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

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. ABSTRACT 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.

distinct organisms (Table 1 and Fig. 1). A geometric mean MIC of 0.122 mg/L was calculated.
The activity of LLCZ against the planktonic growth of L. prolificans and S. apiospermum/P. boydii over time was analyzed with an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] growth kinetics assay, revealing that various concentrations of the antifungal agent had a significant effect (Fig. 2). The inhibition started after 6 h, and a decrease in metabolic activity could be observed until 48 h, which was also the last measured time point.
The effect of LLCZ against biofilm was also analyzed via XTT and crystal violet (CV) assays at different formation stages. While the effect of the drug was similar for all three organisms, the results of the two assays differed. The XTT assay (Fig. 3) showed a decrease in metabolic activity of about 75% when the drug was added in the early stages of biofilm formation (0 h and 2 h), whereas the CV assay (see Fig. S1 in the supplemental material) showed a decrease in biofilm mass of 22% (0 h) and 50% (2 h). Similar results were recorded for late-stage biofilm. In both the XTT and CV assays, a biofilm inhibition of about 30% was demonstrated.
The effect of the antifungal agent could be visualized using confocal laser scanning microscopy (CLSM). L. prolificans isolate 2229 was treated with an LLCZ concentration of 0.03 mg/L (Fig. 4B) and half of this concentration (Fig. 4C). In both cases, the biofilm formation of L. prolificans was inhibited. Figure 4A shows the biofilm formation of the untreated L. prolificans isolate. Hyphal development is visible in Fig. 4C, where a sub-MIC was used, while the formation of hyphae was inhibited when treated with LLCZ at the MIC. It was determined previously that LLCZ has no toxic effect on Galleria mellonella (23). An infection assay (Fig. S2) showed that the optimal fungal inoculum for all strains was 2 Â 10 7 CFU/mL, with a survival outcome of 21% for L. prolificans, 10% for S. apiospermum, and 25% for P. boydii after a total incubation period of 14 days at 37°C. The larvae started to die 1 to 2 days after infection. When treated with a dose of 152 mg/L LLCZ, the survival outcome of the larvae increased significantly, compared to that of the placebo group. The treated larvae infected with L. prolificans showed a 40% higher survival outcome and those infected with S. apiospermum and P. boydii around 15% and 20% higher, respectively. VCZ was used as a negative control for L. prolificans and as a positive control for S. apiospermum and P. boydii. While treatment with VCZ  Through incubation with 50 mL XTT (2.5 mg/mL) plus menadione (125 mg/mL) per well (the XTT was added 2 h prior to the incubation end), the metabolic activity of the cells was determined. Statistical significance was determined using Dunnett's multiple-comparison test. Significance was set at P , 0.05; *, P , 0.05; **, P , 0.01. yielded survival increases of 15% in S. apiospermum and 25% in P. boydii, no significant effect of this antifungal agent could be seen in L. prolificans (Fig. 5).

DISCUSSION
This study is the first to report the in vitro effect of LLCZ against both planktonic growth and biofilm formation of the azole-resistant mold L. prolificans. Furthermore, the effects of this antifungal agent were assessed in an in vivo invertebrate treatment model.
In the study, 37 isolates were tested according to the EUCAST protocol, showing a wide MIC range of between 0.004 and .0.25 mg/L. No significant differences or distinct patterns were observable in the MIC distribution of the three organisms. Although no comparable data exist for L. prolificans, LLCZ is known to have relatively low MIC values for Scedosporium spp.; other molds, such as A. fumigatus and non-fumigatus Aspergillus spp.; and also for yeasts, such as Candida albicans (21, 23-25, 31). An XTT growth kinetics assay was used to determine the effect of different LLCZ concentrations against the planktonic growth of the organisms (Fig. 2). The metabolic activity of the LLCZ-treated cells was measured and compared to that of the untreated cells. The results showed a steady inhibition of planktonic growth between 6 h and 48 h. A stronger effect was observable at higher LLCZ concentrations. However, the MIC did significantly inhibit the metabolic growth of all organisms, showing the concentration-dependent activity of LLCZ. In contrast to the results from growth inhibition experiments of A. fumigatus with LLCZ, the growth inhibition of L. prolificans and S. apiospermum/P. boydii appeared to be continuous, and no evident activity recovery was recorded (23). This regenerative effect was also observable with A. fumigatus when applying other antifungal drugs, such as amphotericin B, VCZ, or olorofim (32). Although the growth inhibition for L. prolificans, S. apiospermum, and P. boydii seemed to be constant, the antifungal agent did not manage to reach an inhibition of .99%, showing that LLCZ has a fungistatic effect against these organisms (33). This is congruent with LLCZ activity against A. fumigatus (23).
The biofilm formation capacity of LLCZ-treated fungi was assessed via two separate assays. While the XTT assay showed a significant decrease in metabolic activity (75%) in all organisms when the drug was directly incubated with the fungal suspension (0 h), this could not be correlated with a decrease in biomass, since the CV revealed a decrease of only 22% of the total generated biofilm mass (see Fig. S1 in the supplemental material). When the drug was added at a later time point (2 h after incubation), the amount of produced biomass in biofilm was reduced by approximately 20%. This decrease could be attributed to the washing step prior to the addition of the drug (see "In vitro assays. [iii] Biofilm assays" in Materials and Methods, below). No difference was recorded in metabolic activity when the drug was added at 0 h or 2 h after incubation. It is possible that LLCZ effectively inhibits the metabolic activity and development of L. prolificans and Scedosporium spp., despite their high biofilm production. This may be the explanation for the significant effect of LLCZ treatment against mature biofilms (48 h after incubation). Comparable to LLCZ, the novel antifungal drug olorofim also showed a partial (40%) inhibition of L. prolificans and Scedosporium spp., but it has been described as ineffective against late-stage biofilm of azole-resistant A. fumigatus isolates (32,34). CLSM imaging confirmed LLCZ activity against biofilm formation in L. prolificans when applied in the early formation stages, even though hyphae could still be detected, most notably when sub-MICs (half the MIC) were used. Due to the nature of the CV assay (staining biomass without distinction between dead or living cells), dead hyphae could be detected. However, according to the XTT assay, these hyphae were metabolically inactive.
In addition to the in vitro effect, this study also investigated the effect of LLCZ in vivo in the invertebrate infection model using G. mellonella. Despite literature recommendations (1 Â 10 6 CFU/mL), an inoculum of 2 Â 10 7 CFU/mL was chosen for all three organisms, due to their rather reduced virulence (35). Treatment with a single dose of LLCZ 48 h after the initial infection showed a 40% increase in the survival of the larvae infected with L. prolificans and a 20% and 15% survival increase for the larvae infected with P. boydii and S. apiospermum, respectively, after 10 days. The therapeutic dose of 152 mg/L was adapted from a study in which rats infected with A. fumigatus were FIG 5 Survival curves for Galleria mellonella larvae infected with L. prolificans (A), S. apiospermum (B), and P. boydii (C). The larvae were treated with PBS as a placebo (blue) and LLCZ (red) during a 10-day period. Voriconazole (VCZ) was used as a control for all organisms (green). Groups of 15 larvae were injected with a fungal inoculum of 2 Â 10 7 CFU/mL and then after 48 h with 152 mg/L LLCZ (treatment), 160 mg/L VCZ (control), or PBS (placebo). The control groups were injected with PBS (gray) and then reinjected after 48 h, in order to rule out any false-positive results caused by the piercing damage of the needle or by LLCZ. A noninjection control (black) was used as an indicator of the quality of the larvae. The larvae were incubated at 37°C, and mortality was checked every 24 h for at least 10 days. Statistical significance of the survival curves was determined using a log-rank (Mantel-Cox) test. *, P , 0.05; **, P , 0.01; ns, not significant.
Antifungal Activity of Novel Anti-Infective Compounds Microbiology Spectrum treated with a daily dose of LLCZ (36). To the best of our knowledge, there are no previous clinical or nonclinical accounts of this drug being used to treat L. prolificans or Scedosporium spp. in vivo. Therefore, it is not known if this is an optimal therapeutic dose for these organisms. For VCZ, the recommended therapeutic dose is 6 mg/kg (i.v.) twice a day for two doses, followed by 4 mg/kg twice a day (37). Due to the nature of our larval model, multiple-dose treatments resulted in an increased mortality of the larvae through the repeated injections. This could have had a masking effect on the data and led to irreproducibility of the experiments. Despite the several advantages that G. mellonella offers, including the possibility of extensively testing a drug against multiple pathogenic organisms, its main drawback is the fact that the invertebrate larvae are physiologically very different from humans (38). Despite this, it is a reliable in vivo model, surpassing Caenorhabditis elegans, where direct injection is impossible, and it could be used prior to in vivo testing in vertebrate animal models (39). Despite the overwhelmingly positive results in in vitro experiments, LLCZ is not yet approved by the FDA for systemic or oral use. In its current topical formulation, LLCZ is effective only against superficial skin mycoses (17). Due to the low solubility of the drug, treatment of a deep-seated infection is challenging because of its retention in the skin (29). This could mean that the development of a systemic LLCZ treatment will not be straightforward. Research is ongoing for the development of novel strategies to improve the drug's penetration and absorption capabilities. These strategies include the use of nanostructured lipid carriers, which show promising results (40).
Oral formulations of the drug have already been successfully tested in an ex vivo murine model and represent a potential solution to this problem (36). Alternatively, respiratory infections could be treated via aerosol inhalation of LLCZ. This approach has already been successfully used for other topical antifungal drugs such as N-chlorotaurine (41,42). LLCZ showed no significant side effects in clinical and nonclinical trials; thus, the future testing and development of an aerosol or oral formulation could represent a potential treatment for L. prolificans and Scedosporium sp. infections (17,28,29).
As a limitation of this study, it must be noted that the number of S. apiospermum (n = 3)/P. boydii (n = 3) isolates was relatively low due to a lack of availability. Similarly, for the in vivo experiments, the larvae were infected with only one isolate of each organism. Further, the invertebrate animal model may also have been impacted by several external factors, such as light sensitivity and weather conditions, which may have impacted the viability of certain larval batches and influenced the reproducibility of the experiment (43). The use of a murine animal model to confirm the data obtained here could solve this issue. A further limitation is the fact that the genetic diversity of the isolates is unknown. It would be preferable for further experiments to include L. prolificans and Scedosporium sp. isolates with a confirmed genetic diversity. In conclusion, for future experiments, it is suggested to increase the total number of isolates, to include more strains/organisms which are confirmed to be genetically diverse, and to use a murine infection model, in order to properly validate the promising first results.
This study is the first account of the antifungal effect of LLCZ against planktonic growth of L. prolificans. Furthermore, it is the first study to show the effect of LLCZ against biofilm formation of S. apiospermum/P. boydii and to offer data for an in vivo treatment model of these fungal pathogens.

MATERIALS AND METHODS
Isolates. The study comprised 31 L. prolificans isolates and 6 S. apiospermum/P. boydii strains (Table 1). These organisms are well known for their panresistance to antifungal drugs. Of the 31 L. prolificans isolates, 8 isolates (shown in bold in Table 1) were used for growth kinetics and biofilm assays. The organisms indicated were obtained from the Dutch CF Fungal Collection Consortium (30). No clinical information regarding the isolates is available.
Fungal suspensions. The fungal suspensions were prepared as described elsewhere (34). In brief, the isolates were plated on Sabouraud agar (Oxoid, Wesel, Germany) and incubated for 5 to 7 days at 30°C. In order to collect viable conidia, a solution of H 2 O plus 0.1% Tween was pipetted onto the plates. The fungal suspension was collected with a syringe and filtered to remove hyphae (pore size, 10 mm; filcon syringe; Becton Dickinson, Franklin Lakes, NJ, USA). After washing once with 1Â phosphate-buffered saline (PBS), the inoculum was adjusted to an appropriate concentration in RPMI 1640 medium.
Luliconazole preparation. LLCZ was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was used for dilution of the drug into stock solutions, which were stored at 220°C until further use. The stock was diluted to various working concentrations, followed by a 1:100 dilution in RPMI 1640 medium. A DMSO concentration of 1% remained in the aliquots. For further information, refer to the corresponding sections below.
In vitro assays. (i) Broth microdilution. The isolate-specific MIC of LLCZ was determined via broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (44). LLCZ was diluted in DMSO and double-concentrated RPMI 1640 medium (2% glucose) to final concentrations ranging between 0.0004 and 0.25 mg/L. After an incubation time of 48 h, the MIC of every individual isolate was read visually.
(ii) Growth kinetics. To assess the effect of LLCZ against the planktonic growth of L. prolificans (8 isolates) and S. apiospermum/P. boydii (6 isolates), an XTT-based colorimetric microbroth assay was used (45). The isolate-specific MIC and concentrations 4-and 16-fold higher than the determined MIC were used. The effect was compared to an untreated growth control. An inoculum of 2 Â 10 6 CFU/mL was incubated together with the LLCZ drug concentrations in flat-bottomed 96-well plates at 35°C (final inoculum concentration, 1 Â 10 6 ). The growth was assessed after 0 h, 6 h, 12 h, 24 h, and 48 h of incubation with the drug. Two hours before the end of incubation, 50 mL of 2.5 mg/mL XTT (Santa Cruz Biotechnology, Dallas, TX, USA) plus 125 mM menadione (Sigma) solution was added to each well. Aliquots (150 mL) of the suspensions were transferred from each well onto new 96-well plates with a U-shaped bottom. The OD 492 was measured using a microplate reader (Sunrise, Tecan, Switzerland).
(iii) Biofilm assays. The biofilm analysis methods were previously described (23,32,34). In brief, a final inoculum concentration of 1 Â 10 6 CFU/mL was used for all biofilm assays. LLCZ was used at the previously determined isolate-specific MICs, as well as at sub-MICs: 0.5Â, 0.25Â, and 0.125Â the specific MIC value. An untreated growth control was also included. Aliquots (1 mL) of the suspensions were incubated in 24-well plates at 35°C. LLCZ was added at different time points after the initial incubation: 0 h (t 0 ), 2 h (t 2 ) and 48 h (t 48 ). The t 0 group was directly incubated with LLCZ for 48 h, while for the t 2 and t 48 groups, the biofilms were washed with 1Â PBS and incubated with LLCZ after initial incubation times of 2 h and 48 h, respectively. Afterward, the biofilms were washed with 1Â PBS and analyzed via XTT and crystal violet (CV) assays. For the XTT assay, 500 mL of a solution of XTT (0.5 mg/mL) plus 25 mM menadione was pipetted into each well of the plates, which were then incubated at 36°C for 3 h in the dark. For the crystal violet assay, the biofilms were stained with a 0.01% CV solution for 20 min, air-dried overnight at room temperature, and then incubated for 30 min in a 30% acetic acid solution. Aliquots (100 mL) from each well were transferred into a U-shaped 96-well plate, and the optical density was measured in a plate reader at 492 nm (XTT assay) and 620 nm (CV).
(iv) Confocal laser scanning microscopy. The effect of LLCZ against biofilm formation was visualized via confocal laser scanning microscopy (CLSM) using the Elyra LSM 710 instrument (Zeiss, Oberkochen, Germany) with a laser at 405 nm and a 20-fold magnification objective. Suspensions were incubated in m-Slide 8-well glass-bottom plates (ibidi GmbH, Gräfelfing, Germany), as described above. The biofilms were fixed with 100% methanol for 1 min. Images were processed using ZEN black software (Zeiss).
In vivo assays. (i) Galleria mellonella larva handling. Galleria mellonella (larval stage) was used as the in vivo model. The standardized protocol for handling of the larvae was previously published (39). Briefly, the larvae were weighed and separated into groups. The groups were injected in the prolegs with a corresponding fungal inoculum or treatment in volumes of 10 mL (for 300 6 50 mg larvae), 13.33 mL (for 400 6 50 mg larvae), and 16.66 mL (for 500 6 50 mg larvae) with the help of a syringe pump (SyringePumpPro model LA-100; Landgraph Laborsysteme HLL GmbH, Langenhagen, Germany). The infected larvae were subsequently incubated at 37°C over 10 days with daily survival monitoring.
(ii) Infection treatment. The treatment of the larvae infected with L. prolificans, S. apiospermum, and P. boydii with a single dose of LLCZ was tested. Based on a previous infection assay (see Fig. S1 in the supplemental material), the caterpillars were injected in the prolegs with an inoculum of 2 Â 10 7 CFU/mL from each pathogenic organism. LLCZ was injected at a concentration of 5 mg/kg body weight, equaling 152 mg/L (36). In parallel, other groups of larvae were treated with VCZ, which served as a control. The concentration used for VCZ was 160 mg/L, which corresponds to a recommended therapeutic dose of 6 mg/kg in humans (37). The larvae were injected with the fungal inoculum, incubated for 48 h, and then treated with the antifungals. At treatment, a different proleg was chosen for injection, to avoid any unnecessary stress of the larvae. Four different control groups were used: a noninjection control; an injection control, where larvae were injected 2 times with PBS; an infection control, where infected larvae were injected with PBS; and a treatment control, where the infected larvae were treated with VCZ.
(iii) Recultivation of the organisms. Recultivation of the pathogens from dead larvae was performed as previously described (39). The dead larvae were disintegrated using the MagNA lyser (Roche, Grenzach-Wyhlen, Germany) and consecutively plated on Sabouraud agar to confirm the reisolation of the pathogen.
Statistical analysis. (i) In vitro. All experiments were performed in triplicate. The growth percentage of each treated well was calculated in comparison to that of the untreated control. A statistical analysis of the growth percentage was carried out using the program GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA). The statistical significance level was determined using Dunnett's multiple-comparison test; significance was determined at P , 0.05 (*, P , 0.05; **, P , 0.01; ***, P , 0.001; and ****, P , 0.0001).
(ii) In vivo. All experiments were performed in triplicate. The survival data were analyzed using the program GraphPad Prism 9 (GraphPad Software Inc.). Survival curves were plotted using the Kaplan-Meier estimator. To compile the results from multiple repetitions, the raw data were calculated as a single experiment on the datasheet. The statistical significance of the survival curves was determined with a log-rank (Mantel-Cox) test; significance was set at P , 0.05 (*, P , 0.05; **, P , 0.01).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.8 MB.

ACKNOWLEDGMENTS
We hereby thank the Medicines for Malaria Venture (MMV) for their generous donation of the Pandemic Response Box; the Imaging Center Essen (IMCES), Faculty of Medicine, University Hospital Essen, University of Duisburg-Essen, for assistance and the use of their imaging facilities; and David Killengray, a native English speaker, for proofreading the manuscript. We further acknowledge support by the Open Access Publication Fund of the University of Duisburg-Essen.