Colistin and Isavuconazole Interact Synergistically In Vitro against Aspergillus nidulans and Aspergillus niger

The in vitro interactions of isavuconazole in combination with colistin were evaluated against 55 clinical Aspergillus species isolates belonging to the five most important species (Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) responsible for human aspergillosis by a microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method for antifungal susceptibility testing. Selected isolates (A. nidulans, n = 10; A. niger, n = 15) were additionally evaluated by an agar diffusion assay using isavuconazole gradient concentration strips with or without colistin incorporated Roswell Parc Memorial Institute (RPMI) agar. Interpretation of the checkerboard results was done by the fractional inhibitory concentration index. Using the checkerboard method, combination isavuconazole–colistin was synergistic for 100% of the 15 A. nidulans isolates and for 60% of the 20 A. niger isolates. No interactions were found for any of the other isolates. By agar diffusion assay, minimal inhibitory concentrations (MICs) in combination decreased compared to isavuconazole alone for 92% of the isolates. No interactions were found for any A. nidulans isolates, but synergy was observed for 40% of the A. niger isolates. A poor essential agreement of EUCAST and gradient concentration strip MICs at ± 2 log2 dilutions with 0% was obtained. Antagonistic interactions were never observed regardless of the technique used.


Introduction
Aspergillus infections are an important problem in human health [1]. In immunocompromised patients, invasive aspergillosis mainly affects those with hematological malignancies, especially those with severe and prolonged neutropenia [2]. In non-immunocompromised patients with prior or current lung disease, chronic pulmonary aspergillosis can aggravate these preexisting lung diseases [3]. Another immunocompetent patient population at risk for Aspergillus infections comprise those with severe influenza. In a retrospective cohort study of seven intensive care units across Belgium and the

Medium Preparation
As the test medium, Roswell Park Memorial Institute (RPMI) 1640 medium (with L-glutamine and pH indicator but without bicarbonate) (Merck, Darmstadt, Germany) containing 2% D-Glucose (Merck), and buffered with 3-(N-morpholino)propanesulfonic acid (Merck) at a final concentration of 0.165 mol/L adjusted to pH 7.0 with 1 M sodium hydroxide (Merck) was used. To allow two-fold dilution, the test medium was prepared in double strength of the desired final concentration. After preparation, the medium was sterilized by vacuum filtration through a 0.22 µm pore-sized filter (Merck).

Drugs and Microplate Preparation
Combination studies were performed using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines for antifungal susceptibility testing of molds with modifications for a broth microdilution checkerboard procedure. Experiments were carried out in Nunclon TM delta surface 96-wells microtiter plates for adherent cells (Thermo Fisher Scientific, Darmstadt, Germany). The included drugs were isavuconazole (Pfizer, Berlin, Germany) and colistin (Merck). The stock solutions of isavuconazole (3200 µg/mL) and colistin (12,800 µg/mL) were prepared in dimethyl sulfoxide (DMSO) and sterile, distilled water, respectively. Drug dilutions were performed at four times the final concentration in double-strength RPMI medium. The combination was studied on a two-dimensional checkerboard with two-fold dilution. The final concentration for isavuconazole ranged from 0.03 to 16 µg/mL. The final concentration for colistin was 1 to 64 µg/mL. Fifty microliters of each concentration were distributed from row 1 to 8 for colistin and from column 1 to 11 for isavuconazole. Column 12 was used as growth control and contained 100 µL of double-strength RMPI medium with 1% DMSO.

Inoculum Preparation and Inoculation of Microplates
Before the preparation of the inoculum, all isolates were subcultured for a second time on Sabouraud dextrose agar slants containing chloramphenicol and gentamycin and incubated at 35 • C and 95% humidity. After incubation for 7 days, the spores were transferred to a sterile tube containing pure sterile water supplemented with 0.1% of Tween 80 (Merck) using a wet cotton swab. The suspension was counted in a hemocytometer and adjusted to 10 6 conidia/mL. This solution was used to inoculate the RPMI agar plates. This solution was further diluted to 2 × 10 5 conidia/mL with sterile water containing 0.1% Tween 80 in order to prevent growth of fungi on the surfaces of the wells [27]. One hundred microliters of the final inoculum was distributed in each well of the microplates. The final inoculum was further diluted and 100 µL was spread once on Sabouraud dextrose agar plates with a sterile Drigalski spatula. After 24-48 h of incubation at 35 • C, the colony-forming units were counted to ensure inoculum size and viability of the conidia. The microplates were incubated at 35 • C and 95% humidity, and the optical densities were read spectrophotometrically at 48 h at a wavelength of 530 nm using the spectrometer MultiSkan FC (Thermo Fisher Scientific). For each set of experiments, a blank microplate was used. Each well of the blank plates were inoculated with 100 µL of sterile distilled water containing 0.1% Tween 80. Blank microplates were also incubated at 35 • C and 95% humidity for 48 h. Before calculation of the MICs, the blank was subtracted from the inoculated microplates. All experiments were run in duplicate.

Interpretation of the Results
Minimal inhibitory concentrations (MICs) alone and in combination were determined as the lowest concentration giving complete inhibition as measured by 90% inhibition compared to the growth control by spectrophotometric reading. High off-scale MICs were converted to the next log 2 dilution. The fractional inhibition concentration index (FICI) was interpreted as FICI ≤ 0.5 = synergy, FICI > 0.5-4 = no interaction, and FICI > 4.0 = antagonism [28].

Preparation of the RPMI Agar Plates
Fifteen grams of agar (Merck) were dissolved in 450 mL of distilled water. After adjustment of the pH to 7 with 0.1 M NaOH (Merck), the solution was set to 500 mL with distilled water and autoclaved for 20 min at 121 • C and 2 bars. The liquid agar and 500 mL of double-strength RPMI medium were put into a water bath. After temperature adjustment to 50 • C, the solutions were mixed under a flow bench and stirred using a stir fish. The colistin RPMI agar plates additionally contained an adequate volume of sterile colistin stock solution to a final concentration of 64 µg/mL for A. niger isolates and 32 µg/mL for A. nidulans isolates, as determined by preliminary experiments. In these preliminary experiments, 64 µg/mL was chosen for both species, but no growth was observed on the agar plates for colistin alone and in combination for any of the A. nidulans isolates. Therefore, 32 µg/mL was chosen for the A. nidulans isolates. Twenty microliters of the liquid RPMI agar or colistin RPMI agar was added to sterile Petri dishes (diameter 90 mm) (Merck) under a flow bench and allowed to cool down.

Inoculation of RPMI Agar Plates
The RPMI agar plates were inoculated using the inoculator retro C80 (bioMérieux, Nürtingen, Germany). Briefly, a sterile cotton swab was soaked in the spore suspension set to 10 6 conidia/mL and excess fluid was removed by pressing the swab against the wall of the tube. A cross was gently drawn over the agar surface using the wet cotton swab. After the inoculator was turned on with maximum rotation, the cotton swab was moved from the edge of the agar surface to the middle of the plate and back while applying slight pressure. After inoculation, the plates were allowed to dry and isavuconazole gradient concentration strips (Liofilchem, Roseto degli Abruzzi, Italy) were placed onto the agar surface. For the activity of isavuconazole alone, plain RPMI agar plates were used. For the activity of colistin alone and for the combination, colistin RPMI agar plates were used. The activity of colistin alone was tested by inoculation of the agar plate without placement of a gradient concentration strip on the surface. Agar plates were incubated at 35 • C and pictures were taken at 48 h. MIC reading was done according to the recommendations of the manufacturer.

Interpretation of the Results of RPMI Agar Plates
A decrease or increase of ≥ 2 log 2 dilutions in MIC compared to the most active drug was defined as synergy or antagonism, respectively. A decrease or increase of less than 2 log 2 dilutions in MIC to the most active drug was defined as no interaction [29].

Comparison of EUCAST and Gradient Concentration Strip MICs
The essential agreement between the two techniques was evaluated by calculating the percentage of MICs, which differed by no more than 2log 2 dilutions.

Results
In the first part of the experiments, the combination of isavuconazole with colistin was screened against 30 Aspergillus isolates belonging to five different species using the EUCAST broth microdilution technique. The results of these experiments are presented in Table 1. Based on these results, 25 additional (10 A. nidulans and 15 A. niger) isolates were tested using the EUCAST methodology and an agar diffusion assay. The results of these experiments are presented in Table 2. A summary of all results is presented in Table 3.
The 55 isolates exhibited MICs for isavuconazole alone ranging from 0.5 to 16 µg/mL (Tables 1  and 2) with MIC50, MIC90, and geometric mean MIC values of 2, 16, and 2.24 µg/mL, respectively. Isavuconazole MICs for A. flavus, A. fumigatus, A. nidulans, A. niger, and A. terreus ranged from 2 to 4 µg/mL, 1 to 16 µg/mL, 0.5 to 1 µg/mL, 4 to 16 µg/mL, and 0.5 to 1 µg/mL, respectively. Between experiments, isavuconazole MICs were within ± 1 log 2 dilutions in 100% of the cases (data not shown). Colistin alone did not exhibit in vitro activity. MICs for all isolates were > 64 µg/mL. In the screening experiment, synergy was achieved for five of five A. nidulans isolates but only three of five A. niger isolates. For all other species, no interaction was obtained. To validate these results, an additional 10 A. nidulans and 15 A. niger isolates were tested. Using the checkerboard methodology, the combination exhibited synergy for 100% and 60% of the isolates of Aspergillus spp., respectively. For the A. nidulans isolates, die fractional inhibitory concentration index (FICI) ranged from 0.3125 to 0.5 with an average FICI of 0.39. For the A. niger isolates, die FICI ranged from 0.2813 to 0.625 with an average FICI of 0.48. According to the agar diffusion assay, MICs of isavuconazole for A. nidulans were 16 to 32-fold lower using the checkerboard method and ranged from 0.012 to 0.047 µg/mL, with MIC50, MIC90, and geometric mean MIC values of 0.016, 0.023, and 0.02 µg/mL, respectively. The correlation between EUCAST and gradient concentration strip MICs was poor, with an essential agreement at ± 2 log 2 dilutions of 0%. There was a 3 to 5 log 2 dilution difference for all A. nidulans isolates. Growth on RPMI agar was not inhibited by incorporation of 32 µg/mL of colistin for any A. nidulans isolates. Although all MICs in combination were lower than the MICs of isavuconazole alone, none of the isolates met the definition of synergy. Gradient concentration strip MICs of A. niger were also lower than those evaluated by checkerboard and ranged from 0.125 to 0.75 µg/mL, with MIC50, MIC90, and geometric mean MIC values of 0.38, 0.5, and 0.32 µg/mL, respectively. As for A. nidulans, the essential agreement between EUCAST and gradient concentration strip MICs for A. niger isolates at ± 2 log 2 dilutions was 0%. The MIC dilution difference for all A. niger isolates was 3 to 5 log 2 . Colistin at 64 µg/mL incorporated in RMPI agar did not inhibit the growth of the tested A. niger isolates. Combination MICs were lower than MICs of isavuconazole alone for 13 of 15 isolates, with 6 isolates (40%) meeting the definition of synergy of a decrease ≥ 2 log 2 dilutions compared to isavuconazole alone. A decrease of 1 log 2 was seen for two additional isolates, whereas for two isolates the MICs in combination and for isavuconazole alone were the same. Figure 1 shows the interaction of isavuconazole and colistin against one A. nidulans and one A. niger isolate evaluated by agar diffusion assay. Antagonistic interactions were never observed, regardless off the in vitro technique used.   Table 3. Summary of in vitro interactions of isavuconazole with colistin against Aspergillus species using EUCAST broth microdilution checkerboard methodology and agar diffusion assay.

Discussion
The problems of primary and acquired azole-resistance in Aspergillus species are alarming [30], presenting an urgent need for new, broad-spectrum, antifungal drugs with new mechanisms of action [31]. However, strategies like combination therapy [20] or drug repurposing [21] could overcome this therapeutic issue. Therefore, we tested the combination of isavuconazole and colistin

Discussion
The problems of primary and acquired azole-resistance in Aspergillus species are alarming [30], presenting an urgent need for new, broad-spectrum, antifungal drugs with new mechanisms of action [31]. However, strategies like combination therapy [20] or drug repurposing [21] could overcome this therapeutic issue. Therefore, we tested the combination of isavuconazole and colistin against a panel of both azole-susceptible and intrinsic or acquired azole-resistant Aspergillus species isolates.
The isavuconazole MICs of the tested Aspergillus isolates determined by ECUAST methodology in this study were in the same range as previously reported [32]. One drawback of EUCAST technique when testing molds is the visual determination of MICs. Previous reports demonstrated that spectrophotometric reading is a good alternative for Aspergillus spp. [33][34][35]. Incorporation of spectrophotometric reading leads to more objective MIC determination and automation of the process. Therefore, we chose spectrophotometric reading for MIC determination in this work.
The essential agreement between EUCAST and the gradient concentration strip MICs for A. nidulans and A. niger was poor. Two other studies evaluated the essential agreement between these two techniques for A. niger isolates, with one study, in contrast to ours, reporting an essential agreement of 93.75% [36]. These contrary results may be related to the different protocols (Clinical and Laboratory Standards Institute (CLSI) vs. EUCAST) used to perform the checkerboard experiments. The other study reported a poor essential agreement of 18.5% between the techniques using EUCAST methodology, similar to our results [37]. For colistin, we found no antifungal activity using the checkerboard method against Aspergillus species. These results were in accordance with previous studies [23,38].
Synergistic in vitro interactions between colistin and antifungal drugs were reported for yeasts [23,25,26,[38][39][40] and filamentous fungi [23,38], as well as no interaction [23,26,40,41] and antagonism [41]. In this study, we found in vitro synergy for the combination of colistin with isavuconazole using the broth microdilution checkerboard technique for all tested A. nidulans isolates and 60% of the A. niger isolates. Colistin can induce cell membrane damage in C. albicans [23]. Azoles, which are inhibitors of ergosterol biosynthesis, also lead to alterations in the fungal membrane [42]. The synergistic effect of this combination may therefore be explained by the combined activity of the two drugs on the same cellular component. Using the agar diffusion assay, no interaction of the combination was seen for the A. nidulans isolates. For the A. niger isolates, synergy was seen in 40% of the isolates. Nevertheless, the combination MICs were reduced for all A. nidulans isolates and for 87% of the A. niger isolates compared to isavuconazole alone. The discrepancy between the results of the two techniques could be considered as evidence that the synergy of the combination is weak. Further evidence for weak synergy of the combination isavuconazole-colistin exists in the relatively high mean FICI values of 0.39 and 0.48 for A. nidulans and A. niger, respectively. To our knowledge, the combination of colistin with isavuconazole was previously only tested against C. auris, with a weak, but still synergistic, interaction with an average FICI of 0.4 reported [25]. Against Aspergillus species, only one study evaluated the in vitro interaction of colistin with antifungals. Interaction of liposomal amphotericin B with colistin exhibited synergy against three A. fumigatus isolates [38]. In our study, the combination of colistin with isavuconazole was inactive against A. fumigatus.
Concerning the potential usability of the combination in patients, peak serum levels of 13 to 32 µg/mL were reported in patients with cystic fibrosis receiving prolonged colistin therapy [43]. These concentrations would be sufficient, as in our study we found MICs of colistin in combination of 8 to 32 µg/mL and 1 to 32 µg/mL for A. nidulans and A. niger, respectively. Nevertheless, due to its nephrotoxicity [44], the use of colistin is limited in critically ill patients and serum peak levels are lower (0.5 to 9.4 µg/mL) [45]. However, it cannot be excluded that in vivo synergy can be present at lower concentrations than those evaluated in vitro, as demonstrated for the combination of tacrolimus with fluconazole or amphotericin in patients with cryptococcosis [46]. For isavuconazole, mean blood concentrations of 3.6 µg/mL (range 0.64-8.13 µg/mL) were measured in patients during the first 30 days of treatment [47], which would be sufficient as we found ranges of 0.03 to 0.25 µg/mL and 1 to 4 µg/mL (geometric mean 2.38 µg/mL) MICs in combination for A. nidulans and A. niger, respectively.
In summary, colistin enhances the in vitro activity of isavuconazole against clinical A. nidulans isolates and A. niger isolates with high in vitro MICs to isavuconazole. These results warrant further in vivo experiments.