Unveiling the role of sulfur nanoparticles as an anti-dermatomycotic agent against Pichia kudriavzevii

doi:10.21203/rs.3.rs-3615679/v1

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Unveiling the role of sulfur nanoparticles as an anti-dermatomycotic agent against Pichia kudriavzevii

https://doi.org/10.21203/rs.3.rs-3615679/v1

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Non-metal nanoparticles are now a days widely applied in different fields. Among various non-metal nanoparticles (NPs), sulfur nanoparticles (SNPs) are one of the most significant and emerging nanoparticles. In this study, sulfur nanoparticles (SNPs) were prepared by eco-friendly method and well characterized by UV-visible spectroscopy, dynamic light scattering, zeta potential, and transmission electron microscopy. The prepared nanoparticles were spherical in shape with mean size of 35 nm and positive surface charge. Here, an attempt has been made to check the in vitro efficacy of sulfur nanoparticles against the dermatomycosis causing fungus Pichia kudriavzevii. Different antimicrobial experiments were performed to check the potential of SNPs against Pichia kudriavzevii. SEM micrographs and growth inhibition assay confirmed the efficacy of SNPs against the studied fungus. The antifungal activity of SNPs evaluated through the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC). The values of MIC were 50µM SNPs for the studied species, and MFC was 250µM for Pichia kudriavzevii. Treatment with SNPs was found to be effective in altering the cell morphology, inhibiting normal growth, and reducing the cell membrane content, mainly ergosterol of Pichia kudriavzevii. The status of present study can further be use to unravel the detailed mechanism of antifungal activity of sulfur nanoparticles against Pichia kudriavzevii for future workers.

Antifungal

Pichia kudriavzevii

Sulfur nanoparticles

Sterol quantitation

The emergence of pathogenic fungi has become a major global public health concern, with fungal infections posing a significant hazard to human health as resistant strains proliferate. Fungal infections are a silent crisis, as decades of antifungal medication prophylaxis and long-term therapeutic use have facilitated the formation of drug-resistant fungi [1]. A study has shown that 150 million people have serious fungal diseases, close to a billion people are believed to have skin, nail, and hair infections, and tens of millions are associated with mucosal candidiasis [2]. One of the opportunistic fungi that is frequently observed, Candida usually causes superficial mucosal infections but has the ability to penetrate tissue, causing infections that are potentially fatal [3]. Candida species are recognised as one of the major agents associated with nosocomial infections in all tertiary care hospitals. In immunocompromised individuals, infections with Candida lead to invasive mycosis like candidemia. Candidemia is a frequent bloodstream infection worldwide and has a high mortality rate [4]. Pichia kudriavzevii, the environmental teleomorph of Candida krusie, is distantly related to other pathogen-infected Candida species. Candida krusie is responsible for 2% of yeast infections in humans and is an anamorphic (asexual reproduction) strain found in 30% of the Amerindian population, acquired from food or the environment [5]. Teleomorph, the sexual reproductive stage of a fungus, occurs, leading to the formation of sexual spores. The sexual reproductive stage of Candida krusie is represented by Pichia kudriavzevii. Pichia kudriavzevii is among the most relevant etiological agents of candidiasis. Candidiasis is a fungal infection that can manifest in various forms, including superficial infections like oral thrush and vaginal infections [6]. It is most often found in patients with haematological malignancies or receiving long-term azole treatment [7, 8]. Pichia kudriavzevii is particularly important because it is less susceptible to common antifungal agents like fluconazole. This reduced susceptibility complicates treatment, especially in immunocompromised patients [9]. It is concerning for healthcare for specific Candida species for appropriate antifungal treatment. It is concerning that the prevalence of Pichia kudriavzevii-associated infections has been reported to be growing in numerous case study reports [10, 11]. Pichia kudriavzevii belongs to the family Pichiaceae and, in the order Saccharomycetales, is a diploid ascomycete yeast. It produces ovoid or elongated ellipsoidal cells, shows dimorphic growth by pseudohyphae growth, and forms floating biofilm [12, 13].

In recent times, nanotechnology has emerged as a promising strategy for the development of new antimicrobial agents. Researchers from all over the world have accepted metal nanoparticles as a medicinal tool that may be employed in the diagnosis and treatment of a variety of ailments. because of the extensive applications of these particles [14]. Among the various nanomaterials are silver, gold, and platinum. Silver nanoparticles are used as novel antimicrobials, gold nanoparticles are used as drug carriers, and platinum nanoparticles are used in dentistry and bone allografts; all three of them are used in various diagnoses and treatments [15]. Sulfur nanoparticles have gained significant attention due to their low cost, eco-friendliness, non-toxicity, and unique physicochemical properties. The aforesaid features have taken sulfur nanoparticles into consideration by different workers in the field of antimicrobial research [14, 16]. Sulfur is a non-metallic element that is found abundantly in nature and has been used for various medicinal purposes, such as in the field of dermatology, maintaining the health of skin, hair, nails, teeth, muscle, bone, and various metabolic processes of the body [17]. Since ancient times, sulfur has been known to have scabicidal, insecticidal, and fungicidal properties [18]. Sulfur has gained a reputation since it is among the most useful medicines known to humans [14]. Sulfur is used alone or in combination with compounds like salicylic acid and sulfa drugs and has demonstrated efficacy for the treatment of various dermatological conditions [19]. In this context, an attempt has been made to check the efficacy of chemically synthesised sulfur nanoparticles on superficial mycosis caused by Pichia kudriavzevii.

2.1. Materials

Sodium thiosulfate was procured from Merck (Mumbai, India). Oxalic acid and cetyltrimethylammonium bromide (CTAB) were procured from SRL (India). Sabouraud dextrose broth (SDB), Sabouraud dextrose agar with chloramphenicol (SDA), and p-iodonitrotetrazolium chloride (INT) or (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium) were procured from Himedia (Mumbai, India). Calcofluor white was used from Sigma Chemicals Co., St. Louis, MO, USA. ‘HiPurA^™ Yeast Genomic DNA Purification Kit’ was purchased from HIMEDIA MolBio^™ (Mumbai, India). The antifungal drugs used for this study were terbinafine, itraconazole, griseofulvin, and fluconazole, purchased from Merck (Mumbai, India). Polystyrene 35-mm radiation-sterile petri plates were purchased from Tarsons (Mumbai, India).

2.2. Characterization synthesized SNPs

Sulfur nanoparticles were chemically synthesised by the acid reduction of sodium thiosulfate (Na₂S₂O₃, 5H₂O) salt in the presence of CTAB surfactant following the established protocol [21]. All chemicals were used as supplied without any further purification. SNPs are characterised through spectrophotometric study (UV-VIS spectrophotometer, LABMAN, India), dynamic light scattering technique (Zetasizer, Ver 7.11, Malvern), and transmission electron microscopy (JEOL, JEM 2100 HR) [22].

2.3. Fungal Culture collection and maintenance

A Pichia kudriavzevii clinical sample was isolated from a sample of patients attending a tertiary care hospital in West Bengal. Culture was maintained in sabouraud dextrose agar (SDA) with chloramphenicol media and stored at 4^°C.

2.4. Fungal DNA isolation, Polymerase Chain Reaction and sequencing

A single colony of the isolated sample was grown overnight in Saboured Dextrose Broth (SDB) media, and culture was used for the extraction of genomic DNA using the HiPurATM yeast genomic DNA purification kit.

The polymerase chain reaction (PCR) of the nuclear ribosomal internal transcribed spacer (nrDNA ITS) was performed by the following method mentioned in Tarafder et al. [23]. The amplified PCR product was purified and analysed by an automated DNA sequencer named the AB13730×1 DNA Analyzer (Applied Biosystems, USA). Identical primers ITS1 and ITS4 [24] were used for this study. The identified nrDNA sequence was used for BLAST searches in the GenBank nucleotide database (https://www.ncbi.nlm.nih.gov) and deposited with a unique accession number.

2.4.1. Dataset representation

The initial BLAST search revealed that the amplified sequences from the obtained material belonged to the Pichia E.C. Hansen, family Saccharomycetaceae G. Winter. So, a combined dataset was prepared comprising of the members of the specimen Pichia kudriavzevii Boidin, Pignal & Besson based on the highest scored hits of BLAST search and samples earlier used in the phylogenetic reconstruction of Pichia [25, 26, 27] Altogether 15 nrDNA ITS sequences were selected for preparing the dataset among which one sequence (viz. Saccharomyces cerevisiae (Desm.) Meyen) represents the outgroup taxa.

2.4.2. Phylogenetic analysis

MAFFT v.7.305 on XSEDE with default parameters as provided in the CIPRES web interface was used to align the dataset with various sequences [28]. In the next step the aligned sequences were manually imported into MEGA v. 6.0 [29] with subsequent additional adjustment. Based on the lowest BIC values, the best fitted nucleotide evolution model for phylogenetic studies was selected using jModeltest 2.1.6 v.20140903 [30] at the CIPRES web portal. Maximum likelihood (ML) analysis was conducted with the stated model (GTR + G; BIC value 7609.486292), as shown by jModeltest 2.1.6, and with 1000 bootstraps replicates in RAxML v.8.2.9 on the CIPRES NSF XSEDE resource. The Bayesian phylogenetic study was conducted using Metropolis-coupled Markov chain Monte Carlo (MCMCMC) technique in MrBayes v.3.2.2, under best BIC model (GTR + G) and other parameters are set to default configuration. For 106 generations, Markov chains were performed and each one in the 100th generation saved a tree. In order to get estimations of posterior probability (PPs) for the group, Mr Bayes was used to calculate 50 percent majority rule for the rest of trees. Bootstrap support (BS) and Bayesian posterior probability estimates are presented in the resultant trees respectively at above 50 per cent and 0.50.

2.4.3. Nucleotide sequence comparison with the closely related taxon

Based on previous publications [25, 26, 27] a total of fifteen well-represented nrDNA ITS sequences were taken from the nuclear database of NCBI GenBank, out of them twelve nrDNA ITS sequences belong to Pichia kudriavzevii Boidin, Pignal & Besson. and matched with the newly amplified taxon sequences, with the help of MAFFT v.7,305 sequence alignment [28].

2.5. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)

The MIC and MFC values of sulphur nanoparticles were determined using a broth microdilution technique with minimal modifications [31]. Briefly, Pichia kudriavzevii was cultured freshly, and 1×10⁵ cells/mL concentration dilutions were made in different tubes. Reactions were performed on a 96-well plate consisting of 200 µL of SDB, 20 µL of inoculum, and various dilutions of SNPs. The plates were then incubated for 24 hours at 37°C. Subsequently, 40 µL of INT dye (0.2 mg/mL) was added to each well and incubated for another 30 min. The concentration that inhibited 90% of the growth of the fungi in comparison with the positive control was calculated as the MIC value. The minimum fungicidal concentration is the lowest concentration of SNPs at which viable fungal cells are absent. To determine the MFC value of SNPs, 100 µL of each well and a positive control were cultured on SDA plates, and the plates were incubated for 48 hours to see the viable cell growth. Both MIC and MFC determinations were performed two times in triplicate.

2.6. Comparative analysis by Agar dilution assay

The antifungal activity of sodium thiosulfate and sulphur nanoparticles was compared by an agar dilution assay [32]. Different aliquots of precursor salt (sodium thio-sulphate) and sulphur nanoparticles were amended with the melted sabouraud dextrose agar (SDA) medium to achieve the desired concentrations (15 mL final volume). Overnight-grown culture of Pichia kudriavzevii was diluted at 1×105 cells/mL, and 100 µL of volume was inoculated in the petri plates. Plates were incubated at 37°C at room temperature for 48 hours. The experiment was carried out twice, in triplicate.

2.7. Fungal growth inhibition assay

The Pichia kudriavzevii strain was sub-cultured in saboured dextrose agar plates, and a single colony was cultured in saboured dextrose broth (SDB) overnight at 37°C with orbital agitation of 200 rpm prior to the main experiment. The freshly prepared autoclaved sterile SDB media in nephelometric flasks were inoculated (1.5×10⁵ cells/mL) with overnight-grown culture for each concentration of SNPs. The culture containing nephelometric flasks (containing 15 mL of the final volume) was incubated at 37^° C with agitation (200 rpm), and growth was monitored for one hour at 600 nm by measuring optical densities (OD) with the help of a “µp Photocolorimeter”. SNP stock was prepared, and SNPs were added when the OD₆₀₀ reached about 0.3. The final concentrations of SNPs were 0 (control), 50 µM, and 100 µM; the optical densities were monitored for 24 hours every two-hour time interval.

2.8. Assessment of membrane damage by Scanning Electron Microscope

SNP-treated membrane damage was visualised by scanning electron microscopy (SEM) [33]. The cells were incubated with SNPs at MIC and 2×MIC concentrations for 24 hours at 37°C. Harvested cells were then washed with PBS and fixed in a glutaraldehyde solution; afterwards, alcohol was dehydrated. The samples were visualised using SEM (ZEISS EVO 18 Special Edition).

2.9. Quantification of total ergosterol

Alterations in ergosterol levels in Pichia kudriavzevii cells upon treatment with SNPs were quantified using a UV-vis spectrophotometer [34]. Cell suspensions at their 1/2×MIC concentrations were prepared and incubated at 37°C for 24 hours in static conditions. The cells were harvested at 2700 rpm (OD₆₀₀ = 1.2) for 5 minutes and washed with distilled water. The fungal cells were suspended in 3 mL of 25% alcoholic potassium hydroxide and vortexed for 1 minute. After one hour of incubation at 80°C, the suspended tubes were then allowed to cool to room temperature. To extract the total sterol content from the cells, 3 mL of n-heptane and 1 mL of distilled water were later added. When a clear layer of n-heptane was visible, the resulting suspensions were continually vortexed for 10 minutes. The transparent layer of heptane was transferred into a new tube, the solution was diluted up to five times with 100% ethanol, and a UV spectrophotometer was used to scan spectrophotometrically between 200 and 300 nm.

2.10. Statistical analysis

All the parameters were repeated at least three times in triplicate. The data was expressed as mean ± SEM, n = 3. Comparisons between the means of the control and treated groups were made by a one-way ANOVA test (using a Statistical Package, Origin 6.1, Northampton, MA) with multiple comparison t tests, with p < 0.05 as a limit of significance.

3.1. Characterization of SNPs

The UV-visible spectra showed a typical absorption band between 240 to 300 nm and a sharp peak was observed at 245 nm (Fig. 1a) without light absorption of visible light above 400 nm. DLS analyses showed the mean hydrodynamic sizes of SNPs at around 38.04 ± 3.96 nm (Fig. 1b) and zeta potential value of SNPs was 34.4 ± 9.28 mV due to cationic capping (Fig. 1c). Average size of SNPs was found to be 35 nm by TEM analysis (Fig. 1d) and spherical in shape (Fig. 1e).

The present results of nanoparticle characterization have confirmed that the synthesis method used to prepare the sulfur nanoparticles was effective. The microscopic examination of prepared nanoparticles confirms the spherical nature with narrow size distribution. The UV-vis results of prepared nanoparticle are in accordance with previous study [35]. The TEM images provide visual evidence of the spherical shape of the nanoparticles, and the uniform size distribution suggests that the synthesised method was reproducible. The DLS study has further provided the confirmation of the particle hydrodynamic size in aqueous solution. The size of the prepared nanoparticles in dried state along with higher positive zeta potential value indicate the stability of the nanoparticles.

3.2. Phylogenetic analyses and nucleotide sequence comparison

Phylogenetic analyses were performed on an ITS nrDNA dataset. All the 16 sequences were aligned and both ends were trimmed to create a dataset of 583 nucleotides that included 301 parsimony informative characters. The phylogenetic tree obtained from ML and MrBayes analyses shows almost similar in topology. Therefore, the tree obtained from ML analysis (-lnL = 3688.7299) has been displayed here (Fig. 2). Bayesian analyses reached a standard deviation of split frequencies of 0.002 after 10⁶ generations and the preliminary 25% trees recovered were excluded. In the phylogenetic tree, altogether, sixteen nrITS sequences representing 3 Genus were used in the analyses and cluster together with strong statistical support values (99% BS and 1.00 PP).

One newly generated sequence of Pichia kudriavzevii Boidin, Pignal & Besson (GenBank accession no. OK299541) falls in a strongly supported clade (100% BS and 1.00 PP) together with the same taxa Pichia kudriavzevii Boidin, Pignal & Besson previously deposited in the GenBank database based on the collections made from Netherlands, China and Japan (GenBank accession no LC389013, JX174414, MH545928). Three sequences of Pichia kudriavzevii, grouped together (100% BS and 1.00 PP) and distinctly similar to the studied sample (Strain: M433; India). Although strains in the same species do not necessarily have identical sequences, the overall sequence similarity of both ITSs 1 and 2 was 99 percent or higher.

Proper identification of this species is essential, which will be more informative for clinicians and different phylogeny tools have been employed for species identification. Phylogenetic studies based on the sequence; partial 26S rDNA, 18S rRNA and ITS1, 5.8S gene have indicated the polyphyletic origin of the genus Pichia. It has been observed that ITS1, 5.8S gene is the best process to differentiate the genus Pichia [36].

3.2. MIC and MFC of SNPs against Pichia kudriavzevii

In the present study, the MIC value of SNPs was checked against Pichia kudriavzevii. The prepared SNPs showed MIC value at 50 µM (Fig. 3). From the experimental results, it was found that MFC value of SNPs was 250 µM. Treatment with 250 µM SNPs caused complete inhibition of growth of tested organism. This concentration was considered as Minimal Fungicidal concentration (MFC) where complete inhibition of growth observed.

This study demonstrated that MIC and MFC values of SNPs against tested sample were different. Broth microdilution and agar dilution assay were consistent with the previous study [37]. Previous study showed that SNPs has antifungal activity against Pichia kudriavzevii at the concentration of 1500 µg/ml [34]. The antifungal activity in respect to concentration was much lower against tested sample organism Pichia kudriavzevii.

3.3. Comparative antifungal activity of precursor salt and sulfur nanoparticles against Pichia kudriavzevii

Elemental sulfur containing salt, sodium thiosulfate which was used as a precursor for the synthesis of SNPs did not show any antifungal activity against clinical sample Pichia kudriavzevii, whereas at 50 µM concentration SNPs exhibit inhibitory effect on the sample organism (Fig. 4).

The results of this study have demonstrated that sulfur nanoparticles are effective in inhibiting the growth of Pichia kudriavzevii, whereas precursor salt unable to inhibit the fungal growth. Previous studies have demonstrated the antimicrobial effects of sodium metabisulfite and elemental sulfur, while sodium thiosulfate has not shown the same activity. [38]. A separate investigation conducted by Kim et al. addressed the antifungal properties of sulfur-containing salts and elemental sulfur [39]. However, their findings showed that the synthesized SNPs exhibited a greater antifungal effect compared to these substances. It is interestingly observed in the results that the precursor sulfur containing salt (sodium thiosulfate) failed to inhibit the growth of the tested organism at MIC and MFC concentration of SNPs.

2.4. Growth inhibition of Pichia kudriavzevii against SNPs

The growth inhibition capacity of SNPs was investigated against Pichia kudriavzevii using growth curve analysis. Normal growth pattern was reduced by SNPs treatment at 50 µM (MIC), and as the concentration of SNPs was increased to 100 µM (2×MIC), Pichia kudriavzevii growth was almost inhibited (Fig. 5). The control set, which did not receive SNPs treatment, exhibited a normal growth pattern. This was confirmed through triplicate experiments performed during the study. In this study, the growth pattern of Pichia kudriavzrvii in liquid media was monitored and compared with growth inhibition effect of different SNPs concentration. Other factors like pH, temperature, time and volume remained constant. Ahmed et al. [40] carried out an earlier experiment on growth inhibition to test the effectiveness of an antifungal component. They measured the length of time and the organism's growth inhibition. According to the findings of the current study, SNPs interfere with development patterns when applied to the model organism Pichia kudriavzevii in a dose-dependent trial. Normal growth in the control group suggests that the effects seen are solely caused by the presence of SNPs and not by changes in the growing environment.

2.5. SNPs induced alterations in the morphology of fungal cells

SEM was used to examine the surface morphological alterations in the Pichia kudriavzevii cells following treatment with 50 and 100 µM SNPs in comparison to untreated control cells (Fig. 6). Pichia kudriavzevii cells’ seemed to have a smooth cell surface and an intact cell wall in the untreated control photos. (Fig. 6A). In contrast, changes in cell structure were noticed after Pichia kudriavzevii was exposed to 50 and 100 µM of SNPs after 24 hours (Fig. 6B & 6C). The SNPs treated sets displayed abnormalities such as localized cell surface invasion, cell shrinkage and rupture of wall material.

It is widely known that yeast cell surface is negatively charged, porous and viscoelastic in nature [41]. Due to anionicness and nanoform, the cationic SNPs caused destructive morphological changes. The degree of morphological alterations increased with the dose of the SNPs, as seen in the SEM micrographs. (Fig. 6). The SEM results are in good agreement with the previous results which shows SNPs inhibitis Pichia kudriavzevii in a dose-dependent manner. Although the exact antifungal mechanism of SNPs is still unknown, there are some additional explanations that have been proposed for fungi and bacteria. The potential mechanisms include interaction of SNPs with bacterial cell surface molecules leads to cell pit and leakage. The generation of sulfur ions for toxic substance like -H₂S leads to ROS generation and interaction of SNPs with cellular lipids leads to lipid reduction. It causes fungal cell death [39, 42, 43].

2.6. Sterol quantitation of SNPs treated Pichia kudriavzevii

This result summarized that Pichia kudriavzevii cell ergosterol content was decreased due to the treatment of SNPs. Pichia kudriavzevii untreated and treated sets total ergosterol content were measured in respect to cell pellet wet mass. We observed that as the treatment at MIC (50 µM) and 2×MIC (100 µM) doses of SNPs the mean decreased of ergosterol content were found to be 92.91% and 93.17%, compared to control or untreated sets. This study represents the quantified total ergosterol content of control and SNPs treated sets of Pichia kudriavzevii (Fig. 7).

Sterols are essential components of eukaryotic cell membranes. Ergosterol being the structural analogue of cholesterol is found in yeast cell membrane. To treat fungal infections many different classes of antifungal agents (allylamines, phenylmorpholines and azole) inhibits ergosterol biosynthesis pathway [40, 44] According to recent study, ergosterol is a biologically active lipid that triggers pyroptosis or the creation of wall spores, which leads to cell exploitation [45]. So, reduction in ergosterol content in sulfur nanoparticles treated set might be associated with one of the mechanisms.

The sodium thiosulfate salt was reduced to produce sulfur nanoparticles (SNPs). The SNPs' reduced particle size offers many benefits, including chemical stability, good conductivity, and antifungal efficacy against Pichia kudriavzevii. The average particle size was 35 nm. In comparative antifungal studies against Pichia kudriavzevii, SNPs outperform conventional antifungal drugs and elemental sulfur. Ergosterol, the primary component of the fungal cell membrane, was inhibited by the SNPs in a dose-dependent manner, interfering with proper growth. Cell membrane rupture, cell wall pitting, and invagination were all visible in the SEM image. To our knowledge, this is the only study looking at how sulfur nanoparticles affect Pichia kudriavzevii. In Pichia kudriavzevii treated with SNPs, the ergosterol content of the cells was inhibited, which may indicate antifungal action that is linked to the reduction of ergosterol biosynthesis. SNPs can be used as an efficient antifungal agent against Pichia kudriavzevii, it can be inferred from the findings of the current investigation. Further investigations are required to dissect more details of the antifungal potential of sulfur nanoparticles against Pichia kudriavzevii.

Declaration of competing interest

All authors state that there is no conflict of interest.

Author Contribution

Mangal Chandra Biswas, Jit Sarkar, and Khushi Mukherjee wrote the main manuscript text. Mangal Chandra Biswas prepared figures 1, 3, 4,6, and 7. Jit Sarkar prepared figures 2 and 5. Reena Ray (Ghosh) helped with the design of the manuscript. Krishnendu Acharya edited the manuscript and supervised the work. All authors reviewed the manuscript.

Acknowledgment

This work was supported by the University Grants Commission of India (UGC) under Grant [June 18-344924].

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Unveiling the role of sulfur nanoparticles as an anti-dermatomycotic agent against Pichia kudriavzevii

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Materials

2.2. Characterization synthesized SNPs

2.3. Fungal Culture collection and maintenance

2.4. Fungal DNA isolation, Polymerase Chain Reaction and sequencing

2.4.1. Dataset representation

2.4.2. Phylogenetic analysis

2.4.3. Nucleotide sequence comparison with the closely related taxon

2.5. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)

2.6. Comparative analysis by Agar dilution assay

2.7. Fungal growth inhibition assay

2.8. Assessment of membrane damage by Scanning Electron Microscope

2.9. Quantification of total ergosterol

2.10. Statistical analysis

3. Results and discussion

3.1. Characterization of SNPs

3.2. Phylogenetic analyses and nucleotide sequence comparison

3.2. MIC and MFC of SNPs against Pichia kudriavzevii

3.3. Comparative antifungal activity of precursor salt and sulfur nanoparticles against Pichia kudriavzevii

2.4. Growth inhibition of Pichia kudriavzevii against SNPs

2.5. SNPs induced alterations in the morphology of fungal cells

2.6. Sterol quantitation of SNPs treated Pichia kudriavzevii

4. Conclusions

Declarations

Declaration of competing interest

Author Contribution

Acknowledgment

References

Additional Declarations

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