Recent developments on the anti-Candida effect of amphotericin B combined with a second drug - a mini-review.

Invasive Candida infections threaten human health due to the increasing incidence of resistance to the currently available antifungal agents. Amphotericin B (AMB) is the gold standard therapy to treat these infections. Nevertheless, the use of such substance in the clinic is aggravated by its toxicity. Since AMB binds to membrane sterols, it forms pores on human plasma membranes, mainly in kidney cells, leading to nephrotoxicity. The combination of this drug to a second substance could allow for the use of smaller concentrations of AMB, consequently lowering the probability of adverse effects. This mini-review summarizes information regarding an array of substances that enhance AMB antifungal activity. It may be noticed that several of these compounds target plasma membrane. Interestingly, substances approved for human use also presented combinatory anti-Candida activity with AMB. These data reinforce the potential of associating AMB to another drug as a promising therapeutical alternative to treat Candida infections. Further studies, regarding mechanism of action, pharmacokinetics and toxicity parameters must be conducted to confirm the role of these substances as adjuvant agents in candidiasis therapy.

The Antileishmanial Activity of Eugenol Associated with Lipid Storage Reduction Rather Than Membrane Properties Alterations.

Leishmaniasis is a neglected tropical disease that still infects thousands of people per year throughout the world. The occurrence of resistance against major treatments for this disease causes a healthcare burden in low-income countries. Eugenol is a phenylpropanoid that has shown in vitro antileishmanial activity against Leishmania mexicana mexicana (Lmm) promastigotes with an IC50 of 2.72 µg/mL and a high selectivity index. Its specific mechanism of action has yet to be studied. We prepared large unilamellar vesicles (LUVs), mimicking Lmm membranes, and observed that eugenol induced an increase in membrane permeability and a decrease in membrane fluidity at concentrations much higher than IC50. The effect of eugenol was similar to the current therapeutic antibiotic, amphotericin B, although the latter was effective at lower concentrations than eugenol. However, unlike amphotericin B, eugenol also affected the permeability of LUVs without sterol. Its effect on the membrane fluidity of Lmm showed that at high concentrations (≥22.5× IC50), eugenol increased membrane fluidity by 20-30%, while no effect was observed at lower concentrations. Furthermore, at concentrations below 10× IC50, a decrease in metabolic activity associated with the maintenance of membrane integrity revealed a leishmaniostatic effect after 24 h of incubation with Lmm promastigotes. While acidocalcisomes distribution and abundance revealed by Trypanosoma brucei vacuolar H+ pyrophosphatase (TbVP1) immunolabeling was not modified by eugenol, a dose-dependent decrease of lipid droplets assessed by the Nile Red assay was observed. We hereby demonstrate that the antileishmanial activity of eugenol might not directly involve plasma membrane sterols such as ergosterol, but rather target the lipid storage of Lmm.

Apigenin is a promising molecule for treatment of visceral leishmaniasis.

Current treatment for visceral leishmaniasis is based on drugs such as pentavalent antimony and amphotericin B. However, this treatment remains mostly ineffective and expensive, resulting in several side effects and generating resistance. Apigenin, a flavonoid present in fruits and vegetables, has demonstrated several biological functions. In the present study, we observed a concentration-dependent inhibition of the L. infantum promastigote in the presence of apigenin, exhibiting an IC50 value of 29.9 µM. Its effect was also evaluated in L. infantum-infected murine peritoneal macrophages, presenting an C50 value against intracellular amastigotes of 2.3 µM and a selectivity index of 34.3. In a murine model of visceral leishmaniasis, the in vivo effect of apigenin was measured using short-term and long-term treatment schemes. Treatment with apigenin demonstrated 99.7% and 94% reductions in the liver parasite load in the short-term and long-term treatment schemes, respectively. Furthermore, no alterations in serological and hematological parameters were observed. Taken together, these results suggest that apigenin is a potential candidate for visceral leishmaniasis chemotherapy by oral administration.

Formulation and Characterization of Bioactive Agent Containing Nanodisks.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle's lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin B as the hydrophobic bioactive agent.

Enhanced Antifungal Activity of Amphotericin B Bound to Albumin: A "Trojan Horse" Effect of the Protein.

Amphotericin B (AmB) is a life-saving and widely used antifungal antibiotic, but its therapeutic applicability is limited due to severe side effects. Here, we report that the formulation of the drug based on a complex with albumin (BSA) is highly effective against Candida albicans at relatively low concentrations, which implies lower toxicity to patients. This was also concluded based on the comparison with antifungal activities of other popular commercial formulations of the drug, such as Fungizone and AmBisome. Several molecular spectroscopy and imaging techniques, e.g., fluorescence lifetime imaging microscopy (FLIM), were applied to understand the phenomenon of enhanced antifungal activity of the AmB-BSA complex. The results show that the drug molecules bound to the protein remain mostly monomeric and are most likely bound in the pocket responsible for the capture of small molecules by this transport protein. The results of molecular imaging of single complex particles indicate that in most cases, the antibiotic-protein stoichiometry is 1:1. All of the analyses of the AmB-BSA system exclude the presence of the antibiotic aggregates potentially toxic to patients. Cell imaging shows that BSA-bound AmB molecules can readily bind to fungal cell membranes, unlike drug molecules present in the aqueous phase, which are effectively retained by the cell wall barrier. The advantages and prospects of pharmacological use of AmB complexed with proteins are discussed.

Regulation of calcium ions on the interaction between amphotericin B and cholesterol-rich phospholipid monolayer in LE phase and LC phase.

Amphotericin B, as a "gold standard", is used to treat invasive fungal infections. The AmB molecule can bind easily to cholesterol and damage cell membranes, so it produces the toxicity on cell membrane, which limits its clinical dose. However, the interaction between AmB and cholesterol-rich membrane is unclear now. The phase state of the membrane and the metal cation outside cell membrane may affect the interaction between AmB and the membrane. In this work, the effects of amphotericin B on the mean molecular area, elastic modulus and stability of mammalian cell membrane rich in cholesterol in the presence of Ca2+ ions were studied using DPPC/Chol mixed Langmuir monolayer as a model. The Langmuir-Blodgett method and AFM test were used to study the effects of this drug on the morphology and height of cholesterol-rich phospholipid membrane in the presence of Ca2+ ions. The influence of calcium ions on the mean molecular area and the limiting molecular area was similar in LE phase and in LC phase. The calcium ions made the monolayer more condensed. However, calcium ions can weaken the shortening effect of AmB on the relaxation time of the DPPC/Chol mixed monolayer in LE phase but enhance it in LC phase. Interestingly, calcium ions caused a LE-LC coexistence phase to occur in the DPPC/Chol/AmB mixed monolayers at 35mN/m, which was confirmed by atomic force microscopy. The results can help to understand the interaction between amphotericin B and cell membrane rich in cholesterol in the calcium ions environment.

Identification and susceptibility testing of oral candidiasis in advanced cancer patients.

BACKGROUND: Patients with advanced cancer are prone to develop different opportunistic oral infection due to anti-cancer treatment or the malignancies themselves. Studies of oral fungal samples show an increased prevalence of non-Candida albicans species in mixed oral infections with Candida albicans. Non-C. albicans and C. albicans are associated with varying degrees of resistance to azoles, which may have implications for treatment. This study aimed to assess the diversity and antifungal susceptibility of Candida species detected in the oral cavity. METHODS: An observational study with microbiological analysis was conducted. Clinical fungal isolates were collected from patients in a hospice unit in 2014-2016. Isolates were re-grown on chromID® Candida plates in 2020. Single colony of each species was re-cultivated and prepared for biochemical identification with a VITEK2® system and verified by gene sequencing. Etest was performed on RPMI agar, and the antifungals fluconazole, amphotericin B, anidulafungin and nystatin were applied. RESULTS: Fifty-six isolates from 45 patients were identified. Seven different Candida species and one Saccharomyces species were detected. The results of biochemical identification were confirmed with sequencing analysis. Thirty-six patients had mono infection, and nine out of 45 patients had 2-3 different species detected. Of C. albicans strains, 39 out of 40 were susceptible to fluconazole. Two non-C. albicans species were resistant to fluconazole, one to amphotericin B and three to anidulafungin. CONCLUSION: C. albicans was the predominant species, with a high susceptibility to antifungal agents. Different Candida species occur in both mono and mixed infections. Identification and susceptibility testing may therefore lead to more effective treatment and may prevent the development of resistance among patients with advanced cancer. TRAIL REGISTRATION: The study Oral Health in Advanced Cancer was registered at ClinicalTrials.gov (#NCT02067572) in 20/02/2014.

Anti-Balamuthia mandrillaris and anti-Naegleria fowleri effects of drugs conjugated with various nanostructures.

Balamuthia mandrillaris and Naegleria fowleri are protist pathogens that can cause fatal infections. Despite mortality rate of > 90%, there is no effective therapy. Treatment remains problematic involving repurposed drugs, e.g., azoles, amphotericin B and miltefosine but requires early diagnosis. In addition to drug discovery, modifying existing drugs using nanotechnology offers promise in the development of therapeutic interventions against these parasitic infections. Herein, various drugs conjugated with nanoparticles were developed and evaluated for their antiprotozoal activities. Characterizations of the drugs' formulations were accomplished utilizing Fourier-transform infrared spectroscopy, efficiency of drug entrapment, polydispersity index, zeta potential, size, and surface morphology. The nanoconjugates were tested against human cells to determine their toxicity in vitro. The majority of drug nanoconjugates exhibited amoebicidal effects against B. mandrillaris and N. fowleri. Amphotericin B-, Sulfamethoxazole-, Metronidazole-based nanoconjugates are of interest since they exhibited significant amoebicidal effects against both parasites (p < 0.05). Furthermore, Sulfamethoxazole and Naproxen significantly diminished host cell death caused by B. mandrillaris by up to 70% (p < 0.05), while Amphotericin B-, Sulfamethoxazole-, Metronidazole-based drug nanoconjugates showed the highest reduction in host cell death caused by N. fowleri by up to 80%. When tested alone, all of the drug nanoconjugates tested in this study showed limited toxic effects against human cells in vitro (less than 20%). Although these are promising findings, prospective work is warranted to comprehend the mechanistic details of nanoconjugates versus amoebae as well as their in vivo testing, to develop antimicrobials against the devastating infections caused by these parasites.

In vitro antifungal and antibiofilm activities of auranofin against itraconazole-resistant Aspergillus fumigatus.

BACKGROUND: Infections caused by azole-resistant Aspergillus are a rising public health threat with high mortality rates, high treatment costs and limited available antifungals, indicating an urgent need for new antifungals or strategies. Our aim was to investigate antifungal and antibiofilm activities of auranofin, an FDA-approved anti-antirheumatic drug. METHODS: Fungal susceptibility testing for auranofin was carried out by the broth-based microdilution methods. Cell viability treated by auranofin was tested by resazurin dye testing. The synergistic effect of auranofin and antifungal drugs was evaluated using checkboard assay. The inhibitory of biofilms were measured by crystal violet staining. Gene expression level analysis and enzyme activity was investigated with qRT-PCR analysis and DTNB assay. The key amino acid residues in the binding of auranofin with A. fumigatus thioredoxin reductase (AfTrxR) were indicated by structural analyses, site-directed mutagenesis, and microscale thermophoresis (MST) assays. RESULTS: Auranofin has fungicidal activity and in vitro antifungal spectrum including Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Aspergillus niger, even itraconazole (ITC)-resistant A. fumigatus. Additionally, it has antibiofilm activities against ITC-resistant A. fumigatus by reducing the expression level of SomA and MedA. Moreover, we discovered a synergistic effect of auranofin and ITC or amphotericin B against ITC-resistant A. fumigatus. Auranofin downregulated the gene transcription of AfTrxR, and strongly inhibited the enzyme activity of AfTrxR through interacting with residues C145 and C148. CONCLUSIONS: Auranofin has fungicidal and antibiofilm activities in Aspergillus spp. and is also a potentiator of ITC or amphotericin B in vitro.

Detection of Naganishia albida Yeasts in Dermatological Patients.

The yeasts Cryptococcus albidus (Naganishia albida) usually occur on natural substrates and rarely are the etiological factor of different mycoses. More than a half of mycosis cases described in the literature were reported during the period from 2004 to 2021. In this regard, evaluation of yeast sensitivity to antimycotic drugs is as important as their identification. In the present study, two yeast isolates from the skin of female patients (age 7 and 74 years) with infective dermatitis (ICD-10-CM Code L30.3) were studied. Common identification of the isolates, MALDI-TOF mass spectrometry, and analysis of the nucleotide sequences of the ITS1-5.8S-ITS2 rDNA region showed that they belong to the species N. albida. The sensitivity of the obtained strains to antimycotics of three different chemical groups, namely itraconazole, naftifine, and amphotericin B, determined by microdilution method in a synthetic medium showed the following minimum inhibitory concentrations: 64-128, 16, and 0.125-4 µg/ml, respectively. It was found that the sensitivity of this yeast to pooled human serum was 30-47%, i.e. lower by 1.9-2.9 times than the sensitivity of the collection strains of C. albicans and C. neoformans. This result could be explained by lower prevalence of N. albida in the human population in comparison with these species. However, the sensitivity of N. albida strains to the low-molecular-weight fraction of serum was approximately the same as in C. albicans and C. neoformans, which indicates their high sensitivity to antimicrobial peptides.

Amphotericin B, itraconazole, posaconazole, and isavuconazole MICs against clinical Mucorales isolates obtained by visual inspection and spectrophotometric reading according to the EUCAST 9.4 procedure.

We compared the antifungal susceptibility of 92 Mucorales isolates obtained by visual inspection and spectrophotometric readings following EUCAST (European Committee on Antimicrobial Susceptibility Testing) testing. Amphotericin B minimum inhibitory concentrations (MICs) were up to 1 mg/l against most isolates and variable among species, except for Cunninghamella bertholletiae. Posaconazole MICs against most isolates were up to 1 mg/l and high against Mucor circinelloides, some Rhizopus arrhizus, and Rhizopus microsporus. Isavuconazole MICs ranged between 1 and 8 mg/l but were invariably >8 mg/l against M. circinelloides and C. bertholletiae. The agreement between MICs obtained by visual endpoint or spectrophotometric readings was moderate and higher when using the ≥90% fungal growth inhibition endpoint.

The agreement between minimum inhibitory concentration (MIC) values obtained by visual inspection or spectrophotometric readings was moderate and higher when the ≥90% fungal growth inhibition endpoint was chosen. Isavuconazole presented higher MICs than posaconazole, regardless of the inhibition endpoint used.

Interactions between antifungals and everolimus against Cryptococcus neoformans.

Cryptococcus is the causal agent of cryptococcosis, a disease with high mortality mainly related to HIV immunosuppression and usually manifests with pneumonia and/or meningoencephalitis. There are very few therapeutic options; thus, innovative approaches are required. Herein, We examined the interaction of everolimus (EVL) with amphotericin B (AmB) and azoles [fluconazole (FLU), posaconazole (POS), voriconazole (VOR), itraconazole (ITR)] against Cryptococcus. Eighteen Cryptococcus neoforman clinical isolates were analyzed. Following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) M27-A4, we conducted a broth microdilution experiment to determine the minimum inhibitory concentrations (MICs) of azoles, EVL, and AmB for assessing antifungal susceptibility. A fractional inhibitory concentration index (FICI) of less than and equal to 0.5 indicated synergy, with a range of 0.5 to 4.0 indicated indifference and a value more than 4.0 indicated antagonism. These experiments revealed that EVL had antifungal activity against C. neoforman. Moreover, EVL, POS, AmB, FLU, ITR, and VOR exhibited MIC values ranging from 0.5-2 µg/mL, 0.03125-2 µg/mL, 0.25-4 µg/mL, 0.5-32µg/mL, 0.0625-4µg/mL and 0.03125-2µg/mL, respectively. The combination of EVL with AmB and azoles (POS, FLU, ITR, and VOR) exhibited synergistic antifungal effects against 16 (88.9%), 9 (50%), 11 (61.1%), 10 (55.6%) or 6 (33.3%) of analyzed Cryptococcus strains. In the presence of EVL, the MIC values of AmB and azoles were significantly lowered. No antagonism was observed. Subsequently, in vivo analyses conducted using the G. mellonella model further confirmed that combination EVL+ POS, EVL+ FLU, and EVL+ITR treatment were associated with significantly improved larval survival following Cryptococcus spp. infection. These findings provide the first published evidence suggesting that a combination of EVL and AmB or azoles exhibit a synergistic effect and may be an effective antifungal disease treatment strategy for infections caused by Cryptococcus spp.

[Expert consensus on rational clinical application of amphotericin B deoxycholate (2022)].

Amphotericin B (AmB) is an antifungal drug with the broadest spectrum and the most robust antifungal activity in clinical practice. Although there are many kinds of lipid formulations which significantly reduce the toxicity and side effects of amphotericin B deoxycholate (AmBd), AmBd will still play an important clinical role in China for a long time since lipid formulations are substantially more expensive. To standardize the clinical application of AmBd, well-known experts in this field were invited to reach a consensus on the antifungal properties, pharmacokinetic characteristics, dosage regimen, clinical application, prevention and treatment of adverse reactions of AmBd.

Post-translational modifications confer amphotericin B resistance in Candida krusei isolated from a neutropenic patient.

Neutropenia is a common complication in the treatment of hematological diseases and the most common predisposing factor for invasion by fungi, such as Candida krusei. Recent studies have shown that C. krusei, a life-threatening pathogen, has developed resistance to amphotericin B (AMB). However, the mechanisms that led to the rapid emergence of this AMB-resistant phenotype are unclear. In this study, we found the sensitivity for AMB could be promoted by inhibiting histone acyltransferase activity and western blot analysis revealed differences in the succinylation levels of C. krusei isolated from immunocompromised patients and of the corresponding AMB-resistant mutant. By comparative succinyl-proteome analysis, we identified a total of 383 differentially expressed succinylated sites in with 344 sites in 134 proteins being upregulated in the AMB-resistant mutant, compared to 39 sites in 23 proteins in the wild-type strain. These differentially succinylated proteins were concentrated in the ribosome and cell wall. The critical pathways associated with these proteins included those involved in glycolysis, gluconeogenesis, the ribosome, and fructose and mannose metabolism. In particular, AMB resistance was found to be associated with enhanced ergosterol synthesis and aberrant amino acid and glucose metabolism. Analysis of whole-cell proteomes, confirmed by parallel reaction monitoring, showed that the key enzyme facilitating lysine acylation was significantly upregulated in the AMB-resistant strain. Our results suggest that lysine succinylation may play an indispensable role in the development of AMB resistance in C. krusei. Our study provides mechanistic insights into the development of drug resistance in fungi and can aid in efforts to stifle the emergence of AMB-resistant pathogenic fungi.

Analysis of the synergistic antifungal activity of everolimus and antifungal drugs against dematiaceous fungi.

Introduction: Chromoblastomycosis (CBM) is a form of chronic mycosis that affects the skin and mucous membranes and is caused by species of dematiaceous fungi including Exophiala spp., Phialophora spp., and Fonsecaea spp. The persistence of this disease and limitations associated with single-drug treatment have complicated efforts to adequately manage this condition. Methods: In this study, a microdilution assay was used to explore the synergistic antifungal activity of everolimus (EVL) in combination with itraconazole (ITC), voriconazole (VRC), posaconazole (POS), and amphotericin B (AMB) against a range of clinical dematiaceous fungal isolates. Results: These analyses revealed that the EVL+POS and EVL+ITC exhibited superior in vitro synergistic efficacy, respectively inhibiting the growth of 64% (14/22) and 59% (13/22) of tested strains. In contrast, the growth of just 9% (2/22) of tested strains was inhibited by a combination of EVL+AMB, and no synergistic efficacy was observed for the combination of EVL+VRC. Discussion: Overall, these findings indicate that EVL holds promise as a novel drug that can be synergistically combined with extant antifungal drugs to improve their efficacy, thereby aiding in the treatment of CBM.

Multicentric Analysis of the Species Distribution and Antifungal Susceptibility of Clinical Isolates from Aspergillus Section Circumdati.

The clinical involvement and antifungal susceptibility of Aspergillus section Circumdati are poorly known. We analyzed 52 isolates, including 48 clinical isolates, belonging to 9 species inside the section Circumdati. The whole section exhibited, by the EUCAST reference method, a poor susceptibility to amphotericin B, but species/series-specific patterns were observed for azole drugs. This underlines the interest in getting an accurate identification inside the section Circumdati to guide the choice of antifungal treatment in clinical practice.

Drug Susceptibility Profiling of Prototheca Species Isolated from Cases of Human Protothecosis.

Prototheca are unicellular, achlorophyllous, yeast-like microalgae that occur in a wide range of natural habitats. At least five species have been implicated as the causative agents of opportunistic infections of men. Human protothecosis typically manifests as cutaneous, articular, or systemic disease. Treatment is largely empirical with poorly predictable and often unsuccessful outcomes. This is largely due to the frequently observed resistance of Prototheca species to conventional antimicrobial agents. This work is the first to perform drug susceptibility profiling exclusively on isolates from human cases of protothecosis. A total of 23 such isolates were tested against amphotericin B and 9 azoles, including efinaconazole and luliconazole, whose activities against Prototheca have never been studied before. Efinaconazole was the most active, with median minimum inhibitory concentration (MIC) and minimum algicidal concentration (MAC) values of 0.031 mg/L and 0.063 mg/L, respectively. Fluconazole and luliconazole had the lowest activity, with median MIC and MAC values of 128 mg/L. To conclude, amphotericin B and most of the azoles showed in vitro activity, with an algicidal rather than algistatic effect, against Prototheca. Still, the activity of individual drugs differed significantly between the species and even between strains of the same species. These differences can be attributed to a species-specific potential for acquiring drug resistance, which, in turn, might be linked to the treatment history of the patient from whom the strain was recovered. The results of this study underscore the potential clinical utility of efinaconazole as a promising therapeutic agent for the treatment of human protothecosis.

Targeting lung macrophages for fungal and parasitic pulmonary infections with innovative amphotericin B dry powder inhalers.

The incidence of fungal pulmonary infections is known to be on the increase, and yet there is an alarming gap in terms of marketed antifungal therapies that are available for pulmonary administration. Amphotericin B (AmB) is a highly efficient broad-spectrum antifungal only marketed as an intravenous formulation. Based on the lack of effective antifungal and antiparasitic pulmonary treatments, the aim of this study was to develop a carbohydrate-based AmB dry powder inhaler (DPI) formulation, prepared by spray drying. Amorphous AmB microparticles were developed by combining 39.7 % AmB with 39.7 % γ-cyclodextrin, 8.1 % mannose and 12.5 % leucine. An increase in the mannose concentration from 8.1 to 29.8 %, led to partial drug crystallisation. Both formulations showed good in vitro lung deposition characteristics (80 % FPF < 5 µm and MMAD < 3 µm) at different air flow rates (60 and 30 L/min) when used with a DPI, but also during nebulisation upon reconstitution in water.

Pharmacodynamic and immunological interactions of amphotericin B formulations and voriconazole with human neutrophils against mature Scedosporium apiospermum and Fusarium spp. biofilms.

BACKGROUND: Mould infections caused by Scedosporium apiospermum and Fusarium solani species complex (FSSC) biofilms are rising among immunocompromised and immunocompetent patients. Little is known about the immunomodulatory effects of antifungal agents against these moulds. We examined the effects of deoxycholate and liposomal amphotericin B (DAmB, LAmB) and voriconazole on antifungal activities and immune responses of neutrophils (PMNs) against mature biofilms compared with their planktonic counterparts. METHODS: Antifungal activity of human PMNs exposed to mature biofilms and planktonic cells for 24 h was determined at effector-to-target ratios of 2:1 and 5:1, alone or combined with DAmB, LAmB and voriconazole, assessed as fungal damage by XTT assay. Cytokine production was evaluated by multiplex ELISA, following PMN stimulation with biofilms in the presence/absence of each drug. RESULTS: All drugs showed additive or synergistic effects with PMNs against S. apiospermum at 0.03-32 mg/L. They showed antagonism primarily against FSSC at 0.06-64 mg/L. Increased IL-8 was produced by PMNs exposed to S. apiospermum biofilms plus DAmB or voriconazole compared with PMNs exposed to biofilms alone (P < 0.01). During combined exposure, IL-1ß was increased, an effect only counteracted by increased levels of IL-10 caused by DAmB (P < 0.01). LAmB and voriconazole caused similar IL-10 levels with those released by biofilm-exposed PMNs. CONCLUSIONS: The synergistic, additive or antagonistic effects of DAmB, LAmB or voriconazole on biofilm-exposed PMNs are organism-specific, with FSSC exhibiting greater resilience than S. apiospermum to antifungals. Biofilms of both moulds caused dampened immune responses. The drug-mediated immunomodulating effect on PMNs, evidenced by IL-1ß, enhanced host protective functions.

Synthetic amino acids-based short amphipathic peptides exhibit antifungal activity by targeting cell membrane disruption.

Availability of a limited number of antifungal drugs created a necessity to develop new antifungals with distinct mode of action. Investigation on a new series of peptides led us to identify Boc-His-Trp-His[1-(4-tert-butylphenyl)] (10g) as the most promising inhibitor exhibiting IC50 value of 4.4 µg/mL against Cryptococcus neoformans. Analog 10g exhibit high selectivity to fungal cells and was nonhemolytic and noncytotoxic at its minimum inhibitory concentration. 10g produced fungicidal effect on growing cryptococcal cells and displayed synergistic effect with amphotericin B. Overall cationic character of 10g resulted in interaction with negatively charged fungal membrane while hydrophobicity enhanced penetration inside the cryptococcal cells causing hole(s) formation and disruption to the membrane as evident by the scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy analyses. Flow cytometric investigation revealed rapid death of fungal cells by apopotic pathway.