In vitro activity of ibrexafungerp against clinically relevant echinocandin-resistant Candida strains.

Invasive candidiasis is a major hospital-acquired infection. Usually, echinocandins are considered first-line treatment. However, resistant phenotypes have emerged. Ibrexafungerp (IBX) is a new antifungal substance with potent anti-Candida activity. We challenged IBX with a library of 192 pheno-/genotypically echinocandin-resistant Candida isolates, focusing on the substance susceptibility, its activity on certain FKS hotspot (HS) mutated strains, and applying WTULs (wild-type upper limits). Therefore, a 9-year-old strain and patient data collection provided by the German National Reference Center for Invasive Fungal Infections were analyzed. Species identification was confirmed through ITS-sequencing. Molecular susceptibility testing was performed by sequencing HS of the FKS gene. Anidulafungin (AND) and IBX EUCAST-broth-microdilution was conducted. The four most common echinocandin-resistance mediating mutations were found in Candida glabrata [112/192 isolates; F659-(43×) and S663-(48×)] and Candida albicans [63/192 isolates; F641-(15×) and S645-(39×)]. Mutations at the HS-start sequence were associated with higher IBX MIC-values (F659 and F641 (MIC 50/90 mg/L: >4/>4 and 2/4 mg/L) in comparison to AND (F659 and F641 (MIC 50/90: 1/4 and 0.25/1 mg/L). MIC-values in HS-center mutations were almost equal [MIC50/90 in S663: 2/4 (AND and IBX); in S645: 0.5/1 (AND) and 0.25/1 (IBX) mg/L]. In total, 61 vs 78 of 192 echinocandin-resistant isolates may be classified as IBX wild type by applying WTULs, whereas the most prominent effect was seen in C. albicans [48% (30/63) vs 70% (44/63)]. IBX shows in vitro activity against echinocandin-resistant Candida and thus is an addition to the antifungal armory. However, our data suggest that this effect is more pronounced in C. albicans and strains harboring mutations, affecting the HS-center.

Polyhexanide based contact lens storage fluids frequently exhibit insufficient antifungal activity against Fusarium species.

PURPOSE: Fusarium keratitis is a severe infection of the anterior eye, frequently leading to keratoplasty or surgical removal of the affected eye. A major risk factor for infection is the use of contact lenses. Inadequate hygiene precautions and mold-growth permissive storage fluids are important risk factors for fungal keratitis. The aim of this study was to comparatively analyze contact lens storage fluids disinfection efficacy against Fusarium species. METHODS: Eleven commercially available storage fluids were tested. The storage fluids were classified according to their active ingredients myristamidopropyldimethylamine (Aldox), polyhexanide and hydrogen peroxide. Efficacy was tested against isolates belonging to the Fusarium solani and Fusarium oxysporum species complexes as the most common agents of mould keratitis. Tests were carried out based on DIN EN ISO 14729. RESULTS: All Aldox and hydrogen peroxide (H2O2) based fluids were effective against Fusarium spp., while the majority of polyhexanide based storage fluids showed only limited or no antifungal effects. Efficacy of polyhexanide could be restored by the addition of the pH-regulating agent tromethamine - an additive component in one commercially available product. CONCLUSIONS: In summary, the use of Aldox- or hydrogen peroxide-based storage fluids may reduce the risk of Fusarium keratitis, while polyhexanide-based agents largely lack efficacy against Fusarium.

Ecological Niche-Inspired Genome Mining Leads to the Discovery of Crop-Protecting Nonribosomal Lipopeptides Featuring a Transient Amino Acid Building Block.

Investigating the ecological context of microbial predator-prey interactions enables the identification of microorganisms, which produce multiple secondary metabolites to evade predation or to kill the predator. In addition, genome mining combined with molecular biology methods can be used to identify further biosynthetic gene clusters that yield new antimicrobials to fight the antimicrobial crisis. In contrast, classical screening-based approaches have limitations since they do not aim to unlock the entire biosynthetic potential of a given organism. Here, we describe the genomics-based identification of keanumycins A-C. These nonribosomal peptides enable bacteria of the genus Pseudomonas to evade amoebal predation. While being amoebicidal at a nanomolar level, these compounds also exhibit a strong antimycotic activity in particular against the devastating plant pathogen Botrytis cinerea and they drastically inhibit the infection of Hydrangea macrophylla leaves using only supernatants of Pseudomonas cultures. The structures of the keanumycins were fully elucidated through a combination of nuclear magnetic resonance, tandem mass spectrometry, and degradation experiments revealing an unprecedented terminal imine motif in keanumycin C extending the family of nonribosomal amino acids by a highly reactive building block. In addition, chemical synthesis unveiled the absolute configuration of the unusual dihydroxylated fatty acid of keanumycin A, which has not yet been reported for this lipodepsipeptide class. Finally, a detailed genome-wide microarray analysis of Candida albicans exposed to keanumycin A shed light on the mode-of-action of this potential natural product lead, which will aid the development of new pharmaceutical and agrochemical antifungals.

In Vitro Activity of Nitroxoline in Antifungal-Resistant Candida Species Isolated from the Urinary Tract.

Infections by drug-resistant fungi are increasingly reported worldwide; however, only few novel antifungals are being developed. The old antimicrobial nitroxoline is currently repurposed for oral treatment of bacterial urinary tract infections (UTI). Previously, antifungal activity has been demonstrated and in contrast to many antifungals nitroxoline reaches high urinary concentrations. In this study, the activity of nitroxoline was assessed in vitro in a collection of yeasts from the German National Reference Centre for Invasive Fungal Infections. Susceptibility was determined by broth microdilution (BMD) and disk diffusion (DD). The collection comprised 45 Candida isolates originating from the urinary tract. MICs of amphotericin, anidulafungin and azoles were analyzed using EUCAST BMD. Among the collection isolates, resistance to antifungals was common, e.g., for fluconazole the MIC50/90 was 16/>64 mg/L; in contrast MIC50/90 of nitroxoline was 2/2 mg/L (MIC range 0.25-4 mg/L), which is at least two dilutions below the EUCAST breakpoint for uncomplicated UTI defined for E. coli (susceptible ≤ 16mg/L). Activity of nitroxoline was high irrespective of resistance to other agents. As BMD is labor-intensive, DD was investigated as an alternative method using three different agars. Nitroxoline disks produced large inhibition zones on all agars (≥19mm), but the correlation of MICs and zone diameters was low, with the highest correlation recorded for the CLSI recommended agar for antifungal DD (Pearson's r = -0,52). In conclusion, isolates of different Candida species are highly susceptible to nitroxoline, which could be a promising antimicrobial to treat candiduria caused by multidrug resistant yeasts.

Bloodstream Infections Caused by Magnusiomyces capitatus and Magnusiomyces clavatus: Epidemiological, Clinical, and Microbiological Features of Two Emerging Yeast Species.

Magnusiomyces clavatus and Magnusiomyces capitatus are emerging yeasts with intrinsic resistance to many commonly used antifungal agents. Identification is difficult, and determination of susceptibility patterns with commercial and reference methods is equally challenging. For this reason, few data on invasive infections by Magnusiomyces spp. are available. Our objectives were to determine the epidemiology and susceptibility of Magnusiomyces isolates from bloodstream infections (BSI) isolated in Germany and Austria from 2001 to 2020. In seven institutions, a total of 34 Magnusiomyces BSI were identified. Identification was done by internal transcribed spacer (ITS) sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antifungal susceptibility was determined by EUCAST broth microdilution and gradient tests. Of the 34 isolates, M. clavatus was more common (n = 24) than M. capitatus (n = 10). BSI by Magnusiomyces spp. were more common in men (62%) and mostly occurred in patients with hemato-oncological malignancies (79%). The highest in vitro antifungal activity against M. clavatus/M. capitatus was observed for voriconazole (MIC50, 0.03/0.125 mg/L), followed by posaconazole (MIC50, 0.125/0.25 mg/L). M. clavatus isolates showed overall lower MICs than M. capitatus. With the exception of amphotericin B, low essential agreement between gradient test and microdilution was recorded for all antifungals (0 to 70%). Both species showed distinct morphologic traits on ChromAgar Orientation medium and Columbia blood agar, which can be used for differentiation if no MALDI-TOF MS or molecular identification is available. In conclusion, most BSI were caused by M. clavatus. The lowest MICs were recorded for voriconazole. Gradient tests demonstrated unacceptably low agreement and should preferably not be used for susceptibility testing of Magnusiomyces spp.

Human mucosal-associated invariant T cells respond to Mucorales species in a MR1-dependent manner.

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales.

Frequency of occurrence, seasonal variation and antifungal susceptibility of opportunistic Mucorales isolated from hospital soils in Iran.

BACKGROUND: Mucorales are opportunistic pathogens that can cause life-threatening diseases predominantly in immunocompromised patients. OBJECTIVES: This study aimed to investigate the frequency, seasonal variation and antifungal susceptibility of pathogenic Mucorales in the soil collected from seven hospitals in Urmia, Iran, between November 2017 and July 2018 in four different seasons. METHODS: Mucorales isolates obtained from soil were characterised based on conventional and molecular assays. In addition, in vitro antifungal susceptibility was performed using the CLSI M38Ed3 procedure. RESULTS: Out of 196 tested soil samples, 80 (40.8%) samples were positive for mucoralean fungi. Rhizopus arrhizus var. arrhizus (n = 47) was the most frequent species followed by Mucor circinelloides (n = 21) and Cunninghamella echinulata (n = 6). A seasonal variation in the frequency of Mucorales in soil was detected with a maximum of culture-positive soil samples detected in wet autumn (43.2%) followed by winter (23.4%), summer (19.7%) and spring (13.6%). In vitro antifungal susceptibility testing for 80 environmental isolates exhibited MIC of ≤2 µg/ml for amphotericin B indicating the smallest range of MIC variation among the tested Mucorales (range: 0.125-2 µg/ml). Among the azoles, posaconazole was the most effective antifungals (GM MIC, 0.724 µg/ml). CONCLUSIONS: We considered associations of species and seasonal frequencies between soil mucoralean fungi and mucormycosis. The effect of opportunistic Mucorales dominating in the soil and prevalent causative agents of mucormycosis in Iran reported in the literatures but more comprehensive studies are needed to confirm this conclusion.

"Indian" strains of Trichophyton mentagrophytes with reduced itraconazole susceptibility in Germany.

We use published reports and three of our own tinea cases as an opportunity to report on "Indian" strains of Trichophyton (T.) mentagrophytes with ITS genotype VIII and reduced susceptibility to itraconazole due to the mutation c.1342G>A in the SQLE gene in Germany. In vitro measurements of resistance revealed normal susceptibility to terbinafine, but markedly reduced susceptibility to itraconazole - although no valid breakpoints are currently defined and minimum inhibitory concentrations (MICs) depend on the methods used. Problems related to the determination and interpretation of MICs are outlined. Our cases show that azole-resistant "Indian" strains of T. mentagrophytes with ITS genotype VIII occurred in Germany as early as 2011, which is earlier than was previously assumed. This variant of the pathogen cannot be phenotypically distinguished from customary strains of T. mentagrophytes; its identification is based on genetics. The taxonomic classification is still under debate. This variant is anthropophilic and causes only mildly inflammatory tinea lesions with many fungal elements. Its further dissemination must therefore be expected. Prerequisites for rapid and valid antimycotic testing against dermatophytes need to be developed.

Low rate of azole resistance in cases of avian aspergillosis in Germany.

Aspergillosis is the most common fungal disease of the avian respiratory tract. Due to delayed diagnosis and treatment failure, the outcome of these infections is often poor. We investigate 159 cases of avian aspergillosis among captive birds in Germany to define clinical features as well as the frequency of in vitro triazole resistance. Adult birds were more likely to present with clinical signs compared to juvenile birds, and dyspnoea was the most common clinical sign, present in 53% of birds. Molecular species identification indicated that all infections were caused by Aspergillus fumigatus. Only one of 159 independent isolates was azole resistant.

Clinical isolates of Trichophyton rubrum are completely inhibited by photochemical treatment with a γ-cyclodextrin formulation of curcuminoids.

INTRODUCTION: It was shown previously that dermatophytes can markedly be inhibited by a photochemical treatment with curcumin. This kind of photo-inactivation needs to be improved, however, because curcumin is poorly water-soluble. Therefore, a new water-soluble γ-cyclodextrin formulation of curcuminoids was tested for its photochemical inactivation of Trichophyton (T.) rubrum. MATERIALS AND METHODS: Conidia were harvested from 6 typical strains of T rubrum and used to inoculate wells of microtiter plates. These wells were also filled with a γ-cyclodextrin curcuminoid formulation with 0.1% DMSO and Sabouraud broth. The assays were then irradiated with visible light (wavelength 420 nm, 45 J/cm2 ). After 24 hours, curcuminoid was added once more, and irradiation was repeated. Fungal growth was monitored photometrically for 8 days and compared with controls. RESULTS: Growth of all 6 T rubrum strains was completely inhibited by the photochemical treatment with the γ-cyclodextrin formulation of curcuminoids. The same curcuminoid formulation applied without irradiation had only a minor inhibitory effect. DISCUSSION: Photo-inactivation of dermatophytes with a γ-cyclodextrin formulation of curcuminoids plus visible light is a very promising procedure with potential for a new treatment of patients with superficial tinea.

Outbreaks of Mucorales and the Species Involved.

The order Mucorales is an ancient group of fungi classified in the subphylum Mucoromycotina. Mucorales are mainly fast-growing saprotrophs that belong to the first colonizers of diverse organic materials and represent a permanent part of the human environment. Several species are able to cause human infections (mucormycoses) predominantly in patients with impaired immune system, diabetes, or deep trauma. In this review, we compiled 32 reports on community- and hospital-acquired outbreaks caused by Mucorales. The most common source of mucoralean outbreaks was contaminated medical devices that are responsible for 40.7% of the outbreaks followed by contaminated air (31.3%), traumatic inoculation of soil or foreign bodies (9.4%), and the contact (6.2%) or the ingestion (6.2%) of contaminated plant material. The most prevalent species were Rhizopus arrhizus and R. microsporus causing 57% of the outbreaks. The genus Rhizomucor was dominating in outbreaks related to contaminated air while outbreaks of Lichtheimia species and Mucor circinelloides were transmitted by direct contact. Outbreaks with the involvement of several species are reported. Subtyping of strains revealed clonality in two outbreaks and no close relation in two other outbreaks. Based on the existing data, outbreaks of Mucorales can be caused by heterogeneous sources consisting of different strains or different species. Person-to-person transmission cannot be excluded because Mucorales can sporulate on wounds. For a better understanding and prevention of outbreaks, we need to increase our knowledge on the physiology, ecology, and population structure of outbreak causing species and more subtyping data.

Candida auris in Germany and Previous Exposure to Foreign Healthcare.

The emerging yeast Candida auris has disseminated worldwide. We report on 7 cases identified in Germany during 2015-2017. In 6 of these cases, C. auris was isolated from patients previously hospitalized abroad. Whole-genome sequencing and epidemiologic analyses revealed that all patients in Germany were infected with different strains.

A Revised Species Concept for Opportunistic Mucor Species Reveals Species-Specific Antifungal Susceptibility Profiles.

Recently, the species concept of opportunistic Mucor circinelloides and its relatives has been revised, resulting in the recognition of its classical formae as independent species and the description of new species. In this study, we used isolates of all clinically relevant Mucor species and performed susceptibility testing using the EUCAST reference method to identify potential species-specific susceptibility patterns. In vitro susceptibility profiles of 101 mucoralean strains belonging to the genus Mucor (72), the closely related species Cokeromyces recurvatus (3), Rhizopus (12), Lichtheimia (10), and Rhizomucor (4) to six antifungals (amphotericin B, natamycin, terbinafine, isavuconazole, itraconazole, and posaconazole) were determined. The most active drug for all Mucorales was amphotericin B. Antifungal susceptibility profiles of pathogenic Mucor species were specific for isavuconazole, itraconazole, and posaconazole. The species formerly united in M. circinelloides showed clear differences in their antifungal susceptibilities. Cokeromyces recurvatus, Mucor ardhlaengiktus, Mucor lusitanicus (M. circinelloides f. lusitanicus), and Mucor ramosissimus exhibited high MICs to all azoles tested. Mucor indicus presented high MICs for isavuconazole and posaconazole, and Mucor amphibiorum and Mucor irregularis showed high MICs for isavuconazole. MIC values of Mucor spp. for posaconazole, isavuconazole, and itraconazole were high compared to those for Rhizopus and the Lichtheimiaceae (Lichtheimia and Rhizomucor). Molecular identification combined with in vitro susceptibility testing is recommended for Mucor species, especially if azoles are applied in treatment.

Comparative Genomics of Serial Candida glabrata Isolates and the Rapid Acquisition of Echinocandin Resistance during Therapy.

The opportunistic pathogen Candida glabrata shows a concerning increase in drug resistance. Here, we present the analysis of two serial bloodstream isolates, obtained 12 days apart. Both isolates show pan-azole resistance and echinocandin resistance was acquired during the sampling interval. Genome sequencing identified nine nonsynonymous SNVs between the strains, including a S663P substitution in FKS2 and previously undescribed SNVs in MDE1 and FPR1, offering insight into how C. glabrata acquires drug resistance and adapts to a human host.

The geographical region of origin determines the phagocytic vulnerability of Lichtheimia strains.

Mucormycoses are life-threatening infections that affect patients suffering from immune deficiencies. We performed phagocytosis assays confronting various strains of Lichtheimia species with alveolar macrophages, which form the first line of defence of the innate immune system. To investigate 17 strains from four different continents in a comparative fashion, transmitted light and confocal fluorescence microscopy was applied in combination with automated image analysis. This interdisciplinary approach enabled the objective and quantitative processing of the big volume of image data. Applying machine-learning supported methods, a spontaneous clustering of the strains was revealed in the space of phagocytic measures. This clustering was not driven by measures of fungal morphology but rather by the geographical origin of the fungal strains. Our study illustrates the crucial contribution of machine-learning supported automated image analysis to the qualitative discovery and quantitative comparison of major factors affecting host-pathogen interactions. We found that the phagocytic vulnerability of Lichtheimia species depends on their geographical origin, where strains within each geographic region behaved similarly, but strongly differed amongst the regions. Based on this clustering, we were able to also classify clinical isolates with regard to their potential geographical origin.

Human MAIT cells are rapidly activated by Aspergillus spp. in an APC-dependent manner.

Mucosal associated invariant T cells (MAIT cells) are innate-like T cells (TC) which are known to be activated by several bacteria and viruses. However, activation of MAIT cells by moulds, such as the opportunistic human pathogen Aspergillus, is not well described. Stimulation of human PBMC with A. fumigatus, A. flavus, or A. terreus conidia revealed that in contrast to conventional CD4+ and CD8+ TC, MAIT cells responded already after 4 h of coincubation with upregulation of CD69. Furthermore, concurrent increase of CD107a expression and reduced intracellular expression of cytolytic proteins like perforin and granzyme indicated degranulation of intracellular vesicles. MAIT cell activation only occurred in the presence of APC and was dependent on cell-cell contact as separation of TC and APC abrogated MAIT cell activation. Furthermore, we observed that MAIT cell activation by moulds requires presentation of riboflavin metabolites and depends on TCR engagement as antibody blocking of MR1, the antigen presenting molecule for MAIT cells, prevented upregulation of CD69 and CD107a. In summary, we could demonstrate that MAIT cells are activated by Aspergillus conidia in a TCR-dependent manner by APC. These findings reveal MAIT cells as an interesting new target in antifungal defense.

FunResDB-A web resource for genotypic susceptibility testing of Aspergillus fumigatus.

Therapy of invasive aspergillosis is becoming more difficult due to the emergence of azole resistance in Aspergillus fumigatus. A majority of resistant strains carries mutations in the CYP51A gene. Due to a lack of sensitivity of culture-based methods, molecular detection of A. fumigatus has become an important diagnostic tool. We set up the database FunResDB (www.nrz-myk.de/funresdb) to gather all available information about CYP51A-dependent azole resistance from published literature. In summary, the screening resulted in 79 CYP51A variants, which are linked to 59 nonsynonymous mutations. A tailor-made online sequence analysis tool allows for genotypic susceptibility testing of A. fumigatus.

Fusarium Keratitis in Germany.

Fusarium keratitis is a destructive eye infection that is difficult to treat and results in poor outcome. In tropical and subtropical areas, the infection is relatively common and associated with trauma or chronic eye diseases. However, in recent years, an increased incidence has been reported in temperate climate regions. At the German National Reference Center, we have observed a steady increase in case numbers since 2014. Here, we present the first German case series of eye infections with Fusarium species. We identified Fusarium isolates from the eye or eye-related material from 22 patients in 2014 and 2015. Thirteen isolates belonged to the Fusarium solani species complex (FSSC), 6 isolates belonged to the Fusarium oxysporum species complex (FOSC), and three isolates belonged to the Fusarium fujikuroi species complex (FFSC). FSSC was isolated in 13 of 15 (85%) definite infections and FOSC in 3 of 4 (75%) definite contaminations. Furthermore, diagnosis from contact lens swabs or a culture of contact lens solution turned out to be highly unreliable. FSSC isolates differed from FOSC and FFSC by a distinctly higher MIC for terbinafine. Outcome was often adverse, with 10 patients requiring keratoplasty or enucleation. The use of natamycin as the most effective agent against keratitis caused by filamentous fungi was rare in Germany, possibly due to restricted availability. Keratitis caused by Fusarium spp. (usually FSSC) appears to be a relevant clinical problem in Germany, with the use of contact lenses as the predominant risk factor. Its outcome is often adverse.

Candida albicans bloodstream isolates in a German university hospital are genetically heterogenous and susceptible to commonly used antifungals.

From an eight-year-span, 99 Candida bloodstream isolates were collected at the University Hospital Wuerzburg, Germany. In this study, all strains were analyzed using molecular and phenotypic typing methods. Confirmatory species identification revealed three isolates that were initially diagnosed as C. albicans to be actually C. dubliniensis. Two isolates contained a mixed culture of C. albicans and C. glabrata, in one of the specimens both species could be separated while it was not possible to recover C. albicans in the other sample. The remaining 95 C. albicans isolates were profiled by multilocus sequence typing (MLST). Phylogenetic analyses showed a highly heterogenous collection of strains, associated with many different clades and constituting a set of new diploid sequence types (DST). For all strains with identical DST, patient data were reviewed for potential nosocomial transmission. In addition, all isolates were tested for their susceptibility to amphotericin B, caspofungin, fluconazole, itraconazole, posaconazole and voriconazole. No clinically relevant resistance could be detected. Furthermore, these data underline that correlation between minimal inhibitory concentrations for caspofungin and anidulafungin is low.