Itraconazole-Induced Apoptosis in Candida glabrata ( Nakaseomyces glabratus ) Biofilms: Role of ROS and CgPDR 1 Mutations (K274N,D1082G,and S343F) in Resistant Clinical Isolates

Abstract

Background:

Nakaseomyces glabratus exhibits intrinsic tolerance to azole antifungals, frequently mediated by gain-of-function (GOF) mutations in the transcription factor CgPDR1. While efflux-mediated resistance is well established, its potential interaction with oxidative stress–dependent cell death pathways in biofilms remains insufficiently characterized.

Materials and Methods:

Fifteen clinical isolates (susceptible, n = 5; resistant, n = 10) were analyzed under planktonic and biofilm conditions. Antifungal susceptibility to itraconazole (0.03–32 µg/mL) was determined, while apoptosis and intracellular reactive oxygen species (ROS) were quantified using Annexin V/PI staining and fluorescence-based assays, respectively. The role of oxidative stress was evaluated using ascorbic acid co-treatment. CgPDR1 mutations were identified by sequencing.

Results:

Resistant isolates exhibited significantly elevated minimum inhibitory concentration (MIC) values (up to 32 µg/mL) and a high prevalence of CgPDR1 mutations (K274N: 100%; D1082G: 30%; S343F: 10%). Itraconazole induced a marked increase in ROS production and apoptosis in susceptible biofilms (Annexin V⁺ ≈ 70%), whereas resistant isolates demonstrated attenuated ROS generation and reduced apoptosis (≈10%, p < 0.01). Although ascorbic acid significantly decreased ROS levels in susceptible isolates, it did not alter MIC values or restore antifungal susceptibility.

Conclusions:

CgPDR1 GOF mutations are associated with a coordinated resistance phenotype in N. glabratus biofilms, characterized by enhanced drug tolerance and reduced ROS-mediated apoptosis. These findings support a multifactorial resistance model and identify K274N as a potential population-specific biomarker.

Keywords

biofilm antifungal resistance itraconazole reactive oxygen species apoptosis onychomycosis

Introduction

Onychomycosis, a fungal infection of the nail apparatus, represents a significant global health concern, accounting for up to 50% of all nail disorders. With a prevalence of approximately 5.5% worldwide, this condition disproportionately affects the elderly, with rates exceeding 20% in this demographic.¹ The etiology of onychomycosis is diverse, involving dermatophytes, yeasts, and non-dermatophyte molds.² While dermatophytes, primarily Trichophyton rubrum and Trichophyton mentagrophytes, are the predominant cause, responsible for roughly 90% of toenail and 75% of fingernail infections,³ infections caused by Candida species are increasingly prevalent, particularly in patients with concurrent conditions such as tinea pedis.³

The clinical presentation of candidal onychomycosis is heterogeneous, typically manifesting as one of four forms: distal subungual onychomycosis, chronic paronychia with secondary nail dystrophy, secondary candidal infection, or as a component of chronic mucocutaneous candidiasis.³ Although Candida albicans remains the primary pathogen, other species, including the Candida parapsilosis sensu lato complex, Candida tropicalis, and Nakaseomyces glabratus (formerly Candida glabrata), are significant contributors to the disease burden.^4,5 This is particularly evident in regions like Iran, where Candida spp. account for over 60% of onychomycosis cases, with a notably rising isolation rate of N. glabratus.⁶

The pathogenicity of N. glabratus is underpinned by key virulence determinants, including adhesion proteins (Epa1-7) that facilitate mucosal colonization,⁷ extracellular matrix production that enhances biofilm formation,⁸ and the overexpression of ATP-binding cassette (ABC) transporters, which confers multidrug resistance (MDR).⁹ Crucially, in N. glabratus, ABC transporter overexpression not only drives MDR but also augments virulence by enabling immune evasion and persistent colonization in hostile host environments.^10,11 Given its limited repertoire of virulence factors compared to C. albicans, these MDR mechanisms are a critical determinant of its clinical persistence.¹²

The triazole antifungal fluconazole is approved for various Candida infections, such as oropharyngeal, esophageal, and systemic candidiasis.¹³ However, its application in onychomycosis remains off-label in many jurisdictions. Its spectrum of activity is primarily confined to Candida spp., with minimal efficacy against dermatophytes, thus necessitating accurate pathogen identification and susceptibility testing for its successful use.^14–16 Fluconazole exerts its fungistatic effect by inhibiting the CYP51A1-encoded enzyme lanosterol 14α-demethylase, thereby depleting ergosterol in the fungal cell membrane of susceptible species like C. albicans and N. glabratus.¹⁷ Current therapeutic guidelines for candidal onychomycosis recommend pulsed or continuous fluconazole regimens, which achieve cure rates of 60–75% for fingernails, though efficacy is lower for toenails.¹⁸ Itraconazole, with its superior keratin affinity and broader spectrum, is often preferred, demonstrating activity against C. tropicalis and the C. parapsilosis complex.¹⁹ However, emerging resistance, linked to ERG11 mutations in species like C. parapsilosis, underscores the necessity of susceptibility testing in refractory cases.²⁰

While terbinafine is first-line for dermatophyte onychomycosis, its utility against Candida is limited and often reserved for synergistic combinations with azoles due to variable in vitro susceptibility.²¹ Adjuvant topical therapies, such as amorolfine 5% or ciclopirox 8%, can enhance penetration and reduce systemic drug exposure,²² with recent meta-analyses reporting a 15–20% higher cure rate with combination therapy compared to monotherapy.²³

A pivotal recent advance has been the elucidation of itraconazole’s fungicidal mechanism, which involves the induction of apoptosis via reactive oxygen species (ROS) in pathogens including N. glabratus, C. albicans, and Aspergillus fumigatus.²⁴ In susceptible strains, itraconazole: (a)

Inhibits CYP51-mediated ergosterol biosynthesis;

(b)

Triggers mitochondrial membrane depolarization;

(c)

Generates cytotoxic ROS (H₂O₂, ^•OH) via Fenton reactions;²⁵

(d)

Induces phosphatidylserine externalization and DNA fragmentation.²⁶

In clinical isolates, gain-of-function (GOF) mutations in the transcription factor CgPDR1 confer resistance to this regulatory mechanism by orchestrating the defensive strategies summarized in Table 1.

Table 1.

Functional Consequences of Clinically Significant CgPDR1 Mutations in N. glabratus

Mutation	Domain	Mechanism	Clinical impact	Reference
K274N	Middle homology	Constitutive efflux pump activation	8–16 × increased itraconazole MIC	²⁷
D1082G	Transactivation	Enhanced β-glucan synthesis	Biofilm-associated resistance	²⁸
S343F	DNA-binding	Altered CgCDR1 promoter affinity	Cross-resistance to azoles	²⁹

These mutations lead to the upregulation of efflux pumps, reduce intracellular drug accumulation, limit ROS production, and enhance antioxidant defenses (e.g., catalase, Superoxide dismutase), thereby enabling biofilm persistence and treatment failure.^30,31

The emergence of N. glabratus as a predominant cause of azole-resistant onychomycosis necessitates a deeper investigation into its resistance mechanisms.³¹ Although CgPDR1-mediated efflux is well-characterized, recent evidence suggests that clinical isolates with specific GOF mutations (e.g., K274N, D1082G, S343F) may concurrently suppress ROS-induced apoptosis—a coordinated resistance mechanism that remains insufficiently characterized in the context of biofilm-associated nail infections.³⁰ This dual resistance mechanism may provide a plausible explanation for the persistent treatment failures observed clinically, particularly in regions like Iran where these mutations demonstrate a high prevalence.⁶

This study was designed to investigate the interplay between oxidative stress, apoptosis, and efflux pump regulation in N. glabratus. Specifically, we aimed to: (i) evaluate the antifungal efficacy of itraconazole against biofilm-forming clinical isolates of N. glabratus; (ii) delineate the role of intracellular ROS in itraconazole-induced apoptosis; and (iii) determine the prevalence of specific CgPDR1 mutations (K274N, D1082G, and S343F) associated with clinical resistance. By elucidating these pathways, this research contributes to the development of targeted strategies to overcome antifungal resistance and improve therapeutic outcomes for onychomycosis.

Materials and Methods

Fungal isolates and culture conditions

A panel of 15 clinical isolates of N. glabratus was utilized in this study, comprising five itraconazole-susceptible (wild-type, WT) and 10 itraconazole-tolerant (non-wild-type, NWT) strains. All strains were maintained on Sabouraud Dextrose Agar (SDA; Merck, Darmstadt, Germany) and cultured at 37°C for 24 hours. For experimental procedures, log-phase cultures were prepared by inoculating Sabouraud Dextrose Broth (SDB; Merck), and cell densities were adjusted to a range of 1 × 10³ to 10⁷ colony-forming units per milliliter (CFU/mL) using a Neubauer-improved hemocytometer (Marienfeld, Germany).

Biofilm formation of WT and NWT N. glabratus strains

Biofilms were established in flat-bottomed 96-well polystyrene microtiter plates (Corning, USA). Each well was inoculated with 1 × 10⁵ CFU suspended in 200 µL of SDB and incubated statically at 37°C for 72 hours to facilitate mature biofilm formation, as established in prior studies. Following incubation, non-adherent cells were removed by gently washing the biofilms twice with phosphate-buffered saline (PBS; pH 7.2).

Determination of the minimum inhibitory concentration of itraconazole against biofilm cells of WT, NWT N. glabratus

The minimum inhibitory concentration (MIC) of itraconazole (Sigma-Aldrich, USA) was assessed against both planktonic and biofilm cells.

For planktonic cells, assays were performed in 96-well plates using inocula of low cell density (LCD; ∼10³ CFU/mL) and high cell density (HCD; ∼10⁷ CFU/mL) for both WT and NWT strains. Two-fold serial dilutions of itraconazole (0.03–32 µg/mL) were prepared. Following 24 hours of incubation at 37°C, microbial growth was quantified by measuring the optical density at 570 nm (OD₅₇₀) using an ELISA microtiter plate reader (Model IMark, BioRad Laboratories). The MIC for planktonic cells was defined as the lowest drug concentration that inhibited growth to ≤10% of the control. For planktonic cells, viability was assessed directly by OD₅₇₀ measurement without MTT reduction, as described above.

For biofilm cells, after the planktonic phase was removed and the established biofilms were washed once with PBS, the adherent biofilm was treated with 100 µL of itraconazole (0.03–32 µg/mL) for 24 hours at 37°C. Biofilm viability was then assessed using a colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, biofilms were incubated with 100 µL of MTT solution for 3 hours at 37°C. The MIC for biofilms was defined as the lowest concentration that resulted in ≥90% reduction in metabolic activity compared to the drug-free control. All susceptibility testing was performed in technical duplicate across three independent biological replicates.

ROS detection and quantification

Intracellular ROS generation in mature biofilms was evaluated using a dual-staining approach with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich, USA) and acridine orange (AO).³² Following 24 hours of exposure to itraconazole, biofilms (WT and NWT isolates) were incubated with 10 µM DCFH-DA and 10 µg/mL AO for 30 minutes at 37°C in the dark.

Fluorescence images were acquired using an Olympus BX43 fluorescence microscope (Olympus, Tokyo, Japan) at excitation/emission wavelengths of 485/530 nm. Fluorescence intensity was quantified using ImageJ software (NIH, USA) by calculating mean pixel intensity values following background subtraction.

All imaging parameters, including exposure time and detector gain, were kept constant across all experimental conditions to ensure comparability. Each experiment was performed in three independent biological replicates.

Effect of ascorbic acid on ROS and biofilm viability

To investigate the contribution of oxidative stress to itraconazole-induced cellular responses, mature biofilms were treated with itraconazole in the presence or absence of ascorbic acid (AA; 10 mM). A control group treated with ascorbic acid alone was included to distinguish antioxidant-specific effects.

Following 24 hours of incubation, intracellular ROS levels were quantified using the DCFH-DA assay as described above. Biofilm viability was assessed by CFU enumeration. Briefly, treated biofilms were mechanically disrupted, serially diluted, and plated onto SDA media, followed by incubation at 37°C for 24 hours prior to colony counting.

To evaluate whether antioxidant treatment influenced antifungal susceptibility, the MIC of itraconazole was also determined in the presence of ascorbic acid under identical conditions. All experiments were conducted in triplicate.

Apoptosis detection

Apoptosis in biofilm cells was assessed using Annexin V-FITC/Propidium Iodide (PI) dual staining (Sigma-Aldrich, USA). Following itraconazole treatment, biofilms were harvested, washed, and incubated with Annexin V-FITC and PI according to the manufacturer’s protocol.

Flow cytometric analysis was performed using a BD FACSCalibur instrument (BD Biosciences, USA), collecting 10,000 events per sample. Data were analyzed using FlowJo software (Tree Star Inc., USA). Cells were classified as viable (Annexin V⁻/PI^-), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), or necrotic (Annexin V⁻/PI⁺). Representative flow cytometry plots are provided in Supplementary Figure S1. Results are expressed as mean ± standard deviation (SD) from three independent biological replicates.

CgPDR1 gene amplification and sequencing

Genomic DNA was extracted from all isolates using a commercial kit (Roche, Germany). The CgPDR1 gene was amplified via polymerase chain reaction (PCR) using a Bio-Rad thermal cycler and gene-specific primers (Cinnagen, Iran): •

Forward: 5′-AGCCTTGCCGATAGTCATAC-3′

•

Reverse: 5′-AAGGTCAGGGCATACTTCAG-3′

The PCR mixture consisted of 12 µL of MasterMix (Ampliqon, Denmark), 1 µL of each primer, 3 µL of DNA template, and 8 µL of nuclease-free water. The thermal cycling conditions were: initial denaturation at 95°C for 4 minutes; 40 cycles of 95°C for 20 seconds, 62°C for 45 seconds, and 72°C for 20 seconds; and a final extension at 72°C for 5 minutes. The resulting amplicons were purified and sequenced commercially (Pishgam Biotech, Iran). Sequence alignment and analysis were performed using the Clustal W algorithm in MEGA v7.0.21, with sequence KU242352 serving as the reference.

Statistical analysis

Statistical analyses were conducted using SPSS software v26.0 (IBM Corp., USA). For comparisons between two groups, the non-parametric Mann–Whitney U test was applied. A p value < 0.05 was considered statistically significant for all tests.

Results

Determination of CFU in log phase biofilm cell cultures of N. glabratus (WT, NWT)

Log-phase cultures of N. glabratus isolates exhibited comparable growth profiles. The WT strains yielded an average of approximately 3.0 × 10⁶ CFU/mL, while NWT strains produced about 2.8 × 10⁶ CFU/mL. Biofilm formation was robust across all isolates, with no significant difference in biomass between WT and NWT strains (p > 0.05), confirming that resistance was not attributable to impaired biofilm capacity.

Antifungal susceptibility of biofilm-forming N. glabratus to itraconazole

Itraconazole exhibited concentration-dependent antifungal activity against both planktonic and biofilm cells. MIC values were significantly influenced by cell density and resistance phenotype (Table 2).

Table 2.

MIC Values of Itraconazole against WT and NWT N. glabratus Isolates

Strain type	Low cell density MIC (µg/mL)	High cell density MIC (µg/mL)
WT (n = 5)	0.10 ± 0.03	1.0 ± 0.00
NWT (n = 10)	12.57 ± 9.9	32.0 ± 0.00

The MTT assay confirmed a significant reduction in metabolic activity of biofilm cells at 0.1% itraconazole concentration, achieving >90% inhibition in WT strains. In contrast, higher concentrations were required for similar effects in NWT strains (p < 0.01). The concentration-dependent reduction in biofilm viability is illustrated in Figure 1.

Biofilm-associated cells required higher drug concentrations compared to planktonic counterparts. The distribution of MIC values differed between WT and NWT isolates, with the majority of NWT strains (9/10) falling within the 8–32 µg/mL range (Table 3).

Table 3.

Distribution of Itraconazole MICs among WT and NWT Biofilm Isolates

MIC (µg/mL)	No. of NWT isolates (n = 10)	No. of WT isolates (n = 5)
0.03–0.25	0	3
0.5–4	1	2
8–32	9	0

Itraconazole-induced ROS generation in N. glabratus biofilm (WT/NWT) cells

Itraconazole treatment resulted in a significant increase in intracellular ROS levels in WT biofilms (160 ± 12 vs. 42 ± 5 AU, p < 0.01). In contrast, NWT isolates exhibited only a modest, non-significant increase (58 ± 7 vs. 45 ± 6 AU, p > 0.05).

All measurements were obtained under standardized imaging conditions and quantified using ImageJ-based analysis. Quantitative ROS data for treated and untreated WT and NWT biofilms are presented in Figure 2.

Effect of AA on ROS accumulation and N. glabratus biofilm (WT/NWT) cells viability

Co-treatment with ascorbic acid significantly reduced ROS levels in WT isolates (approximately 75–80% reduction, p < 0.01), whereas only minor changes were observed in NWT isolates (p > 0.05).

Despite ROS suppression, no significant differences were observed in CFU counts between itraconazole-treated and itraconazole + ascorbic acid groups (p > 0.05). Importantly, MIC values remained unchanged in the presence of ascorbic acid across all isolates, indicating that ROS modulation alone does not restore antifungal susceptibility.

Quantitative analysis of ROS levels and biofilm viability, including untreated controls and AA-only conditions, is shown in Figure 3.

Apoptosis induction in biofilm N. glabratus cells (WT/NWT) by itraconazole

Itraconazole induced a pronounced apoptotic response in WT biofilms, with approximately 70% Annexin V-positive cells. In contrast, NWT isolates exhibited significantly lower apoptosis levels (∼10%, p < 0.01).

Data represent mean ± SD from three independent biological replicates, with 10,000 events analyzed per condition by flow cytometry. Representative staining patterns are shown in Supplementary Figure S1.

Statistical analysis was performed using the Mann–Whitney U test. Quantitative apoptosis data are presented in Figure 4.

CgPDR1 mutations in resistant isolates

Our investigation focused on the mutations found in tested resistant and sensitive isolates to assess the impact of the CgPDR1 gene on itraconazole resistance. After amplifying the genomic DNA of N. glabratus, which is resistant to itraconazole, with CgPDR1 gene primers, a 1107 bp product was obtained. A, G to T change at the 822nd base of the gene leads to the substitution of K274N in the amino acid sequence (isolates R1–R10). Additionally, it has been noted that mutations resulting in a D1082G amino acid replacement (in isolates R1, R4, and R5) and an S343F replacement (in isolate R5) have been observed.

Sequencing analysis identified three mutations in resistant isolates: 1

K274N (100% prevalence): G→T substitution at nucleotide position 822

D1082G (30%): Associated with hyperactivation of efflux pumps.

S343F (10%): Unique to isolate R5, potentially synergistic with D1082G.

These mutations were absent in susceptible isolates. The K274N substitution was consistently detected across all resistant strains, while D1082G and S343F were observed in a subset of isolates.

Discussion

This study provides evidence for an association between CgPDR1 GOF mutations and a multifactorial resistance phenotype in N. glabratus biofilms. In addition to the expected increase in itraconazole tolerance, resistant isolates demonstrated attenuated ROS generation and a markedly reduced apoptotic response following antifungal exposure. These findings suggest that resistance in biofilm-associated cells may extend beyond classical efflux-mediated mechanisms and involve alterations in cellular stress responses.^9,11,31

Itraconazole has been increasingly recognized to exert fungicidal activity, at least in part, through the induction of oxidative stress and apoptosis.^24,32 In susceptible isolates, our data show a substantial increase in intracellular ROS levels accompanied by a high proportion of Annexin V-positive cells, consistent with oxidative stress–mediated cell death pathways.^24,26 In contrast, resistant isolates exhibited minimal ROS induction and significantly reduced apoptosis under identical treatment conditions. This divergence supports the notion that resistant strains may effectively mitigate drug-induced oxidative stress, thereby enhancing survival within biofilm structures.^30,31

A plausible explanation for this phenotype is the reduced intracellular accumulation of itraconazole resulting from CgPDR1-driven efflux pump overexpression.^9,27 Lower intracellular drug concentrations would be expected to limit mitochondrial dysfunction and subsequent ROS generation. Within this framework, the observed reduction in ROS may represent a downstream consequence of efflux activity rather than an independently regulated resistance mechanism. This interpretation is supported by the finding that pharmacological suppression of ROS using ascorbic acid did not restore antifungal susceptibility or alter MIC values, indicating that modulation of oxidative stress alone is insufficient to reverse resistance.³¹

The mutation analysis further supports a heterogeneous but coordinated resistance landscape. The K274N substitution was identified in all resistant isolates, suggesting a potential population-specific enrichment. However, given the limited sample size and geographic origin of the isolates, this mutation cannot be considered universally representative.²⁷ Additional mutations, including D1082G and S343F, were observed at lower frequencies and may contribute to phenotypic variability through distinct regulatory effects on transcriptional activation, efflux capacity, or biofilm-associated pathways.^28,29 The coexistence of multiple mutations within individual isolates suggests that resistance may arise through cumulative or synergistic genetic adaptations rather than a single dominant alteration.^9,29

The lack of a direct correlation between ROS suppression and restoration of susceptibility highlights the complexity of antifungal resistance in N. glabratus. While oxidative stress appears to play a role in itraconazole-induced cell death, it is unlikely to function as a primary determinant of resistance in the absence of sufficient intracellular drug accumulation.^24,31 Instead, these findings support a hierarchical model in which efflux-mediated drug exclusion represents the dominant mechanism, with downstream effects on ROS generation and apoptosis.

Study limitations

Several limitations should be considered when interpreting these results. First, the relatively small number of clinical isolates may limit the generalizability of the findings. Second, ROS measurements were based on fluorescence microscopy and image-based quantification, which, although standardized, provide semi-quantitative data.³² More precise approaches, such as flow cytometry or fluorometric assays, could improve quantitative accuracy. Third, the absence of functional genetic validation, including CgPDR1 knockout or overexpression models, precludes definitive conclusions regarding causality. Finally, apoptosis was assessed at a single drug concentration, and dose-dependent responses were not explored. Furthermore, while flow cytometry was used for apoptosis quantification, we did not perform additional confirmatory assays such as caspase activity measurement or TUNEL staining, which could further strengthen the findings.

Despite these limitations, the study provides a coherent framework linking genetic, cellular, and phenotypic aspects of antifungal resistance in biofilm-forming N. glabratus. The findings underscore the importance of considering both drug transport mechanisms and stress response pathways in the evaluation of antifungal efficacy.^30,31

Conclusion

The present study indicates that CgPDR1 GOF mutations are associated with a coordinated resistance phenotype in N. glabratus biofilms, characterized by increased itraconazole tolerance and reduced ROS-mediated apoptosis. The high prevalence of the K274N mutation within the studied isolates suggests a potential population-specific association; however, broader validation is required to determine its general applicability.²⁷

The findings support a model in which efflux-mediated reduction of intracellular drug accumulation limits oxidative stress induction and subsequent apoptotic cell death.^9,31 Importantly, pharmacological suppression of ROS did not restore antifungal susceptibility, indicating that oxidative stress modulation alone is insufficient to overcome resistance.³¹

Overall, these results highlight the multifactorial nature of antifungal resistance in biofilm-associated infections and emphasize the need for therapeutic strategies targeting both drug efflux and downstream cellular responses.^30,31 Further studies incorporating functional genetic approaches and expanded clinical cohorts are warranted to validate these observations and refine their clinical relevance.

Authors’ Contributions

Study concept and design and technical supervision: F.V., and M.F.; Obtaining the specimens from patients and interpretation: F.V., M.F., and E.L.; Acquisition of data and drafting of the article: F.V., and M.F.; Critical revision of the article: N.M.; Procedure: F.V., M.F., and E.L.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Supplemental Material

sj-docx-1-mdr-10.1177_10766294261470531 — Supplemental material for Itraconazole-Induced Apoptosis in Candida glabrata (Nakaseomyces glabratus) Biofilms: Role of ROS and CgPDR1 Mutations (K274N, D1082G, and S343F) in Resistant Clinical Isolates

Supplemental material, sj-docx-1-mdr-10.1177_10766294261470531 for Itraconazole-Induced Apoptosis in Candida glabrata (Nakaseomyces glabratus) Biofilms: Role of ROS and CgPDR1 Mutations (K274N, D1082G, and S343F) in Resistant Clinical Isolates by Farnaz Valizadeh, Mahsa Fattahi, Ensieh Lotfali, and Nasrin Motamed

Footnotes

Disclosure Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding Information

The author(s) declare that no financial support was received for the research, writing and/or publication of this article.

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