Indifferent effect of nonsteroidal anti-inflammatory drugs (NSAIDs) combined with fluconazole against multidrug-resistant Candida auris

Background and Purpose: Emergence and development of antifungal drug resistance in Candida species constitute a serious concern. Candida auris as an emerging multidrug-resistant fungus is the most important public health threat with high levels of mortality and morbidity. Almost all C. auris isolates are resistant to fluconazole, and there have been reports of elevated minimum inhibitory concentrations (MICs) to amphotericin B and echinocandins. To overcome the growing challenge of antifungal resistance, a valuable alternative option would be the use of drug combination. Materials and Methods: The present study evaluated the in vitro combination of nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, diclofenac and aspirin with fluconazole against fluconazole-resistant C. auris in comparison to other fluconazole-resistant Candida species, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei originating from patients with candidiasis. Results: The MIC ranges of fluconazole-ibuprofen and fluconazole-diclofenac decreased from 32-256 to 32-128 and 16-256 µg/ml, respectively and remained the same for fluconazole-aspirin against C. auris. However, the combination of fluconazole with ibuprofen resulted in a synergistic effect for 5 strains, including C. albicans (n=2), C. tropicalis (n=1), C. glabrata (n=1), and C. krusei (n=1), by decreasing the MIC of fluconazole by 2-3 log2 dilutions. Conclusion: Although the interaction of NSAIDs with fluconazole was not synergistic against fluconazole-resistant C. auris isolates, no antagonism was observed for any combinations. Therefore, combination with newer azole agents needs to be conducted.


Introduction
luconazole-resistance has emerged among Candida species since the beginning of the fluconazole era as a result of the selective pressure caused by prophylaxis and therapeutic management [1]. Emergence and development of antifungal drug resistance in Candida species isolates have serious concerns in terms of therapeutic failures mainly related to echinocandins and azoles resistance [2]. Candida auris has become an emerging serious public health threat [3] since the recognition of the first case from Japan in 2009 [4].
During the last ten years, the number of reported cases has drastically increased, including six outbreaks during 2013 and 2017 [5][6][7][8][9][10]. Candida auris infection has recently been reported from all continents. However, sporadic cases of C. auris have been reported from Austria, Belgium, Malaysia, the Netherlands, Norway, Russia, Switzerland, United Arab Emirates, and Iran [11][12]. Challenges in laboratory detection, unique predisposition to cause nosocomial outbreaks, and multidrug-resistant phenotypes of C. auris are the rule [13]. As a consequence of its ability to overcome antifungal therapy, C. auris continuously increases its ecological niche and resistance to environmental conditions. Accordingly, the multidrug-resistant phenotypes of C. auris may be associated with hospital-acquired infections [14][15]. Although most C. auris isolates are resistant to fluconazole and elevated the minimum inhibitory concentrations (MICs) of amphotericin B and echinocandins have been reported, echinocandins have been the most active drugs up to now [16][17][18]. Therefore, to overcome the growing challenge of antifungal resistance, a valuable alternative option would be the use of drug combinations [19].
Successful combination therapy for the treatment of fungal infectious diseases can achieve broader antifungal coverage and potentially reduce acquired resistance. Nonsteroidal anti-inflammatory drugs (NSAIDs), i.e., ibuprofen, diclofenac, and aspirin, show antifungal activity against Candida species alone or in combination with antifungal agents [20][21][22][23][24][25]. Antifungal activity of NSAIDs is related to the inhibition of the cyclooxygenase enzyme (COX) that leads to decreased prostaglandin levels. It seems that the suppression of oxylipins, oxygenated fatty acid metabolites derived from arachidonic acid, by NSAIDs can reduce the hyphal formation of C. albicans [20,25].
Although the potential antifungal activities of NSAIDs against Candida species, such as the changes in prostaglandin production, reduction of extracellular polysaccharide, decrease in hyphal and biofilm formations, which may provide evidence for a combination strategy against pathogenic yeast, are known [20][21][22][23][24][25], no study has investigated the combination of NSAIDs and fluconazole against multi drug resistant C. auris. Therefore, the current study evaluated the in vitro combination of NSAIDs (i.e., ibuprofen, diclofenac, and aspirin) with fluconazole against fluconazole-resistant C. auris in comparison to other fluconazole-resistant Candida species.

In vitro antifungal susceptibility testing
Fluconazole MICs were determined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) ( Table 1) [27,28]. Candida albicans (ATCC 64124) was used as a reference strain for azole resistance with mutation in the azole target Erg11 [29].

In vitro combination testing
A checkerboard microdilution assay based on the CLSI reference technique was performed in 96-well microtiter plates to evaluate the in vitro interactions between NSAIDs (i.e., ibuprofen, diclofenac, and aspirin) and fluconazole against azole-resistant Candida species [30]. The drugs were dissolved in 100% dimethyl sulfoxide (DMSO), and the concentrations were within the range of 0.5 to 128 µg/ml for fluconazole (FLZ; Pfizer, Groton, CT, USA), 32 to 2048 µg/ml for ibuprofen (IBR, Sigma), and aspirin (ASA, Sigma) and 64 to 4096 µg/ml for diclofenac (DIC, Sigma). Briefly, 50 µl of each concentration of fluconazole was dispensed into the columns of 1 to 10, and 50 µl of NSAIDs (IBR or DIC or ASA) was added to the rows of A to G of 96-well microplates. The H row and column 11 contained fluconazole and NSAIDs alone, respectively. In addition, column 12 was used as the drug-free growth control. The inoculum was prepared using fresh colonies, and their density was adjusted to 1-3 × 10 3 CFU/ml at 530 nm wavelength to a percentage transmission within the range of 75-77% according to CLSI M27-S4 guide [26].
For each drug combination plate, 100 µl of inoculum was added to all the wells, and the plates were incubated at 35 °C for 24 h. The test was performed in duplicate on two separate days. The MICs were visually determined as the lowest concentration of drug that reduced growth (≥50% inhibition) in comparison to growth controls. For the determination of drug interactions, fractional inhibitory concentration index (FICI) was calculated as MIC drug A in combination/MIC drug A alone plus MIC drug B in combination/MIC drug B alone and interpreted as synergism (FICI≤0.5), indifference (0.5<FICI≤4), and antagonism (FICI>4) [30]. Table 1 summarizes the results of the MICs of fluconazole and NSAIDs (i.e., ibuprofen, diclofenac, and aspirin) alone and in combination. The MIC ranges of the individual tested NSAIDs against C. auris isolates were 256-4096, 512-8192, and 1024-4096 µg/ml for ibuprofen, diclofenac, and aspirin, respectively. However, the MIC range of fluconazole against C. auris was 32-256 µg/ml. Based on MIC values, all tested strains were fully resistant to fluconazole, except for one strain of C. glabrata that was susceptible dose-dependent, and the MIC range of fluconazole against other non-albicans species was 16-256 µg/ml. Checkerboard microdilution assays of C. auris showed that when fluconazole was combined with NSAIDs, the MIC ranges of fluconazole-ibuprofen and fluconazole-diclofenac decreased from 32-256 to 32-128 and 16-256 µg/ml, respectively. In addition, it remained the same for fluconazole-aspirin (Table 1) suggestive of the indifferent combinations between fluconazole with ibuprofen, diclofenac, or aspirin (FICI>0.5 to ≤4).
Synergistic interactions between fluconazole and aspirin were recorded for three strains and observed in only one instance synergism when fluconazole was combined with diclofenac. None of the analyzed data sets had FICI higher than 4, indicating that no antagonism was observed except for one C. tropicalis strain in which the combinations of fluconazole with ibuprofen and fluconazole with diclofenac showed an antagonistic effect.

Discussion
Candida auris is one of the top-ten most serious fungal pathogens to date [31]. Despite the application of antifungal therapy, management is a big challenge because C. auris is a persistent colonizer and difficult to eradicate from the hospital environment [32,33]. Moreover, the ability to form biofilms, adherence to catheter material, and resistance to the major antifungal drugs may have contributed to its persistence and nosocomial transmission [7,10]. Pan-resistance of C. auris has been reported with the high MICs of fluconazole (90%), echinocandins (2%), amphotericin B (8%), and voriconazole (2.3%) [16].
Our previous experience on the interaction of caspofungin with voriconazole and micafungin with fluconazole against multidrug-resistant C. auris revealed indifferent activities against all strains (FICI, 0.62 to 2). Nonetheless, the synergistic effects of micafungin with voriconazole were observed (FICI, 0.15 to 0.5) and no antagonism was identified for any combinations [35].
In a study carried out by Eldesouky et al., it was reported that sulfamethoxazole had the highest synergistic activity with fluconazole against both C. albicans and C. auris [36]. In this regard, the fluconazole MICs of C. auris were reduced by 8 log2 dilutions resulting in an FICI of 0.156 [37].
In our previous study, geldanamycin interacted synergistically with both fluconazole and itraconazole against C. albicans, C. glabrata, and C. parapsilosis.

Curr Med Mycol, 2019, 5(3): 26-30 29
However these combinations were indifferent against C. auris [38]. Significant inhibitory effects of NSAIDs on the growth of Candida species, Trichosporon asahii, and Cryptococcus species have been previously reported [20]. Furthermore, there have been reports of the in vitro synergistic effect of fluconazole with ibuprofen, sodium salicylate, or propylparaben against C. albicans [21,23]. In a study carried out by Scott et al. a synergistic interaction was observed between NSAIDs and fluconazole against azole-resistant C. albicans. Moreover, NSAIDs were able to revert the azole resistance of C. albicans at clinically relevant concentrations, in vitro [23]. In this regard, the results of the aforementioned study are in line with the findings of the present study.
In the present study, the combination of fluconazole with ibuprofen resulted in a synergistic effect against C. albicans (n=2), C. tropicalis (n=1), C. glabrata (n=1), and C. krusei (n=1). Similar to the findings of the present study, Pina-Vaz et al. demonstrated that the combination of ibuprofen with fluconazole had synergic activity in four fluconazole-resistant strains, including C. glabrata (n=2), C. albicans (n=1), and C. krusei (n=1). Furthermore, they recommended the practicability of using ibuprofen alone or in combination with azoles in the treatment of candidiasis, particularly when topically applied [21].

Conclusion
In summary, the results of the present study showed that the interaction of NSAIDs and fluconazole had a synergistic activity against azole-resistant C. albicans and C. tropicalis, but indifferent effects were observed for both resistant C. auris and C. krusei. Although the interaction of NSAIDs and fluconazole was not synergistic against resistant C. auris isolates, the combination with newer azole agents would be another option. However, the significance of these in vitro findings remains elusive and requires consideration.