Evaluation of the synergistic antifungal activity of micafungin and voriconazole plus sertraline against Candida auris.

Candida auris is an emerging global public health threat. It is an opportunistic yeast that usually affects critically ill patients in healthcare settings and is characterized by reduced susceptibility to multiple antifungal classes. Combination therapy with antifungals and repurposed drugs is a feasible alternative to overcome this problem. The aim of this study was to examine the in vitro interactions and potential synergy of micafungin (MFG) and voriconazole (VRC) plus the antidepressant sertraline (SRT) against clinical isolates of C. auris. Conventional antifungal testing was first performed with the three drugs according to the CLSI methodology. Drug interactions were determined by the checkerboard microdilution assay using the fractional inhibitory concentration (FIC) index. Synergistic interactions were noted with the combination of MFG and SRT plus VRC with FIC values of 0.37 to 0.49 for some strains. Indifferent interactions were observed when MFG was combined with SRT with just one exception (FIC 0.53). No antagonism was observed for any combination. The combination of VRC with MCF or SRT may be relevant for treating C. auris infections.

Introduction

Initially isolated in 2009 from otic drainage of a Japanese patient, Candida auris is an opportunistic nosocomial yeast pathogen that emerged simultaneously on five continents, spreading to over 30 countries since its first description [1-3]. Major risk factors for developing C. auris infections are longer hospital stays, particularly in intensive care units (ICU) [4, 5], current exposure to indwelling medical devices, and having undergone invasive procedures [6, 7]. Candidemia is the most common fungal infection, especially in low-immunity patients in the ICU; it is associated with a poor outcome and overall mortality approaching 68% [6].

As a result of its easy transmission, provoking global outbreaks, and exceptional multidrug resistance, C. auris was recently classified as an “urgent health threat” by the US Centers for Disease Control and Prevention (CDC) [8]. This fungus is characterized by reduced susceptibility to azoles, polyenes, and echinocandins [9]. Even if the latter are considered first-line therapy for C. auris infections [10], resistance to these agents and therapeutic failures have been reported [11], severely limiting available treatment options. Thus, alternative strategies are urgently needed to overcome this alarming crisis.

Combination therapy is a convenient approach defined as the co-application of two or more drugs with distinct biological targets to achieve a synergistic interaction, increasing the probability of therapeutic success and limiting the emergence of drug resistance [12, 13]. Few antifungal combinations have been evaluated against multidrug-resistant C. auris strains. A good example is the recently reported in vitro synergistic interaction between micafungin and voriconazole [14]. Alternatively, the combination of antifungals with “off-patent” non-antifungal drugs has also been investigated in the context of drug repurposing for treating invasive infections [15]. In this sense, sertraline, the most frequently prescribed antidepressant, exhibited in vitro antifungal activity against C. auris [16], but this effect has not been evaluated in combination with antifungals. This study examined the in vitro interactions and potential synergy between sertraline and two antifungals with different modes of action (micafungin and voriconazole) against clinical isolates of C. auris. Therefore, the tested hypothesis of the work was the following: the in vitro combinations of micafungin and voriconazole plus sertraline exert synergistic antifungal activity against C. auris.

Material and methods

Ethics statement

This study was evaluated and approved by the local Ethics Committee of the Universidad Autónoma de Nuevo León (registration number: MB22-00001), and was conducted in agreement with Good Laboratory Practices.

Clinical isolates

Twelve strains previously identified by DNA multilocus sequence typing as C. auris from an outbreak of COVID-19-associated C. auris infections in a tertiary care hospital in Monterrey, Mexico [5] were included in this study. Isolates were recovered from blood (6/12), urine (8/12), and both (2/12). All belong to the clade IV (South American). Fungal strains were retrieved from frozen stocks on 15% glycerol suspensions maintained at −70 °C and cultivated in Sabouraud dextrose agar (SDA) at 37 °C for 24 h before each experiment.

Agents

The drugs tested included micafungin (MFG) (Mycamine®; Astellas Pharma, Deerfield, IL, USA), voriconazole (VRC) (Pfizer, New York, NY, USA), and sertraline (SRT) (TCI Chemicals Inc., New York, NY, USA). The drugs were dissolved in 100% dimethyl sulfoxide (DMSO) (Bio Basic, USA) [17, 18] to obtain stock solutions of 3200 µg/mL and kept at −80 °C until use.

Conventional antifungal testing

Broth microdilution testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 [17]. In brief, serial two-fold dilutions were prepared for each agent. Further dilutions were made in RPMI-1640 with L-glutamine and buffered with 165 mM MOPS (Hardy Diagnostics, USA), reaching final concentrations ranging from 0.03 to 16 µg/mL for VRC and from 0.015 to 8 µg/mL for MFG. Finally, the inoculated plates were incubated at 35 °C and read after 24 h. The minimum inhibitory concentration (MIC) endpoint was visually determined as the lowest drug concentration that produced a significant decrease (≥ 50%) in growth compared to the growth of the drug-free control. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were quality-control strains. MIC breakpoints for interpreting results were those recommended by the CDC (http://www.cdc.gov/fungal/candida-auris/c-auris-antifungal.html). Experiments were conducted in duplicate on different days.

To obtain the minimum fungicidal concentration (MFC), 0.1 mL of each serial drug dilution was taken from each well with no visible growth and poured onto SDA [19]. Plates were incubated at 37 °C for 24–48 h. The MFC was the lowest drug concentration yielding less than five yeast colonies.

Antifungal combination testing

Interactions between MFG and VRC, and these with SRT, were investigated using the checkerboard microdilution assay based on the CLSI reference method with 96-well microtiter plates (Corning, USA) [20, 21]. Broadly, drug dilutions were prepared at four times the final concentration ranging from 0.25 to 32 µg/mL for SRT, 0.03 to 4 µg/mL for VRC, and 0.06 to 4 µg/mL for MFG. These ranges depended on MIC results previously obtained for each strain. For two-dimensional microplate preparation, 50 µL of each concentration of SRT was added into the wells of columns 2 through 9; then, 50 µL of the partner drug (MFG or VRC) was added into the wells of rows A through G, respectively. For the combination MFG-VRC, the echinocandin was collocated from rows A to G and the triazole from columns 2 to 9. The wells of column 1 and row H contained the respective drugs alone. Column 10 was the drug-free well and the growth control, while column 11 was the uninoculated well corresponding to the sterile control. Trays were kept at −80 °C until use.

Once trays were defrosted, the inoculum was adjusted spectrophotometrically and further diluted with RPMI 1640; 100 μL of the suspension was added to each plate well, except for the sterile control. Trays were incubated at 37 °C for 24 h, and a 100% inhibition reading was recorded with a convex mirror [22, 23]. Experiments were performed in duplicate on different days. Drug combination interactions were evaluated based on the fractional inhibitory concentration (FIC) index: FIC A (MIC drug A in combination/MIC drug A alone) + FIC B (MIC drug B in combination/MIC drug B alone). The summation of the FIC (∑FIC) index was calculated for each drug combination and strain and its interpretation was synergy, ≤ 0.5; partial synergy, > 0.5 to < 1.0; additivity, 1.0; indifference, > 1.0 to < 4.0; antagonism, ≥ 4.0 [17].

Results

All C. auris strains tested in this study were susceptible to MFG (range: 0.125–0.5 µg/mL, MIC50: 0.25 µg/mL, MIC90: 0.5 µg/mL) and VRC (range: 0.03–1 µg/mL, MIC50: 0.25 µg/mL, MIC90: 1 µg/mL), with a median of 2 µg/mL and 1 µg/mL for MFG and VRC, respectively. Regarding SRT, MICs ranged from 4 to 8 µg/mL, with an MIC50 and MIC90 of 8 µg/mL and a median MFC of 16 µg/mL.

The results of the drug combinations evaluated in this study are summarized in Table 1. When SRT was combined with MFG, the ∑FIC ranged from 0.53 to 1.03, indicative of no-interaction (or indifferent activity) except for strain #3, for which a partial synergy was observed (∑FICmin 0.53). When this drug was combined with VRC, a synergistic interaction was noted in three strains (∑FIC 0.37 to 0.49) in addition to a partial synergy evidenced in strain #12 (∑FICmin 0.54); moreover, the MIC ranges of SRT and VRC were reduced to 0.5 to 4 µg/mL and 0.03 to 0.06 µg/mL, respectively. On the other hand, synergistic effects of VRC plus MFG were shown against four C. auris isolates (∑FIC 0.37 to 0.49) with a reduction in MIC ranges of VRC and MFG to 0.03 to 0.125 µg/mL and 0.25 to 0.5 µg/mL, correspondingly. No antagonistic activity was observed for any combination tested.

Table 1

Synergy results for the antifungal combinations tested in this study against 12 strains of C. auris

		SRT + MFG					SRT + VRC					VRC + MFG
		MIC (µg/mL)					MIC (µg/mL)					MIC (µg/mL)
Strain	Sourcea	SRT	MFG	SRT/MFG	∑FICminb	INTc	SRT	VRC	SRT/VRC	∑FICminb	INTc	VRC	MFG	VRC/MFG	∑FICminb	INTc
1	B	8	0.5	0.25/0.5	1.03	IND	8	0.25	4/0.03	0.62	IND	0.25	0.5	0.125/0.25	1	IND
2	U	8	0.5	0.25/0.5	1.03	IND	8	0.25	4/0.03	0.62	IND	0.25	0.5	0.125/0.25	1	IND
3	B/U	8	2	0.25/1	0.53	PSYN	8	0.25	2/0.06	0.49	SYN	0.25	2	0.06/0.25	0.37	SYN
4	U	4	1	4/0.06	1.06	IND	4	0.25	2/0.03	0.62	IND	0.25	1	0.03/0.5	0.62	IND
5	B	8	1	0.25/1	1.03	IND	8	0.25	2/0.06	0.49	SYN	0.25	1	0.03/0.5	0.62	IND
6	B	8	1	0.25/1	1.03	IND	8	0.25	4/0.06	0.74	IND	0.25	1	0.06/0.25	0.49	SYN
7	B/U	8	1	0.25/1	1.03	IND	8	0.25	2/0.03	0.37	SYN	0.25	1	0.06/0.25	0.49	SYN
8	U	8	1	0.25/1	1.03	IND	8	0.125	2/0.06	0.74	IND	0.125	1	0.03/0.5	0.74	IND
9	U	8	1	0.25/1	1.03	IND	8	0.125	2/0.06	0.71	IND	0.125	1	0.06/0.25	0.71	IND
10	U	8	1	0.25/1	1.03	IND	8	0.125	2/0.06	0.71	IND	0.125	0.5	0.06/0.25	0.71	IND
11	B	8	1	2/0.5	0.75	IND	8	0.125	1/0.06	0.61	IND	0.125	0.5	0.03/0.25	0.49	SYN
12	U	8	1	0.25/1	1.03	IND	8	0.125	0.5/0.06	0.54	PSYN	0.125	1	0.03/0.5	0.74	IND

MIC minimum inhibitory concentration, SRT sertraline, MFG micafungin, VRC voriconazole

aSource: B blood, U urine

bLowest fractional inhibitory concentration

cINT interaction, IND indifference, PSYN partial synergy, SYN synergy

Discussion

C. auris is an emerging global multidrug-resistant nosocomial pathogen considered a major threat to healthcare settings. It exhibits a clade-specific resistance to fluconazole (FLC) but varying susceptibility to other triazoles, amphotericin B (AMB), and echinocandins [6, 24]. Resistance rates for FLC, VRC and AMB were nearly 90%, 3–73%, and 13–35%, respectively [6, 24, 25]. Roughly 4% of C. auris isolates resistant to echinocandins have been reported in the USA [26]. In this context, combination drug therapy is an attractive strategy to fight and overcome antifungal resistance in C. auris and presents known benefits over the single use of drugs [27].

Limited antifungal combinations have been evaluated against C. auris. One of the most promising combinations is triazoles plus echinocandins [14, 28, 29]. Pfaller et al. [28] evaluated the interaction of VRC or isavuconazole (ISA) in combination with anidulafungin (AFG) against isolates of C. auris using the FIC index analysis. They reported synergism or partial synergism against most isolates, mainly for the combination of ISA with AFG. Later, Fakhim et al. [14] communicated that the combination of MFG plus VRC exhibited synergistic activity against all ten multidrug-resistant strains of C. auris belonging to the South Asian clade, determined by the FIC index. More recently, Caballero et al. [29] observed the synergism of ISA and echinocandins against six C. auris bloodstream isolates from an outbreak in a Spanish hospital. Their findings were consistent with the two previous reports. In our study, we found a synergistic interaction of VRC plus MFG in four C. auris strains in accordance with Fakhim et al. [14]. The proportion of synergic isolates they reported is higher than what we encountered, this finding may be because the strains tested belonged to different clades and had strain-specific behaviors.

Drug repurposing is another increasingly interesting alternative approach to search for new potential antifungal candidates. Several reports have described good in vitro antifungal activity of the antidepressant SRT for Cryptococcus spp. [30] Moreover, in vivo studies have revealed that this repurposed drug has a role alone or in combination for treating invasive fungal infections [31, 32]. Recently, Gowri et al. [16] reported that SRT inhibited the yeast to hyphae conversion of C. auris and biofilm formation upon treatment. In our study, SRT exhibited antifungal activity against all the C. auris isolates tested with MICs ranging from 4 to 8 µg/mL. These levels are considerably lower than those previously communicated by Gowri et al. [16] Additionally, while the combination of SRT plus MFG overall redounded in indifference except for strain #3, the combination of SRT plus VRC resulted in synergism for three strains and partial synergy for strain #12.

Even though our study shares some limitations with the reports mentioned above, such as the reduced number of isolates tested, all belonging to the clade IV (South American), and the absence of strains resistant to the antifungals used, this work fills a gap as there is no report on the efficacy of antifungal combinations against C. auris from the Monterrey, Mexico outbreak. Furthermore, to our knowledge, this is the first attempt to use SRT in combination against C. auris with encouraging results.

Conclusions

The tested hypothesis of the work was corroborated. We have shown that interaction between SRT or MFG plus VCZ exhibited synergistic antifungal activity against some strains of C. auris.