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. 2021 Oct 18;65(11):e00638-21. doi: 10.1128/AAC.00638-21

Activity of Amphotericin B Formulations and Voriconazole, Alone or in Combination, against Biofilms of Scedosporium and Fusarium spp.

Katerina Vikelouda a, Maria Simitsopoulou a, Lemonia Skoura b, Charalampos Antachopoulos a, Emmanuel Roilides a,✉
PMCID: PMC8522719  PMID: 34370583

ABSTRACT

Scedosporium and Fusarium species are emerging opportunistic pathogens, causing invasive fungal diseases in humans, particularly in immunocompromised patients. Biofilm-related infections are associated with increased morbidity and mortality. Here, we assessed the ability of Scedosporium apiospermum and Fusarium solani species complex (FSSC) isolates to form biofilms and evaluated the efficacy of deoxycholate amphotericin B (D-AMB), liposomal amphotericin B (L-AMB), and voriconazole (VRC), alone or in combination, against mature biofilms. Biofilm formation was assessed by safranin staining and spectrophotometric measurement of optical density. Planktonic and biofilm damage was assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] reduction assay. Planktonic cell and biofilm MIC50s were determined as the minimum concentrations that caused ≥50% fungal damage compared to untreated controls. The combined activity of L-AMB (0.5 to 32 mg/liter) and VRC (0.125 to 64 mg/liter) against biofilms was determined by the checkerboard microdilution method and analyzed by the Bliss independence model. Biofilm MIC50s of D-AMB and L-AMB against S. apiospermum isolates were 1 and 2 mg/liter and against FSSC isolates were 0.5 and 1 mg/liter, respectively. Biofilm MIC50s of VRC against S. apiospermum and FSSC were 32 mg/liter and >256 mg/liter, respectively. Synergistic effects were observed at 2 to 4 mg/liter of L-AMB combined with 4 to 16 mg/liter of VRC against S. apiospermum biofilms (mean ΔE ± standard error, 17% ± 3.7%). Antagonistic interactions were found at 0.5 to 4 mg/liter of L-AMB combined with 0.125 to 16 mg/liter of VRC against FSSC isolates, at −28% ± 2%. D-AMB and L-AMB were more efficacious against S. apiospermum and FSSC biofilms than VRC.

KEYWORDS: biofilms, Scedosporium, Fusarium, synergy, antagonism

INTRODUCTION

Over the last decade, the epidemiology of filamentous fungi causing life-threatening invasive infections has shifted, leading to an increase in non-Aspergillus species (1, 2). In some countries, Scedosporium spp. have emerged as the second most frequent mold pathogen associated with respiratory infections following Aspergillus spp. (3, 4), while Fusarium spp. have evolved to be the second most common nosocomial fungal pathogen among immunocompromised patients, with a mortality rate greater than 75% (1, 5).

Scedosporium and Fusarium spp. can cause infections ranging from superficial (onychomycosis and otitis) to locally invasive (sinusitis, keratitis, brain abscess, and osteomyelitis) and disseminated infections depending on the host immune status (6–8). While voriconazole is more effective than amphotericin B formulations against some of these fungi, both are recommended therapeutic agents used in clinical practice (9). However, the multidrug resistance profiles of these molds, along with the exhibited species-specific concentration-dependent activities of the drugs and their actual bioavailability to the target site, have not provided an established benefit of one drug over the other (10–14). Furthermore, in vitro combination studies between antifungal agents have shown a range of interactions, with limited clinical cases supporting benefit of survival against Scedosporium apiospermum or Fusarium sp. infections (15–18).

Another attribute of non-Aspergillus molds that contributes to their resistant profile is the ability to form biofilms. Biofilms are composed of microbial cells embedded in a self-secreted gelatinous matrix composed of extracellular polymeric substances. The presence of the extracellular matrix has been shown to impair antifungal penetration, making them highly resistant to most antimicrobial agents (19, 20). Biofilm formation has been demonstrated for both S. apiospermum and Fusarium spp. (6, 21–23).

To the best of our knowledge, while there are limited data on the activity of single antifungal agents against biofilms formed by S. apiospermum (21, 22) and Fusarium spp. (24–26), no data exist on the combined activity of antifungal agents used to treat biofilms formed by S. apiospermum and Fusarium spp. The aim of our study was to evaluate the antibiofilm activities of voriconazole (VRC) and the amphotericin B formulations deoxycholate amphotericin B (D-AMB) and liposomal amphotericin B (L-AMB) alone and of VRC in combination with an amphotericin B formulation against clinical isolates of S. apiospermum and Fusarium solani species complex (FSSC), the most common organisms causing non-Aspergillus hyaline mold infections.

(This work was presented in part at the 28th European Congress of Clinical Microbiology and Infectious Diseases [ECCMID], Madrid, Spain, 21 to 24 April 2018, and at the 29th ECCMID, Amsterdam, Netherlands, 13 to 16 April 2019.)

RESULTS

Biofilm formation.

All the preliminary experiments to study biofilm formation indicated that, when incubated at 105 CFU/ml in RPMI medium at 37°C for 48h, all S. apiospermum and all FSSC isolates produced stably adherent biofilms (S. apiospermum optical density [OD], 0.238 ± 0.004, and FSSC OD, 0.195 ± 0.015, versus a control OD of 0.035 ± 0.0012).

Antifungal susceptibility of planktonic cells to D-AMB, L-AMB, and VRC.

The planktonic cells of both organisms showed comparable susceptibility profiles to the three antifungals, with MICs ranging from 0.125 to 0.5 mg/liter against S. apiospermum and from 0.06 to 0.125 mg/liter against FSSC isolates, respectively (Table 1).

TABLE 1.

MICs of D-AMB, L-AMB, and VRC for planktonic cells and biofilms of S. apiospermum and FSSC determined by the XTT assay

Organism (n) MIC (mg/liter)
D-AMB
 L-AMB
 VRC
Planktonic cells Biofilms Planktonic cells Biofilms Planktonic cells Biofilms
S. apiospermum (3) 0.125 to 0.5 1 to 4 0.25 to 1 1 to 2 0.125 32 to 128a,b
FSSCc (3) 0.25 to 0.5 0.25 to 1 0.06 to 0.25 0.5 to 8a 0.03 to 0.25 128 to >256a,b
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a

Significant differences between MIC50s of antifungal agents against planktonic cells versus biofilms of each organism (P < 0.05).

b

Significant differences between MICs of amphotericin B formulations compared to those of VRC against biofilms of each organism. These values were derived from data presented in detail in Fig. 1 and 2.

c

FSSC includes F. solani, F. metavorans, and F. petroliphilum.

Antifungal susceptibility of biofilms to D-AMB, L-AMB, and VRC.

Voriconazole showed diminished activity against biofilms of all isolates compared to that of amphotericin B formulations. Specifically, while the biofilm MIC50s of D-AMB and L-AMB ranged from 0.5 to 2 mg/liter against the isolates of both organisms, the MIC50 of VRC was 32 mg/liter against S. apiospermum biofilms and >256 mg/liter against FSSC isolates (Fig. 1A to C and 2A to C) (between VRC and amphotericin B formulations, P < 0.01).

FIG 1.

FIG 1

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Fungal damage of biofilms of S. apiospermum isolates caused by different concentrations of deoxycholate amphotericin B (A), liposomal amphotericin B (B), and voriconazole (C). The striped column represents the MIC50 of each drug. Each concentration of each antifungal agent was tested in quintuplicate per clinical isolate, and each drug-free control was tested in 16 replicates per experiment. All isolates were retested three times in independent experiments. The average values for these replicates were used in the data analysis to determine the mean and SE under each condition.

FIG 2.

FIG 2

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Fungal damage of biofilms of F. solani isolates caused by different concentrations of deoxycholate amphotericin B (A), liposomal amphotericin B (B), and voriconazole (C). The striped column represents the MIC50 of each drug. Each concentration of each antifungal agent was tested in quintuplicate per clinical isolate, and each drug-free control was tested in 16 replicates per experiment. All isolates were retested three times in independent experiments. The average values for these replicates were used in the data analysis to determine the mean and SE under each condition.

The biofilm damage caused by amphotericin B formulations increased with increasing drug concentrations, with D-AMB showing a tendency toward better antibiofilm activity than that observed with L-AMB at comparable concentrations against S. apiospermum. Such an effect was most pronounced against FSSC isolates. Indeed, the biofilm damage caused by D-AMB against FSSC isolates was 90% at 2 mg/liter, reaching levels of eradication at ≥8 mg/liter compared to those seen with L-AMB, which caused 52% damage at 2 mg/liter, with maximum biofilm damage reaching 77% at 256 mg/liter (Fig. 1A and B and 2A and B). In contrast, VRC showed poor antibiofilm effects, as even at the highest concentration, the biofilm damage observed was 62% and 48% against S. apiospermum and FSSC isolates, respectively (Fig. 1C and 2C).

Antibiofilm interactions between L-AMB and VRC.

The Bliss independence drug interactions of the combined treatment of S. apiospermum (Fig. 3) and FSSC (Fig. 4) biofilms with L-AMB and VRC are summarized in Table 2. Synergistic effects were observed at 2 to 4 mg/liter of L-AMB combined with 4 to 16 mg/liter of VRC against Scedosporium biofilms. The mean ΔE value of significant interactions was 17% (range, 14% to 20%), and the mean standard error (SE) was 3.7% (range, 3.0% to 4.3%). None of the combinations exhibited antagonism (Fig. 3; Table 2). In contrast, combination treatment of Fusarium biofilms resulted in antagonistic effects at concentrations of 0.5 to 4 mg/liter of L-AMB combined with 0.125 to 16 mg/liter of VRC, demonstrating a mean ΔE value of significant interactions of −28% (range, −11% to −55%) with a mean SE of 4.6% (range, 2.5 to 7.9). All other combined concentrations exhibited indifferent interactions (Fig. 4; Table 2).

FIG 3.

FIG 3

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In vitro interaction between liposomal amphotericin B (L-AMB; 0.5 to 32 mg/liter) and voriconazole (VRC; 0.125 to 64 mg/liter) against S. apiospermum biofilms. The x axis and y axis represent the concentrations of VRC and L-AMB, and the z axis represents the percent ΔE (see the text). The zero plane (ΔE = 0) represents indifferent interactions, whereas volumes above (ΔE > 0) represent statistically significant synergistic interactions. The magnitude of the synergistic interactions is directly related to positive ΔE values. The different colors in the three-dimensional plots represent different percentile bands of synergy. The percent ΔE ± SE was 17% ± 3.7% for the VRC–L-AMB combination (synergistic interaction).

FIG 4.

FIG 4

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In vitro interaction between liposomal amphotericin B (L-AMB; 0.5 to 32 mg/liter) and voriconazole (VRC, 0.125 to 64 mg/liter) against F. solani biofilms. The x axis and y axis represent the concentrations of VRC and L-AMB, and the z axis represents the percent ΔE (see the text). The zero plane (ΔE = 0) represents indifferent interactions, whereas volumes above (ΔE > 0) represent statistically significant synergistic interactions. The magnitude of the synergistic interactions is directly related to positive ΔE values. The different colors in the three-dimensional plots represent different percentile bands of synergy. The percent ΔE ± SE was −28% ± 2% for the VRC–L-AMB combination (antagonistic interaction).

TABLE 2.

In vitro interactions assessed by Bliss independence analysis of L-AMB in combination with VRC against biofilms of S. apiospermum and FSSC

Organism Type of interaction Concn (mg/liter) of:
 Mean % ΔΕ (range) Mean % SE (range)
L-AMB VRC
S. apiospermum Synergism 2 to 4 4 to 16 17 (14 to 20) 3.7 (3 to 4.3)
FSSC Antagonism 0.5 to 4 0.125 to 16 −28 (−11 to −55) 4.6 (2.5 to 7.9)
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DISCUSSION

As more non-Aspergillus mold species, such as Scedosporium and Fusarium spp., increasingly cause biofilm-associated diseases, such as superficial (onychomycosis and otitis) or locally invasive (sinusitis, keratitis, brain abscess, and osteomyelitis) or disseminated infections depending on the host immune status (8–11), the in vitro assessment of drug efficacy against these emerging pathogens remains an important antifungal susceptibility tool to guide optimal therapeutic regimens. Fungal cells growing as biofilms and often found on implanted medical devices or chronic infections are difficult to treat due to increased resistance to antifungal agents. Antifungal lock therapy, using single or combined agents, is considered a promising strategy to treat biofilm-related bloodstream infections (27). Both Scedosporium and Fusarium species have been shown to form protective biofilm structures with variable resistance profiles to azoles or polyenes, often requiring high drug concentrations to achieve biofilm eradication (21–24).

In our study, all six clinical isolates of S. apiospermum and FSSC were biofilm producers after incubation under static conditions for 48 h. While planktonic cells showed comparable susceptibility profiles to D-AMB, L-AMB, and VRC, biofilms exhibited significantly higher resistance to VRC (32 mg/liter to >256 mg/liter) than that shown for amphotericin B formulations. As we demonstrated similar antifungal activities between D-AMB and L-AMB against biofilms of both Scedosporium and Fusarium species, and keeping in mind that D-AMB is shown to exhibit adverse side effects in therapeutic regimens, we chose to investigate double combinations using the lipid formulation of AMB. The combination of L-AMB plus VRC showed synergy against S. apiospermum biofilms, but their interaction produced antagonistic effects against Fusarium biofilms.

Our results regarding the single-drug activities against S. apiospermum are in agreement with another in vitro comparative biofilm study of Scedosporium species by Rollin-Pinheiro et al. (21) investigating the susceptibility profile of Scedosporium boydii and Scedosporium aurantiacum to fluconazole, itraconazole, and voriconazole. They showed that Scedosporium biofilms were resistant to all azoles (MIC >128 mg/liter), compared to their planktonic counterparts, against which voriconazole was mainly active (0.5 to 1 mg/liter) (21). The poor activity of voriconazole against S. apiospermum, S. aurantiacum, and Scedosporium minutisporum biofilms was also demonstrated by another study, where the biofilm MICs of voriconazole ranged from 128-fold to 1,024-fold compared to that for planktonic forms (22). Biofilm resistance was attributed to the presence of a viable, densely compacted mycelial biomass and extracellular matrix.

In the present study, the efficacy of amphotericin B and the intrinsic resistance to voriconazole was also demonstrated against Fusarium biofilms. Sav et al. investigated the antifungal activity of polyenes and azoles against 38 Fusarium isolates and found that amphotericin B had the highest in vitro activity against both planktonic cells (MIC, 0.25 to 2 mg/liter) and biofilms (MIC, 2 to 8 mg/liter), whereas interspecies variability was observed for voriconazole, with MICs ranging up to >16 mg/liter (26). Similarly, Zhang et al. reported that mature biofilms of Fusarium species isolated from patients with keratitis were intrinsically resistant to VRC, suggesting that the extracellular matrix of biofilms deters antifungal diffusion or that biofilms induce increased expression of components making up the efflux pump system (24). It appears that for Fusarium species, there also exists species-dependent resistance to antifungal agents, as, in contrast to our results, Mukherjee et al. reported that VRC was more active against biofilms formed by FSSC than amphotericin B (25). The above results suggest that, when these agents are used in single schemes against Fusarium biofilms, the variability demonstrated for amphotericin B and VRC may be due to additional virulence factors not yet identified. It is worth noting the potential utility of both amphotericin B formulations as antifungal therapy against Scedosporium and Fusarium biofilm-based infections, as we observed >80% biofilm damage at concentrations of >2 mg/liter for D-AMB and >70% biofilm damage at >16 mg/liter for L-AMB.

Combining antifungal agents to treat biofilm-based invasive infections especially among immunocompromised patients has been shown to be a potential promising strategy for increasing antifungal potency and efficacy (28, 29). In the present study, the combination of amphotericin B formulations with voriconazole was synergistic against mature S. apiospermum biofilms, but their interaction produced antagonistic effects against Fusarium biofilms. To date, data on in vitro combination studies against Scedosporium (30) and Fusarium planktonic cells (17, 31) are scarce, and results differ even between different clinical isolates of the same species. A single murine study of disseminated infections by Scedosporium prolificans (currently named Lomentospora prolificans) assessing the efficacy of double combinations of amphotericin B with VRC or micafungin showed that all treatments were able to prolong the survival compared to monotherapies (27).

Based on the proposed mechanisms of interactions between azoles and amphotericin B, synergy could be produced when increased azole concentrations reach the intracellular target site due to cell membrane deconstruction by the action of amphotericin B. Alternatively, an antagonistic effect may result because both antifungals exert competitive antifungal action at the same target site, since the azoles block ergosterol synthesis and amphotericin B needs to bind to ergosterol to exert its antifungal activity (32). The concentration-dependent attribute of amphotericin B-voriconazole interactions was shown in an in vitro pharmacokinetic-pharmacodynamic model against Aspergillus fumigatus, where synergy was demonstrated at low amphotericin B-high voriconazole concentrations and antagonism was shown at high amphotericin B-low voriconazole concentrations (33). In clinical practice, liposomal amphotericin B and voriconazole are the preferred agents against Fusarium infections, possibly in combination, but controversial results exist, mainly due to disease severity, dose regimens, site of infection, and underlying disease (34–36). Based on the mechanism of action of amphotericin B and voriconazole, the possibility of antagonism between the polyenes and the azoles cannot be excluded, but there is no firm clinical evidence of antagonism between these two classes of drugs in fusariosis.

In the present study, biofilms add an additional layer of complexity to the concentration-dependent nature of the antifungal agents under investigation, since it is unknown to what extent the extracellular matrix, the regulation of efflux pumps, and the metabolic state of biofilm cells affect drug diffusion and net antifungal activity. Nevertheless, synergistic interactions against other biofilm-forming fungal organisms grown in intravenous catheters have been identified in vitro, such as amphotericin B-caspofungin or voriconazole-caspofungin for Aspergillus spp. (37) and amphotericin B-posaconazole for Candida albicans (38). As the resistant phenotype of biofilms precludes the use of high antifungal concentrations, the synergistic interactions observed in the present study against S. apiospermum biofilms would find clinical application in antifungal therapy for the treatment of biofilm-based infections.

A possible limitation of the present study, mostly due to the rarity of scedosporiosis and fusariosis, is the small number of isolates investigated, since only six were biofilm formers. A multicenter study with additional biofilm-producing isolates is expected to validate our results.

In conclusion, the present study provides further insight into susceptibility of biofilms of Scedosporium and Fusarium spp. to antifungal agents. In addition, we show for the first time that the combination of L-AMB and VRC produces synergy against Scedosporium biofilms but antagonism against Fusarium biofilms in specific drug concentration ranges. These results may be the basis for further understanding the antifungal susceptibility of biofilms of these difficult-to-treat organisms and the interactions of antifungal agents frequently used against them.

MATERIALS AND METHODS

Clinical isolates and growth conditions.

Scedosporium apiospermum (n = 3) and Fusarium solani species complex (FSSC, n = 3) isolates were recovered from lung tissue, bronchial secretions, trauma sites, and corneas of adult patients with proven invasive infections. Species identification for all clinical isolates was performed by multilocus sequence typing using internal transcribed spacer regions ITS1 and ITS4, elongation factor 1 alpha and the RNA polymerase II subunit (39). Molecular identification recognized the three FSSC isolates as Fusarium petroliphilum, Fusarium metavorans, and Fusarium solani. Stocks were maintained on potato dextrose agar (PDA; Sigma-Aldrich, Darmstadt, Germany) slants at −30°C. Clinical isolates were revived by subculture on PDA agar plates at 37°C for 4 to 6 days, and the sporangiospores were subsequently harvested in PBS (0.02 M phosphate, 0.15 M NaCl; pH 7.2) containing 0.05% Tween 80. The resulting suspensions were centrifuged at 4,000 rpm for 10 min, washed, suspended in PBS, and counted on a hemocytometer. For all experiments, the final sporangiospore concentration used was 105/ml for biofilm formation and 2 × 105/ml for planktonic cells.

Biofilm formation.

Biofilms were formed in 96-well presterilized flat-bottomed polystyrene plates as previously described (40, 41). In order to assess the optimum cell concentration for biofilm formation, sporangiospore concentrations from 104 to 107 CFU/ml were prepared in RPMI 1640 medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) buffered to pH 7.2 with 3-(N-morpholino)propanesulfonic acid (Sigma-Aldrich). Briefly, 200 μl of each suspension was inoculated into each well of a microtiter plate and incubated under static conditions at 37°C for 24 and 48 h. Formed biofilms were washed once with sterile distilled water to remove unattached cells, and the polysaccharide structure of the extracellular matrix was stained with 1% safranin for 3 min, washed once, and allowed to air dry. Biofilm formation was quantitated spectrophotometrically at 490 nm with a microplate reader (Chromate 4300; Awareness Technology Inc, Palm City, FL). The experimental wells were compared with safranin-stained control wells containing RPMI 1640 without organisms. Isolates were classified arbitrarily as previously described (42) as good or no biofilm formers based on the optical density (OD) of biofilm-containing wells compared to that of control wells. A clinical isolate with an OD of >0.106 {three times the average OD of the control plus 1 standard deviation: [(3 × 0.035) + 0.0012] = 0.106} was considered a biofilm producer. All the preliminary experiments to study biofilm formation were performed three times, and each concentration for each isolate was measured in 40 wells per experiment.

Antifungal agents.

The antifungal agents studied were D-AMB (Sigma-Aldrich), L-AMB (Gilead Sciences, Inc.), and VRC (Sigma-Aldrich). D-AMB was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) to a stock concentration of 5 mg/ml, L-AMB was reconstituted in sterile water to 5 mg/ml (the powder containing 50 mg of amphotericin B was dissolved in 10 ml sterile water and filtered), and VRC was dissolved in DMSO to 18 mg/ml. An appropriate amount of each agent was further diluted to 1,024 mg/liter in RPMI 1640 medium and used to prepare a series of 2-fold dilutions ranging from 0.007 to 256 mg/liter.

Antifungal susceptibility assessment.

One hundred microliters of the above-mentioned series of double-strength 2-fold dilutions of each drug was added to 96-well plates containing 100 μl of 48-h biofilms (105 CFU/ml) or planktonic cells (2 × 105 CFU/ml). The plates were incubated at 37°C for 24 h. Experiments were performed three times on three independent days. Each concentration of antifungal agents was tested in quintuplicate for each clinical isolate, and each untreated control was tested in 16 replicates per experiment. The average values for these replicates were used in the data analysis to determine the mean and standard error (SE) for each condition. The antifungal activity of each drug against planktonic cells or biofilms was assessed by the XTT reduction assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide salt (XTT) (0.25 mg/ml; Sigma-Aldrich) and coenzyme Q0 (40 μg/ml; Sigma-Aldrich); the OD was measured spectrophotometrically at 450 nm with a reference wavelength of 690 nm (41, 43, 44). Percent fungal damage was expressed by the formula 100 × (1 − X/C), where X is the average absorbance of treated biofilms and C is the average value of control wells (untreated biofilms). The MIC for planktonic cells and biofilms (MIC50) was determined as the minimum antifungal drug concentration that caused ≥50% damage of planktonic cells or biofilms compared to untreated controls.

Combinational antifungal treatment against biofilms.

The interaction of L-AMB with VRC against mature biofilms was determined using a two-dimensional (8-by-12) checkerboard microdilution method. Mature biofilms were coincubated at 37°C for 24 h with serially 2-fold-diluted concentrations of L-AMB ranging from 0.5 to 32 mg/liter and VRC ranging from 0.125 to 64 mg/liter. The combinational experiments were performed 10 times for each isolate. The range of drug concentrations investigated was based on the biofilm MIC50 determined for each antifungal agent. The combined effects were assessed by the XTT reduction assay as described above. Drug interactions were analyzed using the Bliss independence model. The combinational effect was defined as synergistic, antagonistic, or indifferent when the observed biofilm damage was significantly higher than, lower than, or equal to the expected damage, respectively (45).

Statistical analysis.

The differences in MIC50 between planktonic cells and biofilms caused by each drug and the differences between MICs of amphotericin B formulations compared to those of VRC against biofilms for each organism were assessed by one-way analysis of variance (ANOVA) with Dunnett’s post-test. A two-tailed P value of <0.05 indicated statistical significance. Data were analyzed using Instat v.3 biostatistics software (GraphPad Inc., San Diego CA).

Analysis of drug antifungal interactions.

The in vitro interactions between L-AMB and VRC were analyzed using the Bliss independence model. According to the Bliss model, the expected theoretical percentage of growth (Eind) (compared to a control without drug) describing the effect of the combination of two antifungal agents was calculated using the equation Eind = EA × EB, where EA and EB are the experimental percentages of growth when each antifungal agent acts alone. For each combination of drug A with drug B, the experimental observed percentage of growth, Eobs, was subtracted from Eind. When ΔE (ΔE = Eind − Eobs) was positive, and its 95% confidence interval (CI) value did not include zero, statistically significant synergy was concluded for the specific combination, while when ΔE was negative and its 95% CI did not include zero, significant antagonism was claimed. In any other case, where the 95% CI of ΔE included zero, the conclusion was Bliss independence. ΔE was calculated for all the combinations of drug concentrations for each of the isolates and for each of the three different experiments. The mean and standard deviation of ΔE of all the combinations were calculated and are reported for the combinations, which are statistically significantly synergistic (ΔE ± 95% CI of >0) or antagonistic (ΔE ± 95% CI of <0). The ΔEs that were statistically different from zero were constructed as a three-dimensional plot, with peaks above and below the zero plane indicating synergistic and antagonistic interactions, respectively, and the zero plane indicating indifferent interactions (45). Comparative analysis between synergistic interactions was performed by ordinary ANOVA with Dunnett’s posttest. A P value of <0.05 indicated statistical significance.

ACKNOWLEDGMENTS

This research was cofunded by Greece and the European Union (European Social Fund [ESF]) through the Operational Program Human Resources Development, Education and Lifelong Learning 2014-2020 in the context of the project “Pharmacodynamic and immunomodulatory activities of antifungal agents against clinically important hyphomycetes” (MIS: 5047921).

We thank Miranda Drogari for kindly donating Scedosporium and Fusarium strains and Ioanna Stamouli for technical assistance.

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