Insights into the degradation of fluconazole in water by some light-based AOPs

Occurrence, seasonal variability, and ecological risk of emerging contaminants in estuarine and nearshore waters around Hainan Island, China.

This study investigates the repurposing potential of the anesthetic ketamine (KET) as a preventive agent, both alone and in combination with the azoles fluconazole (FLC) and itraconazole (ITR), against Candida spp. biofilms adhering to fragments of peripheral venous catheters. The activity was assessed in vitro through cell viability and colonization assays of impregnated catheter segments, while morphological alterations were analyzed using scanning electron microscopy (SEM). KET, both alone and in association with ITR, significantly reduced (p < 0.05) the adherence of C. albicans to the impregnated fragments. Furthermore, the combinations KET + FLC and KET + ITR frequently exhibited greater efficacy in reducing biofilm viability than the agents used individually, suggesting additive/synergistic interactions. SEM revealed structural damage to the treated fungal cells. These findings indicate that KET exhibits preventive activity against Candida spp. biofilms, including on catheter surfaces, and may enhance the activity of azoles, positioning it as a promising candidate for repurposing in the treatment of biofilm-associated fungal infections.

The transcriptional upregulation of drug efflux pumps constitutes a primary mechanism of azole resistance acquired by diverse fungal pathogens. This overexpression diminishes intracellular azole accumulation, thereby compromising drug efficacy and contributing to therapeutic failure. The coadministration of azole antifungals with efflux pump inhibitors presents a promising therapeutic strategy to combat resistant fungal infections. In the search for novel antifungal drugs, we obtained 49 isoxazoline derivatives through structural modification of the isoxazolin-3-one skeleton. Among them, 6ao showed potent synergistic activity with fluconazole (FLC) against azole-resistant Candida albicans (FICI = 0.094). In the Galleria mellonella larvae and mice model, 6ao exhibited potent in vivo therapeutic efficacy. Interestingly, mechanistic investigations revealed that 6ao, when combined with FLC, reduced the expression of MDR1, an efflux pump gene, in the multidrug-resistant clinical isolate CA632. Taken together, these results identify 6ao as a promising lead candidate for combating azole-resistant Candida infections.

Introduction. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used for rapid micro-organism identification and has recently been explored for antifungal susceptibility testing (AFST).Hypothesis. Although MALDI-TOF MS has emerged as a promising tool for AFST, simplified and clinically applicable strategies for rapid fluconazole (FLZ) susceptibility detection in Candida spp. remain insufficiently validated. We hypothesized that a streamlined AFST-MS approach would demonstrate good categorical agreement (CA) with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method while significantly reducing turnaround time.Aim. To establish a simplified MALDI-TOF MS-based AFST approach for detecting FLZ resistance in Candida species.Methodology. Fifty-one clinical isolates and reference strains were incubated for 3 h in the presence of FLZ at two concentrations (32 and 4 µg ml−1) and in drug-free controls. Spectral profiles were compared with the EUCAST reference method.Results. Overall CA between AFST-MS and EUCAST was 85.2% (κ=0.7306). Species-specific accuracy was 100% for Candida auris, Pichia kudriavzevii (formerly Candida krusei), Candida tropicalis and Candida parapsilosis; 92.9% for Candida albicans and 40% for Nakaseomyces glabrata (formerly Candida glabrata); however, these estimates should be interpreted cautiously given the limited number of isolates per species. All discrepancies were minor errors, with no major or very major errors observed. The method reduced analysis time from 24 to 3 h and enabled presumptive FLZ susceptibility detection with good overall agreement with the reference methodConclusion. These findings support the potential of MALDI-TOF MS as a rapid adjunct tool for antifungal susceptibility assessment and may contribute to earlier therapeutic decision-making.

Azole resistance in Candida albicans is an increasing clinical challenge. Upc2 is a key transcription factor regulating ergosterol biosynthesis, but its additional roles in azole tolerance remain unclear. This study investigated whether Upc2 contributes to azole resistance through pathways beyond ergosterol synthesis. Chemical sensitivity screening, RNA sequencing, flow cytometry, and molecular assays were performed to compare wild-type C. albicans and the upc2Δ/upc2Δ mutant under fluconazole (FLC) treatment. The UPC2 gene deletion affected physiological processes that are dependent on the calcineurin signaling pathway and led to an overall negative enrichment trend in the unfolded protein response (UPR) pathway gene set. Mechanistically, the UPC2 gene deletion impaired unconventional splicing of HAC1 mRNA, leading to accumulation of unfolded proteins and phenotypically its deletion enhanced sensitivity of C. albicans to FLC in planktonic growth, hyphal development, and biofilm formation. Our findings reveal that Upc2 regulates proteostasis in C. albicans, and its absence enhances FLC efficacy by disrupting the UPR pathway. Targeting Upc2-mediated UPR signaling may represent a promising strategy to combat azole resistance.

The emerging threat of drug-resistant Candida albicans, especially in recurrent vulvovaginal candidiasis (VVC) biofilms, has revealed weaknesses in existing monotherapeutic antifungal treatments. In this study, we investigated the antifungal activity of a new dual-drug in situ mucoadhesive gel system containing acarbose (AC), a pseudo-oligosaccharide sugar and FDA-approved α-glucosidase inhibitor, together with fluconazole (FLC). Although AC showed mild antifungal activity by itself, when combined with FLC, it dramatically potentiated fungicidal activity, disrupted biofilms, blocked morphogenetic switching, and arrested post-treatment hyphal regrowth. In checkerboard assays and time-kill kinetics, AC-FLC combination was highly synergistic against Candida spp., including clinical isolates, with > 2-log₁₀ decrease in CFU/mL. The thermosensitive gel (PCH16), formulated using poloxamer 407 along with the carbohydrate-based polymers chitosan and HPMC, exhibited sustained drug release, favorable rheological properties, and strong mucoadhesion suitable for vaginal delivery. Notably, the formulation retained Lactobacillus species viability, suggesting microbiome compatibility. These results affirm the promise of repurposing acarbose as a novel, non-traditional antifungal adjuvant that, in co-administration with FLC in vaginal gel, represents an encouraging, localized, and microbiota-conserving therapy for treating recurrent and resistant VVC.

Metastable phase diagram, mobility, and kinetic stability of amorphous mixtures of two mutually compatible APIs.

ABSTRACTBackgroundNakaseomyces species, particularly Nakaseomyces glabratus and Pichia kudriavzevii, pose remarkable treatment challenges due to their resistance to common antifungal agents like fluconazole (FCZ). Essential oils, such as Cinnamomum cassia essential oil (CC‐EO), offer a promising alternative, especially when enhanced with nanotechnology to improve stability and bioavailability.AimThis study aims to evaluate the antifungal efficacy of CC‐EO and its nano‐encapsulated form (nano‐CC‐EO) against Nakaseomyces spp. to explore their potential as alternative treatments for resistant candidiasis.MethodsTen clinical isolates of N. glabratus and P. kudriavzevii were tested using CC‐EO and nano‐CC‐EO, which were prepared via nanoprecipitation. Antifungal efficacy was evaluated through disk diffusion, microdilution and checkerboard assays, whereas nano‐CC‐EO's physicochemical properties were characterized using dynamic light scattering, scanning electron microscopy and zeta potential analysis.ResultsCharacterization of nano‐CC‐EO showed an average particle size of 174.8 nm and a stable zeta potential of −18.6 mV, confirming uniformity and stability. In disk diffusion screening, nano‐CC‐EO produced larger inhibition zones than CC‐EO at 2000 ppm, providing a qualitative indication of stronger growth inhibition. Microdilution tests revealed minimum inhibitory concentration (MIC) values of 250–500 ppm for nano‐CC‐EO and 125–500 ppm for CC‐EO, highlighting strong inhibitory effects against Nakaseomyces isolates. Checkerboard assays demonstrated synergistic and additive interactions between nano‐CC‐EO and FCZ, with several isolates showing fractional inhibitory concentration index (FICI) values below 0.5.ConclusionThis study highlights the enhanced antifungal potential of nano‐CC‐EO, offering a promising alternative for treating resistant Nakaseomyces infections. Nano‐CC‐EO formulations demonstrated superior efficacy and targeted delivery, suggesting their potential as effective natural antifungal agents.

Antifungal drug resistance is a growing global health concern, yet treatment options remain limited to just four classes: polyenes, azoles, allylamines, and echinocandins. Cryptococcus species are responsible for life-threatening meningoencephalitis and pulmonary infections, which cause over 140,000 deaths annually. The high mortality of Cryptococcus is driven by limited drug access, toxicity, persistence, and emerging resistance. To better understand antifungal resistance and identify novel therapeutics, we profiled a genetically diverse panel of Cryptococcus isolates spanning all major lineages (VNI, VNIII, VNIV, VGI-VGVI). Phenotypic assays were used to assess susceptibility to fluconazole (FLZ), 5-fluorocytosine (5-FC), and amphotericin B (AmpB), as well as the epigenetic inhibitors CPTH2 (a histone acetyltransferase inhibitor) and SAHA (a histone deacetylase inhibitor). We found lineage- and strain-specific variation in antifungal susceptibility, with disc-diffusion and microdilution assays yielding consistent patterns. Pharmacodynamic modelling revealed potent fungicidal activity of CPTH2 across all tested lineages. DNA content and chitin levels varied significantly by lineage and treatment: FLZ and CPTH2 induced DNA content increases in susceptible strains, suggesting a stress response, while 5-FC-induced chitin remodelling was most pronounced in resistant strains. Multivariate analysis identified chitin induction as a principal driver of phenotypic variance under antifungal stress. Our findings reveal lineage-specific antifungal responses in Cryptococcus, highlight the antifungal potential of epigenetic inhibitors such as CPTH2, and underscore the utility of phenotypic profiling to inform drug development and resistance surveillance.

Fluconazole (FZL) is a broad-spectrum antifungal drug associated with certain serious adverse effects such as polymorphic ventricular arrhythmia due to QT prolongation. Torsade de Pointes (TdP) is a unique type of polymorphic ventricular arrhythmia due to QT prolongation. It has been shown that the calcium-channel blocker verapamil is effective in the management of TdP. Therefore, the present critical review aims to discuss and explain the possible role of verapamil in the prevention of FZL-induced TdP. Databases of Scopus, Cochran, Embase, PubMed, and CENTRAL were examined to recognize suitable publications. Screened articles were designated rendering to exact eligibility criteria including original articles such as prospective and retrospective studies recognizing the role verapamil in the prevention of FZL-induced TdP. The use of FZL is linked with the development of TdP and prolongation of QT, and verapamil use could be effective in the management of TdP. In addition, verapamil has antifungal effects, affects the pharmacokinetics and pharmacodynamics of FZL. Verapamil inhibits the propagation of TdP by inhibiting the deregulation of repolarization early after depolarization in the heart. This critical review suggests that combination of verapamil with FZL leads to more beneficial effects by increasing the antifungal activity of FZL with a significant reduction the development of TdP which is a serious adverse effect of FZL.

Introduction. Candida spp. are opportunistic pathogens frequently associated with nosocomial infections, and their incidence in urinary tract infections (UTIs) has increased significantly in recent decades, representing a serious public health concern.Hypothesis/Gap Statement. While fungal UTIs are gaining attention, there is a lack of comparative studies linking gender and specific niches to the pathogenic potential and antifungal resistance of Candida isolates in the genitourinary tract.Aim. The aim of this study is to characterize Candida spp. isolated from the urinary tract by investigating their incidence, antifungal resistance patterns and phenotypic virulence factors - including biofilm formation, germ tube production and adhesion to human epithelial cells - while specifically comparing these characteristics between male and female patients.Methodology. A total of 37 urinary isolates were collected from patients at a Portuguese public hospital between December 2022 and June 2023. Antifungal susceptibility to fluconazole (FLC) and clotrimazole (CLT) was determined via European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution. Virulence was assessed through crystal violet biofilm biomass quantification, germ tube formation assays for Candida albicans and adhesion assays using HeLa cells.Results. C. albicans was the most prevalent species, while Nakaseomyces glabratus was exclusively found in female patients (30%). All isolates were susceptible to FLC (MIC ≤2 µg ml-1) and CLT (MIC ≤1 µg ml-1). High biofilm biomass was particularly noted in non-albicans species and isolates from hospitalized patients. While isolates from male patients exhibited higher germ tube formation (P<0.05), those from female patients demonstrated a potentially greater capacity for adhesion to HeLa cells (P<0.05).Conclusion. Candida isolates from the urinary tract demonstrate a potential virulence trait that varies by patient gender and clinical setting. The findings suggest that hospitalized female patients and elderly patients harbour isolates with greater pathogenic potential, highlighting the need for continuous epidemiological surveillance to improve UTI diagnosis and treatment.

Fungal infections are combatted using three main classes of antifungals, of which the azoles, considered to be fungistatic, are the most widely used. Slow growth of Candida albicans at supra-minimal inhibitory concentrations (MIC) of fluconazole (FLC), termed tolerance, is routinely observed. A combination therapy resulting in the eradication of this fungistatic character would be a valid therapeutic strategy, and indeed, the synergistic combination of the antibiotic doxycycline and FLC has such an effect. We hypothesized that iron-requiring mitochondrial functions may be the targets of the synergistic combination. The proteome enriched for mitochondria obtained from FLC + Fe-treated cells hinted that iron alleviated the FLC stress and that intracellular iron homeostasis, more specifically the vacuolar iron exporter Smf3, might be a key factor during FLC treatment, as its expression was induced. Moreover, a ROS assay revealed that a smf3Δ/Δ strain treated with FLC accumulated ROS to a similar extent as that displayed by the WT undergoing a FLC+DOX combination treatment. Thus, deletion of SMF3 mimics the addition of doxycycline in wild-type cells. The ROS accumulation can be attenuated through overexpression of the mitochondrial superoxide dismutase SOD2, and this restored the synergy between DOX and FLC in the smf3Δ/Δ background. ROS accumulation, in part through altered iron availability from the vacuolar storage pool, is thus the molecular mechanism underlying the synergy between doxycycline and FLC. Furthermore, no effect on either cidality or tolerance was observed in the smf3Δ/Δ strain, highlighting that synergy is not necessarily an indication of cidal therapies.IMPORTANCEAzoles are widely used against Candida albicans, yet many cells survive above the minimal inhibitory concentrations (MIC) by growing slowly, which can prolong infection and foster resistance. We show that intracellular iron homeostasis alters the fluconazole characteristics by affecting ROS accumulation in mitochondria, and that this is the molecular mechanism underlying the combination therapy of fluconazole and doxycycline. These results place iron release from the vacuole at the center of azole responses, suggesting novel ways to boost azole efficacy.

The rise of resistant Candida albicans (C. albicans) poses a significant challenge to fluconazole (FCZ) therapy. Restoring FCZ sensitivity offers a therapeutic strategy for resistant isolates. Atractylodin (ATL), a polyacetylene fromAtractylodis Rhizoma, possesses antifungal activity. This study aimed to investigate the synergistic efficacy of ATL and FCZ against drug-resistant C. albicans and explore the underlying mechanisms. Fractional inhibitory concentration index (FICI) for synergy evaluation, scanning electron microscopy, and reactive oxygen species (ROS) production were conducted to investigate the antifungal effect on FCZ-resistant C. albicans 04. Transcriptome sequencing was performed to confirm the differentially expressed genes between ATL + FCZ and FCZ treatment. Meanwhile, the effect of ATL and FCZ combination on the virulence factors was explored through hyphae formation and antibiofilm experiment. Microbial metabolomics was used to identify the mechanism of the synergistic biofilm inhibition. ATL had significant synergistic effect with FCZ against FCZ-resistant C. albicans, and reduced the MICs of FCZ by 32 or 64 times, with FICI < 0.5. The ATL and FCZ combination caused damage to C. albicans cells and significantly increased ROS levels. Transcriptome sequencing also revealed that the gene involved in ROS inculding SOD2 and HSP90 were upregulated, while cell wall-related genes CHS7 and CWH8 were downregulated. Meanwhile, ATL strongly synergized with FCZ against hyphae and biofilm formation of FCZ resistant C. albicans. Metabolomics revealed that key metabolites (e.g., glucose 6-phosphate, isocitric acid, most lipid species, leucylproline, 3'-adenylic acid) were significantly downregulated in the ATL + FCZ combination group versus control and monotherapies. In conclusion, we demonstrated for the first time that the combination of ATL and FCZ may produce a synergistic antifungal effect against FCZ-resistantC. albicans, potentially through the induction of ROS accumulation. The alterations of metabolites mainly involved in glycolysis, TCA cycle, amino acids, lipid, and nucleotide metabolism may be responsible for the inhibition of C. albicans biofilm formation. ATL exhibited a strong synergist with FCZ against C. albicans, highlighting the potential of ATL as a sensitizer in clinical antifungal therapy.

Chronic wounds, burns and ulceration of dermal and mucosal tissues are extremely common and can arise for a wide variety of reasons causing extreme pain and reducing patient quality of life. Current treatment regimens involve the use of topical corticosteroids for prolonged treatment periods. Due to issues surrounding the use of topical ointments there is inadequate drug contact with the wound site and non-specific tissue interaction, potentially leading to significant development of fungal infections as a side effect to corticosteroid treatment. Medium chain length (MCL) and short chain length (SCL) polyhydroxyalkanoates (PHAs) may be applicable to optimise material properties for wound dressing applications. Initial work focussed on defining the optimal electrospinning parameters for suitably elastic fibres whilst subsequent work focussed on achieving an optimised dosing of clobetasol propionate (CP) and fluconazole (FLU) for incorporation with the electrospun fibres without detrimentally compromising the properties of the scaffolds for wound healing applications. Physical and mechanical analysis showed that the 80:20 blend of MCL:SCL polymer at an electrospinning solution concentration of 10% (w/v) gave defect-free fibres with the best elastic properties for wound dressing applications. CP and FLU incorporation into the electrospun fibres did not cause any significant decrease in oral mucosal cell viability. Following in vitro wound healing study promising formulations containing 2% and 10% CP and FLU, respectively, were identified.

The rapid growth of fungal infections caused by Candida spp. associated with antimicrobial resistance requiresthe developmentof new therapeutic approaches to combat these infections. Campheneis a compound derived from the essential oils of various plants andhas antimicrobial properties. Based on this, the objective of thisstudy was to carry out the late-stage diversification of campheneby means of highly stereoselective Heck–Matsuda (HM) arylationand to evaluate the antifungal activity of these derivatives against Candida spp. The reaction provided direct access to sevenaryl-camphene derivatives (adducts HM 3a-g) with good to excellent yields (48–98%) in short reactiontimes under moderate and aerobic conditions. For the antifungal tests,camphene and the HM adducts 3a and 3b, withthe highest yields, were selected. Of the compounds, camphene showedthe best intrinsic antifungal activity, outperforming fluconazole(FCZ) againstC. albicans. In associationwith FCZ, camphene and HM adduct 3a significantly potentiatedthe drug’s action, resulting in a notable reduction in theaverage inhibitory concentration (IC50). Tests to determinethe minimum fungicidal concentration showed that only the combinationof camphene and FCZ showed fungicidal action againstC. albicans. ForC. tropicalis, there was fungicidal activity with the combinations of campheneand FCZ, and HM adduct 3a and FCZ. The morphologicaltransition from yeast to hyphae and/or pseudohyphae inC. albicans was inhibited only by camphene. InC. tropicalis, filamentation was completely inhibitedby the action of camphene and its derivatives at the concentrationsevaluated. Taken together, these results suggest that camphene andits HM adducts (3a–b) evaluated maybe promising candidates for the development of antifungal agents,especially in combination therapies with fluconazole.

Ethanolic extract of Withania somnifera (EEWS) augments the activity of fluconazole against drug-resistant vulvovaginal candidiasis

The incidence of infections by non-albicans Candida species, including Pichia kudriavzevii (formerly Candida krusei) has been increasing. P. kudriavzevii exhibits intrinsic resistance to fluconazole (FLU) and rapidly develops acquired resistance to other antifungals. However, large-scale epidemiological, antifungal susceptibility, and genotyping data from mainland China remain limited. We analyzed the clinical and laboratory data of 203 P. kudriavzevii isolates spanning from 2017 to 2023. In vitro antifungal susceptibility testing was performed by the Sensititre YeastOne microdilution method. The genotyping of P. kudriavzevii isolates was analysed by multilocus sequence typing (MLST). The results showed that P. kudriavzevii were predominantly isolated from sputum (132, 65.02%) and urine (21, 10.34%). Patients with P. kudriavzevii isolation were mainly from the Infectious Diseases Department (50, 24.63%), Intensive Care Unit (lCU) (45, 22.17%) and General Surgery (32, 15.76%), and the pathogen was highly prevalent in individuals over 60years old. All isolates exhibited intrinsic resistance to FLU. The resistance rate to voriconazole (VOR) was 9.4% (19/203), showing an increasing trend. 80 isolates (39.41%) were non-wild-type (NWT) to amphotericin B (AMB) and posaconazole (POS). Excluding FLU, 31 isolates (15.27%) exhibited a resistant or NWT phenotype to at least two distinct antifungal agent classes, and the MLST analysis of them revealed 16 distinct diploid sequence types (DST) profiles. Among these, ST195 (7, 22.6%) and ST268 (6, 19.4%) were the predominant genotypes associated with antifungal resistance, and 58.1% of the resistant sequence types exhibited resistance to VOR. Continuous monitoring of antifungal susceptibility and molecular typing is essential. This study provides valuable insights for managing P. kudriavzevii infections and guiding clinical treatment strategies.

Background: Given the escalating resistance of Candida albicans biofilms to conventional antifungal agents, in this research, we evaluated the potential of combination therapy comprising hinokitiol (HNK) and fluconazole (FLZ), as an alternative, for combating biofilm-associated infections. Methods: To assess their synergistic effects, the fractional inhibitory concentration index was calculated. The viability of biofilms and intracellular oxidative stress were quantified by XTT and reactive oxygen species assays. Structural integrity and viability were further investigated by fluorescence and scanning electron microscopy; gene expression levels were measured by real-time fluorescence analysis. Results: Treatment with HNK combined with FLZ led to noticeable disruptions in the biofilm architecture of Candida albicans and resulted in significant modulation of multiple biofilm-associated genes, such as ALS3 and HWP1. The observed increase in oxidative stress, together with a marked decline in biofilm viability, provided strong support for a synergistic effect between the two compounds. Conclusions: The findings in this study revealed a potential synergistic effect between HNK and FLZ, with influences on fungal biofilm architecture and modulation of genetic pathways associated with biofilm development.

Fluconazole (FLC)-resistant Candidozyma auris isolates have reduced drug accumulation compared to azole-susceptible isolates. Of 119 C. auris isolates, 83 out of 87 resistant isolates (~95%) had extremely low fluconazole uptake, whereas 30 out of 32 susceptible isolates (~93%) had high fluconazole uptake. In search of a genetic explanation for this phenomenon, we compared metadata for TAC1B and CDR1 single-nucleotide polymorphisms (SNPs) and found overlap with many but not all isolates that are FLC resistant. We found that CDR1 mutations are common in resistant isolates from Clade 1, and TAC1B mutations are commonly found in resistant isolates from clades 1 and 3. There is clearly an association between FLC resistance and certain CDR1 and TAC1B polymorphisms, but mutations in these genes do not account for all mechanisms of resistance in this species and do not account for the difference in FLC accumulation. However, when ERG11 SNPs were included in the analysis, there is a clear correlation between low FLC accumulation and isolates that have one of five ERG11 variants and also high FLC accumulation and isolates that have non-variant ERG11 sequences. The ERG11 mutations F126L, K143R, V125/F126L, Y132F, or Y501H are correlated to fluconazole resistance and reduced fluconazole accumulation. This is a unique characteristic of C. auris, suggesting mutations in ERG11 can cause changes in the ergosterol biosynthesis pathway and membrane composition, organization, and permeability.IMPORTANCECandidozyma auris is a global human health threat because of its near-universal resistance to the antifungal fluconazole as well as a predisposition to multidrug resistance among clinical isolates. The underlying mechanisms of antifungal drug resistance in this species are still largely under investigation, and these efforts are significantly supported by research that increase our understanding of unique aspects of C. auris biology. We have identified a correlation between C. auris isolates' susceptibility to fluconazole and intracellular drug accumulation in which drug-resistant isolates have significantly reduced intracellular fluconazole compared to isolates that are susceptible to fluconazole. We have proposed a mechanism for this phenomenon and demonstrated important roles for mutations in ERG11, TAC1B, and CDR1 gene sequences for drug resistance.