Fusarium keratitis in a Brazilian tropical semi-arid area: Clinical-epidemiological features, molecular identification and antifungal susceptibility.

BACKGROUND: Fungal keratitis is a severe eye infection that can result in blindness and visual impairment, particularly in developing countries. Fusarium spp. are the primary causative agents of this condition. Diagnosis of Fusarium keratitis (FK) is challenging, and delayed treatment can lead to serious complications. However, there is limited epidemiological data on FK, especially in tropical areas. OBJECTIVES: This study aimed to describe the clinical, laboratorial and epidemiological characteristics of FK in a tropical semi-arid region of Brazil. PATIENTS/METHODS: Adult patients with laboratory-confirmed FK diagnosed between October 2019 and March 2022 were evaluated. Fusarium isolates were characterized at molecular level and evaluated regarding antifungal susceptibility. RESULTS: A total of 226 clinical samples from patients suspected of keratitis were evaluated; fungal growth was detected in 50 samples (22.12%); out of which 42 were suggestive of Fusarium spp. (84%). Molecular analysis of a randomly selected set of 27 isolates identified F. solani species complex (n = 14); F. fujikuroi sensu lato (n = 6) and F. dimerum sensu lato (n = 7); a total of 10 haplotypes were identified among the strains. All but one Fusarium strains were inhibited by amphotericin B, natamycin and fluconazole. Most patients were male (71.42%; 30 out of 42), aged from 27 to 73 years old. Trauma was the most important risk factor for FK (40.47%; 17 out of 42). Patients were treated with antifungals, corticoids and antibiotics; keratoplasty and eye enucleation were also performed. CONCLUSIONS: The study provided insights into the characteristics of FK in tropical regions and emphasized the importance of enhanced surveillance and management strategies.

Synergistic activity of gold nanoparticles with amphotericin B on persister cells of Candida tropicalis biofilms.

AIM: The antifungal activity was studied on sessile and persister cells (PCs) of Candida tropicalis biofilms of gold nanoparticles (AuNPs) stabilized with cetyltrimethylammonium bromide (CTAB-AuNPs) and those conjugated with cysteine, in combination with Amphotericin B (AmB). MATERIALS/METHODS: The PC model was used and synergistic activity was tested by the checkerboard assay. Biofilms were studied by crystal violet and scanning electron microscopy. RESULTS/CONCLUSIONS: After the combination of both AuNPs and AmB the biofilm biomass was reduced, with significant differences in architecture being observed with a reduced biofilm matrix. In addition, the CTAB-AuNPs-AmB combination significantly reduced PCs. Understanding how these AuNPs aid in the fight against biofilms and the development of new approaches to eradicate PCs has relevance for chronic infection treatment.

Oral candidiasis in liver transplant patients: species identification and antifungal susceptibility profile.

OBJECTIVE: This study aimed to verify oral candidiasis, identify the causative species, and investigate the antifungal susceptibility of yeasts isolated from liver transplant patients. METHODS: A descriptive analysis of 97 patients who underwent liver transplantation was conducted at a hospital. Two clinical examinations (Collections A and B) of the oral cavity were performed. Oral material was collected from all patients, inoculated in Sabouraud Dextrose Agar, and incubated at 35℃ for 48 hours. Samples were identified by molecular sequencing of the internal trascribed space region of rDNA. RESULTS: An antifungal susceptibility test with fluconazole, amphotericin B, and micafungin was performed using the Clinical and Laboratory Standards Institute yeast broth microdilution method. Among the patients, 15 presented with oral candidiasis: eight in Collection A and seven in Collection B. The primary type of candidiasis was atrophic, followed by pseudomembranous candidiasis. The most prevalent species was Candida albicans (nine), followed by Candida glabrata (three), Candida tropicalis (two), and Candida dubliniensis (one). Regarding susceptibility to fluconazole, of the 15 samples, 11 were susceptible, three were susceptible in a dose-dependent manner, and one was resistant. CONCLUSION: The most commonly identified type of candidiasis was atrophic, with C. albicans and C. glabrata being the most prevalent causative species. One fluconazole-resistant isolate each of C. tropicalis and C. albicans were identified.

Amphotericin B resistance in Leishmania amazonensis: In vitro and in vivo characterization of a Brazilian clinical isolate.

In Brazil, Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The state of Maranhão in the Northeast of Brazil is prevalent for these clinical forms of the disease and also has high rates of HIV infection. Here, we characterized the drug susceptibility of a L. amazonensis clinical isolate from a 46-year-old man with diffuse cutaneous leishmaniasis coinfected with HIV from this endemic area. This patient underwent several therapeutic regimens with meglumine antimoniate, liposomal amphotericin B, and pentamidine, without success. In vitro susceptibility assays against promastigotes and intracellular amastigotes demonstrated that this isolate had low susceptibility to amphotericin B, when compared with the reference strain of this species that is considered susceptible to antileishmanial drugs. Additionally, we investigated whether the low in vitro susceptibility would affect the in vivo response to amphotericin B treatment. The drug was effective in reducing the lesion size and parasite burden in mice infected with the reference strain, whereas those infected with the clinical isolate and a resistant line (generated experimentally by stepwise selection) were refractory to amphotericin B treatment. To evaluate whether the isolate was intrinsically resistant to amphotericin B in animals, infected mice were treated with other drugs that had not been used in the treatment of the patient (miltefosine, paromomycin, and a combination of both). Our findings demonstrated that all drug schemes were able to reduce lesion size and parasite burden in animals infected with the clinical isolate, confirming the amphotericin B-resistance phenotype. These findings indicate that the treatment failure observed in the patient may be associated with amphotericin B resistance, and demonstrate the potential emergence of amphotericin B-resistant L. amazonensis isolates in an area of Brazil endemic for cutaneous leishmaniasis.

The interaction of plant flavones with amphotericin B: Consequences for its pore-forming ability.

The growth of antibiotic resistance to antifungal drugs contributes to the search for new ways to enhance their effectiveness and reduce toxicity. The undeniable advantage of polyene macrolide antibiotic amphotericin B (AmB) which ensures low pathogen resistance is its mechanism of action related to the formation of transmembrane pores in target lipid membranes. Here, we investigated the effects of plant flavones, chrysin, wogonin, baicalein, apigenin, scutellarein, luteolin, morin and fisetin on the pore-forming activity of AmB in the sterol-enriched membranes by electrophysiological assays. Сhrysin, wogonin, baicalein, apigenin, scutellarein, and luteolin were shown to decrease the AmB pore-forming activity in the bilayers composed of palmitoyloleylphosphocholine independently of their sterol composition. Morin and fisetin led to the increase and decrease in the AmB pore-forming activity in the ergosterol- and cholesterol-containing bilayers respectively. Differential scanning microcalorimetry of the gel-to-liquid crystalline phase transition of membrane forming lipids, molecular dynamics simulations, and absorbance spectroscopy revealed the possibility of direct interactions between AmB and some flavones in the water and/or in the lipid bilayer. The influence of these interactions on the antibiotic partitioning between aqueous solution and membrane and/or its transition between different states in the bilayer was discussed.

Notable enhancement of Amphotericin B channel activity by applied pressures in the range of MS channel activation.

The mechanism of Amphotericin B at the membrane is still subject of debate, with the prevailing hypothesis being the formation of pores. The activity of these pores is influenced by various factors. Recently aggregation in solution and insertion in the membrane had been highlighted as crucial for action of the drug Here we investigated the effect of applied pressure on the activity of Amphotericin B. Our findings demonstrate that applied pressure of 50 mmHg is sufficient to enhance the activity. We interpreted the results as supporting the idea that pressure fractures the membrane and promotes the insertion of the polyene.

Regulation of copper uptake by the SWI/SNF chromatin remodeling complex in Candida albicans affects susceptibility to antifungal and oxidative stresses under hypoxia.

Candida albicans is a human colonizer and also an opportunistic yeast occupying different niches that are mostly hypoxic. While hypoxia is the prevalent condition within the host, the machinery that integrates oxygen status to tune the fitness of fungal pathogens remains poorly characterized. Here, we uncovered that Snf5, a subunit of the chromatin remodeling complex SWI/SNF, is required to tolerate antifungal stress particularly under hypoxia. RNA-seq profiling of snf5 mutant exposed to amphotericin B and fluconazole under hypoxic conditions uncovered a signature that is reminiscent of copper (Cu) starvation. We found that under hypoxic and Cu-starved environments, Snf5 is critical for preserving Cu homeostasis and the transcriptional modulation of the Cu regulon. Furthermore, snf5 exhibits elevated levels of reactive oxygen species and an increased sensitivity to oxidative stress principally under hypoxia. Supplementing growth medium with Cu or increasing gene dosage of the Cu transporter CTR1 alleviated snf5 growth defect and attenuated reactive oxygen species levels in response to antifungal challenge. Genetic interaction analysis suggests that Snf5 and the bona fide Cu homeostasis regulator Mac1 function in separate pathways. Together, our data underlined a unique role of SWI/SNF complex as a potent regulator of Cu metabolism and antifungal stress under hypoxia.

Antimony susceptible Leishmania donovani: evidence from in vitro drug susceptibility of parasites isolated from patients of post-kala-azar dermal leishmaniasis in pre- and post-miltefosine era.

Post-kala-azar dermal leishmaniasis (PKDL) patients are a key source of Leishmania donovani parasites, hindering the goal of eliminating visceral leishmaniasis (VL). Monitoring treatment response and parasite susceptibility is essential due to increasing drug resistance. We assessed the drug susceptibility of PKDL isolates (n = 18) from pre-miltefosine (MIL) era (1997-2004) with isolates (n = 16) from the post-miltefosine era (2010-2019) and post-miltefosine treatment relapse isolates (n = 5) towards miltefosine and amphotericin B (AmB) at promastigote stage and towards sodium antimony gluconate (SAG) at amastigote stage. PKDL isolates were examined for mutation in gene-encoding AQP1 transporter, C26882T mutation on chromosome 24, and miltefosine-transporter (MT). PKDL isolates from the post-miltefosine era were significantly more susceptible to SAG than SAG-resistant isolates from the pre-miltefosine era (P = 0.0002). There was no significant difference in the susceptibility of parasites to miltefosine between pre- and post-miltefosine era isolates. The susceptibility of PKDL isolates towards AmB remained unchanged between the pre- and post-miltefosine era. However, the post-miltefosine era isolates had a higher IC50 value towards AmB compared with PKDL relapse isolates. We did not find any association between AQP1 gene sequence variation and susceptibility to SAG, or between miltefosine susceptibility and single nucleotide polymorphisms (SNPs in the MT gene. This study demonstrates that recent isolates of Leishmania have resumed susceptibility to antimonials in vitro. The study also offers significant insights into the intrinsic drug susceptibility of Leishmania parasites over the past two decades, covering the period before the introduction of miltefosine and after its extensive use. IMPORTANCE: Post-kala-azar dermal leishmaniasis (PKDL) patients, a key source of Leishmania donovani parasites, hinder eliminating visceral-leishmaniasis. Assessment of the susceptibility of PKDL isolates to antimony, miltefosine (MIL), and amphotericin-B indicated that recent isolates remain susceptible to antimony, enabling its use with other drugs for treating PKDL.

Low toxicity contributes to Sporothrix globosa invade the skin of patients in low-epidemic areas of China.

OBJECTIVE: This study aims to assess the clinical characteristics of sporotrichosis in low-endemic areas of China, including the prevalence geography, genotypic traits of patients, clinical manifestations, and strain virulence and drug sensitivities. The objective is to improve the currently used clinical management strategies for sporotrichosis. METHODS: Retrospective data were collected from patients diagnosed with sporotrichosis through fungal culture identification. The isolates from purified cultures underwent identification using CAL (Calmodulin) gene sequencing. Virulence of each strain was assessed using a Galleria mellonella (G. mellonella) larvae infection model. In vitro susceptibility testing against commonly used clinical antifungal agents for sporotrichosis was conducted following CLSI criteria. RESULTS: In our low-endemic region for sporotrichosis, the majority of cases (23) were observed in middle-aged and elderly women with a history of trauma, with a higher incidence during winter and spring. All clinical isolates were identified as Sporothrix globosa (S. globosa). The G. mellonella larvae infection model indicated independent and dose-dependent virulence among strains, with varying toxicity levels demonstrated by the degree of melanization of the G. mellonella. Surprisingly, lymphocutaneous types caused by S. globosa exhibited lower in vitro virulence but were more common in affected skin. In addition, all S.globosa strains displayed high resistances to fluconazole, while remaining highly susceptible to terbinafine, itraconazole and amphotericin B. CONCLUSION: Given the predominance of elderly women engaged in agricultural labour in our region, which is a low-epidemic areas, they should be considered as crucial targets for sporotrichosis monitoring. S. globosa appears to be the sole causative agent locally. However, varying degrees of melanization in larvae were observed among these isolates, indicating a divergence in their virulence. Itraconazole, terbinafine and amphotericin B remain viable first-line antifungal options for treating S.globosa infection.

Synergistic action of synthetic peptides and amphotericin B causes disruption of the plasma membrane and cell wall in Candida albicans.

The objective of this work was to evaluate the combination of synthetic peptides based on the γ-core motif of defensin PvD1 with amphotericin B (AmB) at different concentrations against Candida albicans. We applied the checkerboard assay using different concentrations of the commercial drug AmB and the synthetic peptides γ31-45PvD1++ and γ33-41PvD1++ against C. albicans, aiming to find combinations with synergistic interactions. Between these two interactions involving γ31-45PvD1++ and AmB, an additive effect was observed. One such interaction occurred at concentrations of 0.009 µM of peptide γ31-45PvD1++ and 13.23 µM of AmB and another condition of 0.019 µM of peptide γ31-45PvD1++ and 6.61 µM of AmB. The other two concentrations of the interaction showed a synergistic effect in the combination of synthetic peptide γ31-45PvD1++ and AmB, where the concentrations were 1.40 µM peptide γ31-45PvD1++ and 0.004 µM AmB and 0.70 µM γ31-45PvD1++ peptide and 0.002 µM AmB. We proceeded with analysis of the mechanism of action involving synergistic effects. This examination unveiled a range of impactful outcomes, including the impairment of mitochondrial functionality, compromise of cell wall integrity, DNA degradation, and a consequential decline in cell viability. We also observed that both synergistic combinations were capable of causing damage to the plasma membrane and cell wall, causing leakage of intracellular components. This discovery demonstrates for the first time that the synergistic combinations found between the synthetic peptide γ31-45PvD1++ and AmB have an antifungal effect against C. albicans, acting on the integrity of the plasma membrane and cell wall.

Discovery of Antifungal Norsesquiterpenoids from a Soil-Derived Streptomyces microflavus: Targeting Biofilm Formation and Synergistic Combination with Amphotericin B against Yeast-like Fungi.

Thirty-five norsesquiterpenoids were isolated from the fermentation broth of Streptomyces microflavus from the forest soil of Ailaoshan in China. The structures of new compounds (1-5, 10-26) were elucidated by comprehensive spectroscopic analysis including data from experimental and calculated ECD spectra, as well as Mosher's reagent derivatives method. Norsesquiterpenoids showed different levels of antifungal activity with MIC80 values ranging from 25 to 200 µg/mL against Candida albicans, Candida parapsilosis, and Cryptococcus neoformans. The combining isolated norsesquiterpenoids with amphotericin B resulted in a synergistic interaction against test yeast-like fungi with a fractional inhibitory concentration index < 0.5. Compound 33 significantly inhibited biofilm formation and destroyed the preformed biofilm of fungi. Moreover, 33 downregulated the expression of adhesion-related genes HWP1, ALS1, ALS3, ECE1, EAP1, and BCR1 to inhibit the adhesion of C. albicans. Findings from the current study highlight the potential usage of norsesquiterpenoids from soil-derived Streptomyces for antifungal leads discovery.

Genome-wide analysis of in vivo-evolved Candida auris reveals multidrug-resistance mechanisms.

Candida auris, an emerging and multidrug-resistant fungal pathogen, has led to numerous outbreaks in China. While the resistance mechanisms against azole and amphotericin B have been studied, the development of drug resistance in this pathogen remains poorly understood, particularly in in vivo-generated drug-resistant strains. This study employed pathogen whole-genome sequencing to investigate the epidemiology and drug-resistance mutations of C. auris using 16 strains isolated from two patients. Identification was conducted through Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and antimicrobial susceptibilities were assessed using broth microdilution and Sensititre YeastOne YO10. Whole-genome sequencing revealed that all isolates belonged to the South Asian lineage, displaying genetic heterogeneity. Despite low genetic variability among patient isolates, notable mutations were identified, including Y132F in ERG11 and A585S in TAC1b, likely linked to increased fluconazole resistance. Strains from patient B also carried F214L in TAC1b, resulting in a consistent voriconazole minimum inhibitory concentration of 4 µg/mL across all isolates. Furthermore, a novel frameshift mutation in the SNG1 gene was observed in amphotericin B-resistant isolates compared to susceptible ones. Our findings suggest the potential transmission of C. auris and emphasize the need to explore variations related to antifungal resistance. This involves analyzing genomic mutations and karyotypes, especially in vivo, to compare sensitive and resistant strains. Further monitoring and validation efforts are crucial for a comprehensive understanding of the mechanisms of drug resistance in C. auris.

Ibrexafungerp is efficacious in a neutropenic murine model of pulmonary mucormycosis as monotherapy and combined with liposomal amphotericin B.

Ibrexafungerp (formerly SCY-078) is the first member of the triterpenoid class that prevents the synthesis of the fungal cell wall polymer ß-(1,3)-D-glucan by inhibiting the enzyme glucan synthase. We evaluated the in vivo efficacy of ibrexafungerp against pulmonary mucormycosis using an established murine model. Neutropenic mice were intratracheally infected with either Rhizopus delemar or Mucor circinelloides. Treatment with placebo (diluent control), ibrexafungerp (30 mg/kg, PO BID), liposomal amphotericin B (LAMB 10 mg/kg IV QD), posaconazole (PSC 30 mg/kg PO QD), or a combination of ibrexafungerp plus LAMB or ibrexafungerp plus PSC began 16 h post-infection and continued for 7 days for ibrexafungerp or PSC and through day 4 for LAMB. Ibrexafungerp was as effective as LAMB or PSC in prolonging median survival (range: 15 days to >21 days) and enhancing overall survival (30%-65%) vs placebo (9 days and 0%; P < 0.001) in mice infected with R. delemar. Furthermore, median survival and overall percent survival resulting from the combination of ibrexafungerp plus LAMB were significantly greater compared to all monotherapies (P ≤ 0.03). Similar survival results were observed in mice infected with M. circinelloides. Monotherapies also reduce the lung and brain fungal burden by ~0.5-1.0log10 conidial equivalents (CE)/g of tissue vs placebo in mice infected with R. delemar (P < 0.05), while a combination of ibrexafungerp plus LAMB lowered the fungal burden by ~0.5-1.5log10 CE/g compared to placebo or any of the monotherapy groups (P < 0.03). These results are promising and warrant continued investigation of ibrexafungerp as a novel treatment option against mucormycosis.

Leishmania blood parasite dynamics during and after treatment of visceral leishmaniasis in Eastern Africa: A pharmacokinetic-pharmacodynamic model.

BACKGROUND: With the current treatment options for visceral leishmaniasis (VL), recrudescence of the parasite is seen in a proportion of patients. Understanding parasite dynamics is crucial to improving treatment efficacy and predicting patient relapse in cases of VL. This study aimed to characterize the kinetics of circulating Leishmania parasites in the blood, during and after different antileishmanial therapies, and to find predictors for clinical relapse of disease. METHODS: Data from three clinical trials, in which Eastern African VL patients received various antileishmanial regimens, were combined in this study. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative PCR (qPCR) before, during, and up to six months after treatment. An integrated population pharmacokinetic-pharmacodynamic model was developed using non-linear mixed effects modelling. RESULTS: Parasite proliferation was best described by an exponential growth model, with an in vivo parasite doubling time of 7.8 days (RSE 12%). Parasite killing by fexinidazole, liposomal amphotericin B, sodium stibogluconate, and miltefosine was best described by linear models directly relating drug concentrations to the parasite elimination rate. After treatment, parasite growth was assumed to be suppressed by the host immune system, described by an Emax model driven by the time after treatment. No predictors for the high variability in onset and magnitude of the immune response could be identified. Model-based individual predictions of blood parasite load on Day 28 and Day 56 after start of treatment were predictive for clinical relapse of disease. CONCLUSION: This semi-mechanistic pharmacokinetic-pharmacodynamic model adequately captured the blood parasite dynamics during and after treatment, and revealed that high blood parasite loads on Day 28 and Day 56 after start of treatment are an early indication for VL relapse, which could be a useful biomarker to assess treatment efficacy of a treatment regimen in a clinical trial setting.

Redox homeostasis in human renal cells that had been treated with amphotericin B in combination with selected 1,3,4-thiadiazole derivatives.

BACKGROUND: The use of amphotericin B (AmB) in the therapy of systemic mycosis is associated with strong side effects, including nephrotoxicity, and hepatotoxicity. Therefore, agents that can reduce the toxic effects of AmB while acting synergistically as antifungal agents are currently being sought. 1,3,4-thiadiazole derivatives are promising compounds that have an antifungal activity and act synergically with AmB. Such combinations might allow the dose of AmB, which is essential for preventing patients from having serious side effects, to be decreased. This might result from the antioxidant properties of 1,3,4-thiadiazoles. Thus, the aim of the study was to investigate redox homeostasis in human renal proximal tubule epithelial cells (RPTEC) after they had been treated with AmB in combination with 1,3,4-thiadiazole derivatives. METHODS: Cellular redox homeostasis was assessed by investigating the total antioxidant capacity (TAC) of cells, the malondialdehyde (MDA) concentration, and the activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). TAC was measured using an ABTS method. The MDA concentration, and the activity of SOD, GPX, and CAT were determined spectrophotometrically using commercially available assays. Additionally, the antioxidant defense system-related gene expression profile was determined using oligonucleotide microarrays (HG-U133A 2.0). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to confirm the microarray results. RESULTS: Amphotericin B and selected 1,3,4-thiadiazole derivatives had a significant effect on the total antioxidant capacity of the RPTEC cells, and the activity of the antioxidant enzymes. We also revealed that the effect of thiadiazoles on the SOD and CAT activities is dependent on the treatment of RPTEC cells with AmB. At the transcriptional level, the expression of several genes was affected by the studied compounds and their combinations. CONCLUSIONS: The results confirmed that thiadiazoles can stimulate the RPTEC cells to defend against the oxidative stress that is generated by AmB. In addition, together with the previously demonstrated synergistic antifungal activity, and low nephrotoxicity, these compounds have the potential to be used in new therapeutic strategies in the treatment of fungal infections.

Synthesis of calix (4) resorcinarene based amphiphilic macrocycle as an efficient nanocarrier for Amphotericin-B to enhance its oral bioavailability.

The supramolecular-based macrocyclic amphiphiles have fascinating attention and find extensive utilization in the pharmaceutical industry for efficient drug delivery. In this study, we designed and synthesized a new supramolecular amphiphilic macrocycle to serve as an efficient nanocarrier, achieved by treating 4-hydroxybenzaldehyde with 1-bromotetradecane. The derivatized product was subsequently treated with resorcinol to cyclize, resulting in the formation of a calix(4)-resorcinarene-based supramolecular amphiphilic macrocycle. The synthesized macrocycle and intermediate products were characterized using mass spectrometry, IR, and 1H NMR spectroscopic techniques. The amphotericin-B (Amph-B)-loaded and unloaded amphiphiles were screened for biocompatibility studies, vesicle formation, particle shape, size, surface charge, drug entrapment, in-vitro release profile, and stability through atomic force microscopy (AFM), Zetasizer, HPLC, and FT-IR. Amph-B -loaded macrocycle-based niosomal vesicles were investigated for in-vivo bioavailability in rabbits. The synthesized macrocycle exhibited no cytotoxicity against normal mouse fibroblast cells and was found to be hemocompatible and safe in mice following an acute toxicity study. The drug-loaded macrocycle-based vesicles appeared spherical, nano-sized, and homogeneous in size, with a notable negative surface charge. The vesicles remained stable after 30 days of storage. The results of Amph-B oral bioavailability and pharmacokinetics revealed that the newly tailored niosomal formulation enhanced drug solubility, protected drug degradation at gastric pH, facilitated sustained drug release at the specific target site, and delayed plasma drug clearance. Incorporating such advanced niosomal formulations in the field of drug delivery systems has the potential to revolutionize therapeutic outcomes and improve the quality of patient well-being.

Characterisation of Modular Polyketide Synthases Designed to Make Pentaene Analogues of Amphotericin B.

Glycosylated polyene macrolides are important antifungal agents that are produced by many actinomycete species. Development of new polyenes may deliver improved antibiotics. Here, Streptomyces nodosus was genetically re-programmed to synthesise pentaene analogues of the heptaene amphotericin B. These pentaenes are of interest as surrogate substrates for enzymes catalysing unusual, late-stage biosynthetic modifications. The previous deletion of amphotericin polyketide synthase modules 5 and 6 generated S. nodosus M57, which produces an inactive pentaene. Here, the chain-terminating thioesterase was fused to module 16 to generate strain M57-16TE, in which cycles 5, 6, 17 and 18 are eliminated from the biosynthetic pathway. Another variant of M57 was obtained by replacing modules 15, 16 and 17 with a single 15-17 hybrid module. This gave strain M57-1517, in which cycles 5, 6, 15 and 16 are deleted. M57-16TE and M57-1517 gave reduced pentaene yields. Only M57-1517 delivered its predicted full-length pentaene macrolactone in low amounts. For both mutants, the major pentaenes were intermediates released from modules 10, 11 and 12. Longer pentaene chains were unstable. The novel pentaenes were not glycosylated and were not active against Candida albicans. However, random mutagenesis and screening may yet deliver new antifungal producers from the M57-16TE and M57-1517 strains.

Drug Design: Do Not Forget the Supramolecular Factor.

A major challenge currently facing medicinal chemists is designing agents that can selectively destroy drug resistant fungi and bacteria that have begun to emerge. One factor that has been overlooked by virtually all drug discovery/development approaches is the supramolecular factor, in which aggregated forms of a drug candidate exhibit low selectivity in destroying targeted cells while the corresponding monomers exhibit high selectivity. This Perspective discusses how we were led to the supramolecular factor through fundamental studies with simple model systems, how we reasoned that the selectivity of monomers of the antifungal agent amphotericin B should be much greater than the selectivity of the corresponding aggregates, and how we confirmed this hypothesis using derivatives of amphotericin B. In a broader context, these findings provide a strong rationale for considering the supramolecular factor in the design of new drug candidates and the testing of virtually all of them.

Antifungal drug resistance in Candida: a special emphasis on amphotericin B.

Invasive fungal infections in humans caused by several Candida species, increased considerably in immunocompromised or critically ill patients, resulting in substantial morbidity and mortality. Candida albicans is the most prevalent species, although the frequency of these organisms varies greatly according to geographic region. Infections with C. albicans and non-albicans Candida species have become more common, especially in the past 20 years, as a result of aging, immunosuppressive medication use, endocrine disorders, malnourishment, extended use of medical equipment, and an increase in immunogenic diseases. Despite C. albicans being the species most frequently associated with human infections, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei also have been identified. Several antifungal drugs with different modes of action are approved for use in clinical settings to treat fungal infections. However, due to the common eukaryotic structure of humans and fungi, only a limited number of antifungal drugs are available for therapeutic use. Furthermore, drug resistance in Candida species has emerged as a result of the growing use of currently available antifungal drugs against fungal infections. Amphotericin B (AmB), a polyene class of antifungal drugs, is mainly used for the treatment of serious systemic fungal infections. AmB interacts with fungal plasma membrane ergosterol, triggering cellular ion leakage via pore formation, or extracting the ergosterol from the plasma membrane inducing cellular death. AmB resistance is primarily caused by changes in the content or structure of ergosterol. This review summarizes the antifungal drug resistance exhibited by Candida species, with a special focus on AmB.