Modeling of intravenous caspofungin administration using an intestine-on-chip reveals altered Candida albicans microcolonies and pathogenicity.

Candida albicans is a commensal yeast of the human intestinal microbiota that, under predisposing conditions, can become pathogenic and cause life-threatening systemic infections (candidiasis). Fungal-host interactions during candidiasis are commonly studied using conventional 2D in vitro models, which have provided critical insights into the pathogenicity. However, microphysiological models with a higher biological complexity may be more suitable to mimic in vivo-like infection processes and antifungal drug efficacy. Therefore, a 3D intestine-on-chip model was used to investigate fungal-host interactions during the onset of invasive candidiasis and evaluate antifungal treatment under clinically relevant conditions. By combining microbiological and image-based analyses we quantified infection processes such as invasiveness and fungal translocation across the epithelial barrier. Additionally, we obtained novel insights into fungal microcolony morphology and association with the tissue. Our results demonstrate that C. albicans microcolonies induce injury to the epithelial tissue by disrupting apical cell-cell contacts and causing inflammation. Caspofungin treatment effectively reduced the fungal biomass and induced substantial alterations in microcolony morphology during infection with a wild-type strain. However, caspofungin showed limited effects after infection with an echinocandin-resistant clinical isolate. Collectively, this organ-on-chip model can be leveraged for in-depth characterization of pathogen-host interactions and alterations due to antimicrobial treatment.

Outcomes by Candida spp. in the ReSTORE Phase 3 trial of rezafungin versus caspofungin for candidemia and/or invasive candidiasis.

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.

Pulmonary surfactant impacts in vitro activity of selected antifungal drugs against Candida krusei and Candida albicans.

PURPOSE: This study investigates how surfactants affect the in-vitro anti-infective efficacy of micafungin, caspofungin, anidulafungin, and amphotericin B in treating pulmonary mycoses. METHODS: MIC values for antifungal agents were determined against Candida krusei (now Pichia kudriavzevii) ATCC 6258, Candida albicans ATCC 90028, and 18 clinical isolates using the broth microdilution method in RPMI medium, following EUCAST recommendations. MIC assays included testing with and without Curosurf® surfactant at 1 mg/mL for C. krusei ATCC 6258 and all C. krusei isolates. Subsequent Time-kill studies in Sabouraud broth involved testing both C. albicans ATCC 90028 and C. krusei ATCC 6258 strains at concentrations equal their respective MIC values, with and without surfactant, using all four antifungals. CFU/mL were assessed at multiple time points up to 24 h. TKCs with different surfactant concentrations for C. krusei ATCC 6258 and mini-TKCs at various concentrations relative to the MIC of C. krusei isolates and the reference strain were conducted with micafungin, anidulafungin, and caspofungin. RESULTS: MIC results showed that 1 µg/mL surfactant reduced killing of micafungin and anidulafungin against C. krusei, while caspofungin was unaffected. Amphotericin B's MIC decreased by half. TKCs demonstrated significant effects of surfactant on micafungin and anidulafungin against C. krusei, with complete abolition of anidulafungin's activity against C. albicans. CONCLUSION: This in-vitro study highlights the concentration-dependent inhibitory effect of surfactant on antifungal activity against C. krusei and, to some extent, C. albicans, necessitating further clinical validation for invasive lung mycoses treatment.

Development of an environmentally sensitive fluorescent peptide probe for MrgX2 and application in ligand screening of peptide antibiotics.

Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.

Rapamycin and caspofungin show synergistic antifungal effects in caspofungin-susceptible and caspofungin-resistant Candida strains in vitro.

OBJECTIVES: Caspofungin is an echinocandin antifungal agent that inhibits synthesis of glucan required for the fungal cell wall. Resistance is mediated by mutation of Fks1 glucan synthase, among which S645P is the most common resistance-associated polymorphism. Rapamycin is a macrolide that inhibits the mechanistic target of rapamycin (mTOR) protein kinase activity. This study investigated the interaction between rapamycin and caspofungin in inhibiting the growth of WT Candida albicans and Fks1 S645P mutant clinical isolate, and WT Candida lusitaniae and genetically engineered isogenic strain with Fks1 S645P mutation at equivalent position. METHODS: Interactions between caspofungin and rapamycin were evaluated using the microdilution chequerboard method in liquid medium. The results were analysed using the Loewe additivity model (FIC index, FICI) and the Bliss independence model (response surface, RS, analysis). RESULTS: Synergy between rapamycin and caspofungin was shown for C. albicans and C. lusitaniae strains by RS analysis of the chequerboard tests. Synergy was observed in strains susceptible and resistant to caspofungin. Weak subinhibitory concentrations of rapamycin were sufficient to restore caspofungin susceptibility. CONCLUSIONS: We report here, for the first time, synergy between caspofungin and rapamycin in Candida species. Synergy was shown for strains susceptible and resistant to caspofungin. This study highlights the possible implication of the TOR pathway in sensing antifungal-mediated cell wall stress and in modulating the cellular response to echinocandins in Candida yeasts.

Helium Cold Atmospheric Plasma Causes Morphological and Biochemical Alterations in Candida albicans Cells.

(1) Background: Previous studies reported the promising inhibitory effect of cold atmospheric plasma (CAP) on Candida albicans. However, the exact mechanisms of CAP's action on the fungal cell are still poorly understood. This study aims to elucidate the CAP effect on C. albicans cell wall, by evaluating the alterations on its structure and biochemical composition; (2) Methods: C. albicans cells treated with Helium-CAP were analyzed by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) in order to detect morphological, topographic and biochemical changes in the fungal cell wall. Cells treated with caspofungin were also analyzed for comparative purposes; (3) Results: Expressive morphological and topographic changes, such as increased roughness and shape modification, were observed in the cells after CAP exposure. The alterations detected were similar to those observed after the treatment with caspofungin. The main biochemical changes occurred in polysaccharides content, and an overall decrease in glucans and an increase in chitin synthesis were detected; (4) Conclusions: Helium-CAP caused morphological and topographic alterations in C. albicans cells and affected the cell wall polysaccharide content.

Susceptibility to caspofungin is regulated by temperature and is dependent on calcineurin in Candida albicans.

IMPORTANCE: Echinocandins are the newest antifungal drugs and are first-line treatment option for life-threatening systemic infections. Due to lack of consensus regarding what temperature should be used when evaluating susceptibility of yeasts to echinocandins, typically either 30°C, 35°C, or 37°C is used. However, the impact of temperature on antifungal efficacy of echinocandins is unexplored. In the current study, we demonstrated that Candida albicans laboratory strain SC5314 was more susceptible to caspofungin at 37°C than at 30°C. We also found that calcineurin was required for temperature-modulated caspofungin susceptibility. Surprisingly, the altered caspofungin susceptibility was not due to differential expression of some canonical genes such as FKS, CHS, or CHT genes. The molecular mechanism of temperature-modulated caspofungin susceptibility is undetermined and deserves further investigations.

Identification of 10 genes on Candida albicans chromosome 5 that control surface exposure of the immunogenic cell wall epitope ß-glucan and cell wall remodeling in caspofungin-adapted mutants.

IMPORTANCE: Candida infections are often fatal in immuno-compromised individuals, resulting in many thousands of deaths per year. Caspofungin has proven to be an excellent anti-Candida drug and is now the frontline treatment for infections. However, as expected, the number of resistant cases is increasing; therefore, new treatment modalities are needed. We are determining metabolic pathways leading to decreased drug susceptibility in order to identify mechanisms facilitating evolution of clinical resistance. This study expands the understanding of genes that modulate drug susceptibility and reveals new targets for the development of novel antifungal drugs.

Whole genome analysis of echinocandin non-susceptible Candida Glabrata clinical isolates: a multi-center study in China.

BACKGROUND: Candida glabrata is an important cause of invasive candidiasis. Echinocandins are the first-line treatment of invasive candidiasis caused by C. glabrata. The epidemiological echinocandin sensitivity requires long-term surveillance and the understanding about whole genome characteristics of echinocandin non-susceptible isolates was limited. RESULTS: The present study investigated the echinocandin susceptibility of 1650 C. glabrata clinical isolates in China from August 2014 to July 2019. The in vitro activity of micafungin was significantly better than those of caspofungin and anidulafungin (P < 0.001), assessed by MIC50/90 values. Whole genome sequencing was conducted on non-susceptible isolates and geography-matched susceptible isolates. Thirteen isolates (0.79%) were resistant to at least one echinocandin. Six isolates (0.36%) were solely intermediate to caspofungin. Common evolutionary analysis of echinocandin-resistant and echinocandin-intermediate isolates revealed genes related with reduced caspofungin sensitivity, including previously identified sphinganine hydroxylase encoding gene SUR2. Genome-wide association study identified SNPs at subtelometric regions that were associated with echinocandin non-susceptibility. In-host evolution of echinocandin resistance of serial isolates revealed an enrichment for non-synonymous mutations in adhesins genes and loss of subtelometric regions containing adhesin genes. CONCLUSIONS: The echinocandins are highly active against C. glabrata in China with a resistant rate of 0.79%. Echinocandin non-susceptible isolates carried common evolved genes which are related with reduced caspofungin sensitivity. In-host evolution of C. glabrata accompanied intensive changing of adhesins profile.

In vitro combination of antifungal drugs with tacrolimus (FK506) holds promise against clinical Candida species, including Candida auris.

In vitro interactions between tacrolimus, a calcineurin inhibitor, and fluconazole, itraconazole, caspofungin, or anidulafungin were evaluated against Candida auris, C. albicans, C. parapsilosis, and C. glabrata (each five strains). Tacrolimus-itraconazole, tacrolimus-caspofungin, and tacrolimus-fluconazole combinations resulted in synergistic interactions against 95%, 90%, and 60% of Candida isolates, respectively. However, tacrolimus-anidulafungin resulted in only a 35% synergistic effect. A combination of tacrolimus and itraconazole was most potent with synergy against 100% of C. auris, C. parapsilosis, and C. glabrata isolates. Of note, no antagonistic interaction was found.

Caspofungin-induced ß(1,3)-glucan exposure in Candida albicans is driven by increased chitin levels.

To successfully induce disease, Candida albicans must effectively evade the host immune system. One mechanism used by C. albicans to achieve this is to mask immunogenic ß(1,3)-glucan epitopes within its cell wall under an outer layer of mannosylated glycoproteins. Consequently, induction of ß(1,3)-glucan exposure (unmasking) via genetic or chemical manipulation increases fungal recognition by host immune cells in vitro and attenuates disease during systemic infection in mice. Treatment with the echinocandin caspofungin is one of the most potent drivers of ß(1,3)-glucan exposure. Several reports using murine infection models suggest a role for the immune system, and specifically host ß(1,3)-glucan receptors, in mediating the efficacy of echinocandin treatment in vivo. However, the mechanism by which caspofungin-induced unmasking occurs is not well understood. In this report, we show that foci of unmasking co-localize with areas of increased chitin within the yeast cell wall in response to caspofungin, and that inhibition of chitin synthesis via nikkomycin Z attenuates caspofungin-induced ß(1,3)-glucan exposure. Furthermore, we find that both the calcineurin and Mkc1 mitogen-activated protein kinase pathways work synergistically to regulate ß(1,3)-glucan exposure and chitin synthesis in response to drug treatment. When either of these pathways are interrupted, it results in a bimodal population of cells containing either high or low chitin content. Importantly, increased unmasking correlates with increased chitin content within these cells. Microscopy further indicates that caspofungin-induced unmasking correlates with actively growing cells. Collectively, our work presents a model in which chitin synthesis induces unmasking within the cell wall in response to caspofungin in growing cells. IMPORTANCE Systemic candidiasis has reported mortality rates ranging from 20% to 40%. The echinocandins, including caspofungin, are first-line antifungals used to treat systemic candidiasis. However, studies in mice have shown that echinocandin efficacy relies on both its cidal impacts on Candida albicans, as well as a functional immune system to successfully clear invading fungi. In addition to direct C. albicans killing, caspofungin increases exposure (unmasking) of immunogenic ß(1,3)-glucan moieties. To evade immune detection, ß(1,3)-glucan is normally masked within the C. albicans cell wall. Consequently, unmasked ß(1,3)-glucan renders these cells more visible to the host immune system and attenuates disease progression. Therefore, discovery of how caspofungin-induced unmasking occurs is needed to elucidate how the drug facilitates host immune system-mediated clearance in vivo. We report a strong and consistent correlation between chitin deposition and unmasking in response to caspofungin and propose a model in which altered chitin synthesis drives increased unmasking during drug exposure.

Rapid automated antifungal susceptibility testing system for yeasts based on growth characteristics.

Fungal pathogens are a major threat to public health, as they are becoming increasingly common and resistant to treatment, with only four classes of antifungal medicines currently available and few candidates in the clinical development pipeline. Most fungal pathogens lack rapid and sensitive diagnostic techniques, and those that exist are not widely available or affordable. In this study, we introduce a novel automated antifungal susceptibility testing system, Droplet 48, which detects the fluorescence of microdilution wells in real time and fits growth characteristics using fluorescence intensity over time. We concluded that all reportable ranges of Droplet 48 were appropriate for clinical fungal isolates in China. Reproducibility within ±2 two-fold dilutions was 100%. Considering the Sensititre YeastOne Colorimetric Broth method as a comparator method, eight antifungal agents (fluconazole, itraconazole, voriconazole, caspofungin, micafungin, anidulafungin, amphotericin B, and 5-flucytosine) showed an essential agreement of >90%, except for posaconazole (86.62%). Category agreement of four antifungal agents (fluconazole, caspofungin, micafungin, and anidulafungin) was >90%, except for voriconazole (87.93% agreement). Two Candida albicans isolates and anidulafungin showed a major discrepancy (MD) (2.60%), and no other MD or very MD agents were found. Therefore, Droplet 48 can be considered as an optional method that is more automated and can obtain results and interpretations faster than previous methods. However, the optimization of the detection performance of posaconazole and voriconazole and promotion of Droplet 48 in clinical microbiology laboratories still require further research involving more clinical isolates in the future.

Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection.

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish-Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo. ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection.

A host defense peptide mimetic, brilacidin, potentiates caspofungin antifungal activity against human pathogenic fungi.

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections.

Pandemic Response Box® library as a source of antifungal drugs against Scedosporium and Lomentospora species.

Scedosporium and Lomentospora species are opportunistic filamentous fungi that cause localized and disseminated infections in immunocompetent and immunocompromised patients. These species are considered resistant fungi due to their low susceptibility to most current antifungal agents used in healthcare settings. The search for new compounds that could work as promising candidate antifungal drugs is an increasing field of interest. In this context, in the present study we screened the Pandemic Response Box® library (Medicines for Malaria Venture [MMV], Switzerland) to identify compounds with antifungal activity against Scedosporium and Lomentospora species. An initial screening of the drugs from this collection at 5 µM was performed using a clinical Scedosporium aurantiacum isolate according to the EUCAST protocol. Compounds with activity against this fungus were also tested against four other species (S. boydii¸ S. dehoogii, S. apiospermum and L. prolificans) at concentrations ranging from 0.078 to 10 µM. Seven compounds inhibited more than 80% of S. aurantiacum growth, three of them (alexidine, amorolfine and olorofim) were selected due to their differences in mechanism of action, especially when compared to drugs from the azole class. These compounds were more active against biofilm formation than against preformed biofilm in Scedosporium and Lomentospora species, except alexidine, which was able to decrease preformed biofilm about 50%. Analysis of the potential synergism of these compounds with voriconazole and caspofungin was performed by the checkerboard method for S. aurantiacum. The analysis by Bliss methodology revealed synergistic effects among selected drugs with caspofungin. When these drugs were combined with voriconazole, only alexidine and amorolfine showed a synergistic effect, whereas olorofim showed an antagonistic effect. Scanning electron microscopy revealed that alexidine induces morphology alterations in S. aurantiacum biofilm grown on a catheter surface. Reactive oxygen species production, mitochondrial activity and surface components were analyzed by fluorescent probes when S. aurantiacum was treated with selected drugs and revealed that some cell parameters are altered by these compounds. In conclusion, alexidine, amorolfine and olorofim were identified as promising compounds to be studied against scedosporiosis and lomentosporiosis.

UDP-Galactopyranose Mutase Mediates Cell Wall Integrity, Polarity Growth, and Virulence in Fusarium graminearum.

CHY1 is a zinc finger protein unique to microorganisms that was found to regulate polarized tip growth in Fusarium graminearum, an important pathogen of wheat and barley. To further characterize its functions, in this study we identified CHY1-interacting proteins by affinity purification and selected UDP-galactofuranose (Galf) mutase (UGMA) for detailed characterization, because UGMA and UDP-Galf are unique to fungi and bacteria and absent in plants and animals. The interaction between CHY1 and UGMA was confirmed by yeast two-hybrid assays. Deletion of UGMA in F. graminearum resulted in significant defects in vegetative growth, reproduction, cell wall integrity, and pathogenicity. Infection with the ΔugmA mutant was restricted to the inoculated floret, and no vomitoxin was detected in kernels inoculated with the ΔugmA strain. Compared to the wild type, the ΔugmA mutant produced wide, highly branched hyphae with thick walls, as visualized by transmission electron microscopy. UGMA tagged with green fluorescent protein (GFP) mainly localized to the cytoplasm, consistent with the synthesis of Galf in the cytoplasm. The Δchy1 mutant was more sensitive, while the ΔugmA mutant was more tolerant, to cell wall-degrading enzymes. The growth of the ΔugmA mutant nearly ceased upon caspofungin treatment. More interestingly, nocodazole treatment of the ΔugmA strain attenuated its highly branched morphology, while caspofungin inhibited the degree of the twisted Δchy1 mycelia, indicating that CHY1 and UGMA probably have opposite effects on cell wall architecture. In conclusion, UGMA is an important pathogenic factor that is specific to fungi and bacteria and required for cell wall architecture, radial growth, and caspofungin tolerance, and it appears to be a promising target for antifungal agent development. IMPORTANCE The long-term use of chemical pesticides has had increasingly negative impacts on the ecological environment and human health. Low-toxicity, high-efficiency and environmentally friendly alternative pesticides are of great significance for maintaining the sustainable development of agriculture and human and environmental health. Using fungus- or microbe-specific genes as candidate targets provides a good foundation for the development of low-toxicity, environmentally friendly pesticides. In this study, we characterized a fungus- and bacterium-specific UDP-galactopyranose mutase gene, ugmA, that contributes to the synthesis of the cell wall component Galf and is required for vegetative growth, cell wall integrity, deoxynivalenol (DON) production, and pathogenicity in F. graminearum. The ugmA deletion mutant exhibited increased sensitivity to caspofungin. These results demonstrate the functional importance of UGMA in F. graminearum, and its absence from mammals and higher plants constitutes a considerable advantage as a low-toxicity target for the development of new anti-Fusarium agents.

Antifungal activity and potential mechanism of action of caspofungin in combination with ribavirin against Candida albicans.

The number of invasive fungal infections has increased dramatically, resulting in high morbidity and mortality among immunocompromised patients. With increasing use of caspofungin (CAS), resistant strains have emerged frequently and led to limitations in the treatment of patients with severe invasive Candida albicans infections. Combination therapy is an important method to deal with this issue. As such, this study investigated the activity of CAS in combination with ribavirin (RBV) against C. albicans. The results of this in-vitro study showed that the minimum inhibitory concentrations (MICs) of CAS and RBV when they were used as monotherapy were 0.5-1 µg/mL and 2-8 µg/mL, respectively, while the MIC of CAS decreased from 0.5-1 µg/mL to 0.0625-0.25 µg/mL when used in combination with RBV, with a fractional inhibitory concentration index (FICI) ≤0.5. In addition, the RBV + CAS combination group displayed synergistic effects against C. albicans biofilm over 4 h; the sessile MIC (sMIC) of CAS decreased from 0.5-1 µg/mL to 0.0625-0.25µg/mL and the sMIC of RBV decreased from 4-16 µg/mL to 1-2 µg/mL, with FICI <0.5. The survival of C. albicans-infected Galleria mellonella was prolonged, the fungal burden was decreased, and the area of tissue damage was reduced after combination therapy. Further study showed that the mechanisms of action of the synergistic effect were related to the inhibition of biofilm formation, the inhibition of hyphal growth, and the activation of metacaspases, but were not related to the accumulation of reactive oxygen species. It is hoped that these findings will contribute to the understanding of drug resistance in C. albicans, and provide new insights for the application of RBV.

Fungicidal activity of human antimicrobial peptides and their synergistic interaction with common antifungals against multidrug-resistant Candida auris.

Emergence of Candida auris, a multidrug-resistant yeast, demonstrates the urgent need for novel antifungal agents. Human antimicrobial peptides (AMPs) are naturally occurring molecules with wide spectrum antimicrobial activity, particularly against a variety of fungi. Therefore, this study examined the antifungal activity of seven different human AMPs against C. auris following the CLSI guidelines. The antifungal activity was further assessed using time kill curve and cell viability assays. For combination interaction, effectiveness of these peptides with three antifungals, fluconazole, amphotericin B, and caspofungin was done following standard protocols. To elucidate the antifungal mechanism, the effects of peptides on membrane permeability were investigated using propidium iodide staining method and confocal imaging. Antifungal susceptibility results showed that all the examined peptides possessed fungicidal effect against C. auris at different levels, with human ß-defensin-3 being the most potent antifungal with MIC values ranging from 3.125 to 12.5 µg/ml. Time kill curves further confirmed the killing effect of all the tested peptides. Viability assay showed a significant decrease in the percentage of viable cells exposed to different inhibitory and fungicidal concentrations of each peptide (p < 0.01). Furthermore, peptides showed mostly synergistic interaction when combined with conventional antifungal drugs, with caspofungin showing 100% synergy when combined with different AMPs. As antifungal mechanism, peptides disrupted the membrane permeability at concentrations that correlated with the inhibition of growth. Overall, the findings of this study point towards the application of the tested peptides as a monotherapy or as a combination therapy with antifungal drugs to treat multidrug-resistant C. auris infections.

Decreased echinocandin susceptibility in Candida parapsilosis causing candidemia and emergence of a pan-echinocandin resistant case in China.

Candida parapsilosis is becoming a predominant non-albicans cause of invasive candidiasis (IC). Echinocandins are the preferred choice for IC treatment and prophylaxis. Resistance to echinocandins in C. parapsilosis has emerged in several countries, but little is known about the susceptibility profile in China or about mechanisms of resistance. Here, we investigated the echinocandin susceptibilities of 2523 C. parapsilosis isolates collected from China and further explored the resistance mechanism among echinocandin-resistant isolates. Anidulafungin exhibited the highest MICs (MIC50/90, 1 and 2 µg/mL; GM, 0.948 µg/mL), while caspofungin showed better activity (0.5 and 1 µg/mL; 0.498 µg/mL). Significantly higher echinocandin MICs were observed among blood-derived isolates compared to others, especially for caspofungin (GM, 1.348 µg/mL vs 0.478 µg/mL). Isolates from ICU and surgical wards also showed higher MICs. Twenty isolates showed intermediate phenotypes for at least one echinocandin. One was resistant to all three echinocandins, fluconazole and voriconazole, which caused breakthrough IC during long-term exposure to micafungin. WGS revealed this isolate carried a mutation S656P in hotspot1 region of Fks1. Bioinformatics analyses suggested that this mutation might lead to an altered protein conformation. CRISPR Cas9-mediated introduction of this mutation into a susceptible reference C. parapsilosis strain increased MICs of all echinocandins 64-fold, with similar results found in the subspecies, C. orthopsilosis and C. metapsilosis. This is the first report of a multi-azole resistant and pan-echinocandin resistant C. parapsilosis isolate, and the identification of a FKS1S656P conferring pan-echinocandin resistance. Our study underscores the necessity of rigorous management of antifungal use and of monitoring for antifungal susceptibility.

Caspofungin Suppresses Brain Cell Necroptosis in Ischemic Stroke Rats via Up-Regulation of Pellino3.

PURPOSE: Pellino3, an ubiquitin E3 ligase, prevents the formation of the death-induced signaling complex in response to TNF-α by targeting receptor-interacting protein kinase 1 (RIPK1), and bioinformatics analysis predicted an interaction between Pellino3 and caspofungin, a common antifungal drug used in clinics. This study aimed to explore the effect of caspofungin on brain injury in ischemic stroke and the underlying mechanisms. METHODS: Ischemic stroke injury was induced in Sprague Dawley rats by occlusion of the middle cerebral artery (MCA) for 2 h, followed by 24 h reperfusion. PC12 cells were deprived of both oxygen and glucose for 8 h and then were cultured for 24 h with oxygen and glucose to mimic an ischemic stroke in vitro. RESULTS: Animal experiments showed brain injury (increase in neurological deficit score and infarct volume) concomitant with a downregulation of Pellino3, a decreased ubiquitination of RIPK1, and an up-regulation of necroptosis-associated proteins [RIPK1, RIPK3, mixed lineage kinase domain-like protein (MLKL), p-RIPK1, p-RIPK3, and p-MLKL]. Administration of caspofungin (6 mg/kg, i.m.) at 1 h and 6 h after ischemia significantly improved neurological function, reduced infarct volume, up-regulated Pellino3 levels, increased RIPK1 ubiquitination, and down-regulated protein levels of RIPK1, p-RIPK1, p-RIPK3, and p-MLKL. PC12 cells deprived of oxygen/glucose developed signs of cellular injury (LDH release and necroptosis) concomitant with downregulation of Pellino3, decreased ubiquitination of RIPK1, and elevated necroptosis-associated proteins. These changes were reversed by overexpression of Pellino3. CONCLUSION: We conclude that Pellino3 has an important role in counteracting necroptosis via ubiquitination of RIPK1 and caspofungin can suppress the brain cell necroptosis in ischemic stroke through upregulation of Pellino3.