Evaluation of the Synergistic Activity of Antibacterial and Antifungal Drugs against Candida auris Using an Inkjet Printer-Assisted Method

ABSTRACT

Candida auris is an emerging multidrug-resistant fungal pathogen that spreads readily in health care settings and has caused numerous hospital outbreaks. Very few treatment options exist for C. auris infections. We evaluated the activity of all two-drug combinations of three antifungal agents (amphotericin B, caspofungin, and voriconazole) and two antibacterial agents (minocycline and rifampin) against a collection of 10 C. auris isolates using an automated, inkjet printer-assisted checkerboard array method. Three antibacterial-antifungal combinations (amphotericin B plus rifampin, amphotericin B plus minocycline, and caspofungin plus minocycline) demonstrated synergistic activity by checkerboard array against ≥90% of strains, with fractional inhibitory concentration index (FIC_I) values of 0.094 to 0.5. The two amphotericin B-containing combinations were also synergistic using the time-kill synergy testing method, with up to a 4.99-log₁₀ decrease in surviving yeast compared to either agent alone. Our results suggest that combinations of antifungal and antibacterial agents provide a promising avenue for treatment of this multidrug-resistant pathogen.

INTRODUCTION

The pathogenic yeast Candida auris, first identified in external ear canal drainage of a woman in Japan in 2009 (1), is classified by the U.S. Centers for Disease Control and Prevention as one of the most urgent antibiotic resistance threats (2), and invasive C. auris infections have recently been observed as a complication of critical SARS-CoV-2 disease (3–5). C. auris is most commonly reported as a cause of bloodstream infections; patients with central venous catheters and recent surgical procedures are at particularly high risk (6). Compared to other Candida species, C. auris is notable for its propensity to spread within health care settings, for a high rate of mortality in infected patients, and for resistance to multiple antifungal drugs (7, 8).

Few drugs are available to treat even the most susceptible of fungal pathogens, with only three classes of systemic antifungal agents in general use: azoles, echinocandins, and the polyene amphotericin B (AMB) (9). Individual patient factors, such as allergies, drug-drug interactions, and the need for penetration into specific tissue sites frequently further constrain the choice of agents, sometimes reducing practical treatment options to few or none. The resistance profiles observed in C. auris isolates are particularly alarming: nearly all are resistant to fluconazole, and some also demonstrate resistance to echinocandins and AMB (6, 10). Isolates resistant to drugs from all three classes have been reported (11).

Unfortunately, antifungal drug development is hampered by intrinsic challenges (fungi, like humans, are eukaryotic organisms; therefore, it is difficult to identify compounds that are active against fungal pathogens but not highly toxic to host cells) and by poor financial incentives for pharmaceutical companies (9). One treatment approach that does not rely on the introduction of novel agents is the repurposing of existing drugs in combination. Combination therapy using two or more antifungal drugs is already an established component of therapy for certain fungal infections (e.g., AMB plus 5-fluorocytosine as induction therapy for Cryptococcus neoformans meningitis [12] and combination regimens used to treat multidrug-resistant molds such as Scedosporium spp. [13]). This strategy, however, still relies on the limited number of currently available antifungal agents.

In 1972, investigators observed synergistic activity in Saccharomyces cerevisiae between AMB and rifampin (RIF), an antibacterial RNA synthesis inhibitor (14), or tetracycline, an antibacterial protein synthesis inhibitor (15). AMB plus RIF was later found to be synergistic against several Candida species (16), as was AMB plus the tetracycline analogue minocycline (MIN) against C. neoformans and Candida species (17). The azole fluconazole was subsequently reported to be synergistic with MIN and another tetracycline analogue, doxycycline, against C. albicans (18, 19). To our knowledge, however, these antibacterial-antifungal combinations have not been evaluated in C. auris. We used a novel inkjet printer-assisted checkerboard array synergy method as well as time-kill synergy studies to investigate synergistic activity of combinations of MIN, RIF, voriconazole (VRC; an azole), caspofungin (CAS; an echinocandin), and AMB against a collection of C. auris isolates with a range of drug resistance patterns.

RESULTS

Single drug MIC testing by BMD and digital dispensing method (DDM).

Broth microdilution (BMD) MICs were prepared in triplicate to determine the modal MIC (i.e., the MIC obtained in ≥2 replicates) at 24 h (all drugs) and 48 h (VRC and AMB) using standard Clinical and Laboratory Standards Institute (CLSI) methodology (20). If three sequential doubling dilution values were obtained in the three replicates (e.g., 2, 4, and 8 μg/ml), the middle value was considered the modal MIC. Modal MICs ranged from 0.5 to 2 μg/ml for AMB, from ≤0.016 to >8 μg/ml for VRC, and from 0.063 to 0.5 μg/ml for CAS. Using the tentative C. auris breakpoints proposed by the U.S. Centers for Disease Control and Prevention (CDC) (21), 2 of the strains were resistant to AMB at 24 h and 3 at 48 h, and no strains were resistant to CAS. The CDC has not proposed VRC breakpoints for C. auris, but if CLSI breakpoints for C. albicans for VRC at 24 h were applied (22), 6 of the strains would be classified as resistant. VRC MICs were higher at 48 than 24 h, while AMB MICs were either the same or one doubling dilution higher at 48 h compared to 24 h for all strains. Neither RIF nor MIN exhibited inhibitory activity at the concentrations tested (Table 1; see also Table S1 in the supplemental material).

TABLE 1.

Essential agreement between modal BMD MIC and on-scale DDM MIC results^a

Drug and time	MIC50 and range (μg/ml)		DDM MIC results within:
	BMD	DDM	±1 twofold dilution of modal MIC (n, %)	±2 twofold dilutions of modal MIC (n, %)
AMB, 24 h	1 (0.25–4)	1 (0.25–2)	50/50 (100.0)	50/50 (100.0)
AMB, 48 h	1 (1–4)	1 (0.5–2)	49/49 (100.0)	49/49 (100.0)
CAS, 24 h	0.125 (0.063–1)	0.25 (≤0.031–2)	40/49 (81.6)	48/49 (98.0)
VRC, 24 h	1 (≤0.0156–>8)	0.5 (≤0.0156–4)	35/37 (94.6)	37/37 (100.0)
VRC, 48 h	8 (0.031–>8)	4 (≤0.031–>8)	21/21 (100.0)	21/21 (100.0)
MIN, 24 h	NA	NA	NA	NA
RIF, 24 h	NA	NA	NA	NA
Total			195/206 (94.7)	205/206 (99.5)

^aAMB, amphotericin B; CAS, caspofungin; VRC, voriconazole; MIN, minocycline; RIF, rifampin. MIC₅₀, MIC inhibiting ≥50% of isolates (calculated using each replicate as a separate result). MIC results were not used when >1 skipped well occurred (n = 6), when modal BMD MIC or DDM result was off-scale (n = 33) or when there was no modal BMD MIC (n = 5). NA, essential agreement could not be calculated due to off-scale high modal BMD MICs; all DDM results were also off-scale high for these drugs.

For each condition (i.e., each drug tested against each strain at 24 or 48 h), five DDM MIC values were also obtained: a dedicated MIC test result and four results from the single-drug titrations of each synergy grid in the checkerboard array testing described below (Table S1). When on-scale DDM MIC results were compared to on-scale modal BMD MICs, 94.7% were within ±1 doubling dilution and 99.5% were within ±2 doubling dilutions of the modal MIC (Table 1). For 5 drug-strain pairings, the BMD modal MIC was off-scale; all DDM results for the same pairing were also off-scale in the same direction. Four pairings with an on-scale BMD MIC included off-scale low DDM MICs; in all cases the corresponding BMD MIC was within one doubling dilution of the lowest on-scale result. In antifungal susceptibility testing, essential agreement between a new method and the reference method is generally defined as a MIC value falling within ±2 doubling dilutions of the reference result. Concordance with the modal BMD MIC was highest for AMB (100% of on-scale results within ±1 doubling dilution) and lowest for CAS (81.6% and 98.0% of on-scale results within ±1 and ±2 doubling dilutions, respectively; one DDM result was 4 doubling dilutions above the modal BMD MIC). The relatively lower concordance of CAS results is consistent with prior investigators’ observations of variability in CAS BMD testing results, a phenomenon that is not well understood (23). Overall, our data indicate that the DDM automation method provides accurate and robust antifungal testing results.

Checkerboard array synergy testing using DDM.

Using the checkerboard array assay, we evaluated all two-drug combinations of MIN, RIF, VRC, CAS, and AMB for evidence of synergy. We found that MIN was synergistic against all 10 strains when combined with either AMB or CAS, and RIF was synergistic with AMB against 9 strains. In synergistic combinations, the concentration of MIN at the minimum FIC_I (FIC_I-MIN) ranged from 4 to 16 μg/ml and was ≤8 μg/ml in 12 of 22 cases, while the concentration of RIF was 8 μg/ml in 5 cases and 16 μg/ml in 4 cases. All other combinations were synergistic against ≤2 strains. Three of the 5 combinations that were not synergistic against any strains were antagonistic against 2 strains. (Table 2 and Table S2).

TABLE 2.

Checkerboard array synergy results^b

Drug combination	% Strains against which combination was synergistic (n = 10)	% Strains against which combination was antagonistic (n = 10)	Minimum FICIa (range)	Concn of drugs at individual MICa (range) (μg/ml)	Concn of drugs at minimum FICIa (range) (μg/ml)
AMB + MIN	100	0	0.094–0.5	0.5–2 (AMB), >64 (MIN)	0.031–0.5 (AMB), 8–16 (MIN)
CAS + MIN	100	0	0.094–0.375	0.125–2 (CAS), >64 (MIN)	0.031–0.125 (CAS), 4–16 (MIN)
AMB + RIF	90	0	0.188–0.5	0.5–2 (AMB), >32 (RIF)	0.031–0.5 (AMB), 8–16 (RIF)
MIN + VOR	20	0	0.156–0.188	>64 (MIN), 0.5–0.5 (VOR)	4–8 (MIN), 0.063–0.063 (VOR)
CAS + VOR	10	0	0.313	0.25 (CAS), 4 (VOR)	0.063 (CAS), 0.5 (VOR)
MIN + RIF	0	0	NA	NA	NA
AMB + CAS	0	0	NA	NA	NA
AMB + VOR	0	20	NA	NA	NA
CAS + RIF	0	20	NA	NA	NA
RIF + VOR	0	20	NA	NA	NA

^aOnly includes values from strains against which the combination was synergistic.

^bFIC, fractional inhibitory concentration; FIC_I, FIC index; AMB, amphotericin B; CAS, caspofungin; VOR, voriconazole; MIN, minocycline; RIF, rifampin.

Time-kill synergy testing.

The three combinations that demonstrated synergy against ≥9 strains by the checkerboard array assay (MIN plus AMB, MIN plus CAS, and RIF plus AMB) were tested using the time-kill method against a subset of 5 of the C. auris strains, which were chosen to represent a range of susceptibility profiles (Table 3, Fig. 1). Each drug pair was tested, at two combinations of concentrations of individual drugs, against each of the 5 isolates and was considered synergistic and/or fungicidal if at least one of these combinations met criteria for synergistic or fungicidal activity, respectively. At 24 h, AMB plus RIF and AMB plus MIN were synergistic against all 5 strains, while at 48 h, AMB plus RIF was synergistic against 4 strains and AMB plus MIN against 3 strains. In combination with RIF, AMB showed fungicidal activity against 2 strains at 24 and 48 h, and against an additional strain at 24 h, at concentrations at which it was not fungicidal alone. In combination with MIN, AMB showed fungicidal activity against 2 strains at 24 and 48 h at concentrations at which it was not fungicidal alone. The combination of CAS plus MIN was not synergistic or fungicidal against any strains.

TABLE 3.

Time-kill results^c

Combination	Synergy				Fungicidal activity
	Combination vs most active single agent (median, range)a (log10 CFU/ml)		% Strains (n = 5) against which combination was synergisticb		Combination vs starting inoculum (median, range)a (log10 CFU/ml)		% Strains (n = 5) against which combination was fungicidalb
	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h
AMB + RIF	−2.40, −4.82–0.0	−1.84, −4.78–0.07	100	60	−2.74, −3.52–1.57	−0.46, −3.21 to −1.91	60	40
AMB + MIN	−2.19, −3.48–0.62	−1.03, −4.99–0.11	100	80	−1.06, −3.30–0.13	0.26, −3.30–2.02	40	40
CAS + MIN	0.51, −0.46–1.07	0.81, −0.34–1.26	0	0	−0.76, 2.46–0.37	−0.91, −2.0–0.44	0	0

^aMedian and range of two concentration combinations for each of 5 strains.

^bAt one or both concentration combinations.

^cAR Bank, CDC & FDA Antibiotic Resistance Isolate Bank; AMB, amphotericin B; CAS, caspofungin; MIN, minocycline; RIF, rifampin. Synergy was a ≥2-log₁₀ reduction in CFU/ml in the combination compared to the most active agent alone at the specified time point. Fungicidal was a ≥3-log₁₀ CFU/ml reduction compared to starting inoculum at the specified time point.

FIG 1.

Time-kill synergy graphs. Strain numbers refer to CDC and FDA Antibiotic Resistance Isolate Bank designations. The dashed line indicates the assay lower limit of detection. Filled (red) symbols indicate synergistic concentration combinations.

DISCUSSION

We identified three combinations of antibacterial and antifungal drugs (AMB plus RIF, AMB plus MIN, and CAS plus MIN) that demonstrated synergistic activity by checkerboard array against ≥90% of C. auris strains evaluated; the two AMB-containing combinations were also synergistic by time-kill synergy testing at one or more concentration combinations against the strains evaluated. We thereby demonstrate synergistic activity between antibacterial and antifungal agents against the emerging and highly multidrug-resistant pathogen C. auris.

We hypothesize that the mechanism of synergy for these combinations involves impairment in cell wall or membrane integrity by the antifungal drug, permitting entry of an antibacterial agent that would otherwise be unable to access the intracellular compartment of a fungal cell. Echinocandins such as CAS act by inhibiting β-(1,3)-glucan synthase, thereby impeding cell wall synthesis and impairing cell wall integrity, resulting in increased vulnerability of the cell to osmotic pressure (24). AMB has traditionally been understood to act by forming ion channels in the lipid bilayer of the fungal cell membrane, thereby permeabilizing the cell and ultimately causing cell death (25, 26). However, recent work suggests that large aggregates of AMB, which assemble outside the membrane and act as “sterol sponges” that kill cells by extracting ergosterol from the lipid bilayer, play a more important role in cell death than do the ion channels (27). It is conceivable that at the subinhibitory concentrations at which it demonstrates synergy with MIN and RIF, AMB could be exerting sublethal poration activity without aggregate-based cytotoxicity, allowing increased entry of the antibacterial drugs.

Our hypothesis is supported by prior observations that tetracycline inhibits protein synthesis in isolated yeast ribosomes (28) and that RIF appears to inhibit RNA polymerase in yeast (14). These drugs, therefore, may have targets in yeast cells analogous to those in bacteria but accessible in yeast only in the setting of disruption of cell membrane or cell wall integrity. A similar phenomenon is well established in bacteria, whereby drugs that are unable to bypass the defenses of the Gram-negative outer membrane under normal circumstances demonstrate activity in the presence of low levels of membrane-permeabilizing agents such as polymyxins (29, 30).

We used two separate synergy testing methods in an effort to increase the robustness of our results and found that while two combinations demonstrated consistent synergy using both methods, a third was synergistic only by the checkerboard array method and not the time-kill method. It is possible that this finding reflects a limitation of the synergistic activity of this combination (CAS plus MIN). Alternatively, the finding may reflect the limitations of the time-kill testing method for evaluation of CAS activity, as our single-drug CAS killing curves did not demonstrate concentration-dependent inhibitory or fungicidal effects (see Fig. S1 in the supplemental material) within the concentration range used in the presented study, a finding that was previously observed with this drug in C. auris (31).

In this study, we also demonstrated the utility of an automated inkjet printer-assisted digital dispensing method for MIC and checkerboard array synergy testing in yeast. Manual synergy testing is an error-prone and time-consuming process, and automation allows for significantly higher throughput of the technique, thereby facilitating more rapid investigation of novel combinations. This use of the DDM for MIC and synergy testing of bacteria, first described in our laboratory (32, 33), has been adopted by the U.S. Centers for Disease Control and Prevention to test drug combinations against multidrug-resistant bacterial pathogens (34) and has the potential for similar use for fungal pathogens such as C. auris.

In vitro synergy testing has certain intrinsic limitations and is not always a direct indicator of in vivo efficacy (35). However, identification of combinations with in vitro activity provides preliminary data to suggest regimens that may ultimately prove to be of therapeutic benefit. Given the paucity of new antifungal drugs in the development pipeline, regimens that involve readily available drugs for which extensive pharmacokinetic and safety data already exist offer the potential for expedited clinical evaluation and implementation. The concentrations of antifungal drugs active in the combinations identified were very low and easily clinically achievable. Although no interpretive criteria (i.e., susceptibility breakpoints) exist for MIN or RIF for yeast, the concentration of MIN in synergistic combinations was ≤8 μg/ml in more than half the instances of synergy we identified; such concentrations would be considered susceptible (4 μg/ml) or intermediate (8 μg/ml) for Gram-positive bacterial pathogens such as Staphylococcus aureus and Enterococcus spp. by CLSI (36), suggesting plausible clinical applicability. In topical or local applications (e.g., ophthalmic drops or catheter coating), antibiotics can often be used at concentrations greater than can be safely achieved systemically (37); thus, the combinations we identified could also have potential use in these scenarios.

The need for new therapeutic options for C. auris has been underscored since the advent of the COVID-19 pandemic, with recent reports from India (3), Colombia (4), and the United States (5) describing C. auris infection as a complication of SARS-CoV-2-related critical illness. In addition to possible direct applicability, if the combinations we evaluated act, as we predict, by allowing access of MIN and RIF to intracellular targets in yeast, this information may guide future antifungal drug development approaches. Evaluation of combinations in animal models and, ultimately, in clinical trials will be critical future steps in establishing clinical activity. In the absence of a predictable timeline for the introduction of novel antifungal agents, repurposing existing drugs may be our best hope in identifying new treatment approaches for patients with infections caused by C. auris and other emerging multidrug-resistant fungal pathogens.

MATERIALS AND METHODS

Fungal isolates.

Ten C. auris isolates were obtained from the CDC and FDA Antibiotic Resistance (AR) Isolate Bank (Atlanta, GA). Five strains belong to clade I (South Asian), 1 to clade II (East Asian), 2 to clade III (African), and 2 to clade IV (South American). Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 29213 were obtained from the American Type Culture Collection (Manassas, VA). All strains were colony purified, minimally passaged, and stored at −80°C in tryptic soy broth (BD Diagnostics, Franklin Lakes, NJ) with 50% glycerol (Sigma-Aldrich, St. Louis, MO).

Antimicrobial agents.

Voriconazole (VRC) was obtained from Acros Organics (Pittsburgh, PA). Caspofungin (CAS) was obtained from Carbosynth (Oakbrook Terrace, IL). Amphotericin B (AMB) was obtained from Sigma-Aldrich (St. Louis, MO). Minocycline (MIN) was obtained from Chem Impex International (Wood Dale, IL). Rifampin (RIF) was obtained from Fisher Scientific (Waltham, MA). Antimicrobial stocks were prepared in dimethyl sulfoxide (Sigma-Aldrich), with the exception of minocycline stock used for time-kill experiments, which was prepared in water. All antimicrobials were quality control (QC) tested with C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 (VRC, CAS, and AMB) or with E. coli ATCC 25922 and S. aureus ATCC 29213 (MIN and RIF) and were used only if they produced a MIC result in the QC range accepted by CLSI (22, 36). After passing QC, stocks were aliquoted and stored at −80°C (antifungal drugs) or −20°C (antibacterial drugs) until use. Aliquots were discarded after a single use.

Antimicrobial susceptibility testing.

Manual broth microdilution (BMD) testing of C. auris isolates was performed in triplicate for each drug according to CLSI guidelines (20). Strains were isolation streaked on Sabouraud dextrose agar plates (Thermo Scientific, Waltham, MA) and incubated for 24 h at 35°C in ambient air. BMD plates were made by preparing serial 2-fold dilutions of antimicrobial agents at twice the desired final concentration in 100 μl RPMI 1640 with l-glutamine (Cytiva, Marlborough, MA), prepared with morpholinepropanesulfonic acid buffer (Fisher Scientific, Waltham, MA) in clear, round-bottom, untreated 96-well plates (Evergreen Scientific, Los Angeles, CA, USA). Fungal inocula were prepared by suspending colonies from the overnight plates in sterile 0.9% sodium chloride and adjusting to a 0.5 McFarland standard, diluting this suspension 1:1,000 in RPMI, and then adding 100 μl of the diluted suspension to each well for a final volume of 200 μl and cell density of 0.5 × 10³ to 2.5 × 10³ CFU/ml. Negative (sterility) control and growth control wells were included in each row. Plates were then incubated at 35°C in ambient air. At 24 h (all drugs) and 48 h (VRC and AMB), plates were removed from the incubator and vortexed on a plate shaker for 4 min, after which optical density at 600 nm (OD₆₀₀) readings were taken with a Tecan Infinite M1000 Pro microplate reader (Tecan, Morrisville, NC) to quantify growth. OD₆₀₀ readings were normalized by subtracting the average reading of the negative-control wells from the same plate, which contained media without yeast, and then the percent inhibition for each well was calculated relative to the average of the positive growth control wells of the same isolate from the same plate. For CAS and VRC, the lowest concentration of drug that reduced growth by at least 50% was considered the MIC; for AMB, the lowest concentration of drug that reduced growth by at least 90% was considered the MIC (20). If a skipped well occurred, MIC testing was repeated.

MIC testing was also performed using an automated inkjet printer-assisted digital dispensing method (DDM) adapted from a method developed in our laboratory for MIC testing of bacteria (32). Initial 0.5 McFarland yeast suspensions were prepared in RPMI as described above and then diluted 1:2,000 in RPMI. The same final volume and cell density in each well was achieved by adding 200 μl of this diluted suspension to each well in a 96-well plate. Antimicrobial drugs were then dispensed by the HP D300 digital dispenser instrument (HP, Inc., Palo Alto, CA) into the yeast suspension in the wells. The volume of antimicrobial stock dispensed into the wells ranged from 53.1 nl to 844 nl, constituting 0.03 to 0.42% of total well volume. Incubation and growth interpretation were carried out as in the BMD method described above.

Checkerboard array synergy testing.

The DDM method described above was used to prepare checkerboard arrays in which two drugs were each dispensed in 7 to 9 2-fold dilutions. Each two-drug combination was tested against every C. auris strain, with growth determinations made after 24 h of incubation. Growth inhibition was determined as described above. For combinations in which both drugs use 90% inhibition for MIC determination (AMB plus MIN, AMB plus RIF, and MIN plus RIF), combination wells were considered inhibitory when growth was inhibited by 90% relative to growth control wells; for all other combinations, combination wells were considered inhibitory when growth was inhibited by at least 50% relative to growth control wells. The fractional inhibitory concentration index (FIC_I) for each drug was calculated as FIC_I = [MIC drug A (in combination)/MIC drug A (alone)] + [MIC drug B (in combination)/MIC drug B (alone)]; if the MIC of a drug was off-scale, the highest concentration tested was assigned an FIC of 0.5. A minimum FIC_I (FIC_I-MIN) of ≤0.5 was considered synergistic, an FIC_I-MIN of >4.0 antagonistic, and an intermediate FIC_I-MIN indifferent (38).

Time-kill synergy testing.

Antimicrobial stocks were diluted in 9.5 ml of RPMI 1640 in 25- by 150-mm glass round-bottom tubes to the appropriate starting concentrations. Antimicrobial concentrations for the time-kill study were based on the checkerboard array results. For a given drug combination and strain, we identified the well in the checkerboard array with the lowest concentration of MIN or RIF among wells with an FIC_I of ≤0.5 and noted the concentration of both drugs in that well. Time-kill drug concentrations were then chosen as follows. For AMB plus RIF, RIF at the concentration in this well was tested with AMB at the concentration in the well and AMB at twice this concentration. For AMB plus MIN, the same formula was used, except that MIN was tested at twice the concentration in the well because there was no effect at lower concentrations in any strain tested. For CAS plus MIN, MIN at the concentration in the well and twice that concentration was tested in combination with CAS at the concentration in the well. Negative (sterility) control and positive growth control tubes containing no antimicrobials were also prepared. A 1.0 McFarland suspension of yeast cells from an overnight plate was prepared in 0.9% sodium chloride, and 0.5 ml of this suspension was added to each tube for a final starting concentration of 1 × 10⁵ to 5 × 10⁵ CFU/ml (39). Cultures were incubated with shaking in ambient air at 35°C for 48 h. At 0, 3, 6, 24, and 48 h, aliquots were removed from the culture tube and a 10-fold dilution series was prepared in 0.9% sodium chloride. A 10-μl drop from each dilution was plated onto Sabouraud dextrose agar and incubated overnight. The colonies within each drop were then counted and cell density was calculated (40). Only dilutions containing 3 to 40 colonies per drop were counted in order to prevent quantification distortion associated with very low numbers of colonies while ensuring that individual colonies could be distinguished by eye (41). If more than one dilution for a given sample was usable, the cell densities of the two drops were averaged. If no drops were usable, the densities for consecutive drops above and below the usable range were averaged. The lower limit of detection with this method is 300 CFU/ml. A combination was considered synergistic if it resulted in a ≥2-log₁₀ reduction in CFU/ml compared to the most active agent alone and fungicidal if it resulted in a ≥3-log₁₀ CFU/ml reduction compared to the starting inoculum. Synergy and fungicidal activity were evaluated at 24 and 48 h.

Data analysis.

Data output from plate readings was visualized using Microsoft Excel (Microsoft Corporation, Redmond, WA). A custom Python script was used to normalize MIC and synergy results and to calculate and visualize growth inhibition.