MIC values of this antimycotic for C. glabrata strains was proved [36]. In rare studies that examined biofilm producing yeasts and their susceptibility to antifungals, it was shown that the strains of C. albicans with the ability to produce biofilms were resistant to FLU [19, 37].

Based on our results and many reports, yeast resistance to commonly used antifungal drugs could be one of the possible causes of relapse of infection. To avoid these problems, a potential treatment option in the future might be new antifungal drugs such as oteseconazole, which is an oral selective inhibitor of fungal lanosterol demethylase, as well as ibrexafungerp with echinocandin-like mechanism and excellent oral bioavailability [2, 38]. Regarding the fact that besides resistance to used antimycotics, yeasts have the ability to produce a biofilm that additionally enhances the resistance of sessile cells and facilitates persistent reinfection, besides the new therapeutic options, further surveys are needed to explore the potential antifungal and antibiofilm synergistic effects of antimycotics and natural products [39].

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors have no conflict of interest.