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.

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.

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.

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.

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.

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.

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.

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.

Antifungal, anti-biofilm, and anti-hyphal properties of N-substituted phthalimide derivatives against Candida species.

Candida species comprise a ubiquitous pathogenic fungal genus responsible for causing candidiasis. They are one of the primary causatives of several mucosal and systemic infections in humans and can survive in various environments. In this study, we investigated the antifungal, anti-biofilm, and anti-hyphal effects of six N-substituted phthalimides against three Candida species. Of the derivatives, N-butylphthalimide (NBP) was the most potent, with a minimum inhibitory concentration (MIC) of 100 µg/ml and which dose-dependently inhibited biofilm at sub-inhibitory concentrations (10-50 µg/ml) in both the fluconazole-resistant and fluconazole-sensitive Candida albicans and Candida parapsilosis. NBP also effectively inhibited biofilm formation in other pathogens including uropathogenic Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, and Vibrio parahaemolyticus, along with the polymicrobial biofilms of S. epidermidis and C. albicans. NBP markedly inhibited the hyphal formation and cell aggregation of C. albicans and altered its colony morphology in a dose-dependent manner. Gene expression analysis showed that NBP significantly downregulated the expression of important hyphal- and biofilm-associated genes, i.e., ECE1, HWP1, and UME6, upon treatment. NBP also exhibited mild toxicity at concentrations ranging from 2 to 20 µg/ml in a nematode model. Therefore, this study suggests that NBP has anti-biofilm and antifungal potential against various Candida strains.

Synergistic and potential antifungal properties of tailored, one pot multicomponent monoterpenes co-delivered with fluconazole encapsulated nanostructure lipid carrier.

Frequent and variant infections are caused by the virtue of opportunistic fungi pathogens. Candidiasis, aspergillosis, and mucormycosis are pathogenic microorganisms that give rise to vast fungal diseases that alternate between moderate to fatal in severity. The use of fluconazole as an antifungal drug was limited due to the acquired resistance in some types of Candida and other fungal species. This study aims to consolidate fluconazole's biological effectiveness against several pathogenic fungi. Six active monoterpenes (MTs) of carvacrol, linalool, geraniol, α-terpinene, citronellal, and nerolidol were selected and encapsulated in nanostructure lipid carrier (NLC) with (NLC-Flu-MTs) and/without (NLC-MTs) fluconazole in one nanoformulation to determine if they will act synergistically or not? The synthesized nanoformulation NLC-Flu-MTs and NLC-MTs exhibited very good particle size of 144.5 nm and 138.6 nm for size and zeta potential values of (- 23.5 mV) and (- 20.3 mV), respectively. Transmission electron microscope investigation confirmed that the synthesized NLCs have regular and spherical shape. The abundance and concentration of the six released monoterpenes were determined, as a novel approach, using GC-MS with very good results and validity. In-vitro antifungal screening was done before and after nano co-delivery against seven pathogenic, and aggressive fungi of Candida tropicalis, Candida krusei, Candida glabrata, Geotrichum Candidum, Candidaalbicans, Aspergillus Niger, and mucor circinelloides. Inhibition Zone diameter (IZD) and the minimum inhibitory concentration (MIC) were measured. Nanoformulations NLC-Flu-MTs and NLC-MTs manifested potential and unique biological susceptibility against all the tested microorganisms with reduced (MIC) values, especially against Candida Tropicalis (MIC = 0.97 µg/ml) which represents 16-fold of the value shown by NLC-MTs (MIC = 15.6 µg/ml) and 64-fold of fluconazole free before nanoformulation (MIC = 62.5 µg/ml). The efficiency of nanomaterials, particularly NLC-Flu-MTs, has become evident in the diminishing value of MIC which affirmed the synergism between fluconazole and the other six monoterpenes.

Activity and mechanism of action of antimicrobial peptide ACPs against Candida albicans.

AIMS: Candida albicans is the most prevalent pathogenic fungus, exhibiting escalating multidrug resistance (MDR). Antimicrobial peptides (AMPs) represent promising candidates for addressing this issue. In this research, five antimicrobial peptides, ACP1 to ACP5 which named ACPs were studied as alternative fungicidal molecules. MAIN METHODS: CD assay was used to analyze the 2D structures, Absorbance method was used to test the antimicrobial activity, haemolytic activity, time-kill kinetics, biofilm inhibition and reduction activity, resistance induction activity and assessment against fluconazole-resistant C. albicans. SEM, TEM, CLSM, flow cytometer and FM were carried out to provide insight into the mechanisms of anti-Candida action. KEY FINDINGS: ACPs possessed an α-helical structure and strong anti-Candida activities, with minimum inhibitory concentrations (MICs) from 3.9 to 15.6 µg/mL. In addition, ACPs did not produce hemolysis at concentrations lower than 10 or 62 × MIC, indicating their low cytotoxicity. Fungicidal kinetics showed that they completely killed C. albicans within 8 h at 2 to 4 × MIC. Notably, ACPs were highly fungicidal against fluconazole-resistant C. albicans and showed low resistance. In addition, they were effective in inhibiting mycelium and biofilm formation. Fluorescence microscopy revealed that while fluconazole had minimal to no inhibitory effect on biofilm-forming cells, ACPs induced apoptosis in all of them. The research on mechanism of action revealed that ACPs disrupted the cell membranes, with ROS increasing and cellular mitochondrial membrane potential decreasing. SIGNIFICANCE: ACPs could be promising candidates for combating fluconazole-resistant C. albicans infections.

Emergence of the novel sixth Candida auris Clade VI in Bangladesh.

Candida auris, initially identified in 2009, has rapidly become a critical concern due to its antifungal resistance and significant mortality rates in healthcare-associated outbreaks. To date, whole-genome sequencing (WGS) has identified five unique clades of C. auris, with some strains displaying resistance to all primary antifungal drug classes. In this study, we presented the first WGS analysis of C. auris from Bangladesh, describing its origins, transmission dynamics, and antifungal susceptibility testing (AFST) profile. Ten C. auris isolates collected from hospital settings in Bangladesh were initially identified by CHROMagar Candida Plus, followed by VITEK2 system, and later sequenced using Illumina NextSeq 550 system. Reference-based phylogenetic analysis and variant calling pipelines were used to classify the isolates in different clades. All isolates aligned ~90% with the Clade I C. auris B11205 reference genome. Of the 10 isolates, 8 were clustered with Clade I isolates, highlighting a South Asian lineage prevalent in Bangladesh. Remarkably, the remaining two isolates formed a distinct cluster, exhibiting >42,447 single-nucleotide polymorphism differences compared to their closest Clade IV counterparts. This significant variation corroborates the emergence of a sixth clade (Clade VI) of C. auris in Bangladesh, with potential for international transmission. AFST results showed that 80% of the C. auris isolates were resistant to fluconazole and voriconazole, whereas Clade VI isolates were susceptible to azoles, echinocandins, and pyrimidine analogue. Genomic sequencing revealed ERG11_Y132F mutation conferring azole resistance while FCY1_S70R mutation found inconsequential in describing 5-flucytosine resistance. Our study underscores the pressing need for comprehensive genomic surveillance in Bangladesh to better understand the emergence, transmission dynamics, and resistance profiles of C. auris infections. Unveiling the discovery of a sixth clade (Clade VI) accentuates the indispensable role of advanced sequencing methodologies.IMPORTANCECandida auris is a nosocomial fungal pathogen that is commonly misidentified as other Candida species. Since its emergence in 2009, this multidrug-resistant fungus has become one of the five urgent antimicrobial threats by 2019. Whole-genome sequencing (WGS) has proven to be the most accurate identification technique of C. auris which also played a crucial role in the initial discovery of this pathogen. WGS analysis of C. auris has revealed five distinct clades where isolates of each clade differ among themselves based on pathogenicity, colonization, infection mechanism, as well as other phenotypic characteristics. In Bangladesh, C. auris was first reported in 2019 from clinical samples of a large hospital in Dhaka city. To understand the origin, transmission dynamics, and antifungal-resistance profile of C. auris isolates circulating in Bangladesh, we conducted a WGS-based surveillance study on two of the largest hospital settings in Dhaka, Bangladesh.

Candida parapsilosis candidemia in children admitted to a tertiary hospital in Turkey: clinical features and antifungal susceptibility.

In recent years, the incidence and drug resistance of Candida parapsilosis have increased. Our study aimed to determine the antifungal sensitivity of C. parapsilosis and the clinical and demographic characteristics of children with candidemia. Two hundred pediatric patients with C. parapsilosis candidemia were included in the study between 1 January 2010 and 1 August 2023. Clinical samples were evaluated on a BACTEC-FX-40 automatic blood culture device (Becton Dickinson, USA). Yeast isolates were identified to the species level via identification cards (YST) using the VITEK 2 Compact (bioMeriéux, France) system. Antifungal susceptibility was performed using antifungal cell cards (AST-YST01). Approval for the study was received from the "University Faculty of Medicine" Hospital Clinical Research Ethics Committee. Non-catheter candidemia was detected in 127 (63.5%) patients, and catheter-related candidemia was detected in 73 (36.5%) patients. It was observed that the patients' history of malignancy, mechanical ventilation, urinary catheter, nasogastric tube, and intensive care unit stay was associated with C. parapsilosis mortality. The mortality rate from candidemia was 9.5%. The most frequently preferred antifungal agents were amphotericin B and fluconazole. The fluconazole drug resistance rate was found to be 6%, and the amphotericin B drug resistance rate was 4%. Because C. parapsilosis candidemia mortality rates can be high depending on risk factors and clinical characteristics, it is important to initiate appropriate and timely antifungal therapy. We think that our study can provide important information about the clinical profiles, distributions, susceptibility profiles, and control of antifungal resistance of C. parapsilosis isolates. IMPORTANCE: It has been observed that the frequency and antifungal resistance of Candida parapsilosis have increased recently. In our study, we aimed to determine the antifungal sensitivity of C. parapsilosis and the clinical and demographic characteristics of children with candidemia. It was observed that the patients' history of malignancy, mechanical ventilation, urinary catheter, nasogastric tube, and intensive care stay was associated with C. parapsilosis mortality. The mortality rate from candidemia was 9.5%. The most frequently preferred antifungal agents were amphotericin B and fluconazole. The fluconazole drug resistance rate was found to be 6%, and the amphotericin B drug resistance rate was 4%. Because C. parapsilosis candidemia mortality rates can be high depending on risk factors and clinical characteristics, it is important to initiate appropriate and timely antifungal therapy.

Antifungal bioactivity of Sarcococca hookeriana var. digyna Franch. against fluconazole-resistant Candida albicans in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Sarcococca hookeriana var. digyna Franch. has been widely utilized in folk medicine by the Miao people in the southwestern region of China for treating skin sores which may be associated with microbial infection. AIM OF THE STUDY: To investigate the antifungal bioactivity of S. hookeriana var. digyna against fluconazole-resistant Candida albicans in vitro and in vivo, as well as its underlying mechanism and the key bioactive component. MATERIALS AND METHODS: The antifungal bioactivity of 80% ethanol extract of S. hookeriana var. digyna (SHE80) was investigated in vitro using the broth microdilution method, time-growth curve, and time-kill assay. Its key functional component and antifungal mechanism were explored with combined approaches including UPLC-Q-TOF-MS, network pharmacology and metabolomics. The antifungal pathway was further supported via microscopic observation of fungal cell morphology and examination of its effects on fungal biofilm and cell membranes using fluorescent staining reagents. In vivo assessment of antifungal bioactivity was conducted using a mouse model infected with C. albicans on the skin. RESULTS: S. hookeriana var. digyna suppressed fluconazole-resistant C. albicans efficiently (MIC = 16 µg/mL, MFC = 64 µg/mL). It removed fungal biofilm, increased cell membrane permeability, induced protein leakage, reduced membrane fluidity, disrupted mitochondrial membrane potential, induced the release of reactive oxygen species, promoted cell apoptosis, and inhibited the transformation of fungi from the yeast state to the hyphal state significantly. In terms of mechanism, it affected sphingolipid metabolism and signaling pathway. Moreover, the predicted bioactive component, sarcovagine D, was supported by antifungal bioactivity evaluation in vitro (MIC = 4 µg/mL, MFC = 16 µg/mL). Furthermore, S. hookeriana var. digyna promoted wound healing, reduced the number of colony-forming units, and reduced inflammation effectively in vivo. CONCLUSIONS: The traditional use of S. hookeriana var. digyna for fungal skin infections was supported by antifungal bioactivity investigated in vitro and in vivo. Its mechanism and bioactive component were predicted and confirmed by experiments, which also provided a new antifungal agent for future research.

Heteroleptic cobalt complex augments antifungal activity with fluconazole and causes membrane disruption in Candida albicans.

Heteroleptic metal complexes containing CuII, CoII, and ZnII, incorporating curcumin and a Schiff base ligand (L), were synthesized and characterized, and their antifungal activity was evaluated. Their antifungal activities were investigated individually and in combination with fluconazole. Utilizing various analytical techniques such as UV-Vis, FT-IR, NMR, ESI-MS, TGA-DTG, elemental analyses, conductance, and magnetic susceptibility measurements, complex C1 ([Cu(Cur)LCl(H2O)]) was assigned a distorted octahedral geometry, while complexes C2 ([Co(Cur)LCl(H2O)]) and C3 ([Zn(Cur)LCl(H2O)]) were assigned octahedral geometries. Among these complexes, C2 exhibited the highest inhibitory activity against both FLC-susceptible and resistant strains of Candida albicans. Furthermore, C2 demonstrated candidicidal activity and synergistic interactions with fluconazole, effectively inhibiting the growth and survival of both FLC-resistant and FLC-sensitive C. albicans strains. The complex displayed a dose-dependent inhibition of drug efflux pumps in FLC-resistant C. albicans strains, indicating its potential to disrupt the cell membrane of these strains. The significant role of membrane efflux transporters in the development of antifungal drug resistance within Candida species has been extensively documented and our findings indicate that complex C2 specifically targets this crucial factor, thereby playing a pivotal role in mitigating drug resistance in C. albicans.

Pan-drug resistance and hypervirulence in a human fungal pathogen are enabled by mutagenesis induced by mammalian body temperature.

The continuing emergence of invasive fungal pathogens poses an increasing threat to public health. Here, through the China Hospital Invasive Fungal Surveillance Net programme, we identified two independent cases of human infection with a previously undescribed invasive fungal pathogen, Rhodosporidiobolus fluvialis, from a genus in which many species are highly resistant to fluconazole and caspofungin. We demonstrate that R. fluvialis can undergo yeast-to-pseudohyphal transition and that pseudohyphal growth enhances its virulence, revealed by the development of a mouse model. Furthermore, we show that mouse infection or mammalian body temperature induces its mutagenesis, allowing the emergence of hypervirulent mutants favouring pseudohyphal growth. Temperature-induced mutagenesis can also elicit the development of pan-resistance to three of the most commonly used first-line antifungals (fluconazole, caspofungin and amphotericin B) in different Rhodosporidiobolus species. Furthermore, polymyxin B was found to exhibit potent activity against the pan-resistant Rhodosporidiobolus mutants. Collectively, by identifying and characterizing a fungal pathogen in the drug-resistant genus Rhodosporidiobolus, we provide evidence that temperature-dependent mutagenesis can enable the development of pan-drug resistance and hypervirulence in fungi, and support the idea that global warming can promote the evolution of new fungal pathogens.

Fluconazole Analogs and Derivatives: An Overview of Synthesis, Chemical Transformations, and Biological Activity.

Fluconazole (2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol), which was patented in 1981 and introduced for commercial use in 1988, is a widely utilized antifungal drug whose mechanism of action involves inhibition of the activity of 14-α lanosterol demethylase. Its safety and effectiveness have established it as one of the most frequently employed antifungal agents. Resistance to azole antifungal drugs is becoming more common. It may be related to a mutation of the gene encoding the enzyme. To address this issue, molecules with modifications in three main regions of fluconazole, namely the hydroxyl group, the aromatic ring, and the 1,2,4-triazole rings, have been synthesized in an attempt to create more potent antifungal drugs. These modifications aim at enhancing the effectiveness against microorganisms and improving pharmacokinetic parameters and safety profiles of the synthesized compounds. The present review explores the synthesis of fluconazole derivatives, accompanied by insights into the results of biological studies evaluating the therapeutic effects of these compounds.

Exploring treatment and antifungal resistance in an outbreak of tinea caused by Microsporum audouinii.

BACKGROUND: Microsporum audouinii has resurged recently. Infections with the dermatophyte are difficult to treat, which raises the question if we treat M. audouinii infections with the most effective antifungal (AF) agent. OBJECTIVES: The aims of this study was to investigate an outbreak of tinea capitis (TC) in Denmark, address the challenges in outbreak management and to conduct two reviews regarding previous outbreaks and minimal inhibitory concentration (MIC). METHODS: We used Wood's light, culture, direct microscopy, and PCR for screening and antifungal susceptibility testing (AFST) for treatment optimization. We performed two reviews to explore M. audouinii outbreaks and MIC values using broth microdilution method. RESULTS: Of 73 screened individuals, 10 had confirmed M. audouinii infections. Clinical resistance to griseofulvin was observed in 4 (66%) cases. While previous outbreaks showed high griseofulvin efficacy, our study favoured terbinafine, fluconazole and itraconazole in our hard-to-treat cases. AFST guided the choice of AF. Through the literature search, we identified five M. audouinii outbreaks, where differences in management included the use of Wood's light and prophylactic topical AF therapy. Terbinafine MIC values from the literature ranged from 0.002 to 0.125 mg/L. CONCLUSION: Use of Wood's light and preventive measurements were important for limiting infection. The literature lacked MIC data for griseofulvin against M. audouinii, but indicated sensitivity for terbinafine. The clinical efficacy for M. audouinii treatment was contradictory favouring both terbinafine and griseofulvin. AFST could have a key role in the treatment of difficult cases, but lack of standardisation of AFST and MIC breakpoints limits its usefulness.

Mechanism and bioinformatics analysis of the effect of berberine-enhanced fluconazole against drug-resistant Candida albicans.

Biofilms produced by Candida albicans present a challenge in treatment with antifungal drug. Enhancing the sensitivity to fluconazole (FLC) is a reasonable method for treating FLC-resistant species. Moreover, several lines of evidence have demonstrated that berberine (BBR) can have antimicrobial effects. The aim of this study was to clarify the underlying mechanism of these effects. We conducted a comparative study of the inhibition of FLC-resistant strain growth by FLC treatment alone, BBR treatment alone, and the synergistic effect of combined FLC and BBR treatment. Twenty-four isolated strains showed distinct biofilm formation capabilities. The antifungal effect of combined FLC and BBR treatment in terms of the growth and biofilm formation of Candida albicans species was determined via checkerboard, time-kill, and fluorescence microscopy assays. The synergistic effect of BBR and FLC downregulated the expression of the efflux pump genes CDR1 and MDR, the hyphal gene HWP1, and the adhesion gene ALS3; however, the gene expression of the transcriptional repressor TUP1 was upregulated following treatment with this drug combination. Furthermore, the addition of BBR led to a marked reduction in cell surface hydrophobicity. To identify resistance-related genes and virulence factors through genome-wide sequencing analysis, we investigated the inhibition of related resistance gene expression by the combination of BBR and FLC, as well as the associated signaling pathways and metabolic pathways. The KEGG metabolic map showed that the metabolic genes in this strain are mainly involved in amino acid and carbon metabolism. The metabolic pathway map showed that several ergosterol (ERG) genes were involved in the synthesis of cell membrane sterols, which may be related to drug resistance. In this study, BBR + FLC combination treatment upregulated the expression of the ERG1, ERG3, ERG4, ERG5, ERG24, and ERG25 genes and downregulated the expression of the ERG6 and ERG9 genes compared with fluconazole treatment alone (p < 0.05).