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Rezafungin is a long-acting, intravenously administered echinocandin for the treatment of candidemia and invasive candidiasis (IC). Non-inferiority of rezafungin vs caspofungin for the treatment of adults with candidemia and/or IC was demonstrated in the Phase 3 ReSTORE study based on the primary endpoints of day 14 global cure and 30-day all-cause mortality. Here, an analysis of ReSTORE data evaluating efficacy outcomes by baseline Candida species is described. Susceptibility testing was performed for Candida species using the Clinical and Laboratory Standards Institute reference broth microdilution method. There were 93 patients in the modified intent-to-treat population who received rezafungin; 94 received caspofungin. Baseline Candida species distribution was similar in the two treatment groups; C. albicans (occurring in 41.9% and 42.6% of patients in the rezafungin and caspofungin groups, respectively), C. glabrata (25.8% and 26.6%), and C. tropicalis (21.5% and 18.1%) were the most common pathogens. Rates of global cure and mycological eradication at day 14 and day 30 all-cause mortality by Candida species were comparable in the rezafungin and caspofungin treatment groups and did not appear to be impacted by minimal inhibitory concentration (MIC) values for either rezafungin or caspofungin. Two patients had baseline isolates with non-susceptible MIC values (both in the rezafungin group: one non-susceptible to rezafungin and one to caspofungin, classified as intermediate); both were candidemia-only patients in whom rezafungin treatment was successful based on the day 30 all-cause mortality endpoint. This analysis of ReSTORE demonstrated the efficacy of rezafungin for candidemia and IC in patients infected with a variety of Candida species.

Epithelial-immune cell communication is pivotal to control microbial infections. We show that glycosylphosphatidylinositol-linked aspartyl proteases (Yapsins) of the human opportunistic pathogenic yeast Candida glabrata (Cg) thwart epithelial cell (EC)-neutrophil signalling by targeting the EC protein, Arpc1B (actin nucleator Arp2/3 complex subunit), which leads to actin disassembly and impeded IL-8 secretion by ECs. Further, the diminished IL-8 secretion inhibits neutrophil migration, and protects Cg from the neutrophil-mediated killing. CgYapsin-dependent Arpc1B degradation requires Arginine-142 in Arpc1B, and leads to reduced Arpc1B-p38 MAPK interaction and downregulated p38 signalling. Consistently, Arpc1B or p38 deletion promotes survival of the Cg aspartyl protease-deficient mutant in ECs. Importantly, kidneys of the protease-deficient mutant-infected mice display elevated immune cell infiltration and cytokine secretion, implicating CgYapsins in immune response suppression in vivo. Besides delineating Cg-EC interplay, our results uncover a novel target, Arpc1B, that pathogens attack to constrain the host signalling networks, and link Arpc1B mechanistically with p38 activation. Synopsis: Candida glabrata (Cg) suppresses IL-8 secretion to escape from neutrophil killing. Cg engages cell surface-associated aspartyl proteases to target Arpc1B, a subunit of the Arp2/3 complex, to restrain p38 MAPK activation and IL-8 secretion. The Yapsin family of cell surface-associated aspartyl proteases targets Arpc1B in host epithelial cells. Arpc1B degradation inhibits p38 signalling activation and IL-8 secretion. Reduced IL-8 levels hamper neutrophil migration towards Candida glabrata. Candida glabrata (Cg) suppresses IL-8 secretion to escape from neutrophil killing. Cg engages cell surface-associated aspartyl proteases to target Arpc1B, a subunit of the Arp2/3 complex to restrain p38 MAPK activation and IL-8 secretion. [ABSTRACT FROM AUTHOR]

Background: Candida glabrata is the second most common cause of invasive candidiasis worldwide. In this study, we determined the clinical characteristics and drug sensitivity of C. glabrata isolates and investigated the associations between MSH2 gene mutations, sequence types (ST), and drug resistance. Methods: A total of 154 C. glabrata isolates were collected from patients being treated in three hospitals in China. The antifungal sensitivity of the strains was assessed using the broth microdilution method. Multilocus sequence typing (MLST) was also performed, followed by MSH2 sequencing. The clinical features and outcomes of C. glabrata infection were analysed for a total of 49 strains, which were collected from patients with invasive Candida infection at Longhua Hospital. Results: All 154 isolates were found to be susceptible to amphotericin, 5-fluorocytosine, anidulafungin, caspofungin, and micafungin, whereas 11.7% were fluconazole-resistant, 18.8% were itraconazole non-wild type, and 35.7% were voriconazole non-wild type. ST7 (62.34%) was the most common ST genotype, followed by ST10 (16.88%) and ST15 (7.79%). The total azole resistance rates for all isolates, ST7, ST10, and other STs were 36.4, 42.7, 34.6, and 18.8%, respectively. The ST7 and ST10 isolates were characterised by a higher drug resistance rate than the other minor ST isolates. Moreover, 59.09% of isolates had one or more MSH2 non-synonymous mutations, with V239L being the most commonly detected mutation. The frequency of MSH2 mutations was significantly higher in azole-resistant isolates than in other isolates, whereas P6L or L87P mutations were associated with the highest azole resistance rates of up to 87.5% and 80%, respectively. Our results indicated that ST7 and ST15 are independent predictors of mortality caused by C. glabrata infection and revealed a higher 30-day mortality in patients infected with these strains than in those infected with other ST isolates. Conclusions: Our findings revealed the relationships between MLST, MSH2 gene mutations, and drug resistance in the common pathogenic fungus C. glabrata, and thereby enabled us to identify strains that are associated with higher rates of mortality. These findings will contribute to enhancing our understanding of the pathogenesis of C. glabrata infection. [ABSTRACT FROM AUTHOR]

Background: Menthol, a natural quorum sensing molecule, is derived from the Mentha species. Combating pathogenicity by inactivating quorum sensing is an emerging approach. Therefore, our objective was to investigate anti-quorum sensing and anti-biofilm potentials of menthol in Candida albicans and Candida glabrata. Methods: The antifungal properties of menthol were evaluated using a broth microdilution assay and a time-kill assay, and its effects on quorum sensing-mediated virulence factors, cellular reactive oxygen species (ROS), and biofilm formation were tested by evaluating TUP1 expression levels in both C. albicans and C. glabrata. Results: Quorum sensing-mediated virulence factors and biofilm formation were inhibited by menthol in both C. albicans and C. glabrata. Furthermore, coinciding with elevated ROS levels, mRNAs of the quorum sensing-related gene TUP1 were upregulated in both C. albicans and C. glabrata. Conclusions: This study highlights the anti-quorum sensing potential of menthol through the inhibition of quorum sensing-mediated virulence factors, ROS generation, and biofilm development by targeting TUP1, which could have potential in the treatment of Candida infections. [ABSTRACT FROM AUTHOR]

Background: The increasing incidence and highmortality rate of Candida glabrata infection in ICU patients is an important issue. Therefore, it is imperative to investigate the antifungal susceptibility profiles and epidemiological characteristics in local regions. Methods: Herein, antifungal susceptibility testing was conducted to determine the minimum inhibitory concentrations (MICs) of eight antifungal drugs. Multilocus sequence typing (MLST) was used to study the strain genotype, geographical distribution, and susceptibility to antifungal agents among C. glabrata isolates. The mechanism of echinocandin resistance was explored by sequencing the FKS1 and FKS2 genes (encoding 1,3-b-D-glucan synthases) of echinocandin-resistant C. glabrata strains. Moreover, we further investigated the clinical manifestations and the various risk factors of patients infected with C. glabrata in the ICU. Results: We selected 234 C. glabrata isolates from 234 patients in the ICU randomly for the follow-up study. Cross-resistance was found among the ICU C. glabrata isolates. Analysis using MLST showed that the genetic diversity among the C. glabrata isolates was low. Furthermore, sequence type showed no correlation with the antifungal resistance profiles, but was associated with geographical distribution. We also revealed novel mutations in FKS1 (S629P) and FKS2 (W1497stop) that mediated high-level echinocandin resistance (MIC >8 µg/mL). More than 14 days' stay in ICU (P=0.007), Acute Physiology and Chronic Health Evaluation II (APACHE-II) score (P=0.024), prior antifungal exposure (P=0.039) and lung disease (P=0.036) were significantly associated with antifungal resistant/non-wild-type C. glabrata infection. Conclusion: Our study shed light on the antifungal susceptibility, molecular epidemiology, and clinical risk factors of C. glabrata in the ICU of a Chinese Tertiary Hospital. Importantly, we revealed the molecular mechanism of echinocandin resistance. These results highlight the significance of continued surveillance in ICUs and provide data support for the treatment of C. glabrata in clinics. [ABSTRACT FROM AUTHOR]

Introduction: With the growing population of immunocompromised individuals, opportunistic fungal pathogens pose a global health threat. Candida species, particularly C. albicans and non-albicans Candida species such as C. glabrata, are the most prevalent pathogenic fungi. Azoles, especially fluconazole, are widely used therapeutic options. Objective: This study investigates how C. glabrata adapts to fluconazole, with a focus on understanding the factors regulating fluconazole tolerance and its relationship to resistance. Methods: This study compared the factors regulating fluconazole tolerance between C. albicans and C. glabrata. We analyzed the impact of temperature on fluconazole tolerance, and requirement of calcineurin and Hsp90 for maintenance of fluconazole tolerance. We isolated colonies from edge, inside and outside of inhibition zone in disk diffusion assays. And we exposed C. glabrata strain to high concentrations of fluconazole and investigated the mutants for development of fluconazole resistance and tolerance. Results: We found temperature modulated tolerance in the opposite way in C. albicans strain YJB-T1891 and C. glabrata strain CG4. Calcineurin and Hsp90 were required for maintenance of fluconazole tolerance in both species. Colonies from inside and outside of inhibition zones did not exhibited mutated phenotype, but colonies isolated from edge of inhibition zone exhibited diverse phenotype changes. Moreover, we discovered that high concentrations (16-128 mg/mL) of fluconazole induce the simultaneous but parallel development of tolerance and resistance in C. glabrata, unlike the sole development of tolerance in C. albicans. Conclusion: This study highlights thatwhile tolerance to fluconazole is a common response in Candida species, the specific molecular mechanisms and evolutionary pathways that lead to this response vary between species. Our findings emphasize the importance of understanding the regulation of fluconazole tolerance in different Candida species to develop effective therapeutic strategies. [ABSTRACT FROM AUTHOR]

The ERG6 gene encodes the sterol C24-methyltransferase converting zymosterol to fecosterol in the ergosterol biosynthetic pathway. Here, we extend the results of functional analysis of the CgERG6 gene, which was previously shown to modulate drug susceptibility in Candida glabrata mutant cells, by demonstrating that its deletion leads to increased susceptibility to cycloheximide, 4-nitroquinoline-N-oxide and weak organic acids, and such effects are associated with attenuated virulence. Together with abrogated efflux of drug substrates by CgCdr1p and CgPdr12p, the Cgerg6Δ mutation leads to reduced cell surface hydrophobicity and decreased virulence of the mutant cells of C. glabrata. The absence of CgErg6p impacts the lipid organization and function of the plasma membrane, resulting in non-specific permeability and abrogation of normal function of membrane-bound proteins accompanied by decreased virulence in Cgerg6Δ cells. Galleria mellonella larvae were used as a non-vertebrate animal host model to determine differences in the virulence potential of C. glabrata strains (parental strain and the Cgerg6Δ deletion mutant). We found that Cgerg6Δ mutant strain attenuated in virulence caused 25–30% survival of larvae compared with parental strain. [ABSTRACT FROM AUTHOR]

Bioethanol production from agro-food industry waste is one of the suitable alternatives to fossil fuels. In this study, six agro-food wastes were evaluated to select those with a high concentration of starch or fermentable sugars for bioethanol processing. Yeast's ability to produce biofuels by fermentation is affected by temperature. In this study, thermotolerant yeasts, obtained from natural sources in Algeria (soil and agro-food waste), were selected for their capacity to produce and tolerate bioethanol. Three wastes were selected, two of which had significant starch content. In order to get fermentable sugars, these wastes underwent chemical and enzymatic hydrolysis. The selected yeasts were identified by morphological, physiological, biochemical, and molecular characterization. Enzymatic and acid hydrolysis of whole potato and durum wheat bran released (190 g/L and 130 g/L) and (138.40 g/L and 90.03 g/L) reducing sugars, respectively. Among the isolated strains, three were found to be able to produce bioethanol, namely Candida tropicalis, Candida glabrata, and Saccharomyces cerevisiae. These strains were identical to those stored in the data bank with 99%, 100%, and 100%, respectively. In addition, C. glabrata and C. tropicalis exhibited an ethanol tolerance of up to 14%, while S. cerevisiae tolerates up to 15%. Interestingly, enzymatic hydrolysis–treated potatoes produced a considerable amount of bioethanol after 48 h of fermentation by S. cerevisiae (7.525% (v/v)), C. glabrata (6.80% (v/v)), and C. tropicalis (4.50% (v/v)). Taken together, our findings suggest that S. cerevisiae and whole potato waste could be considered good candidates for industrial bioethanol production at high temperatures. [ABSTRACT FROM AUTHOR]

Fungal infections have become an important public health problem. Currently, there are only three available classes of antifungals for the treatment of invasive infections. Two of them, azoles and polyenes, target the synthesis of ergosterol or bind to sterols. A promising strategy to improve current therapies is the use of natural compounds in combinational therapies with the existing antifungals. In this work, we analyzed the changes in the susceptibility of the mutant strain of Nakaseomyces glabratus (Candida glabrata) lacking the ERG6 gene (encoding the sterol C-24 methyltransferase in ergosterol biosynthesis) in the presence of catechin and antifungal azoles. The reduced content of ergosterol in the Cgerg6Δ mutant resulted in the increased tolerance of the mutant cells to both azoles and polyenes. The combination of catechin with fluconazole or miconazole led to the growth inhibition of the azole-resistant Cgerg6Δ mutant strain. In the presence of catechin and miconazole, the Cgerg6Δ mutant fails to properly activate the expression of genes encoding the transcription factors CgYap1p and CgMsn4p, as well as the gene expression of CgCTA1, which are involved in oxidative stress response and lead to the intracellular accumulation of ROS. Finally, we show that catechin administration reduces mortality in a Galleria mellonella model infected with C. glabrata. Our work thus supports the use of catechin in combination therapies for fungal infections and shows that the CgERG6 gene could be a potential new drug target. [ABSTRACT FROM AUTHOR]

Background and purpose: Oral candidiasis is one of the most common infections affecting the mucosal surfaces and skin, specifically involving the oral mucosa. This disease is primarily caused by various species of Candida, including Candida albicans. Current treatments with polyene and azole antifungal drugs can affect patient cells, and there are also several limitations in treating fungal infections. These limitations include the shortage and high cost of antifungal drugs, their side effects, and the increasing resistance to azole drugs among Candida species. Given these challenges, researchers are exploring new antifungal drugs, particularly heterocyclic compounds. Heterocyclic compounds are widely used in the pharmaceutical industry. Thiazolidinone derivatives can weaken the fungal and bacterial resistance caused by many antibiotics and drugs. Additionally, oxindoles show significant pharmacological and biological activity. Given these factors and the notable biological properties of thiazolidinones and oxindoles, their potential for developing new antifungal drugs has gained interest among researchers. Therefore, this study aims to investigate the effectiveness of synthetic indole-hydrazono-thiazolidinones on Candida isolates obtained from oral infections. Materials and methods: Antifungal susceptibility testing was conducted on 100 clinical isolates of Candida, including 53 Candida albicans isolates, 22 Candida tropicalis isolates, 17 Candida parapsilosis isolates, and 8 Candida glabrata isolates. The susceptibility of these isolates to 4a and 4b indole-hydrazonothiazolidinone derivatives and nystatin was assessed using the broth microdilution method, based on CLSIM27S4 guidelines for determining drug sensitivity in yeasts. Species identification was performed using phenotypic chrome agar and genetic PCR-RFLP methods. The compounds and antifungal drugs were tested at a dilution range of 0.016–16 μg/ml. The minimum inhibitory concentration (MIC) was defined as the concentration that resulted in at least 50% inhibition of growth compared to the positive control group. Data were analyzed using SPSS version 20 software, with a significance level of P<0.05. Results: The results showed a significant difference in MIC values between the indole-hydrazonothiazolidinone derivatives 4a and 4b, and nystatin against Candida albicans, Candida tropicalis, and Candida parapsilosis. There was also a significant difference in the average MIC values of the derivatives. However, no significant difference was observed in MIC values between these derivatives and nystatin against Candida glabrata. Conclusion: The antifungal activity of the indole-hydrazono-thiazolidinone derivatives was lower than that of nystatin against Candida albicans, Candida tropicalis, and Candida parapsilosis. However, the activity against Candida glabrata was comparable to that of nystatin. Further studies on the cytotoxicity and side effects of these derivatives may pave the way for the development of new antifungal drugs and treatment options. [ABSTRACT FROM AUTHOR]

The rise in antifungal resistance among medically important fungi causing severe infectious diseases has underscored the urgent need for developing more effective antifungal agents. Growing evidence suggests that compounds combining functional antifungal groups with metals are promising candidates and may well be the key to addressing this global challenge. In this work, a range of new azole-containing tertiary amine compounds were prepared from three N-alkylamine azole skeletons appended with a 2,4-dihalogenobenzene function and one of the five different metal-binding motifs pyridine, quinoline, 8-hydroxyquinoline, 2-methoxyphenol, and 4-bromophenol. The copper(II) binding of these azole compounds was studied by spectrophotometric titrations in buffered aqueous medium to determine the metal binding equilibria and to comparatively characterize the copper(II)-binding ability of the compounds. The activity of all compounds against the opportunistic fungal pathogen Candida glabrata was also evaluated, allowing us to draw important conclusions about structure–activity relationships that will guide the future design of more effective metal-binding antifungal compounds. [ABSTRACT FROM AUTHOR]

Candida glabrata (also called Nakaseomyces glabratus) is an opportunistic pathogen that can resist common antifungals and rapidly acquire multidrug resistance. A large amount of genetic variation exists between isolates, which complicates generalizations. Portable transposon-sequencing (Tn-seq) methods can efficiently provide genome-wide information on strain differences and genetic mechanisms. Using the Hermes transposon, the CBS138 reference strain and a commonly studied derivative termed 2001 were subjected to Tn-seq in control conditions and after exposure to varying doses of the clinical antifungal micafungin. The approach revealed large differences between these strains, including a 131-kb tandem duplication and a variety of fitness differences. Additionally, both strains exhibited up to 1,000-fold increased transposon accessibility in subtelomeric regions relative to the BG2 strain, indicative of open subtelomeric chromatin in these isolates and large epigenetic variation within the species. Unexpectedly, the Pdr1 transcription factor conferred resistance to micafungin through targets other than CDR1. Other micafungin resistance pathways were also revealed including mannosyltransferase activity and biosynthesis of the lipid precursor sphingosine, the inhibition of which by SDZ 90–215 and myriocin enhanced the potency of micafungin in vitro. These findings provide insights into the complexity of the C. glabrata species as well as strategies for improving antifungal efficacy. [ABSTRACT FROM AUTHOR]

Azole resistance in the pathogenic yeast Candida glabrata is a serious clinical complication and increasing in frequency. The majority of resistant organisms have been found to contain a substitution mutation in the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. These mutations typically lead to this factor driving high, constitutive expression of target genes like the ATP-binding cassette transporter-encoding gene CDR1. Overexpression of Cdr1 is required for the observed elevated fluconazole resistance exhibited by strains containing one of these hyperactive PDR1 alleles. While the identity of hyperactive PDR1 alleles has been extensively documented, the mechanisms underlying how these gain-of-function (GOF) forms of Pdr1 lead to elevated target gene transcription are not well understood. We have used a tandem affinity purification-tagged form of Pdr1 to identify coactivator proteins that biochemically purify with the wild-type and 2 different GOF forms of Pdr1. Three coactivator proteins were found to associate with Pdr1: the SWI/SNF complex Snf2 chromatin remodeling protein and 2 different components of the SAGA complex, Spt7 and Ngg1. We found that deletion mutants lacking either SNF2 or SPT7 exhibited growth defects, even in the absence of fluconazole challenge. To overcome these issues, we employed a conditional degradation system to acutely deplete these coactivators and determined that loss of either coactivator complex, SWI/SNF or SAGA, caused defects in Pdr1-dependent transcription. A double degron strain that could be depleted for both SWI/SNF and SAGA exhibited a profound defect in PDR1 autoregulation, revealing that these complexes work together to ensure high-level Pdr1-dependent gene transcription. [ABSTRACT FROM AUTHOR]

The emergence of non-albicans Candida as a potential causative agent of candidiasis following Candida albicans is occurring globally. It is known that phenolic cresols, thymol, and carvacrol have anti-Candida characteristics. However, there are few papers that disclose the mechanism of action, which is characterized by a damaged cell wall and ergosterol synthesis, as well as calcium-induced death in C. albicans. This work utilized in-silico docking analysis to examine the molecular targets of thymol and carvacrol in Candida glabrata. The results were then validated in vitro by antifungal susceptibility testing and growth curve analysis. The study incorporated C. glabrata deletion mutants for the target proteins to gain understanding of the function of these proteins in the antifungal effects of the two monoterpenoids. After conducting molecular docking and in silico toxicity evaluations, thymol and carvacrol were chosen for in vitro investigations. Experimental tests conducted in a laboratory setting have shown that both phytocompounds exhibit strong binding affinity towards cell wall synthesis proteins (Kre1p, Kre2p, Ecm33p), calcium channel proteins (Mid1p, Ecm7p), and proteins involved in the ergosterol synthesis pathway (Erg5p). These proteins are likely to be the specific targets for the anti-Candida properties of the two isomeric monoterpenoids in C. glabrata. Minimum inhibitory concentration (MIC50) values for thymol and carvacrol range from 50 µg/ml to 75 µg/ml. The wild-type strains exhibit a minimum fungicidal concentration of 100 µg/ml for thymol and 125 µg/ml for carvacrol. The results underscore the significance of these proteins in the fungal reaction to thymol and carvacrol and also indicate a promising opportunity for the development of novel antifungal treatment methods utilizing these proteins. [ABSTRACT FROM AUTHOR]

Abstract Background Candida glabrata is the second most common cause of invasive candidiasis worldwide. In this study, we determined the clinical characteristics and drug sensitivity of C. glabrata isolates and investigated the associations between MSH2 gene mutations, sequence types (ST), and drug resistance. Methods A total of 154 C. glabrata isolates were collected from patients being treated in three hospitals in China. The antifungal sensitivity of the strains was assessed using the broth microdilution method. Multilocus sequence typing (MLST) was also performed, followed by MSH2 sequencing. The clinical features and outcomes of C. glabrata infection were analysed for a total of 49 strains, which were collected from patients with invasive Candida infection at Longhua Hospital. Results All 154 isolates were found to be susceptible to amphotericin, 5-fluorocytosine, anidulafungin, caspofungin, and micafungin, whereas 11.7% were fluconazole-resistant, 18.8% were itraconazole non-wild type, and 35.7% were voriconazole non-wild type. ST7 (62.34%) was the most common ST genotype, followed by ST10 (16.88%) and ST15 (7.79%). The total azole resistance rates for all isolates, ST7, ST10, and other STs were 36.4, 42.7, 34.6, and 18.8%, respectively. The ST7 and ST10 isolates were characterised by a higher drug resistance rate than the other minor ST isolates. Moreover, 59.09% of isolates had one or more MSH2 non-synonymous mutations, with V239L being the most commonly detected mutation. The frequency of MSH2 mutations was significantly higher in azole-resistant isolates than in other isolates, whereas P6L or L87P mutations were associated with the highest azole resistance rates of up to 87.5% and 80%, respectively. Our results indicated that ST7 and ST15 are independent predictors of mortality caused by C. glabrata infection and revealed a higher 30-day mortality in patients infected with these strains than in those infected with other ST isolates. Conclusions Our findings revealed the relationships between MLST, MSH2 gene mutations, and drug resistance in the common pathogenic fungus C. glabrata, and thereby enabled us to identify strains that are associated with higher rates of mortality. These findings will contribute to enhancing our understanding of the pathogenesis of C. glabrata infection.

Background and purpose: Oral candidiasis is one of the most common infections affecting the mucosal surfaces and skin, specifically involving the oral mucosa. This disease is primarily caused by various species of Candida, including Candida albicans. Current treatments with polyene and azole antifungal drugs can affect patient cells, and there are also several limitations in treating fungal infections. These limitations include the shortage and high cost of antifungal drugs, their side effects, and the increasing resistance to azole drugs among Candida species. Given these challenges, researchers are exploring new antifungal drugs, particularly heterocyclic compounds. Heterocyclic compounds are widely used in the pharmaceutical industry. Thiazolidinone derivatives can weaken the fungal and bacterial resistance caused by many antibiotics and drugs. Additionally, oxindoles show significant pharmacological and biological activity. Given these factors and the notable biological properties of thiazolidinones and oxindoles, their potential for developing new antifungal drugs has gained interest among researchers. Therefore, this study aims to investigate the effectiveness of synthetic indole-hydrazono-thiazolidinones on Candida isolates obtained from oral infections. Materials and methods: Antifungal susceptibility testing was conducted on 100 clinical isolates of Candida, including 53 Candida albicans isolates, 22 Candida tropicalis isolates, 17 Candida parapsilosis isolates, and 8 Candida glabrata isolates. The susceptibility of these isolates to 4a and 4b indole-hydrazono-thiazolidinone derivatives and nystatin was assessed using the broth microdilution method, based on CLSI-M27S4 guidelines for determining drug sensitivity in yeasts. Species identification was performed using phenotypic chrome agar and genetic PCR-RFLP methods. The compounds and antifungal drugs were tested at a dilution range of 0.016–16 μg/ml. The minimum inhibitory concentration (MIC) was defined as the concentration that resulted in at least 50% inhibition of growth compared to the positive control group. Data were analyzed using SPSS version 20 software, with a significance level of P

ABSTRACT: Objectives: This study aimed to identify the resistance mechanisms to micafungin and fluconazole in a clinical isolate of Candida glabrata. Methods: The isolate was whole-genome sequenced to identify amino acid changes in key proteins involved in antifungal resistance, and the isolate was further characterised by pathogenicity-related phenotypic assays that supported the sequencing results. Results: Amino acid substitutions were detected in 8 of 17 protein candidates. Many of these substitutions were novel, including in CHS3, CHS3B, and KRE5, which are involved in the development of micafungin resistance. Regarding fluconazole resistance, overexpression of efflux pumps was observed. Our isolate did not exhibit an increased virulence potential compared with the control strain; however, a significant increase in chitin content and potential to resist the cell surface disruptant sodium dodecyl sulphate was observed. Conclusions: This clinical Candida glabrata isolate experienced a change in cell wall architecture, which correlates with the development of micafungin resistance.

Introduction: The study of Candida glabrata genes associated with fluconazole resistance, from a molecular perspective, increases the understanding of the phenomenon with a view to its clinical applicability. Objective: We sought to establish the predictive molecular profile of fluconazole resistance in Candida glabrata by analyzing the ERG11, ERG3, CgCDR1, and CgSNQ2 genes. Method: Expression was quantified using RT-qPCR. Metrics were obtained through molecular docking and Fisher discriminant functions. Additionally, a predictive classification was made against the susceptibility of C. glabrata to fluconazole. Results: The relative expression of the ERG3, CgCDR1, and CgSNQ2 genes was higher in the fluconazole-resistant strains than in the fluconazole-susceptible, dose-dependent strains. The gene with the highest relative expression in the fluconazole-exposed strains was CgCDR1, and in both the resistant and susceptible, dose-dependent strains exposed to fluconazole, this was also the case. The molecular docking model generated a median number of contacts between fluconazole and ERG11 that was lower than the median number of contacts between fluconazole and ERG3, -CgCDR1, and -CgSNQ2. The predicted classification through the multivariate model for fluconazole susceptibility achieved an accuracy of 73.5%. Conclusion: The resistant strains had significant expression levels of genes encoding efflux pumps and the ERG3 gene. Molecular analysis makes the identification of a low affinity between fluconazole and its pharmacological target possible, which may explain the lower intrinsic susceptibility of the fungus to fluconazole. [ABSTRACT FROM AUTHOR]

The arginine transporter Can1 is a multifunctional protein of the conventional yeast Saccharomyces cerevisiae. Apart from facilitating arginine uptake, Can1 plays a pivotal role in regulating proline metabolism and maintaining cellular redox balance. Here, we report a novel function of Can1 in the control of yeast biofilm formation. First, the S. cerevisiae CAN1 gene knockout strain displayed a significant growth delay compared to the wild-type strain. Our genetic screening revealed that the slow growth of the CAN1 knockout strain is rescued by a functional deficiency of the FLO8 gene, which encodes the master transcription factor associated with biofilm formation, indicating that Can1 is involved in biofilm formation. Intriguingly, the CAN1 knockout strain promoted the Flo11-dependent aggregation, leading to higher biofilm formation. Furthermore, the CAN1 knockout strain of the pathogenic yeast Candida glabrata exhibited slower growth and higher biofilm formation, similar to S. cerevisiae. More importantly, the C. glabrata CAN1 gene knockout strain showed severe toxicity to macrophage-like cells and nematodes. The present results could help to elucidate both the molecular mechanism underlying yeast biofilm formation and the role it plays. Future investigations may offer insights that contribute to development of antibiofilm agents. [ABSTRACT FROM AUTHOR]

Background: The prevalence of Candida glabrata healthcare‐associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms. Objectives: The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates. Methods: A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole‐resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA‐sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays. Results: All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved— in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP‐binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1. Conclusions: In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de‐repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1. [ABSTRACT FROM AUTHOR]

Recognising the growing global burden of fungal infections, the World Health Organization (WHO) established an advisory group consisting of experts in fungal diseases to develop a Fungal Priority Pathogen List. Pathogens were ranked based on their research and development needs and perceived public health importance using a series of global surveys and pathogen characteristics derived from systematic reviews. This systematic review evaluates the features and global impact of invasive disease caused by Candida glabrata (Nakaseomyces glabrata). PubMed and Web of Science were searched for studies reporting on mortality, morbidity (hospitalization and disability), drug resistance (including isolates from sterile and non-sterile sites, since these reflect the same organisms causing invasive infections), preventability, yearly incidence, diagnostics, treatability, and distribution/emergence in the last 10 years. Candida glabrata (N. glabrata) causes difficult-to-treat invasive infections, particularly in patients with underlying conditions such as immunodeficiency, diabetes, or those who have received broad-spectrum antibiotics or chemotherapy. Beyond standard infection prevention and control measures, no specific preventative measures have been described. We found that infection is associated with high mortality rates and that there is a lack of data on complications and sequelae. Resistance to azoles is common and well described in echinocandins—in both cases, the resistance rates are increasing. Candida glabrata remains mostly susceptible to amphotericin and flucytosine. However, the incidence of the disease is increasing, both at the population level and as a proportion of all invasive yeast infections, and the increases appear related to the use of antifungal agents. [ABSTRACT FROM AUTHOR]

Candida glabrata can thrive inside macrophages and tolerate high levels of azole antifungals. These innate abilities render infections by this human pathogen a clinical challenge. How C. glabrata reacts inside macrophages and what is the molecular basis of its drug tolerance are not well understood. Here, we mapped genome-wide RNA polymerase II (RNAPII) occupancy in C. glabrata to delineate its transcriptional responses during macrophage infection in high temporal resolution. RNAPII profiles revealed dynamic C. glabrata responses to macrophages with genes of specialized pathways activated chronologically at different times of infection. We identified an uncharacterized transcription factor (CgXbp1) important for the chronological macrophage response, survival in macrophages, and virulence. Genome-wide mapping of CgXbp1 direct targets further revealed its multi-faceted functions, regulating not only virulence-related genes but also genes associated with drug resistance. Finally, we showed that CgXbp1 indeed also affects fluconazole resistance. Overall, this work presents a powerful approach for examining host-pathogen interaction and uncovers a novel transcription factor important for C. glabrata’s survival in macrophages and drug tolerance.

ABSTRACT The clonal transmission of fluconazole-resistant Candida glabrata isolates within hospitals has seldom been analyzed by whole-genome sequencing (WGS). We performed WGS on 79 C. glabrata isolates, comprising 31 isolates from three premature infants with persistent C. glabrata bloodstream infection despite antifungal treatment in the same neonatal intensive care unit (NICU) in 2022 and 48 (27 fluconazole-resistant and 21 fluconazole-susceptible dose-dependent) bloodstream isolates from 48 patients in 15 South Korean hospitals from 2010 to 2022. Phylogenetic analysis based on WGS single-nucleotide polymorphisms (SNPs) distinguished the 79 isolates according to multilocus sequence typing (MLST) (17 sequence type [ST]3, 13 ST7, two ST22, 41 ST26, four ST55, and two ST59 isolates) and unveiled two possible clusters of nosocomial transmission among ST26 isolates. One cluster from two premature infants with overlapping NICU hospitalizations in 2022 encompassed 15 fluconazole-resistant isolates harboring pleiotropic drug-resistance transcription factor (Pdr1p) P258L (13 isolates) or N1086I (two isolates), together with 10 fluconazole-susceptible dose-dependent isolates lacking Pdr1p SNPs. The other cluster indicated unforeseen clonal transmission of fluconazole-resistant bloodstream isolates among five patients (four post-lung transplantation and one with diffuse interstitial lung disease) in the same hospital over 8 months. Among these five isolates, four obtained after exposure to azole antifungals harbored distinct Pdr1p SNPs (N1091D, E388Q, K365E, and R376Q). The findings reveal the transmission patterns of clonal bloodstream isolates of C. glabrata among patients undergoing antifungal treatment, exhibiting different levels of fluconazole susceptibility or distinct Pdr1p SNP profiles.IMPORTANCEThe prevalence of fluconazole-resistant bloodstream infections caused by Candida glabrata is increasing globally, but the transmission of these resistant strains within hospitals has rarely been documented. Through whole-genome sequencing and epidemiological analyses, this study identified two potential clusters of C. glabrata bloodstream infections within the same hospital, revealing the transmission of clonal C. glabrata strains with different levels of fluconazole susceptibility or distinct transcription factor pleiotropic drug resistance protein 1 (Pdr1p) single-nucleotide polymorphism profiles among patients receiving antifungal therapy.

Yixin Liu,1 Feifei Ren,1 Shan Li,1 Xiangchen Li,1 Dongyan Shi,2 Zhiqing Zhang1 1Department of Pharmacy, the Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 2Department of Clinical Laboratory, the Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of ChinaCorrespondence: Zhiqing Zhang, Department of pharmacy, the Second Hospital of Hebei Medical University, 215, Heping Road, Xinhua District, Shijiazhuang, Hebei, People’s Republic of China, Email 26500007@hebmu.edu.cn Dongyan Shi, Department of clinical laboratory, the Second Hospital of Hebei Medical University, 215, Heping Road, Xinhua District, Shijiazhuang, Hebei, People’s Republic of China, Email shidongyan73@126.comObjective: To define the antifungal activity of n-butylphthalide alone or in combination with fluconazole in Candida glabrata and Candida tropicalis.Methods: The antifungal activity of n-butylphthalide alone and in combination with fluconazole was investigated by the classical broth microdilution method and the time-killing curve method. The QRT-PCR method was used to determine gene expressions changes of mitochondrial respiratory chain enzymes, drug efflux pumps and drug target enzymes in Candida glabrata and Candida tropicalis after n-butylphthalide treatment.Results: The MIC values of n-butylphthalide against Candida glabrata and Candida tropicalis ranged from 16 to 64 μg·mL-1. The FICI value of the combination of n-butylphthalide and fluconazole against drug-resistant Candida glabrata and Candida tropicalis ranged from 0.5001 to 0.5315 with partial synergism. Time-killing curves showed that 256 μg·mL-1 n-butylphthalide significantly inhibited the growth of drug-resistant colonies of Candida glabrata and Candida tropicalis, and 128 μg·mL-1 n-butylphthalide combined with 1 μg·mL-1 fluconazole had an additive effect. N-butylphthalide could alter the expression of mitochondrial respiratory chain enzymes COX1, COX2, COX3, and CYTB genes in Candida glabrata and Candida tropicalis (P< 0.05) and downregulate the expression of the drug efflux pump genes CDR1 and CDR2 in drug-resistant Candida glabrata to 3.36% and 3.65%, respectively (P< 0.001), but did not affect the drug target enzyme ERG11 in drug-resistant Candida tropicalis.Conclusion: N-butylphthalide had antifungal activity against Candida glabrata and Candida tropicalis. N-butylphthalide improved the activity of fluconazole against drug-resistant Candida glabrata by affecting the expression of mitochondrial respiratory chain enzyme genes and reversing the high expression of drug efflux pump genes CDR1 and CDR2.Keywords: n-butylphthalide, fluconazole, drug resistance, Candida glabrata, Candida tropicalis, potentiation

Abstract Background Side effects associated with antimicrobial drugs, as well as their high cost, have prompted a search for low-cost herbal medicinal substances with fewer side effects. These substances can be used as supplements to medicine or to strengthen their effects. The current study investigated the effect of oleuropein on the inhibition of fungal and bacterial biofilm in-vitro and at the molecular level. Materials and methods In this experimental study, antimicrobial properties were evaluated using microbroth dilution method. The effect of oleuropein on the formation and eradication of biofilm was assessed on 96-well flat bottom microtiter plates and their effects were observed through scanning electron microscopy (SEM). Its effect on key genes (Hwp1, Als3, Epa1, Epa6, LuxS, Pfs) involved in biofilm formation was investigated using the quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) method. Results The minimum inhibitory concentration (MIC) and minimum fungicidal/bactericidal concentration (MFC/MBC) for oleuropein were found to be 65 mg/ml and 130 mg/ml, respectively. Oleuropein significantly inhibited biofilm formation at MIC/2 (32.5 mg/ml), MIC/4 (16.25 mg/ml), MIC/8 (8.125 mg/ml) and MIC/16 (4.062 mg/ml) (p

The interactions between pathogens during infection and the impact of these interactions on drug effectiveness are poorly understood, making polymicrobial infections challenging to treat. During an infection, cross-interactions between bacteria and fungi can strengthen virulence mechanisms and affect how the disease develops. The purpose of this study is to determine how Pseudomonas aeruginosa interacts with Candida glabrata, Candida albicans, Candida krusei, Candida parapsilosis, and Candida tropicalis in the development of polymicrobial biofilms. Pseudomonas aeruginosa, Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis isolates were used in this experimental investigation. After preparing a 0.5 Mc Farland suspension of each isolate, the gold standard for measuring biofilm was applied: the Tissue Plate Culture (TCP) method. After that, an ELISA reader with a wavelength of 595 nm was used to measure the optical density (OD) of the biofilm. SPSS 26.0 was then used for statistical analysis to compare the OD values between Pseudomonas aeruginosa that had not been exposed to Candida and those that had. Pseudomonas aeruginosa and Candida are found to interact synergistically if there is an increase in OD, and antagonistic interaction is discovered if there is a decrease in OD. In comparison to the group that was not exposed to Candida, Pseudomonas aeruginosa exposed to Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis showed an increase in the OD value of biofilm. Pseudomonas aeruginosa and Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis interact synergistically.

BackgroundCandidemia is increasing in frequency and is associated with high mortality. We sought to determine the burden of illness, the population it affects and its resistance profile in our region.MethodsThe Calgary Zone (CZ) provides all care for residents of Calgary and surrounding communities (~ 1.69 million) via five tertiary hospitals each served by a common single laboratory for acute care microbiology. All adult patients in the CZ with at least one Candida spp.-positive blood culture between January 1, 2010, and December 31, 2018, were identified using microbiological data from Calgary Lab Services, the laboratory that processes > 95% of all blood culture samples in the CZ, were reviewed for the study.ResultsThe overall annual incidence of candidemia among individuals living in the CZ was 3.8 per 100,000 persons (Median age 61 years (IQR 48-72) and 221/455 (47.4%) were female). C. albicans was the most common species (50.6%), followed by C. glabrata, (24.0%). No other species accounted for more than 7% of cases. Overall mortality at 30, 90, and 365 days was 32.2, 40.1, and 48.1% respectively. Mortality rate did not differ by Candida species. Of individuals who developed candidemia, more than 50% died within the next year. No new resistance pattern has emerged in the most common Candida species in Calgary, Alberta.ConclusionsIn Calgary, Alberta, the incidence of candidemia has not increased in the last decade. C. albicans was the most common species and it remains susceptible to fluconazole.

Background: Side effects associated with antimicrobial drugs, as well as their high cost, have prompted a search for low-cost herbal medicinal substances with fewer side effects. These substances can be used as supplements to medicine or to strengthen their effects. The current study investigated the effect of oleuropein on the inhibition of fungal and bacterial biofilm in-vitro and at the molecular level. Materials and methods: In this experimental study, antimicrobial properties were evaluated using microbroth dilution method. The effect of oleuropein on the formation and eradication of biofilm was assessed on 96-well flat bottom microtiter plates and their effects were observed through scanning electron microscopy (SEM). Its effect on key genes (Hwp1, Als3, Epa1, Epa6, LuxS, Pfs) involved in biofilm formation was investigated using the quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) method. Results: The minimum inhibitory concentration (MIC) and minimum fungicidal/bactericidal concentration (MFC/MBC) for oleuropein were found to be 65 mg/ml and 130 mg/ml, respectively. Oleuropein significantly inhibited biofilm formation at MIC/2 (32.5 mg/ml), MIC/4 (16.25 mg/ml), MIC/8 (8.125 mg/ml) and MIC/16 (4.062 mg/ml) (p < 0.0001). The anti-biofilm effect of oleuropein was confirmed by SEM. RT-qPCR indicated significant down regulation of expression genes involved in biofilm formation in Candida albicans (Hwp1, Als3) and Candida glabrata (Epa1, Epa6) as well as Escherichia coli (LuxS, Pfs) genes after culture with a MIC/2 of oleuropein (p < 0.0001). Conclusions: The results indicate that oleuropein has antifungal and antibacterial properties that enable it to inhibit or destroy the formation of fungal and bacterial biofilm. [ABSTRACT FROM AUTHOR]

Introduction: Candida species are endowed with the ability to produce biofilms, which is one of the causes of pathogenicity, as biofilms protect yeasts from antifungal drugs. Candida glabrata (Nakaseomyces glabrata) is one of the most prevalent pathogenic yeasts in humans and a biofilm producer. Methods: The study was aimed at evaluating the combined effects of two highly promising antifungal biomolecules (AF4 and AF5) lipopeptide in nature, chromatographically purified to homogeneity from Bacillus subtilis (B. subtilis) and the standard antifungal fluconazole (at different concentrations) to demonstrate C. glabrata biofilm formation inhibition. Biofilm production and inhibition were evaluated by quantification of the biofilm biomass and metabolic activity using crystal violet (CV) staining and XTT reduction assays, respectively. Microscopic techniques such as confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM) were employed to visualize biofilm formation and inhibition. Results and Discussion: Compared to untreated and fluconazole-treated biofilms, an enhanced in vitro anti-biofilm effect of the antifungal lipopeptides AF4/AF5 alone and their combinations with fluconazole was established. The lipopeptides AF4/AF5 alone at 8 and 16 µg/mL exhibited significant biomass and metabolic activity reductions. SEM and CSLM images provided evidence that the lipopeptide exposure results in architectural alterations and a significant reduction of C. glabrata biofilms, whereas (2', 7'-dichlorofluorescin diacetate (DCFDA) and propidium iodide (PI) analyses showed reactive oxygen species (ROS) generation along with membrane permeabilization. The estimation of exopolysaccharides (EPS) in AF4/AF5-treated biofilms indicated EPS reduction. The combinations of fluconazole (64/128 µg/mL) and AF4/AF5 lipopeptide (16 µg/mL) were found to significantly disrupt the mature (24 h) biofilms as revealed by CSLM and SEM studies. The CSLM images of biofilms were validated using COMSTAT. The FTIR-analyses indicate the antibiofilm effects of both lipopeptides on 24 h biofilms to support CSLM and SEM observations. The combinations of fluconazole (64/128 µg/mL) and AF4/AF5 lipopeptide were found to disrupt the mature biofilms; the study also showed that the lipopeptides alone have the potentials to combat C. glabrata biofilms. Taken together, it may be suggested that these lipopeptide leads can be optimized to potentially apply on various surfaces to either reduce or nearly eradicate yeast biofilms. [ABSTRACT FROM AUTHOR]

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Objective: In the defense against microorganisms like Candida albicans, macrophages recruit LC3(Microtubule-associated protein 1A/1B-light chain 3) to the periplasm, engaging in the elimination process through the formation of a single-membrane phagosome known as LC3-associated phagocytosis (LAP). Building on this, we propose the hypothesis that glucocorticoids may hinder macrophage phagocytosis of Candida glabrata by suppressing LAP, and rapamycin could potentially reverse this inhibitory effect. Methods: RAW264.7 cells were employed for investigating the immune response to Candida glabrata infection. Various reagents, including dexamethasone, rapamycin, and specific antibodies, were utilized in experimental setups. Assays, such as fluorescence microscopy, flow cytometry, ELISA (Enzyme- Linked Immunosorbent Assay), Western blot, and confocal microscopy, were conducted to assess phagocytosis, cytokine levels, protein expression, viability, and autophagy dynamics. Results: Glucocorticoids significantly inhibited macrophage autophagy, impairing the cells' ability to combat Candida glabrata. Conversely, rapamycin exhibited a dual role, initially inhibiting and subsequently promoting phagocytosis of Candida glabrata by macrophages. Glucocorticoids hinder macrophage autophagy in Candida glabrata infection by suppressing the MTOR pathway (mammalian target of rapamycin pathway), while the activation of MTOR pathway by Candida glabrata diminishes over time. Conclusion: Our study elucidates the intricate interplay between glucocorticoids, rapamycin, and macrophage autophagy during Candida glabrata infection. Understanding the implications of these interactions not only sheds light on the host immune response dynamics but also unveils potential therapeutic avenues for managing fungal infections. [ABSTRACT FROM AUTHOR]

Background and Objectives: The presence of fungi in the respiratory tract as mycobiome, particularly Candida species (spp.), remains a serious problem due to increasing numbers of immunocompromised patients. The confirmed reliable existence of these pathogens due to frequent colonization is essential. This investigation aimed to recognize Candida spp. among isolates from bronchoalveolar lavage of immunocompromised and critically ill patients and to evaluate their susceptibility to antimycotic drugs. Materials and Methods: Bronchoalveolar lavage fluid was collected from 161 hospitalized patients presenting with suspected respiratory fungal infection/colonization. The specimens were examined by standard molecular and mycological assays. Candida spp. were recognized with sequence assessment of the D1-D2 section of the large subunit ribosomal DNA. The susceptibility of Candida isolates to common antimycotic drugs was distinguished by standard broth microdilution. Results: Seventy-one clinical isolates of Candida spp. were recognized. Candida albicans was the most frequent, followed by C. glabrata, C. krusei (Pichia kudriavzevii), C. dubliniensis, C. parapsilosis, and C. tropicalis. We found 5.1% of C. albicans isolates and 8% of C. glabrata isolates to show resistance to fluconazole. The whole of the Candida spp. were sensitive to amphotericin B and caspofungin. Conclusion: This study demonstrated that C. albicans and C. glabrata are the most common isolates of bronchoalveolar lavage fluid in patients, and the drug susceptibility screening confirmed that amphotericin B and caspofungin are effective against Candida spp. but some C. glabrata and C. albicans isolates showed resistance to fluconazole. [ABSTRACT FROM AUTHOR]

Candida albicans has the capacity to neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids. The molecular components underlying alkalization and its physiological significance remain poorly understood. Here, we present an integrative model with the cytosolic NAD+‐dependent glutamate dehydrogenase (Gdh2) as the principal ammonia‐generating component. We show that alkalization is dependent on the SPS‐sensor‐regulated transcription factor STP2 and the proline‐responsive activator Put3. These factors function in parallel to derepress GDH2 and the two proline catabolic enzymes PUT1 and PUT2. Consistently, a double mutant lacking STP2 and PUT3 exhibits a severe alkalization defect that nearly phenocopies that of a gdh2‐/‐ strain. Alkalization is dependent on mitochondrial activity and in wild‐type cells occurs as long as the conditions permit respiratory growth. Strikingly, Gdh2 levels decrease and cells transiently extrude glutamate as the environment becomes more alkaline. Together, these processes constitute a rudimentary regulatory system that counters and limits the negative effects associated with ammonia generation. These findings align with Gdh2 being dispensable for virulence, and based on a whole human blood virulence assay, the same is true for C. glabrata and C. auris. Using a transwell co‐culture system, we observed that the growth and proliferation of Lactobacillus crispatus, a common component of the acidic vaginal microenvironment and a potent antagonist of C. albicans, is unaffected by fungal‐induced alkalization. Consequently, although Candida spp. can alkalinize their growth environments, other fungal‐associated processes are more critical in promoting dysbiosis and virulent fungal growth. [ABSTRACT FROM AUTHOR]

Invasive candidiasis caused by non‐albicans species has been on the rise, with Candida glabrata emerging as the second most common etiological agent. Candida glabrata possesses an intrinsically lower susceptibility to azoles and an alarming propensity to rapidly develop high‐level azole resistance during treatment. In this study, we have developed an efficient piggyBac (PB) transposon‐mediated mutagenesis system in C. glabrata to conduct genome‐wide genetic screens and applied it to profile genes that contribute to azole resistance. When challenged with the antifungal drug fluconazole, PB insertion into 270 genes led to significant resistance. A large subset of these genes has a role in the mitochondria, including almost all genes encoding the subunits of the F1F0 ATPase complex. We show that deleting ATP3 or ATP22 results in increased azole resistance but does not affect susceptibility to polyenes and echinocandins. The increased azole resistance is due to increased expression of PDR1 that encodes a transcription factor known to promote drug efflux pump expression. Deleting PDR1 in the atp3Δ or atp22Δ mutant resulted in hypersensitivity to fluconazole. Our results shed light on the mechanisms contributing to azole resistance in C. glabrata. This PB transposon‐mediated mutagenesis system can significantly facilitate future genome‐wide genetic screens. [ABSTRACT FROM AUTHOR]

The interactions between pathogens during infection and the impact of these interactions on drug effectiveness are poorly understood, making polymicrobial infections challenging to treat. During an infection, cross-interactions between bacteria and fungi can strengthen virulence mechanisms and affect how the disease develops. The purpose of this study is to determine how Pseudomonas aeruginosa interacts with Candida glabrata, Candida albicans, Candida krusei, Candida parapsilosis, and Candida tropicalis in the development of polymicrobial biofilms. Pseudomonas aeruginosa, Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis isolates were used in this experimental investigation. After preparing a 0.5 Mc Farland suspension of each isolate, the gold standard for measuring biofilm was applied: the Tissue Plate Culture (TCP) method. After that, an ELISA reader with a wavelength of 595 nm was used to measure the optical density (OD) of the biofilm. SPSS 26.0 was then used for statistical analysis to compare the OD values between Pseudomonas aeruginosa that had not been exposed to Candida and those that had. Pseudomonas aeruginosa and Candida are found to interact synergistically if there is an increase in OD, and antagonistic interaction is discovered if there is a decrease in OD. In comparison to the group that was not exposed to Candida, Pseudomonas aeruginosa exposed to Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis showed an increase in the OD value of biofilm. Pseudomonas aeruginosa and Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis interact synergistically. [ABSTRACT FROM AUTHOR]

The current trend in microbiological research aimed at limiting the development of biofilms of multidrug-resistant microorganisms is increasingly towards the search for possible synergistic effects between various compounds. This work presents a combination of a naturally occurring compound, β-aescin, newly synthesized alkylamidobetaines (AABs) with a general structure—CnTMDAB, and antifungal drugs. The research we conducted consists of several stages. The first stage concerns determining biological activity (antifungal) against selected multidrug-resistant strains of Candida glabrata (C. glabrata) with the highest ability to form biofilms. The second stage of this study determined the activity of β-aescin combinations with antifungal compounds and alkylamidobetaines. In the next stage of this study, the ability to eradicate a biofilm on the polystyrene surface of the combination of β-aescin with alkylamidobetaines was examined. It has been shown that the combination of β-aescin and alkylamidobetaine can firmly remove biofilms and reduce their viability. The last stage of this research was to determine the safety regarding the cytotoxicity of both β-aescin and alkylamidobetaines. Previous studies on the fibroblast cell line have shown that C9 alkylamidobetaine can be safely used as a component of anti-biofilm compounds. This research increases the level of knowledge about the practical possibilities of using anti-biofilm compounds in combined therapies against C. glabrata. [ABSTRACT FROM AUTHOR]

The emergence of non-albicans Candida as a potential causative agent of candidiasis following Candida albicans is occurring globally. It is known that phenolic cresols, thymol, and carvacrol have anti-Candida characteristics. However, there are few papers that disclose the mechanism of action, which is characterized by a damaged cell wall and ergosterol synthesis, as well as calcium-induced death in C. albicans. This work utilized in-silico docking analysis to examine the molecular targets of thymol and carvacrol in Candida glabrata. The results were then validated in vitro by antifungal susceptibility testing and growth curve analysis. The study incorporated C. glabrata deletion mutants for the target proteins to gain understanding of the function of these proteins in the antifungal effects of the two monoterpenoids. After conducting molecular docking and in silico toxicity evaluations, thymol and carvacrol were chosen for in vitro investigations. Experimental tests conducted in a laboratory setting have shown that both phytocompounds exhibit strong binding affinity towards cell wall synthesis proteins (Kre1p, Kre2p, Ecm33p), calcium channel proteins (Mid1p, Ecm7p), and proteins involved in the ergosterol synthesis pathway (Erg5p). These proteins are likely to be the specific targets for the anti-Candida properties of the two isomeric monoterpenoids in C. glabrata. Minimum inhibitory concentration (MIC50) values for thymol and carvacrol range from 50 µg/ml to 75 µg/ml. The wild-type strains exhibit a minimum fungicidal concentration of 100 µg/ml for thymol and 125 µg/ml for carvacrol. The results underscore the significance of these proteins in the fungal reaction to thymol and carvacrol and also indicate a promising opportunity for the development of novel antifungal treatment methods utilizing these proteins.

Purpose: Oral candidiasis is one of the most common fungal opportunistic infections in the mouth caused by a fungus called Candida, and it presents as commensals in all humans. Over the last few decades, the increasing incidence of Candida infections parallels the growing numbers of immunocompromised patients. This trend is aggravated by the fact that there is a reckless use of antifungals. To combat this trend, the use of natural antifungals which have fewer side effects is recommended. Black grape seed extract (GSE) has shown a promise in this regard. Materials and Methods: Candida species such as Candida krusei, Candida glabrata, Candida tropicalis, and Candida albicans were used in the study. Fungal strains were passaged in the agar Sabouraud dextrose environment from 24 h to have live and fresh strains for the test. Black GSE was obtained. Minimum inhibitory concentration (MIC), of GSE, amphotericin B and fluconazole drugs on the included fungi was examined using disc diffusion method. Then, the plates were incubated for 24 h at 37°C. Finally, growth inhibitory zone was examined. Results: The analysis of the results revealed that the growth of C. albicans and C. krusei had been stopped at the concentration of 6.25 μg/mL, but the growth of C. glabrata and C. tropicalis was inhibited at a concentration of 12.25 μg/mL and 0.8 μg/mL respectively. Conclusion: The results obtained from the present study showed that both disc diffusion method and the maximum inhibitory concentration of MIC (Macro dilution) confirms the antimicrobial susceptibility of four species of candida to GSE. We conclude the possibility of GSE as an effective alternative measure for commercial antifungal agents, especially among immunocompromised patients, However, more laboratory and clinical studies need to be conducted to validate our findings.

The ERG6 gene encodes the sterol C24-methyltransferase converting zymosterol to fecosterol in the ergosterol biosynthetic pathway. Here, we extend the results of functional analysis of the CgERG6 gene, which was previously shown to modulate drug susceptibility in Candida glabrata mutant cells, by demonstrating that its deletion leads to increased susceptibility to cycloheximide, 4-nitroquinoline-N-oxide and weak organic acids, and such effects are associated with attenuated virulence. Together with abrogated efflux of drug substrates by CgCdr1p and CgPdr12p, the Cgerg6Δ mutation leads to reduced cell surface hydrophobicity and decreased virulence of the mutant cells of C. glabrata. The absence of CgErg6p impacts the lipid organization and function of the plasma membrane, resulting in non-specific permeability and abrogation of normal function of membrane-bound proteins accompanied by decreased virulence in Cgerg6Δ cells. Galleria mellonella larvae were used as a non-vertebrate animal host model to determine differences in the virulence potential of C. glabrata strains (parental strain and the Cgerg6Δ deletion mutant). We found that Cgerg6Δ mutant strain attenuated in virulence caused 25–30% survival of larvae compared with parental strain.

Fungal infections have become an important public health problem. Currently, there are only three available classes of antifungals for the treatment of invasive infections. Two of them, azoles and polyenes, target the synthesis of ergosterol or bind to sterols. A promising strategy to improve current therapies is the use of natural compounds in combinational therapies with the existing antifungals. In this work, we analyzed the changes in the susceptibility of the mutant strain of Nakaseomyces glabratus (Candida glabrata) lacking the ERG6 gene (encoding the sterol C-24 methyltransferase in ergosterol biosynthesis) in the presence of catechin and antifungal azoles. The reduced content of ergosterol in the Cgerg6Δ mutant resulted in the increased tolerance of the mutant cells to both azoles and polyenes. The combination of catechin with fluconazole or miconazole led to the growth inhibition of the azole-resistant Cgerg6Δ mutant strain. In the presence of catechin and miconazole, the Cgerg6Δ mutant fails to properly activate the expression of genes encoding the transcription factors CgYap1p and CgMsn4p, as well as the gene expression of CgCTA1, which are involved in oxidative stress response and lead to the intracellular accumulation of ROS. Finally, we show that catechin administration reduces mortality in a Galleria mellonella model infected with C. glabrata. Our work thus supports the use of catechin in combination therapies for fungal infections and shows that the CgERG6 gene could be a potential new drug target.

The rise in antifungal resistance among medically important fungi causing severe infectious diseases has underscored the urgent need for developing more effective antifungal agents. Growing evidence suggests that compounds combining functional antifungal groups with metals are promising candidates and may well be the key to addressing this global challenge. In this work, a range of new azole-containing tertiary amine compounds were prepared from three N-alkylamine azole skeletons appended with a 2,4-dihalogenobenzene function and one of the five different metal-binding motifs pyridine, quinoline, 8-hydroxyquinoline, 2-methoxyphenol, and 4-bromophenol. The copper(II) binding of these azole compounds was studied by spectrophotometric titrations in buffered aqueous medium to determine the metal binding equilibria and to comparatively characterize the copper(II)-binding ability of the compounds. The activity of all compounds against the opportunistic fungal pathogen Candida glabrata was also evaluated, allowing us to draw important conclusions about structure–activity relationships that will guide the future design of more effective metal-binding antifungal compounds.

Infections caused by Candida species increased, as well as species resistant to the most conventional antimicrobials. Thus, the search for new natural sources of antimicrobials without adverse effects also increased. In this case, Astronium urundeuva Engl., a medicinal plant with several pharmacological activities, including antifungal activity, emerges as a source of antifungal compounds. Here, we evaluated the antifungal activity of A. urundeuva and its derivatives free or loaded into a nanostructured lipid system and combination studies against Candida species. A. urundeuva component's purification and identification were performed through chromatographic and spectroscopic techniques, respectively. The major components identified were gallic acid (1), methyl gallate (2), ethyl gallate (3), and 1,2,3,4,6-penta-O-galoyl-β-D-glucose (PGG) (4). A. urundeuva extract, its aqueous fraction, and PGG (4) exhibited the same MIC values between 0.2 and 62.5 µg/mL against Candida species being classified as significant activity, while the amphotericin B MIC was 0.1–1.2 µg/mL. Combination assay demonstrated that may occur synergism (0.37) between gallic acid (1) x methyl gallate (2). Thus, the results demonstrated as PGG (4) much as the gallic acid (1) x methyl gallate (2) combined might be the main responsible for the antifungal activity against Candida species, mainly Candida glabrata ATCC 2001 and that the tested nanostructured system maintained the antifungal activity. Through the antifungal activity of the extract and purified compounds, the extract may be employed in the development of antifungal herbal medicine, because its purification process is simple and greener than pure compounds. [ABSTRACT FROM AUTHOR]

Candida glabrata is the most common non-albicans Candida species that causes vulvovaginal candidiasis (VVC). Given the intrinsically low susceptibility of C. glabrata to azole drugs, investigations into C. glabrata prevalence, fungal susceptibility profile, and molecular epidemiology are necessary to optimise the treatment of VVC. This molecular epidemiological study was conducted to determine antifungal drug profile, single nucleotide polymorphisms (SNPs) associated with phenotypic antifungal resistance and epidemic diversity of C. glabrata isolates from women with VVC in Namibia. Candida glabrata isolates were identified using phenotypic and molecular methods. Antifungal susceptibility of strains was determined for fluconazole, itraconazole, amphotericin B, and anidulafungin. Whole genome sequencing was used to determine SNPs in antifungal resistance genes and sequence type (ST) allocation. Among C. glabrata isolates, all (20/20; 100%) exhibited phenotypic resistance to the azole class antifungal drug, (fluconazole), and phenotypic susceptibility to the polyene class (amphotericin B), and the echinocandins (anidulafungin). Non-synonymous SNPs were identified in antifungal resistance genes of all fluconazole-resistant C. glabrata isolates including ERG6 (15%), ERG7 (15%), CgCDR1 (25%) , CgPDR1 (60%) , SNQ2 (10%) , FKS1 (5.0%) , FKS2 (5.0%) , CgFPS1 (5.0%), and MSH2 (15%). ST15 (n  = 8/20, 40%) was predominant. This study provides important insight into phenotypic and genotypic antifungal resistance across C. glabrata isolates from women with VVC in Namibia. In this study, azole resistance is determined by an extensive range of SNPs, while the observed polyene and echinocandin resistance-associated SNPs despite phenotypic susceptibility require further investigation. [ABSTRACT FROM AUTHOR]

Followed by Candida albicans, Candida glabrata ranks as the second major species contributing to invasive candidiasis. Given the higher medical burden and lower susceptibility to azoles in C. glabrata infections, identifying these infections is critical. From 2016 to 2021, patients with deep-seated candidiasis due to C. glabrata and non- glabrata Candida met the criteria to be enrolled in the study. Clinical data were randomly divided into training and validation cohorts. A predictive model and nomogram were constructed using R software based on the stepwise algorithm and logistic regression. The performance of the model was assessed by the area under the receiver operating characteristic curve and decision curve analysis (DCA). A total of 197 patients were included in the study, 134 of them infected with non- glabrata Candida and 63 with C. glabrata. The predictive model for C. glabrata infection consisted of gastrointestinal cancer, co-infected with bacteria, diabetes mellitus, and kidney dysfunction. The specificity was 84.1% and the sensitivity was 61.5% in the validation cohort when the cutoff value was set to the same as the training cohort. Based on the model, treatment for patients with a high-risk threshold was better than 'treatment for all' in DCA, while opting low-risk patients out of treatment was also better than 'treatment for none' in opt-out DCA. The predictive model provides a rapid method for judging the probability of infections due to C. glabrata and will be of benefit to clinicians making decisions about therapy strategies. [ABSTRACT FROM AUTHOR]

Introduction: Candida glabrata has emerged as a fungal pathogen with high infection and mortality rates, and its primary virulence factors are related to adhesion and biofilm formation. These virulence factors in C.glabrata are primarily mediated by epithelial adhesins (Epas), most of which are encoded in subtelomeric regions and regulated by subtelomeric silencing mechanisms. The transcription factor Mss11, known for its regulatory role in adhesion, biofilm formation, and filamentous growth in Saccharomyces cerevisiae and Candida albicans, has also been implicated in the expression of EPA6, suggesting its potential influence on C.glabrata virulence. The present study aims to determine the regulatory role of Mss11 in the virulence of C. glabrata. Methods: In this work, a Δmss11 null mutant and its complemented strain were constructed from a C.glabrata standard strain. The impact of the transcription factor Mss11 on the virulence of C.glabrata was investigated through a series of phenotypic experiments, including the microbial adhesion to hydrocarbons (MATH) test, adherence assay, biofilm assay, scanning electron microscopy and Galleria mellonella virulence assay. Furthermore, transcriptome sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and chromatin immunoprecipitation sequencing (ChIP-seq) were employed to investigate the molecular mechanisms behind the regulation of Mss11. Results: In C.glabrata, the loss of MSS11 led to a significant reduction in several virulence factors including cell surface hydrophobicity, epithelial cell adhesion, and biofilm formation. These observations were consistent with the decreased virulence of the Δmss11 mutant observed in the Galleria mellonella infection model. Further exploration demonstrated that Mss11 modulates C. glabrata virulence by regulating EPA1 and EPA6 expression. It binds to the upstream regions of EPA1 and EPA6, as well as the promoter regions of the subtelomeric silencing-related genes SIR4, RIF1, and RAP1, indicating the dual regulatory role of Mss11. Conclusion: Mss11 plays a crucial role in C. glabrata adhesion and biofilm formation, and thus has a broad influence on virulence. This regulation is achieved by regulating the expression of EPA1 and EPA6 through both promoter-specific regulation and subtelomeric silencing. [ABSTRACT FROM AUTHOR]

Background and Aim: Side effects associated with antimicrobial drugs, as well as their high cost, have stimulated the search for inexpensive herbal medicinals with fewer side effects. These substances can be used as a medicinal supplement or to increase the antimicrobial and antifungal effects of drugs. The aim of this study is to investigate the inhibitory effect of oleuropein on fungal and bacterial pathogens isolated from nail infection in laboratory condition. Methods: Antifungal and antibacterial properties of oleuropein by determining the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC) of this substance on yeasts Candida albicans, Candida glabrata and Escherichia coli by microbroth dilution method using CLSI protocols were evaluated. Results: The results showed that the Minimum Inhibitory Concentration (MIC) against all three studied microorganisms was equal to 65 mg/ml and the Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC) of oleuropein was equal to 130 mg/ml. Conclusion: due to the high antifungal and antibacterial activity of oleuropein, the present study introduces oleuropein as a natural antimicrobial drug. This substance can also be used to increase the antimicrobial power of existing antibiotics. [ABSTRACT FROM AUTHOR]

Aims Candidiasis is the most opportunistic infection with a high rate of recurrent infection. Salvia macrosiphon has an antibacterial effect; however, its antifungal effect was not studied. This study aimed to investigate the chemical composition of essential oil from leaves of Salvia macrosiphon and its antifungal activity compared with fluconazole. Materials & Methods Salvia macrosiphon leaves, a native plant of Kogiluyeh and Boyerahmad province, were collected from Zagros Heights and used in this study. Then, the essential oil of this plant was tested for antibacterial and antifungal properties and compared with fluconazole. The chemical composition of the essential oil was analyzed by GC/MS (Gas Chromatography Mass Spectrometry), and the antifungal activity of the plant’s essential oil was compared with that of fluconazole. Findings The results of GC-MS analysis proved the presence of at least 29 compounds in the essential oil of Salvia macrosiphon. Amon these constitute butyl benzoate (49.16%), n-hexyl benzoate (7%), and isopatolenol (4.83%) were the main compounds. The minimum inhibitory concentration (MIC) of the essential oil (μl/ml) of Salvia macrosiphon and fluconazole (μg/ ml) were 0.44 and 0.7 for Candida albicans, 0.056 and 0.7 for C. glabrata, and 0.1 and 0.088 for C. parapsilosis, respectively. Also, statistical analysis demonstrated a significant difference between the mean of fluconazole and essential oil in total Candida isolates (p=0.001). Conclusion The essential oil of Salvia macrosiphon has stronger antifungal activity than fluconazole. [ABSTRACT FROM AUTHOR]

The growing threat of antimicrobial resistance (AMR) is a global concern. With AMR directly causing 1.27 million deaths in 2019 and projections of up to 10 million annual deaths by 2050, optimising infectious disease treatments is imperative. Prudent antimicrobial use, including treatment duration, can mitigate AMR emergence. This is particularly critical in candidemia, a severe condition with a 45% crude mortality rate, as the 14-day minimum treatment period has not been challenged in randomised comparison. A comprehensive literature search was conducted in August 2023, revealing seven original articles and two case series discussing treatment durations of less than 14 days for candidemia. No interventional trials or prospective observational studies assessing shorter durations were found. Historical studies showed varying candidemia treatment durations, questioning the current 14-day minimum recommendation. Recent research observed no significant survival differences between patients receiving shorter or longer treatment, emphasising the need for evidence-based guidance. Treatment duration reduction post-blood culture clearance could decrease exposure to antifungal drugs, limiting selection pressure, especially in the context of emerging multiresistant Candida species. Candidemia's complexity, emerging resistance and potential for shorter in-hospital stays underscore the urgency of refining treatment strategies. Evidence-driven candidemia treatment durations are imperative to balance efficacy with resistance prevention and ensure the longevity of antifungal therapies. Further research and clinical trials are needed to establish evidence-based guidelines for candidemia treatment duration. [ABSTRACT FROM AUTHOR]

Most previous studies on fungal NADPH oxidases (Nox) focused on multicellular fungi and highlighted the important roles of Nox-derived reactive oxygen species (ROS) in cellular differentiation and signaling communication. However, there are few reports about Nox in unicellular fungi. A novel NOX ortholog, CAGL0K05863g (named CgNOX1), in Candida glabrata was investigated in this study. Deletion of CgNOX1 led to a decrease in both intracellular and extracellular ROS production. In addition, the Cgnox1∆ mutant exhibited hypersensitivity to hydrogen peroxide and menadione. Also, the wild-type strain showed higher levels of both CgNOX1 mRNA expression and ROS production under oxidative stress. Moreover, the absence of CgNOX1 resulted in impaired ferric reductase activity. Although there was no effect on in vitro biofilm formation, the CgNOX1 mutant did not produce hepatic apoptosis, which might be mediated by fungal Nox-derived ROS during co-incubation. Together, these results indicated that the novel NOX gene plays important roles in unicellular pathogenic C. glabrata and its interaction with host cells. [ABSTRACT FROM AUTHOR]