Whole transcriptome analysis of schinifoline treatment in Caenorhabditis elegans infected with Candida albicans.

Candida albicans is an opportunistic fungal human pathogen that has been causing an increasing number of deaths each year. Due to the widespread use of broad-spectrum antibiotics and immunosuppressants, C. albicans resistance to these therapies has increased. Thus, natural plant inhibitors are being investigated for treating C. albicans infections. Schinifoline is a 4-quinolinone alkaloid with antibacterial, insecticidal, antitumor, and other biological activities. Here, we explored the effects of schinifoline on C. albicans in C. elegans and extracted RNA from uninfected C. elegans, C. elegans infected with C. albicans, and C. elegans infected with C. albicans and treated with 100 mg/l schinifoline. Our results showed that there were significant differences among the three groups. The GO and KEGG pathway analysis suggested that the pathogenicity of C. albicans to C. elegans was caused by abnormal protein function. Schinifoline regulates lysosomal pathway related genes that accelerate the metabolism and degradation of abnormal proteins, thereby inhibiting the negative effects of C. albicans in vivo. These findings advance our understanding of the molecular mechanisms underlying schinifoline inhibition of C. albicans.

Lead optimization generates selenium-containing miconazole CYP51 inhibitors with improved pharmacological profile for the treatment of fungal infections.

A series of selenium-containing miconazole derivatives were identified as potent antifungal drugs in our previous study. Representative compound A03 (MIC = 0.01 µg/mL against C.alb. 5314) proved efficacious in inhibiting the growth of fungal pathogens. However, further study showed lead compound A03 exhibited potential hemolysis, significant cytotoxic effect and unfavorable metabolic stability and was therefore modified to overcome these drawbacks. In this article, the further optimization of selenium-containing miconazole derivatives resulted in the discovery of similarly potent compound B17 (MIC = 0.02 µg/mL against C.alb. 5314), exhibiting a superior pharmacological profile with decreased rate of metabolism, cytotoxic effect and hemolysis. Furthermore, compound B17 showed fungicidal activity against Candida albicans and significant effects on the treatment of resistant Candida albicans infections. Meanwhile, compound B17 not only could reduce the ergosterol biosynthesis pathway by inhibiting CYP51, but also inhibited biofilm formation. More importantly, compound B17 also shows promising in vivo efficacy after intraperitoneal injection and the PK study of compound B17 was evaluated. In addition, molecular docking studies provide a model for the interaction between the compound B17 and the CYP51 protein. Overall, we believe that these selenium-containing miconazole compounds can be further developed for the potential treatment of fungal infections.

Active neutrophil responses counteract Candida albicans burn wound infection of ex vivo human skin explants.

Burn wounds are highly susceptible sites for colonization and infection by bacteria and fungi. Large wound surface, impaired local immunity, and broad-spectrum antibiotic therapy support growth of opportunistic fungi such as Candida albicans, which may lead to invasive candidiasis. Currently, it remains unknown whether depressed host defenses or fungal virulence drive the progression of burn wound candidiasis. Here we established an ex vivo burn wound model, where wounds were inflicted by applying preheated soldering iron to human skin explants, resulting in highly reproducible deep second-degree burn wounds. Eschar removal by debridement allowed for deeper C. albicans penetration into the burned tissue associated with prominent filamentation. Active migration of resident tissue neutrophils towards the damaged tissue and release of pro-inflammatory cytokine IL-1ß accompanied the burn. The neutrophil recruitment was further increased upon supplementation of the model with fresh immune cells. Wound area and depth decreased over time, indicating healing of the damaged tissue. Importantly, prominent neutrophil presence at the infected site correlated to the limited penetration of C. albicans into the burned tissue. Altogether, we established a reproducible burn wound model of candidiasis using ex vivo human skin explants, where immune responses actively control the progression of infection and promote tissue healing.

A lipase-responsive antifungal nanoplatform for synergistic photodynamic/photothermal/pharmaco-therapy of azole-resistant Candida albicans infections.

A lipase-triggered drug release nanoplatform (PGL-DPP-FLU NPs) for multi-modal antifungal therapy is developed. The lipases secreted by C. albicans can accelerate FLU release. The ROS and heat produced by PGL-DPP-FLU NPs make C. albicans more vulnerable to FLU, thereby PGL-DPP-FLU NPs exhibit high performance for combating azole-resistant C. albicans biofilms and wound infection.

Enhanced Killing of Candida krusei by Polymorphonuclear Leucocytes in the Presence of Subinhibitory Concentrations of Melaleuca alternifolia and "Mentha of Pancalieri" Essential Oils.

The aim of this study was to evaluate the influence of tea tree oil (TTO) and "Mentha of Pancalieri" essential oil (MPP) on intracellular killing of Candida krusei, often resistant to conventional drugs, by polymorphonuclear leucocytes (PMNs). Intracellular killing was investigated by incubating yeasts and PMNs with essential oils (EOs) at 1/4 and 1/8 × MIC (Minimal Inhibitory Concentration), in comparison with anidulafungin, used as a reference drug. Killing values were expressed as Survival Index (SI) values. The cytotoxicity of EOs was evaluated by 3-[4,-5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Both EOs were more efficaceous at 1/8 × MIC than 1/4 × MIC, with killing values higher than observed in EO-free systems and in presence of anidulafungin, indicating that the decreasing concentrations did not cause lower candidacidal activity. This better activity at 1/8 × MIC is probably due to the EOs' toxicity at 1/4 × MIC, suggesting that at higher concentrations EOs might interfere with PMNs functionality. TTO and MPP at 1/8 × MIC significantly increased intracellular killing by PMNs through their direct action on the yeasts (both EOs) or on phagocytic cells (MPP), suggesting a positive interaction between EOs and PMNs to eradicate intracellular C. krusei. These data showed a promising potential application of TTO and "Mentha of Pancalieri" EO as natural adjuvants in C. krusei infection management.

Candida albicans Infection Model in Zebrafish (Danio rerio) for Screening Anticandidal Drugs.

BACKGROUND: Candida albicans is an opportunistic fungal pathogen which causes systemic infections in human. In this study, C. albicans infection model was developed in zebrafish to understand the host-pathogen interactions for straightforward anticandidal drug screening. METHODS: To develop the infection, 1 × 106 cells of C. albicans suspended in phosphate-buffered saline were deposited in zebrafish dorsal muscle by manually operated syringe. The infection progression was externally assessed by a scale of wound-healing events, based on visible changes of yeast deposited in the muscle tissues. Chemotherapy was carried out with known antifungal drugs (fluconazole, nystatin, and amphotericin B) and a potential antifungal agent, chitosan silver nanocomposites (CAgNC), after the infection as direct exposure in the water. Histopathological analysis was performed to identify the pathogen virulence and the host-pathogen interaction during the infection. RESULTS: The light microscopic observations and histopathological analysis revealed the yeast-hyphae transition at the site of infection (at 72 hpi) and progression of the infection in the host tissues. The larval survival rate under fluconazole (up to 80 µg mL-1) and nystatin (up to 20 µg mL-1) was > 90% and for CAgNC it was 40% at 36 h post-exposure (hpe). The infection progression was suppressed with the fungicidal treatments. Among inflammatory genes, il-1ß has been highly upregulated (14.68-fold) at 24 h post infection (hpi). Both il-1ß and tnf-α were moderately upregulated in infected fish gills at 72 hpi. Among the C. albicans antioxidant genes, cat1 and sod2 have been upregulated during the infection, and relative expression folds were increased from low to moderate levels with the time. DISCUSSION: We demonstrate the approach for the development of artificial infection model of zebrafish with C. albicans. By this mini vertebrate zebrafish model, researchers will be able to study novel anticandidal compounds in vivo with respect to the host, pathogen, and their interactions.

Drosophila melanogaster as a model to study virulence and azole treatment of the emerging pathogen Candida auris.

OBJECTIVES: Candida auris is an emerging, often MDR, yeast pathogen. Efficient animal models are needed to study its pathogenicity and treatment. Therefore, we developed a C. auris fruit fly infection model. METHODS: TollI-RXA/Tollr632 female flies were infected with 10 different C. auris strains from the CDC Antimicrobial Resistance bank panel. We used three clinical Candida albicans strains as controls. For drug protection assays, fly survival was assessed along with measurement of fungal burden (cfu/g tissue) and histopathology in C. auris-infected flies fed with fluconazole- or posaconazole-containing food. RESULTS: Despite slower in vitro growth, all 10 C. auris isolates caused significantly greater mortality than C. albicans in infected flies, with >80% of C. auris-infected flies dying by day 7 post-infection (versus 67% with C. albicans, P < 0.001-0.005). Comparison of C. auris isolates from different geographical clades revealed more rapid in vitro growth of South American isolates and greater virulence in infected flies, whereas the aggregative capacity of C. auris strains had minimal impact on their growth and pathogenicity. Survival protection and decreased fungal burden of fluconazole- or posaconazole-fed flies infected with two C. auris strains were in line with the isolates' disparate in vitro azole susceptibility. High reproducibility of survival curves for both non-treated and antifungal-treated infected flies was seen, with coefficients of variation of 0.00-0.31 for 7 day mortality. CONCLUSIONS: Toll-deficient flies could provide a fast, reliable and inexpensive model to study pathogenesis and drug activity in C. auris candidiasis.

Antifungal activity of Camellia sinensis crude extracts against four species of Candida and Microsporum persicolor.

OBJECTIVE OF THE STUDY: Candidiasis and dermatophytoses are benign infections in humans and animals, but they are very dreaded diseases in immunocompromised individuals. These infections become resistant to different treatments which make them more dangerous. In this work, we tried to find a new way for treating them. So we were interested in the antifungal activity of Camellia sinensis (tea); this plant is known to have many health benefits. MATERIALS AND METHODS: We tested the ability of the acetone and aqueous crude extracts of the plant to inhibit in vitro the growth of Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei and Microsporum persicolor. Then, the antifungal activity against these species was tested in vivo in mice. RESULTS: The results showed that the acetone crude extract had the most important in vitro activity against all the fungi. But in vivo it was only the most active against Candida albicans, Candida glabrata, Candida tropicalis and Microsporum persicolor. Candida krusei was more sensitive to the aqueous crude extract. CONCLUSION: These results indicated that tea could be considered to treat infections caused by the five tested species.

Isolation of Candida auris from Ear of Otherwise Healthy Patient, Austria, 2018.

The emerging pathogen Candida auris is isolated mostly from hospitalized patients and often shows multidrug resistance. We report on the isolation of this yeast in Austria from an outpatient's auditory canal. The isolate showed good susceptibility against antifungals except for echinocandins; the patient was treated successfully with topical administration of nystatin.

A comparison of the effects of Lawsonia inermis (Iranian henna) and clotrimazole on Candida albicans in rats.

BACKGROUND: Candida albicans may cause vaginal infections in women. The aim of this study was to compare the antifungal effect of Lawsonia inermis with that of clotrimazole on rats. METHODS: A total of 35female Wistar rats were randomly assigned into 5groups. Four groups were infected vaginally with C. albicans and one group was not (negative control). The four infected groups received the following treatments: two groups received vaginal creams of 2% or 4% of L. inermis, one group received 1% clotrimazole and one infected group did not receive any treatment (positive control). The hydro-ethanolic henna extract was prepared from the powder of henna leaves using maceration method. Samples were taken for culture from the vaginae of all rats before the treatment, one and two weeks after treatment. An ANOVA test was used to analyze the data. RESULTS: Before the treatment, the mean colony forming units (CFU) was 213.6±10.08 and 334.42±20.32 in the 2% and 4% henna groups, respectively, 312.7±28.32 in the clotrimazole group, 233.85±8.15 in the positive control group, and zero in the negative control group. The mean CFUs were zero for all groups except for the 2% henna and positive control groups (P<0.001) one week after the treatment and zero in all groups except for the positive control group two weeks after the treatment (P<0.001). CONCLUSION: L. inermis (henna) in form of vaginal cream could treat C. albicans infections in female rats; however, 4% henna was more effective and had an effect similar to that of clotrimazole.

Paeoniflorin augments systemic Candida albicans infection through inhibiting Th1 and Th17 cell expression in a mouse model.

Paeoniflorin (PF), a Chinese herbal medicine, has been widely used in clinical practice in China because of its dual immunoregulatory effects. A previous study found that PF inhibited the biofilm formation of Candida albicans (C. albicans) in vitro; however, whether PF plays an antifungal role in vivo is still unexplored. In this study, we sought to examine the effect of PF alone or in combination with an antifungal agent, fluconazole (FCZ), using a mouse model of systemic candidiasis. The results showed that the survival time of mice treated with PF alone or PF + FCZ decreased compared with the Infected alone and FCZ treated groups, respectively (8.20 ±â€¯1.75 vs 10.40 ±â€¯2.50 days, P < 0.05; 24.60 ±â€¯6.55 vs 29.00 ±â€¯3.16 days, P < 0.05). The fungal burden in the kidney of mice increased in the PF alone and PF + FCZ treated groups compared with the Infected alone or FCZ treated group. Furthermore, it was found that the PF and PF + FCZ treated groups showed significantly decreased levels of serum interferon gamma (IFN-γ), interleukin (IL)-17, and IL-22, and an increased level of serum IL-4; PF had no effect on the production of tumor necrosis factor alpha (TNF-α). PF alone or in combination with FCZ decreased the proliferation of Th1 (IFN-γ+CD4+) and Th17 cells (IL-17+CD4+) and increased the expression of Th2 cells (IL-4+CD4+). These results suggested that PF treatment could be detrimental to the host response to systemic C. albicans infection in mice. Thus, caution might be required for clinical use of PF in patients with fungal infection.

Fungal-derived cues promote ocular autoimmunity through a Dectin-2/Card9-mediated mechanism.

Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic-environmental interactions that break immune tolerance to generate eye-specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria-containing complete Freund's adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4+ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye-specific T cells remain poorly defined. In this study, we found that fungal-derived antigens possess autoimmune uveitis-promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP-triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C-type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9-activating CLRs. Using single receptor-deficient mice we identified Dectin-2, but not Mincle or Dectin-1, as a predominant mediator of fungal-promoted uveitis. Conversely, Dectin-2 activation by α-mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9-coupled signalling axis and interleukin (IL)-17 production. Taken together, this report relates the potential of the Dectin-2/Card9-coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell-mediated autoimmunity, it also reveals a previously unappreciated ability of fungal-derived signals to promote autoimmunity.

Drug development challenges and strategies to address emerging and resistant fungal pathogens.

INTRODUCTION: Invasive fungal infections represent an expanding threat to public health. The recent emergence of Candida auris, which is often resistant to existing antifungal agents and is associated with a high mortality rate, underscores the urgent need for novel drug development strategies. Areas covered: In this paper, we examine both challenges and opportunities associated with antifungal drug development and explore potential avenues to accelerate the development pipeline, including data sharing, surrogate endpoints, and the role of historical controls in clinical trials. Expert commentary: We review important lessons learned from the study of other rare diseases, including mitochondrial storage diseases and certain forms of cancer that may inform strategies to develop new antifungal agents while highlighting promising new compounds such as SCY-078 for the treatment of invasive fungal infections.

Oral candidal speciation, virulence and antifungal susceptibility in type 2 diabetes mellitus.

OBJECTIVES: To determine the oral candidal carriage (OCC), activity of virulent factors and fluconazole susceptibility in subjects with type 2 diabetes mellitus (T2DM) and investigate their association with HbA1c measurements. MATERIALS AND METHODS: A cross sectional study was conducted on 100 diabetics and 100 healthy volunteers. The virulence was assessed by measuring the phospholipase activity and proteolysis index. Fluconazole susceptibility was performed using the gradient diffusion method. The OCC, virulence factors and antifungal susceptibility were correlated with patients' HbA1c measurements. RESULTS: The OCC and candidal density carriage was significantly higher in diabetics. Candida albicans (C. albicans) was the most frequently isolated species followed by Candida tropicalis (C. tropicalis). Relatively uncommon species, Candida lusitaniae (C. lusitaniae) and Candida lipolytica (C. lipolytica) were isolated from the diabetics. Prevalence of virulence factor, proteinase, was greater in diabetic group (p<0.05). Reduced fluconazole susceptibility was noted among the isolates from diabetics; however it was not statistically significant (p=0.593). Except one, all the susceptible-dose dependent and resistant isolates were Candida no-albicans (C. non-albicans). CONCLUSION: C. albicans remains the predominant pathogen in diabetics, although other species are on the rise. Compared to control group, the isolated species from T2DM group had higher proteinase activity. Resistance to fluconazole was considerably greater among the C. non-albicans isolates from T2DM group. These findings warrant effective treatment modalities to reduce the occurrence of oropharyngeal candidiasis.

Novel nanoscale bacteriophage-based single-domain antibodies for the therapy of systemic infection caused by Candida albicans.

Candida albicans (C. albicans) is an important human commensal and opportunistic fungal pathogen. Secreted aspartyl proteinases (Saps) are a major virulence trait of C. albicans, and among these proteases Sap2 has the highest expression levels. It is possible that antibodies against Sap2 could provide an antifungal effect. In this study, two phages displaying anti-rSap2 single chain variable fragments (scFvs) were screened from human single fold scFv libraries, and their potential therapeutic roles were evaluated using a murine model infected by C. albicans. The in vivo efficacies were assessed by mortality rates, fungal burden and histological examination. Overall survival rates were significantly increased while the colony counts and infectious foci were significantly decreased after treatment with the scFv-phages relative to the control groups. In order to investigate the immune response provoked by scFv-phages, three kinds of cytokines (Th1, Th2 and Th17 types) were measured and a clear immune response was observed. These findings suggest that anti-rSap2 scFv-phages have potential in the therapy of systemic infection caused by C. albicans.

Cytopiloyne, a polyacetylenic glucoside from Bidens pilosa, acts as a novel anticandidal agent via regulation of macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Bidens pilosa, a tropical and sub-tropical herbal plant, is used as an ethnomedicine for bacterial infection or immune modulation in Asia, America and Africa. It has been demonstrated that cytopiloyne (CP), a bioactive polyacetylenic glucoside purified from B. pilosa, increases the percentage of macrophages in the spleen but the specific effects on macrophages remain unclear. AIM OF THE STUDY: The aim of this study was to evaluate the effects of CP on macrophage activity and host defense in BALB/c mice with Candida parapsilosis infection and investigate the likely mechanisms. MATERIALS AND METHODS: RAW264.7 cells, a mouse macrophage cell line, were used to assess the effects of CP on macrophage activity by phagocytosis assay, colony forming assay and acridine orange/crystal violet stain. To evaluate the activity of CP against C. parapsilosis, BALB/c mouse infection models were treated with/without CP and histopathological examination was performed. The role of macrophages in the infection model was clarified by treatment with carrageenan, a selective macrophage-toxic agent. RAW264.7 macrophage activities influenced by CP were further investigated by lysosome staining, phagosomal acidification assay, lysosome enzyme activity and PKC inhibitor GF109203X. RESULTS: The results showed that CP in vitro enhances the ability of RAW264.7 macrophages to engulf and clear C. parapsilosis. In the mouse model, CP treatment improved the survival rate of Candida-infected mice and lowered the severity of microscopic lesions in livers and spleens via a macrophage-dependent mechanism. Furthermore, with CP treatment, the fusion and acidification of phagolysosomes were accelerated and the lysosome enzyme activity of RAW264.7 macrophages was elevated. PKC inhibitor GF109203X reversed the increase in phagocytic activity by CP demonstrating that the PKC pathway is involved in the macrophage-mediated phagocytosis of C. parapsilosis. CONCLUSIONS: Our data suggested that CP, as an immunomodulator, enhances macrophage activity against C. parapsilosis infections.

Basis for selecting optimum antibiotic regimens for secondary peritonitis.

Adequate management of severely ill patients with secondary peritonitis requires supportive therapy of organ dysfunction, source control of infection and antimicrobial therapy. Since secondary peritonitis is polymicrobial, appropriate empiric therapy requires combination therapy in order to achieve the needed coverage for both common and more unusual organisms. This article reviews etiological agents, resistance mechanisms and their prevalence, how and when to cover them and guidelines for treatment in the literature. Local surveillances are the basis for the selection of compounds in antibiotic regimens, which should be further adapted to the increasing number of patients with risk factors for resistance (clinical setting, comorbidities, previous antibiotic treatments, previous colonization, severity…). Inadequate antimicrobial regimens are strongly associated with unfavorable outcomes. Awareness of resistance epidemiology and of clinical consequences of inadequate therapy against resistant bacteria is crucial for clinicians treating secondary peritonitis, with delicate balance between optimization of empirical therapy (improving outcomes) and antimicrobial overuse (increasing resistance emergence).

Acquired Flucytosine Resistance during Combination Therapy with Caspofungin and Flucytosine for Candida glabrata Cystitis.

Treatment of Candida glabrata cystitis remains a therapeutic challenge, and an antifungal combination using flucytosine is one option. We describe two patients with refractory C. glabrata cystitis who failed flucytosine combined with caspofungin with early-acquired high-level resistance to flucytosine due to nonsense mutations in the FUR1 gene. Rapidly acquired flucytosine resistance with microbiological failure should discourage combination of caspofungin and flucytosine during urinary candidiasis.

Abdominal candidiasis is a hidden reservoir of echinocandin resistance.

FKS mutant Candida isolates were recovered from 24% (6/25) of abdominal candidiasis patients exposed to echinocandin. Candida glabrata (29%) and Candida albicans (14%) mutants were identified. Multidrug-resistant bacteria were recovered from 83% of FKS mutant infections. Mutations were associated with prolonged echinocandin exposure (P = 0.01), breakthrough infections (P = 0.03), and therapeutic failures despite source control interventions (100%). Abdominal candidiasis is a hidden reservoir for the emergence of echinocandin-resistant Candida.

Photodynamic therapy has antifungal effect and reduces inflammatory signals in Candida albicans-induced murine vaginitis.

BACKGROUND: Vaginal candidiasis (VC) is a disease that affects thousands of women of childbearing age, mainly caused by Candida albicans fungus. Photodynamic therapy (PDT) uses photosensitizing substances that are nontoxic in the dark, but able to produce reactive oxygen species when they are subjected to a light source. In this work our purpose was to investigate PDT effects on fungal burden and inflammatory cells in a murine model of C. albicans-induced vaginal candidiasis. METHODS: Female BALB/c mice 6-10 weeks were estrogenized and maintained in this state during all experiment. After 72h, mices were inoculated intravaginally (IV) with 20µL of 2×10(5)C. albicans cells suspension. Mice were separated into 5 groups after five days: H (healthy), PBS (control), laser, MB (methylene blue) and PDT. PDT and MB groups received IV 20µL solution with 1mM of MB, others received PBS. PDT and laser groups were irradiated with a red laser (100mW, 660nm) in one (36J, 6min) or two sessions (18J, 3min). After the end of treatment, mice were submitted to microbiological and histomorphometric analysis with ImageJ software. Data were plotted by mean values and standard deviations of CFU/mL and percentage of inflammatory cells area. ANOVA and Bonferroni post-test were used and data were considered significant when p<0.05. RESULTS: PDT significantly reduced C. albicans after the two tested protocols, however, percentage area of inflammatory cells was significantly reduced just with two sessions of PDT. CONCLUSIONS: PDT with MB and red laser is a promising therapy for VC. It is able to reduce fungal infection in biofilm and inflammatory signals associated with VC in a murine model of vaginitis.