Rational selection of morphological phenotypic traits to extract essential similarities in chemical perturbation in the ergosterol pathway.

Terbinafine, fluconazole, and amorolfine inhibit fungal ergosterol synthesis by acting on their target enzymes at different steps in the synthetic pathway, causing the accumulation of various intermediates. We found that the effects of these three in- hibitors on yeast morphology were different. The number of morphological parameters commonly altered by these drugs was only approximately 6% of the total. Using a rational strategy to find commonly changed parameters,we focused on hidden essential similarities in the phenotypes possibly due to decreased ergosterol levels. This resulted in higher apparent morphological similarity. Improvements in morphological similarity were observed even when canonical correlation analysis was used to select biologically meaningful morphological parameters related to gene function. In addition to changes in cell morphology, we also observed differences in the synergistic effects among the three inhibitors and in their fungicidal effects against pathogenic fungi possibly due to the accumulation of different intermediates. This study provided a comprehensive understanding of the properties of inhibitors acting in the same biosynthetic pathway.

Genotypic and phenotypic characterisation of a nosocomial outbreak of Candida auris in Spain during 5 years.

OBJECTIVES: The investigation of Candida auris outbreaks is needed to provide insights into its population structure and transmission dynamics. We genotypically and phenotypically characterised a C. auris nosocomial outbreak occurred in Consorcio Hospital General Universitario de Valencia (CHGUV), Spain. METHODS: Data and isolates were collected from CHGUV from September 2017 (first case) until September 2021. Thirty-five isolates, including one from an environmental source, were randomly selected for whole genome sequencing (WGS), and the genomes were analysed along with a database with 335 publicly available genomes, assigning them to one of the five major clades. In order to identify polymorphisms associated with drug resistance, we used the fully susceptible GCA_003014415.1 strain as reference sequence. Known mutations in genes ERG11 and FKS1 conferring resistance to fluconazole and echinocandins, respectively, were investigated. Isolates were classified into aggregating or non-aggregating. RESULTS: All isolates belonged to clade III and were from an outbreak with a single origin. They clustered close to three publicly available genomes from a hospital from where the first patient was transferred, being the probable origin. The mutation VF125AL in the ERG11 gene, conferring resistance to fluconazole, was present in all the isolates and one isolate also carried the mutation S639Y in the FKS1 gene. All the isolates had a non-aggregating phenotype (potentially more virulent). CONCLUSIONS: Isolates are genotypically related and phenotypically identical but one with resistance to echinocandins, which seems to indicate that they all belong to an outbreak originated from a single isolate, remaining largely invariable over the years. This result stresses the importance of implementing infection control practices as soon as the first case is detected or when a patient is transferred from a setting with known cases.

Deletion of the Candida albicans TLO gene family results in alterations in membrane sterol composition and fluconazole tolerance.

Development of resistance and tolerance to antifungal drugs in Candida albicans can compromise treatment of infections caused by this pathogenic yeast species. The uniquely expanded C. albicans TLO gene family is comprised of 14 paralogous genes which encode Med2, a subunit of the multiprotein Mediator complex which is involved in the global control of transcription. This study investigates the acquisition of fluconazole tolerance in a mutant in which the entire TLO gene family has been deleted. This phenotype was reversed to varying degrees upon reintroduction of representative members of the alpha- and beta-TLO clades (i.e. TLO1 and TLO2), but not by TLO11, a gamma-clade representative. Comparative RNA sequencing analysis revealed changes in the expression of genes involved in a range of cellular functions, including ergosterol biosynthesis, mitochondrial function, and redox homeostasis. This was supported by the results of mass spectrometry analysis, which revealed alterations in sterol composition of the mutant cell membrane. Our data suggest that members of the C. albicans TLO gene family are involved in the control of ergosterol biosynthesis and mitochondrial function and may play a role in the responses of C. albicans to azole antifungal agents.

Emerging trends in pediatric candidemia: mapping the rise in Candida parapsilosis incidence and antifungal resistance in Turkey.

Candidemia is emerging as a significant concern in children, particularly among those with underlying conditions like malignancies or prematurity. The interpretation of epidemiological data on candidemias and their antifungal resistance plays a vital role in aiding diagnosis and guiding clinicians in treatment decisions. From 2014 to 2021, a retrospective analysis was conducted in Istanbul, Turkey; comparing Candida albicans and non-albicans (NAC) spp in both surviving and deceased groups. Furthermore, an examination of Candida parapsilosis and other species was performed, assessing various clinical and laboratory parameters. Among 93 patients, with a median age of 17 months, C. parapsilosis emerged as the predominant isolated species (44%), followed by C. albicans (34.4%). Resistance to fluconazole, voricanozole, and echinocandins, along with a history of broad-spectrum antibiotic use were found to be significantly higher in the non-albicans Candida group compared to C. albicans group. In the C. parapsilosis group, statistically lower age was identified in comparison to the other groups (P = .018). In addition, high fluconazole and voriconazole resistance was detected in Candida parapsilosis spp. Our study highlights a notable prevalence of C. parapsilosis, particularly in younger children, which is different from similar studies in childhood. This trend may be attributed to the common use of total parenteral nutrition and central venous catheter in gastrointestinal disorders and metabolic diseases. Furthermore, as anticipated, high azole resistance is noted in C. parapsilosis and other non-albicans Candida species. Interestingly, resistance to both amphotericin B and echinocandins within this group has been notably high. It is crucial to emphasize the considerable antifungal resistance seen in C. parapsilosis isolates.

Mechanism of action and synergistic effect of Eugenia uniflora extract in Candida spp.

The limited arsenal of antifungal drugs have prompted the search for novel molecules with biological activity. This study aimed to characterize the antifungal mechanism of action of Eugenia uniflora extract and its synergistic activity with commercially available antifungal drugs on the following Candida species: C. albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. dubliniensis. In silico analysis was performed to predict antifungal activity of the major compounds present in the extract. Minimal inhibitory concentrations (MICs) were determined in the presence of exogenous ergosterol and sorbitol. Yeast cells were grown in the presence of stressors. The loss of membrane integrity was assessed using propidium iodide staining (fluorescence emission). Synergism between the extract and antifungal compounds (in addition to time kill-curves) was determined. Molecular docking revealed possible interactions between myricitrin and acid gallic and enzymes involved in ergosterol and cell wall biosynthesis. Candida cells grown in the presence of the extract with addition of exogenous ergosterol and sorbitol showed 2 to 8-fold increased MICs. Strains treated with the extract revealed greater loss of membrane integrity when compared to their Fluconazole counterparts, but this effect was less pronounced than the membrane damage caused by Amphotericin B. The extract also made the strains more susceptible to Congo red and Calcofluor white. A synergistic action of the extract with Fluconazole and Micafungin was observed. The E. uniflora extract may be a viable option for the treatment of Candida infections.

Long-term stability of acquired drug resistance and resistance associated mutations in the fungal pathogen Nakaseomyces glabratus (Candida glabrata).

The limited number of available antifungal drugs and the increasing number of fungal isolates that show drug or multidrug resistance pose a serious medical threat. Several yeast pathogens, such as Nakaseomyces glabratus (Candida glabrata), show a remarkable ability to develop drug resistance during treatment through the acquisition of genetic mutations. However, how stable this resistance and the underlying mutations are in non-selective conditions remains poorly characterized. The stability of acquired drug resistance has fundamental implications for our understanding of the appearance and spread of drug-resistant outbreaks and for defining efficient strategies to combat them. Here, we used an in vitro evolution approach to assess the stability under optimal growth conditions of resistance phenotypes and resistance-associated mutations that were previously acquired under exposure to antifungals. Our results reveal a remarkable stability of the resistant phenotype and the underlying mutations in a significant number of evolved populations, which conserved their phenotype for at least two months in the absence of drug-selective pressure. We observed a higher stability of anidulafungin resistance over fluconazole resistance, and of resistance-conferring point mutations as compared with aneuploidies. In addition, we detected accumulation of novel mutations in previously altered resistance-associated genes in non-selective conditions, which suggest a possible compensatory role. We conclude that acquired resistance, particularly to anidulafungin, is a long-lasting phenotype, which has important implications for the persistence and propagation of drug-resistant clinical outbreaks.

Clinico-mycological study of onychomycosis and in vitro antifungal susceptibility of Trichophyton rubrum.

Onychomycosis, a fungal nail infection, is primarily caused by dermatophytes, yeasts, and non-dermatophyte moulds (NDMs). The incidence of this disease and the predominance of specific pathogens vary across different regions and evolve. This study aimed to elucidate the epidemiology of onychomycosis and the pattern of causative pathogens in Beijing, and to ascertain the in vitro antifungal susceptibility profiles of Trichophyton rubrum against itraconazole (ITR), terbinafine (TER), and fluconazole (FLU). Involving 245 patients of onychomycosis with positive fungal culture results, the study implemented internal transcribed spacer (ITS) sequencing of ribosomal DNA (rDNA) on all collected samples. The mean age of the participants was 37.93 ± 13.73 years, with a male-to-female ratio of 1.53:1. The prevalence of toenail infections was significantly higher than that of fingernails. Distal and lateral subungual onychomycosis (DLSO) were the most frequent clinical classifications. PCR results indicated that dermatophytes were the most prevalent pathogens, followed by yeasts and NDMs, among which T. rubrum was the most dominant dermatophyte. TER demonstrated high sensitivity to T. rubrum. However, in clinical settings, some patients with onychomycosis exhibit a poor response to TER treatment. The relationship between in vitro antifungal sensitivity and clinical effectiveness is complex, and understanding the link between in vitro MIC values and clinical efficacy requires further investigation.

Epidemiology of cryptococcal meningitis and fluconazole heteroresistance in Cryptococcus neoformans isolates from a teaching hospital in southwestern China.

Cryptococcal meningitis (CM), a common and serious opportunistic infection mostly caused by Cryptococcus neoformans, is primarily treated with fluconazole. Nevertheless, Cryptococcus neoformans strains that undergo repeated exposure to azoles can gradually acquire heteroresistance to fluconazole. The management of this specific CM infection poses a substantial challenge. Determining a globally accepted definition for fluconazole heteroresistance and developing effective and prompt methods for identifying heteroresistance is of utmost importance. We collected data on the clinical and epidemiological characteristics of patients diagnosed with CM. All the available Cryptococcus neoformans strains isolated from these patients were collected and subjected to antifungal susceptibility testing and evaluation of fluconazole heteroresistance. AIDS was present in 40.5% of the patients, whereas 24.1% did not have any underlying diseases. Patients with chronic diseases or impaired immune systems are susceptible to infection by Cryptococcus neoformans, a fungus that frequently (39.6%, 19/48) shows heteroresistance to fluconazole, as confirmed by population analysis profile (PAP).IMPORTANCEFluconazole heteroresistance poses a significant threat to the efficacy of fluconazole in treating cryptococcal meningitis (CM). Unfortunately, the standard broth microdilution method often misses the subtle percentages of subpopulations exhibiting heteroresistance. While the population analysis profile (PAP) method is esteemed as the gold standard, its time-consuming and labor-intensive nature makes it impractical for routine clinical use. In contrast, the Kirby-Bauer (KB) disk diffusion method offers a simple and effective screening solution. Our study highlights the value of KB over PAP and minimum inhibitory concentration (MIC) by demonstrating that when adjusting the inoculum concentration to 1.0 McFarland and subjecting samples to a 72-hour incubation period at 35°C, the KB method closely mirrors the outcomes of the PAP approach in detecting fluconazole heteroresistance. This optimization of the KB method not only enhances assay efficiency but also provides a blueprint for developing a timely and effective strategy for identifying heteroresistance.

Synergic Effect of the Antimicrobial Peptide ToAP2 and Fluconazole on Candida albicans Biofilms.

Candida albicans is one of the agents of invasive candidiasis, a life-threatening disease strongly associated with hospitalization, particularly among patients in intensive care units with central venous catheters. This study aimed to evaluate the synergistic activity of the antifungal peptide ToAP2 combined with fluconazole against C. albicans biofilms grown on various materials. We tested combinations of different concentrations of the peptide ToAP2 with fluconazole on C. albicans biofilms. These biofilms were generated on 96-well plates, intravenous catheters, and infusion tubes in RPMI medium at two maturation stages. Scanning electron microscopy and atomic force microscopy were employed to assess the biofilm structure. We also evaluated the expression of genes previously proven to be involved in C. albicans biofilm formation in planktonic and biofilm cells after treatment with the peptide ToAP2 using qPCR. ToAP2 demonstrated a synergistic effect with fluconazole at concentrations up to 25 µM during both the early and mature stages of biofilm formation in 96-well plates and on medical devices. Combinations of 50, 25, and 12.5 µM of ToAP2 with 52 µM of fluconazole significantly reduced the biofilm viability compared to individual treatments and untreated controls. These results were supported by substantial structural changes in the biofilms observed through both scanning and atomic force microscopy. The gene expression analysis of C. albicans cells treated with 25 µM of ToAP2 revealed a decrease in the expression of genes associated with membrane synthesis, along with an increase in the expression of genes involved in efflux pumps, adhesins, and filamentation. Our results highlight the efficacy of the combined ToAP2 and fluconazole treatment against C. albicans biofilms. This combination not only shows therapeutic potential but also suggests its utility in developing preventive biofilm tools for intravenous catheters.

Transcriptome Analysis of Human Dermal Cells Infected with Candida auris Identified Unique Pathogenesis/Defensive Mechanisms Particularly Ferroptosis.

Candida auris is an emerging multi-drug resistant yeast that can cause life-threatening infections. A recent report clarified the ability of C. auris to form a biofilm with enhanced drug resistance properties in the host skin's deep layers. The formed biofilm may initiate further bloodstream spread and immune escape. Therefore, we propose that secreted chemicals from the biofilm may facilitate fungal pathogenesis. In response to this interaction, the host skin may develop potential defensive mechanisms. Comparative transcriptomics was performed on the host dermal cells in response to indirect interaction with C. auris biofilm through Transwell inserts compared to planktonic cells. Furthermore, the effect of antifungals including caspofungin and fluconazole was studied. The obtained data showed that the dermal cells exhibited different transcriptional responses. Kyoto Encyclopedia of Genes and Genomes and Reactome analyses identified potential defensive responses employed by the dermal cells and potential toxicity induced by C. auris. Additionally, our data indicated that the dominating toxic effect was mediated by ferroptosis; which was validated by qRT-PCR, cytotoxicity assay, and flow cytometry. On the other hand, the viability of C. auris biofilm was enhanced and accompanied by upregulation of MDR1, and KRE6 upon interaction with dermal cells; both genes play significant roles in drug resistance and biofilm maturation, respectively. This study for the first-time shed light on the dominating defensive responses of human dermal cells, microbe colonization site, to C. auris biofilm and its toxic effects. Further, it demonstrates how C. auris biofilm responds to the defensive mechanisms developed by the human dermal cells.

Aneuploidy underlies brefeldin A-induced antifungal drug resistance in Cryptococcus neoformans.

Cryptococcus neoformans is at the top of the list of "most wanted" human pathogens. Only three classes of antifungal drugs are available for the treatment of cryptococcosis. Studies on antifungal resistance mechanisms are limited to the investigation of how a particular antifungal drug induces resistance to a particular drug, and the impact of stresses other than antifungals on the development of antifungal resistance and even cross-resistance is largely unexplored. The endoplasmic reticulum (ER) is a ubiquitous subcellular organelle of eukaryotic cells. Brefeldin A (BFA) is a widely used chemical inducer of ER stress. Here, we found that both weak and strong selection by BFA caused aneuploidy formation in C. neoformans, mainly disomy of chromosome 1, chromosome 3, and chromosome 7. Disomy of chromosome 1 conferred cross-resistance to two classes of antifungal drugs: fluconazole and 5-flucytosine, as well as hypersensitivity to amphotericin B. However, drug resistance was unstable, due to the intrinsic instability of aneuploidy. We found overexpression of AFR1 on Chr1 and GEA2 on Chr3 phenocopied BFA resistance conferred by chromosome disomy. Overexpression of AFR1 also caused resistance to fluconazole and hypersensitivity to amphotericin B. Furthermore, a strain with a deletion of AFR1 failed to form chromosome 1 disomy upon BFA treatment. Transcriptome analysis indicated that chromosome 1 disomy simultaneously upregulated AFR1, ERG11, and other efflux and ERG genes. Thus, we posit that BFA has the potential to drive the rapid development of drug resistance and even cross-resistance in C. neoformans, with genome plasticity as the accomplice.

Dietary Moringa oleifera mitigates Fluconazole-Induced immunological and spleen-histological alterations in Catfish (Clarias gariepinus).

Fluconazole (FCZ), an antifungal from the azole family, causes several detrimental effects in fish. In recent times, there has been a notable surge in interest regarding the utilization of Moringa oleifera (Mo) as a dietary antioxidant. This research aimed to evaluate the potential protective effects of dietary Moringa oleifera (MO) against the adverse impacts of fluconazole in the African catfish (Clarias gariepinus). The fish were allocated into four groups as follows: a control group fed a basal diet, an FCZ - exposed (200 ng/L) fed basal diet, 1% MO fed through basal diet, and an FCZ-exposed (200 ng/L) and 1% MO fed through basal diet fed group. The results showed that FCZ exposure decreased superoxide dismutase, total antioxidant capacity, and acetylcholine esterase levels. On the other hand, FCZ exposure increased malonaldehyde and cortisol levels as compared to control (P < 0.05). FCZ caused immunosuppressive effects in C. gariepinus as revealed by lower immunity indices (lysozyme and phagocytic activity and immunoglobulin level) and increased cytokine levels (IL-6 IL-1ß). Histological examination of the spleen from fish exposed to FCZ showed several splenic changes. We conclude that dietary MO supplementation has the potential to alleviate the oxidative stress, restore immune response balance, and mitigate histological damage induced by FCZ exposure, thus positioning MO as an immunostimulant in C. gariepinus when administered alongside FCZ.

Increase in candidemia cases and emergence of fluconazole-resistant Candida parapsilosis and C. auris isolates in a tertiary care academic hospital during the COVID-19 pandemic, Greece, 2020 to 2023.

BackgroundThe COVID-19 pandemic and the emergence of Candida auris have changed the epidemiological landscape of candidaemia worldwide.AimWe compared the epidemiological trends of candidaemia in a Greek tertiary academic hospital before (2009-2018) and during the early COVID-19 (2020-2021) and late COVID-19/early post-pandemic (2022-2023) era.MethodsIncidence rates, species distribution, antifungal susceptibility profile and antifungal consumption were recorded, and one-way ANOVA or Fisher's exact test performed. Species were identified by MALDI-ToF MS, and in vitro susceptibility determined with CLSI M27-Ed4 for C. auris and the EUCAST-E.DEF 7.3.2 for other Candida spp.ResultsIn total, 370 candidaemia episodes were recorded during the COVID-19 pandemic. Infection incidence (2.0 episodes/10,000 hospital bed days before, 3.9 during the early and 5.1 during the late COVID-19 era, p < 0.0001), C. auris (0%, 9% and 33%, p < 0.0001) and fluconazole-resistant C. parapsilosis species complex (SC) (20%, 24% and 33%, p = 0.06) infections increased over time, with the latter not associated with increase in fluconazole/voriconazole consumption. A significant increase over time was observed in fluconazole-resistant isolates regardless of species (8%, 17% and 41%, p < 0.0001). Resistance to amphotericin B or echinocandins was not recorded, with the exception of a single pan-echinocandin-resistant C. auris strain.ConclusionCandidaemia incidence nearly tripled during the COVID-19 era, with C. auris among the major causative agents and increasing fluconazole resistance in C. parapsilosis SC. Almost half of Candida isolates were fluconazole-resistant, underscoring the need for increased awareness and strict implementation of infection control measures.

Synergistic Interaction of Certain Essential Oils and Their Active Compounds with Fluconazole against Azole-resistant Strains of Cryptococcus neoformans.

OBJECTIVES: This study investigated the anti-cryptococcal potential of certain essential oils (EOs)/compounds alone and in combination with fluconazole. MATERIALS AND METHODS: We investigated the antifungal activity of oils of Cinnamomum verum, Cymbopogon citratus, Cymbopogon martini, and Syzygium aromaticum, and their major active ingredients cinnamaldehyde, citral, eugenol, and geraniol against clinical and standard strains of Cryptococcus neoformans (CN). Disc diffusion, broth microdilution, checkerboard methods, and transmission electron microscopy were employed to determine growth inhibition, synergistic interaction, and mechanism of action of test compounds. RESULTS: EOs/compounds showed pronounced antifungal efficacy against azole-resistant CN in the order of cinnamaldehyde > eugenol > S. aromaticum > C. verum > citral > C. citratus > geraniol ≥ C. martini, each exhibiting zone of inhibition >15 mm. These oils/compounds were highly cidal compared to fluconazole. Eugenol and cinnamaldehyde showed the strongest synergy with fluconazole against CN by lowering their MICs up to 32-fold. Transmission electron microscopy indicated damage of the fungal cell wall, cell membrane, and other endomembranous organelles. CONCLUSION: Test oils and their active compounds exhibited potential anti-cryptococcus activity against the azole-resistant strains of CN. Moreover, eugenol and cinnamaldehyde significantly potentiated the anti-cryptococcal activity of fluconazole. It is suggested that multiple sites of action from oils/compounds could turn static fluconazole into a cidal drug combination in combating cryptococcosis.

RésuméObjectifs: Cette étude a étudié le potentiel anti-cryptocoque de certaines huiles essentielles (HE)/composés seuls et en combinaison avec fluconazole. Matériels et méthodes: Nous avons étudié l'activité antifongique des huiles de Cinnamomum verum, Cymbopogon citratus, Cymbopogon martini et Syzygium spiceum , et leurs principaux ingrédients actifs, le cinnamaldéhyde, le citral, l'eugénol et le géraniol, contre les normes cliniques et standards. souches de Cryptococcus neoformans (CN). Diffusion sur disque, microdilution en bouillon, méthodes en damier et microscopie électronique à transmission ont été utilisés pour déterminer l'inhibition de la croissance, l'interaction synergique et le mécanisme d'action des composés testés. Résultats: HE/composés a montré une efficacité antifongique prononcée contre les CN résistantes aux azoles dans l'ordre suivant: cinnamaldéhyde > eugénol > S. spiceum > C. verum > citral > C. citratus > géraniol ≥ C. martini , chacun présentant une zone d'inhibition > 15 mm. Ces huiles/composés étaient hautement cides par rapport au fluconazole. L'eugénol et le cinnamaldéhyde ont montré la synergie la plus forte avec le fluconazole contre le CN en abaissant leurs CMI jusqu'à 32 fois. La microscopie électronique à transmission a indiqué des dommages à la paroi cellulaire fongique, à la membrane cellulaire et à d'autres organites endomembranaires. Conclusion: Les huiles testées et leurs composés actifs ont montré une activité anti-cryptocoque potentielle contre les souches de CN résistantes aux azoles. De plus, l'eugénol et le cinnamaldéhyde ont significativement potentialisé l'activité anticryptococcique du fluconazole. Il est suggéré que plusieurs Les sites d'action des huiles/composés pourraient transformer le fluconazole statique en une combinaison médicamenteuse cide pour lutter contre la cryptococcose.

Erg251 has complex and pleiotropic effects on sterol composition, azole susceptibility, filamentation, and stress response phenotypes.

Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Homozygous deletions of ERG251 resulted in accumulation of ergosterol intermediates consistent with the fitness defect in rich medium. Dysfunction of ERG251, together with FLC exposure, resulted in decreased accumulation of the toxic sterol (14-ɑ-methylergosta-8,24(28)-dien-3ß,6α-diol) and increased accumulation of non-toxic alternative sterols. The altered sterol composition of the ERG251 mutants had pleiotropic effects on transcription, filamentation, and stress responses including cell membrane, osmotic and oxidative stress. Interestingly, while dysfunction of ERG251 resulted in azole tolerance, it also led to transcriptional upregulation of ZRT2, a membrane-bound Zinc transporter, in the presence of FLC, and overexpression of ZRT2 is sufficient to increase azole tolerance in wild-type C. albicans. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study demonstrates that single allele dysfunction of ERG251 is a recurrent and effective mechanism of acquired azole tolerance. We propose that altered sterol composition resulting from ERG251 dysfunction mediates azole tolerance as well as pleiotropic effects on stress response, filamentation and virulence.

Ketamine metabolism via hepatic CYP450 isoforms contributes to its sustained antidepressant actions.

(R,S)-ketamine (ketamine) has rapid and sustained antidepressant (AD) efficacy at sub-anesthetic doses in depressed patients. A metabolite of ketamine, including (2R,6R)-hydroxynorketamine ((6)-HNKs) has been reported to exert antidepressant actions in rodent model of anxiety/depression. To further understand the specific role of ketamine's metabolism in the AD actions of the drug, we evaluated the effects of inhibiting hepatic cytochrome P450 enzymes on AD responses. We assessed whether pre-treatment with fluconazole (10 and 20 mg/kg, i. p.) 1 h prior to ketamine or HNKs (10 mg/kg, i. p.) administration would alter behavioral and neurochemical actions of the drugs in male BALB/cJ mice with a highly anxious phenotype. Extracellular microdialysate levels of glutamate and GABA (Gluext, GABAext) were also measured in the medial prefrontal cortex (mPFC). Pre-treatment with fluconazole altered the pharmacokinetic profile of ketamine, by increasing both plasma and brain levels of ketamine and (R,S)-norketamine, while robustly reducing those of (6)-HNKs. At 24 h post-injection (t24 h), fluconazole prevented the sustained AD-like response of ketamine responses in the forced swim test and splash test, as well as the enhanced cortical GABA levels produced by ketamine. A single (2R,6R)-HNK administration resulted in prevention of the effects of fluconazole on the antidepressant-like activity of ketamine in mice. Overall, these findings are consistent with an essential contribution of (6)-HNK to the sustained antidepressant-like effects of ketamine and suggest potential interactions between pharmacological CYPIs and ketamine during antidepressant treatment in patients.

Mangiferin potentiates the activity of antifungal agents against fluconazole-resistant Candida spp.

Aim: To evaluate the antifungal activity of mangiferin against Candida spp. resistant to fluconazole.Materials & methods: The antifungal activity of mangiferin was assessed using broth microdilution and its interaction with azoles and amphotericin B was evaluated by checkerboard. The activity of mangiferin against Candida spp. biofilms was assessed using the MTT colorimetric assay and its possible mechanism of action was evaluated using flow cytometry.Results: Mangiferin showed activity against Candida albicans, Candida tropicalis and Candida parapsilosis resistant to fluconazole and showed synergism with azoles and amphotericin B. Mangiferin increased the activity of antifungals against Candida biofilms and caused depolarization of the mitochondrial membrane and externalization of phosphatidylserine, suggesting apoptosis.Conclusion: mangiferin combined with antifungals has potential against Candida spp.

Candida is a type of fungus that can make people ill. Over time, many species of Candida have found ways to resist the drugs used to kill them. It is important to find new drugs. We decided to see if a substance called mangiferin works against Candida. We found that mangiferin works against Candida and may help other drugs to work better. We still need to do more studies to find out whether mangiferin can help prevent diseases caused by Candida in the future.

Implication of genotypes for prognosis of Candida glabrata bloodstream infections.

BACKGROUND: Genotyping isolates of a specific pathogen may demonstrate unique patterns of antimicrobial resistance, virulence or outcomes. However, evidence for genotype-outcome association in Candida glabrata is scarce. We aimed to characterize the mycological and clinical relevance of genotypes on C. glabrata bloodstream infections (BSIs). METHODS: Non-duplicated C. glabrata blood isolates from hospitalized adults were genotyped by MLST, and further clustered by the unweighted pair group method with arithmetic averages (UPGMA). A clonal complex (CC) was defined by UPGMA similarities of >90%. Antifungal susceptibility testing was performed by a colorimetric microdilution method and interpreted following CLSI criteria. RESULTS: Of 48 blood isolates evaluated, 13 STs were identified. CC7 was the leading CC (n = 14; 29.2%), including 13 ST7. The overall fluconazole and echinocandin resistance rates were 6.6% and 0%, respectively. No specific resistance patterns were associated with CC7 or other CCs. Charlson comorbidity index (adjusted OR, 1.49; 95% CI, 1.05-3.11) was the only predictor for CC7. By multivariable Cox regression analyses, CC7 was independently associated with 28 day mortality [adjusted HR (aHR), 3.28; 95% CI, 1.31-8.23], even after considering potential interaction with neutropenia (aHR, 3.41; 95% CI, 1.23-9.42; P for interaction, 0.24) or limited to 34 patients with monomicrobial BSIs (aHR, 2.85; 95% CI, 1.15-7.08). Also, the Kaplan-Meier estimate showed greater mortality with CC7 (P = 0.003). Fluconazole resistance or echinocandin therapy had no significant impact on mortality. CONCLUSIONS: Our data suggested comorbid patients were at risk of developing CC7 BSIs. Further, CC7 was independently associated with worse outcomes.

Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.

The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast Candida albicans, because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, caSAT1 and HygB, which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of C. albicans gene deletion mutants, which demands a thorough examination of recombination events at the target site. IMPORTANCE: Candida albicans is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in C. albicans.

Development of biopolymer films loaded with fluconazole and thymol for resistant vaginal candidiasis.

Vulvovaginal candidiasis (VVC) is an opportunistic infection caused by a fungus of the Candida genus, affecting approximately 75 % of women during their lifetime. Fungal resistance cases and adverse effects have been the main challenges of oral therapies. In this study, the topical application of thin films containing fluconazole (FLU) and thymol (THY) was proposed to overcome these problems. Vaginal films based only on chitosan (CH) or combining this biopolymer with pectin (PEC) or hydroxypropylmethylcellulose acetate succinate (HPMCAS) were developed by the solvent casting method. In addition to a higher swelling index, CH/HPMCAS films showed to be more plastic and flexible than systems prepared with CH/PEC or only chitosan. Biopolymers and FLU were found in an amorphous state, contributing to explaining the rapid gel formation after contact with vaginal fluid. High permeability rates of FLU were also found after its immobilization into thin films. The presence of THY in polymer films increased the distribution of FLU in vaginal tissues and resulted in improved anti-Candida activity. A significant activity against the resistant C. glabrata was achieved, reducing the required FLU dose by 50 %. These results suggest that the developed polymer films represent a promising alternative for the treatment of resistant vulvovaginal candidiasis, encouraging further studies in this context.