In Vitro and In Vivo Effect of the Imidazole Luliconazole against Lomentospora prolificans and Scedosporium spp.

Infections with Scedosporium spp. and Lomentospora prolificans have become a serious threat in clinical settings. The high mortality rates associated with these infections can be correlated with their multidrug resistance. The development of alternative treatment strategies has become crucial. Here, we investigate the in vitro and in vivo activity of luliconazole (LLCZ) against Scedosporium apiospermum (including its teleomorph Pseudallescheria boydii) and Lomentospora prolificans. The LLCZ MICs were determined for a total of 37 isolates (31 L. prolificans isolates, 6 Scedosporium apiospermum/P. boydii strains) according to EUCAST. Furthermore, the LLCZ antifungal activity was tested in vitro, using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] growth kinetics assay and biofilm assays (crystal violet and XTT assay). In addition, a Galleria mellonella infection model was used for in vivo treatment assays. The MIC90 of LLCZ was determined to be 0.25 mg/L for all tested pathogens. Growth was inhibited within 6 to 48 h of the start of incubation. LLCZ inhibited biofilm formation in both preadhesion stages and late-stage adhesion. In vivo, a single dose of LLCZ increased the survival rate of the larvae by 40% and 20% for L. prolificans and Scedosporium spp., respectively. This is the first study demonstrating LLCZ activity against Lomentospora prolificans in vitro and in vivo and the first study showing the antibiofilm effect of LLCZ in Scedosporium spp. IMPORTANCE Lomentospora prolificans and S. apiospermum/P. boydii are opportunistic, multidrug-resistant pathogens causing invasive infections in immunosuppressed patients and sometimes in healthy persons. Lomentospora prolificans is panresistant against the currently available antifungals, and both species are associated with high mortality rates. Thus, the discovery of novel antifungal drugs exhibiting an effect against these resistant fungi is crucial. Our study shows the effect of luliconazole (LLCZ) against L. prolificans and Scedosporium spp. in vitro, as well as in an in vivo infection model. These data reveal the previously unknown inhibitory effect of LLCZ against L. prolificans and its antibiofilm effect in Scedosporium spp. It represents an extension of the literature regarding azole-resistant fungi and could potentially lead to the development of future treatment strategies against these opportunistic fungal pathogens.

In Vitro and In Vivo Activity of Luliconazole (NND-502) against Planktonic Cells and Biofilms of Azole Resistant Aspergillus fumigatus.

Aspergillus fumigatus has become a significant threat in clinical settings. Cases of invasive infections with azole-resistant A. fumigatus isolates (ARAF) increased recently. Developing strategies for dealing with ARAF has become crucial. We here investigated the in-vitro and in-vivo activity of the imidazole luliconazole (LLCZ) against clinical ARAF. In total, the LLCZ minimum inhibitory concentrations (MICs) were tested for 101 A. fumigatus isolates (84 ARAF and 17 azole-susceptible A. fumigatus as wild-type controls) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Additionally, antifungal activity was assessed in vitro, including an XTT planktonic growth kinetics assay and biofilm assays (crystal violet and XTT assay). Further, a single-dose LLCZ treatment (152 mg/L) was tested for seven days in vivo in a Galleria mellonella infection model. LLCZ showed an MIC50 of 0.002 mg/L and no significant difference was found between triazole-resistant and wild-type isolates. Growth inhibition took place between 6 and 12 h after the start of incubation. LLCZ inhibited biofilm formation when added in the pre-adhesion stages. In vivo, single-dose LLCZ-treated larvae show a significantly higher survival percentage than the control group (20%). In conclusion, LLCZ has activity against planktonic cells and early biofilms of ARAF.

Inhibition of azole-resistant Aspergillus fumigatus biofilm at various formation stages by antifungal drugs, including olorofim.

OBJECTIVES: Interest in aspergillosis has increased over the past decades. An increase in the incidence of azole-resistant Aspergillus fumigatus strains has been reported; therefore, the need for novel therapeutic approaches is urgent. The formation of biofilms contributes to pathogen resistance. We investigated the biofilm formation capabilities of azole-resistant A. fumigatus and analysed the susceptibility of biofilms at various developmental stages to three antifungal agents. METHODS: Biofilm formation of 19 clinical A. fumigatus strains (3 azole-susceptible and 16 azole-resistant strains) was determined by crystal violet staining and by an XTT assay over a period of 48 h. We measured antibiofilm activity of voriconazole, amphotericin B and olorofim. These agents were added before adhesion, after adhesion, after germination and to mature fungal biofilm. Antibiofilm activity was assessed in an XTT assay and in confocal laser scan microscopy. Additionally, a growth-kinetic assay with planktonic A. fumigatus was performed. RESULTS: Each of the antifungal agents inhibited the metabolic activity of A. fumigatus biofilms when applied at early stages of biofilm formation. The mature biofilms were more resistant. Olorofim and voriconazole showed promising effects against A. fumigatus adhesion and germination, whereas the mature biofilm was not affected by treatment. In contrast, the biofilm of A. fumigatus showed amphotericin B susceptibility throughout the entire developmental process. The planktonic cells were susceptible to all three antifungal drug classes with an inhibition peak at 12 h after incubation. CONCLUSIONS: This is the first known study to demonstrate the antibiofilm activity of olorofim, voriconazole and amphotericin B against azole-resistant A. fumigatus.

COVID-19-associated pulmonary aspergillosis in ICU patients in a German reference centre: Phenotypic and molecular characterisation of Aspergillus fumigatus isolates.

BACKGROUND: COVID-19-associated invasive pulmonary aspergillosis (CAPA) is associated with increased mortality. Cases of CAPA caused by azole-resistant Aspergillus fumigatus strains have been reported. OBJECTIVES: To analyse the twelve-month CAPA prevalence in a German tertiary care hospital and to characterise clinical A. fumigatus isolates from two German hospitals by antifungal susceptibility testing and microsatellite genotyping. PATIENTS/METHODS: Retrospective observational study in critically ill adults from intensive care units with COVID-19 from 17 February 2020 until 16 February 2021 and collection of A. fumigatus isolates from two German centres. EUCAST broth microdilution for four azole compounds and microsatellite PCR with nine markers were performed for each collected isolate (N = 27) and additional for three non-COVID A. fumigatus isolates. RESULTS: welve-month CAPA prevalence was 7.2% (30/414), and the rate of azole-resistant A. fumigatus isolates from patients with CAPA was 3.7% with detection of one TR34/L98H mutation. The microsatellite analysis revealed no major clustering of the isolates. Sequential isolates mainly showed the same genotype over time. CONCLUSIONS: Our findings demonstrate similar CAPA prevalence to other reports and a low azole-resistance rate. Genotyping of A. fumigatus showed polyclonal distribution except for sequential isolates.

In vitro activity of olorofim (F901318) against fungi of the genus, Scedosporium and Rasamsonia as well as against Lomentospora prolificans, Exophiala dermatitidis and azole-resistant Aspergillus fumigatus.

In recent decades, invasive infections caused by fungal pathogens have been reported with increasing frequency. Concurrently, the rates of detected resistance mechanisms against commonly used antifungal agents in fungi are increasing. The need for novel antifungal drugs is thus imminent. In this study, the novel drug olorofim (F901318) was tested for its antifungal activity against the human fungal pathogens Lomentospora prolificans (n = 20), Scedosporium aurantiacum (n = 2), Scedosporium apiospermum (n = 6), Rasamsonia argillacea species complex (n = 23), Exophiala dermatitidis (n = 10) and azole-resistant Aspergillus fumigatus (ARAF) (n = 25) in an in vitro broth microdilution assay according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. Whilst olorofim was ascertained to be effective against R. argillacea species complex [minimum inhibitory concentrations (MICs) of ≤0.008 mg/L], Scedosporium spp. (MICs of 0.032-0.5 mg/L), L. prolificans (MICs of 0.032-0.5 mg/L) and ARAF (MICs of ≤0.008-0.032 mg/L), the drug had an MIC of >4 mg/L against E. dermatitidis. These data demonstrate the antifungal activity of olorofim against a broad range of filamentous fungal pathogens.

Antibiofilm activity of antifungal drugs, including the novel drug olorofim, against Lomentospora prolificans.

OBJECTIVES: Patients with immunodeficiency or cystic fibrosis frequently suffer from respiratory fungal infections. In particular, biofilm-associated fungi cause refractory infection manifestations, linked to increased resistance to anti-infective agents. One emerging filamentous fungus is Lomentospora prolificans. Here, the biofilm-formation capabilities of L. prolificans isolates were investigated and the susceptibility of biofilms to various antifungal agents was analysed. METHODS: Biofilm formation of L. prolificans (n = 11) was estimated by crystal violet stain and antibiofilm activity was additionally determined via detection of metabolically active biofilm using an XTT assay. Amphotericin B, micafungin, voriconazole and olorofim were compared with regard to their antibiofilm effects when added prior to adhesion, after adhesion and on mature and preformed fungal biofilms. Imaging via confocal laser scanning microscopy was carried out to demonstrate the effect of drug treatment on the fungal biofilm. RESULTS: Antibiofilm activities of the tested antifungal agents were shown to be most effective on adherent cells whilst mature biofilm was the most resistant. The most promising antibiofilm effects were detected with voriconazole and olorofim. Olorofim showed an average minimum biofilm eradication concentration (MBEC) of 0.06 mg/L, when added prior to and after adhesion. The MBECs of voriconazole were ≤4 mg/L. On mature biofilm the MBECs of olorofim and voriconazole were higher than the previously determined MICs against planktonic cultures. In contrast, amphotericin B and especially micafungin did not exhibit sufficient antibiofilm activity against L. prolificans. CONCLUSIONS: To our knowledge, this is the first study demonstrating the antibiofilm potential of olorofim against the human pathogenic fungus L. prolificans.

Biofilm formation of the black yeast-like fungus Exophiala dermatitidis and its susceptibility to antiinfective agents.

Various fungi have the ability to colonize surfaces and to form biofilms. Fungal biofilm-associated infections are frequently refractory to targeted treatment because of resistance to antifungal drugs. One fungus that frequently colonises the respiratory tract of cystic fibrosis (CF) patients is the opportunistic black yeast-like fungus Exophiala dermatitidis. We investigated the biofilm-forming ability of E. dermatitidis and its susceptibility to various antiinfective agents and natural compounds. We tested 58 E. dermatitidis isolates with a biofilm assay based on crystal violet staining. In addition, we used three isolates to examine the antibiofilm activity of voriconazole, micafungin, colistin, farnesol, and the plant derivatives 1,2,3,4,6-penta-O-galloyl-b-D-glucopyranose (PGG) and epigallocatechin-3-gallate (EGCG) with an XTT reduction assay. We analysed the effect of the agents on cell to surface adhesion, biofilm formation, and the mature biofilm. The biofilms were also investigated by confocal laser scan microscopy. We found that E. dermatitidis builds biofilm in a strain-specific manner. Invasive E. dermatitidis isolates form most biomass in biofilm. The antiinfective agents and the natural compounds exhibited poor antibiofilm activity. The greatest impact of the compounds was detected when they were added prior cell adhesion. These findings suggest that prevention may be more effective than treatment of biofilm-associated E. dermatitidis infections.