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Colistin interacts synergistically with echinocandins against Candida auris
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Colistin interacts synergistically with echinocandins against Candida auris Bidaud A.L. , Djenontin E , Botterel F. , Chowdhary A. , Dannaoui E. PII: DOI: Reference:
S0924-8579(20)30040-6 https://doi.org/10.1016/j.ijantimicag.2020.105901 ANTAGE 105901
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
1 October 2019 11 January 2020
Please cite this article as: Bidaud A.L. , Djenontin E , Botterel F. , Chowdhary A. , Dannaoui E. , Colistin interacts synergistically with echinocandins against Candida auris, International Journal of Antimicrobial Agents (2020), doi: https://doi.org/10.1016/j.ijantimicag.2020.105901
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Highlights
Candida auris is an emerging, multi-resistant pathogen
Colistin alone has no antifungal activity
The in vitro combination of colistin with caspofungin is strongly synergistic
Colistin interacts synergistically with echinocandins against Candida auris
Bidaud A.L.1, Djenontin E1. Botterel F.2, Chowdhary A.3, Dannaoui E.1,2*
1
Paris-Descartes University, Faculty of Medicine, APHP, European Georges Pompidou
Hospital, Parasitology-Mycology Unit, Microbiology department, Paris, France 2
Dynamyc Research Group, Paris Est Créteil University (UPEC, EnvA), France
3
Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi,
Delhi, India
Running heading: Colistin-echinocandins combination against Candida auris Word count: Abstract: 227; Text: 2952 Key words: Candida auris, antifungals, combination, caspofungin, micafungin, colistin, synergy, checkerboard
*Corresponding author: Eric DANNAOUI, MD, PhD, FECMM Hôpital Européen Georges Pompidou, Unité de Parasitologie-Mycologie, Service de Microbiologie, Paris, France Phone: +33 1 56 09 39 48 Fax:
+33 1 56 09 24 46
Email: [email protected]
Abstract Antifungal combination is an interesting approach for the treatment of several fungal infections but there is currently little evidence to support combined therapy in C. auris infections. Recently, it has been shown that the antibacterial colistin could interact synergistically with antifungals against Candida spp., including azole-resistant isolates. In this study, we evaluated the in vitro interaction between colistin and either caspofungin or micafungin against C. auris isolates. Interaction between colistin and either caspofungin or micafungin was evaluated against 15 C. auris isolates by a checkerboard methodology based on the EUCAST reference method. Results were analysed by two approaches: calculation of the fractional inhibitory concentration index (FICI) and response surface analysis based on the Bliss model. The MIC range (Gmean) of caspofungin and micafungin were 0.25 to 1 µg/mL (0.691 µg/mL) and 0.03 to 0.125 µg/mL (0.114 µg/mL), respectively. No activity of colistin alone was observed with MIC of >64 µg/mL for all the isolates. When colistin was combined with caspofungin, synergistic interactions were observed for all strains with FICI values of 0.08 to 0.14. In contrast, indifferent interactions were observed for the combination of colistin with micafungin with FICI values of 0.51 to 1.01. Synergy was also demonstrated by using the Bliss model against all isolates for the colistin-caspofungin combination and in 60% of the isolates for the colistin-micafungin combination. Antagonism was never observed, for any combination.
Introduction Candida auris is an emerging, multi-resistant pathogen responsible for invasive hospitalacquired infections [1–3]. Isolated for the first time from an ear sample in Japan [4] and initially described as a rare pathogen, this species was increasingly detected worldwide in less than a decade after its first isolation. The first known C. auris infections were documented in South Korea in 1996 [5]. Infections due to C. auris have been reported in several countries across all four continents [6–18]. Initially, C. auris was probably underestimated due to misidentification of C. auris as C. haemulonii, Candida famata, Candida sake, Saccharomyces cerevisiae or Rhodotorula glutinis by commercial identification techniques in clinical laboratories [19–22]. To enable correct identification, MALDI-TOF MS is required [19]. This method performs well when a sufficient number of Candida auris reference spectra from different geographical origins are included in the database. The in vitro susceptibility profile of C. auris isolates has been explored by using several antifungal susceptibility testing methods, including commercial kits. Nevertheless, to correctly interpret the results, confirmation by the reference dilution method is necessary [19,20]. The resistance of C. auris to antifungals is worrying for the treatment of infections caused by this yeast. Although few C. auris strains exhibit elevated MIC for all three major classes of antifungal drugs i.e. azoles, polyenes and echinocandins [23,24], resistance can evolve quite rapidly in this species especially to echinocandins [25]. Therefore, continuous monitoring of the emergence of resistance is recommended in patients infected or colonized with C. auris. Echinocandins are the first line of treatment for C. auris infections [26] and amphotericin B is an alternative choice, depending on the in vitro susceptibility of the isolate [2,3]. In the face of this multi-resistance and the lack of evidence to support combined therapies, it seems
interesting to explore antifungal interactions against C. auris. In a previous study, it has been shown that combination of flucytosine with amphotericin B, voriconazole or micafungin didn’t show any synergism but could be nevertheless of interest due to the absence of antagonism [27]. Another combination study showed that the interaction between micafungin and voriconazole was synergistic against Candida auris [28]. Because the treatment of fungal infections is limited by the existence of resistance and the low number of antifungal families, drug repurposing could be a good alternative for managing difficult-to-treat infections [29]. This strategy has already allowed to identify off-patent compounds against Candida species including C. auris [30,31]. Another interesting possibility is the combination of polymyxin B, an antibiotic of the family of polymyxins, with an antifungal of the family of echinocandins, as these compounds have different cellular targets. The first evaluation of colistin against yeasts was published in 1970 and showed the in vitro fungicidal activity of colistin against C. tropicalis [32]. Combinations of polymyxin B or colistin with caspofungin have been shown to be synergistic against several Candida spp. including azole-resistant isolates [33–35]. The purpose of the present study was to evaluate the in vitro interaction between colistin, and either caspofungin or micafungin, against C. auris isolates.
Materials and Methods Isolates Fifteen strains previously identified as C. auris by DNA sequencing [4,6,36,37] were used. Strains were from different geographical origin (India (n=12), Korea (n=2), Japan (n=1)), and included the type strain of the species (CBS 10913). Isolates were retrieved from frozen stock on Sabouraud dextrose agar supplemented with chloramphenicol and gentamicin to ensure purity. The two reference strains Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality controls. Drugs and medium RPMI 1640 medium with l-glutamine but without sodium bicarbonate (Sigma-Aldrich, Saint Quentin Fallavier, France) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (VWR, Fontenay-sous-Bois, France) and supplemented with 2% of glucose was used as the test medium. The drug tested included caspofungin (Sigma-Aldrich), micafungin (Astellas Pharma, Tokyo, Japan), and colistin (Acros Organics BVBA, Geel, Belgium). Stock solutions were prepared at 1600 µg/mL in DMSO for both echinocandins and at 12800 µg/mL in water for colistin. Stock solutions were kept at -80°C until used. Microplate preparation for checkerboard The interactions of colistin with either caspofungin or micafungin were investigated by using the guidelines of the Antifungal Susceptibility Testing subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST) reference technique, modified for a broth microdilution checkerboard procedure [38]. For that, 96-well flat-bottom microtiter plates (VWR) were used. Drugs dilutions were prepared at four times the final concentration by following the drug dilution scheme recommended by EUCAST [39]. Final concentrations ranged from 1 to 64 µg/mL for colistin, 0.004 to 2 µg/mL for caspofungin and micafungin. For two-dimensional microplate preparation, 50 µL of each concentration of
colistin was added into wells 1 to 11 of each column, and then 50 µL of the echinocandin was added into wells A to H of each line. The wells of column 11 and the wells of line H contained colistin and the echinocandin alone, respectively. Colum 12 served as the growth control and contained only the RPMI medium with the solvent but without antifungal drugs. Microplates were kept at −20°C until the day of testing. Inoculum and incubation Isolates were grown on Sabouraud dextrose agar for 24h at 37°C and yeast cell suspension were prepared in sterile water and adjusted to 0.5 McFarland. After a 1/10 dilution step in water, each well of the plate was inoculated with 100 µl of the yeast cell suspension resulting in a final inoculum size of 0.5-2.5x105 CFU/ml. Microplates were incubated at 37°C and read spectrophotometrically after 24h of incubation. The experiments were performed in triplicate. For each combination, an uninoculated blank plate (100µl of sterile water was added to each well) was incubated in the same conditions. Reading Spectrophotometric
reading
was
performed
with
an
automated
Dynex
MRX
spectrophotometer (Dynex Technology, Chantilly, VA, USA) at 550 nm. After subtraction of the blank optical density values of the uninoculated plate, the percentage of growth for each well was calculated by comparison with the growth for the drug-free control wells. A growth inhibition endpoint of 50% was used both for the drugs tested alone and in combination. Analysis of results High off-scale MICs were converted to the next highest concentration. Two different methods were used to analyse the drug interactions: one based on the Loewe additivity model (by calculation of the fractional inhibitory concentration index (FICI)) and one based on the Bliss independence model (by response surface modelling).
FICI. For calculation of the FICI, the FIC of each drug was first calculated as follow. In all wells corresponding to an MIC, the FICA was obtained by dividing the concentration of drug A when used in combination (CA) by the MIC of the drug when used alone (MICA). Similarly, FICB was obtained by dividing the concentration of drug B when used in combination (C B) by the MIC of the drug when used alone (MICB). FICI was then calculated by the following equation: FICI = FICA + FICB = (CA / MICA) + (CB / MICB) Interactions were interpreted as follows: synergy for FICI ≤ 0,5, indifference for FICI >0.5 to ≤ 4,0, and antagonism for FICI >4 [40]. Response surface modelling. There are several problems with the FICI approach. First, not all the data generated by the checkerboard experiments are used (only those data corresponding to an MIC are used for the calculation of the FICI). Second, the MIC determination is dependent on the breakpoint used (complete or partial inhibition) and therefore the final results may be different based on the choice of the breakpoint. Therefore, a response surface approach, independent of an endpoint and using all the data, was also used. This kind of approach has been previously used for testing drug interactions against yeasts [41]. Briefly, from the experimental data expressed as percentage of growth for each well of the microplate, the dose-response curve of each drug alone is fitted to a Hill equation [42]. From the two dose-response curves, a theoretical response surface of the combination corresponding to an indifferent interaction is calculated based on the Bliss model. The experimental response surface is then compared to the modelled response surface to calculate the synergy distribution. For visualization, the synergy levels can be mapped on the experimental combination dose-response surface. To summarize the synergy distribution, the SUM-SYN-ANT metric was used. This metric represent the sum of synergy and antagonism observed in the concentration space. For interpreting the SUM-SYN-ANT metric, an
experimental plate of a combination of an antifungal with itself (micafungin combined with micafungin) was performed. The SUM-SYN-ANT of this experimental plate was 24.5%. Synergy and antagonism were assumed when the SUM-SYN-ANT was >24.5% and <-24.5%, respectively. Between -24.5 and 24.5%, a no-interaction was concluded. All the calculations were performed with the Combenefit software [42,43].
Results The results of one of the three replicates, for the tested drugs alone and in combination against C. auris isolates are summarized in Tables 1 and 2. The MIC ranges (and GM) of drugs alone against the strains were 0.25 to 1 µg/mL (0.691 µg/mL) and 0.03 to 0.125 µg/mL (0.114 µg/mL) for caspofungin and micafungin, respectively. There was no activity of colistin alone with MIC of >64 µg/mL for all the isolates. Similar results were obtained for the three replicates: MIC values for drugs alone were within +/-2log2 dilutions in 100% of the cases. Analysis of interactions was evaluated by two different approaches: calculation of the FIC indices based on Loewe additivity and by a response surface method based on Bliss independence. By FIC indices calculation, when caspofungin was combined with colistin, the MIC ranges decreased to 0.015 to 0.125 µg/mL and 1 to 8 µg/mL for caspofungin and colistin, respectively. A strong synergy was observed for all the isolates with FICI ranging from 0.08 to 0.14. When micafungin was combined with colistin the MIC ranges of micafungin remained unchanged from 0.03 to 0.125 µg/mL while colistin decreased to 1 µg/mL. FICI ranged from 0.51 to 1.01 which was indicative of no-interaction against all Candida auris isolates. When the data were analysed with the response surface approach based on the Bliss model, similar results were obtained. For the combination of colistin with caspofungin the SUMSYN-ANT metric ranged from 92.8 to 185.07 indicative of strong synergy for all the strains (Table 1). An example of the synergy mapped on the dose-response surface, along with the matrix of synergy for the strain CBS 10913 is shown in Figure 1. When micafungin was combined with colistin, the SUM-SYN-ANT metric ranged from 8.82 to 40.87. With a significant threshold at 24.5%, synergy was observed for 9 isolates out of 15 (60%). Among these 9 isolates, 8/9 belongs to the Indian clade and the last belongs to the east Asian clade.
The dose-response surface, along with the matrix of interaction for the strain CBS 10913 is shown in Figure 2. Antagonism was never observed for any isolates, whatever the method of analysis.
Discussion C. auris is a multi-drug resistant organism that emerged across the five continents [3]. Therapeutic options for C. auris are limited. According to the recommendations, echinocandins are the best choice for initial treatment [44]. Nevertheless, the mortality remains high and echinocandins resistance has been reported [45,46]. Therefore, new therapeutic options are needed. In this context, combination therapy may be of interest. Currently, few antifungal combinations have been evaluated against C. auris. Interactions between flucytosine and amphotericin B, voriconazole, or micafungin have been recently tested against a collection of 15 clinical isolates of C. auris and showed the absence of antagonism. Several other combinations between an azole and an echinocandins have been evaluated against 10 C. auris isolates [28]. Synergy was observed for the combination of micafungin with voriconazole against all the isolates tested while indifferent interactions were seen for the combinations micafungin + fluconazole, caspofungin + fluconazole, and caspofungin + voriconazole. Combinations of antifungals with non-antifungal drugs have also been tested in the context of drug repurposing. Indeed, since a few years, the drug repurposing of “off-patent” molecules is an alternative for the treatment of invasive infections [29]. The advantages of this approach are a lower cost and a shorter development timeline compared to the development of new molecules. In a study of 1280 molecules belonging to the Prestwick Chemical library, two drugs (ebselen, an anti-inflammatory and suloctidil, a vasodilator) showed synergistic interactions with anidulafungin and voriconazole (FICI <0.44 and <0.5, respectively) on C. auris [29,30]. These new drugs should be evaluated as therapeutic alternative to treat C. auris infections. In another study of 10 isolates of C. auris it was shown that the combination of sulfamethoxazole with voriconazole restored the fungistatic activity of voriconazole against voriconazole-resistant isolates. The same results were found for the combination of
sulfamethoxazole with itraconazole. But in the study, combinations were effective according to the underlying mechanism of resistance to azoles. Combinations were effective if the mechanism was an overproduction of or decreased affinity for the azole target (ERG 11). Combinations were ineffective when the mechanism of azole resistance was due to efflux pump overexpression [47]. In the present study, we tested the combination of colistin with two antifungals belonging to the family of the echinocandins (caspofungin and micafungin). When colistin was combined with caspofungin, FICI was <0.15 indicative of a strong synergy for all the strains. For the combination of colistin with micafungin, FICI was >0.5 and no antagonism was observed. Synergy was nevertheless observed in 60% of the isolates when the checkerboard results were analysed by the response surface approach. The differential effect between caspofungin and micafungin when there are tested in combination has been previously reported [28,48,49]. The explanation of the differential behaviour between this two echinocandins remains unclear. With these encouraging results of combination of colistin with caspofungin, it would be interesting to further explore combinations of colistin with other classes of antifungals. To our knowledge, this is the first attempt to use colistin in combination against C. auris and our results are in agreement with previous studies that evaluate combinations including colistin as a drug partner against Candida spp. As for colistin, it belongs to the family of polymyxins, from the group of polymyxins E. This class of antibiotic was rejected in the 1980s because of its neuro and nephrotoxicity. Currently, it is being re-used as an option for treating multidrug-resistant gram negative bacteria [50]. It targets the external membrane of the bacteria and more precisely the lipid A of lipopolysaccharide. There is a displacement of the Ca2+ and Mg2+ who belong to the lipid A, which results in alteration of the external membrane and then an increase in membrane permeability leading to cell death. Colistin could favour the penetration of other antibiotics or
antifungals as suggested in the study of Yousfi and al. that showed synergistic interaction between azoles and colistin [50]. In this same study, the combination of colistin with amphotericin B was evaluated against 11 multi-drug resistant yeasts. The permeabilization of the fungal membrane induced by amphotericin B plus the damage of the membrane induced by colistin would explain the synergistic effect of the two combined [50]. Combination of colistin with caspofungin has also been evaluated in several studies. Notably one from 2013, highlighting a synergy of this combination against Candida albicans both in vitro and in vivo in a Galleria mellonella model [33]. Another study focused on the synergy between colistin and caspofungin. It was suggested that the alteration of the cell wall by the echinocandin could facilitate the access of colistin to the fungal membrane. In addition, the study showed that for the association to be synergistic, the strains had to be previously susceptible to echinocandins, otherwise the combination was ineffective [34]. In present study, we used the checkerboard method to generate drug interaction data and the results were primarily analysed by the FICI calculation (based on the Loewe additivity model). Nevertheless, it is noteworthy that this kind of analysis may be difficult, particularly for the choice of the most suitable inhibition endpoint which could be different for testing the drug alone and the drugs in combinations. For this reason, we also employed an alternative method, namely the response-surface modelling (based on the Bliss independence model). This approach is not dependent of an MIC endpoint and all the concentrations tested in combination can be used for the calculations. Moreover, with this method, the synergy can be visualized on the three-dimensional response surface. In the present study, there are two main limits. The first and most important is the resistance profile of our strains. Indeed, none of the strains were resistant to echinocandins and only 5/15 had a resistance to voriconazole (if we consider a breakpoint 2 µg/mL) and all strains were susceptible to other antifungals. Combination of colistin with caspofungin should be tested on
echinocandin-resistant strains and multi-resistant strains. The second is the representativeness of our study cohort, which has only two clades out of the existing four. It is known that there is a strong phylogeographic structure within the same clade. It would be interesting to test isolates belonging to the other clades. It has been shown that the inter-laboratory reproducibility for caspofungin susceptibility testing by reference methods was not optimal [51]. This may be a problem for categorization of strains as susceptible or resistant but certainly not in assessing the interaction between caspofungin and another antimicrobial. In summary we demonstrated that colistin in combination with caspofungin is synergistic against C. auris and that there is no antagonism when colistin is combined with micafungin. The development of new antifungal drugs being active against C. auris will be essential to eradicate multidrug-resistant isolates. Before new drugs are available on the market, the emergence of this species and its disturbing resistance to antifungals encourage the study of new therapeutic strategies, including the combination of antibiotics and antifungals.
Declarations Funding: No specific funding has been received for this study. Competing Interests: During the past 5 years, Eric Dannaoui has received research grants from MSD and Gilead; travel grants from Gilead, MSD, Pfizer, and Astellas, and speaker’s fee from Gilead, MSD, and Astellas. Françoise Botterel has received research grants from MSD; travel grants from Gilead, MSD, Pfizer, and speaker’s fee from Gilead, MSD, and Pfizer. Elie Djenontin: no conflict; Anne-Laure Bidaud: no conflict; Anuradha Chowdhary: no conflict Ethical Approval: Not required
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Table 1: In vitro interaction of caspofungin with colistin against 15 isolates of Candida auris
Isolate
MIC (µg/mL) of drug alone
MIC (µg/mL) of the drugs in combination
CAS CST CAS CST CBS 12372 0.25 128 0.015 8 CBS 12373 0.50 128 0.03 4 CBS 12766 1 128 0.06 2 CBS 12767 1 128 0.06 2 CBS 12768 1 128 0.06 4 CBS 12769 1 128 0.06 2 CBS 12770 1 128 0.125 2 CBS 12771 1 128 0.125 1 CBS 12772 1 128 0.125 1 CBS 12773 1 128 0.06 4 CBS 12774 0.50 128 0.06 2 CBS 12775 0.50 128 0.06 1 CBS 12776 0.50 128 0.06 2 CBS 12777 1 128 0.06 2 CBS 10913 0.25 128 0.015 4 CAS Caspofungin; CST Colistin; N: No interaction; S: Synergy
Lowest FICI for the combination CAS/CST 0.125 0.09 0.08 0.08 0.09 0.08 0.14 0.13 0.13 0.09 0.14 0.13 0.14 0.08 0.09
Response surface analysis
Interpretation SUM-SYN-ANT Interpretation S 169.11 S S 158.51 S S 109.25 S S 123.68 S S 112.08 S S 92.80 S S 103.89 S S 108.37 S S 121.19 S S 124.16 S S 96.04 S S 102.39 S S 130.20 S S 97.76 S S 185.07 S
Table 2: In vitro interaction of micafungin with colistin against 15 isolates of Candida auris
Isolate
MIC (µg/mL) of drug alone MICA
CST
MIC (µg/mL) of the drugs in combination MICA
CST
CBS 12372 0.125 128 0.06 1 CBS 12373 0.125 128 0.125 1 CBS 12766 0.125 128 0.125 1 CBS 12767 0.125 128 0.125 1 CBS 12768 0.125 128 0.125 1 CBS 12769 0.125 128 0.125 1 CBS 12770 0.125 128 0.06 1 CBS 12771 0.125 128 0.125 1 CBS 12772 0.125 128 0.125 1 CBS 12773 0.125 128 0.125 1 CBS 12774 0.125 128 0.125 1 CBS 12775 0.125 128 0.06 1 CBS 12776 0.125 128 0.125 1 CBS 12777 0.125 128 0.06 1 CBS 10913 0.03 128 0.03 1 MICA Micafungin; CST Colistin; N: No interaction; S: Synergy
Figure legends:
Lowest FIC index for the combination MICA/CST
Interpretation
0.51 1.01 1.01 1.01 1.01 1.01 0.51 1.01 1.01 1.01 1.01 0.51 1.01 0.51 1.01
N N N N N N N N N N N N N N N
Response surface analysis SUM-SYN- Interpretation ANT 20.53 N 21.06 N 34.17 S 34.33 S 34.14 S 26.39 S 20.34 N 8.82 N 40.87 S 23.48 N 17.19 N 33.61 S 30.99 S 25.15 S 28.34 S
Figure 1: Combination of caspofungin with colistin against C. auris CBS 10913 analysed by response surface modelling based on the Bliss model. (A) Synergy mapped on the experimental response surface, (B) Matrix of the synergy distribution, (C, D) Dose-response curve of each drug alone. Combined results from three independent experiments were used for analysis.
Figure 2: Combination of micafungin with colistin against C. auris CBS 10913 analysed by response surface modelling based on the Bliss model. (A) Synergy mapped on the experimental response surface, (B) Matrix of the synergy distribution, (C, D) Dose-response curve of each drug alone. Combined results from three independent experiments were used for analysis.
Colistin interacts synergistically with echinocandins against Candida auris Bidaud A.L. , Djenontin E , Botterel F. , Chowdhary A. , Dannaoui E. PII: DOI: Reference:
S0924-8579(20)30040-6 https://doi.org/10.1016/j.ijantimicag.2020.105901 ANTAGE 105901
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
1 October 2019 11 January 2020
Please cite this article as: Bidaud A.L. , Djenontin E , Botterel F. , Chowdhary A. , Dannaoui E. , Colistin interacts synergistically with echinocandins against Candida auris, International Journal of Antimicrobial Agents (2020), doi: https://doi.org/10.1016/j.ijantimicag.2020.105901
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Highlights
Candida auris is an emerging, multi-resistant pathogen
Colistin alone has no antifungal activity
The in vitro combination of colistin with caspofungin is strongly synergistic
Colistin interacts synergistically with echinocandins against Candida auris
Bidaud A.L.1, Djenontin E1. Botterel F.2, Chowdhary A.3, Dannaoui E.1,2*
1
Paris-Descartes University, Faculty of Medicine, APHP, European Georges Pompidou
Hospital, Parasitology-Mycology Unit, Microbiology department, Paris, France 2
Dynamyc Research Group, Paris Est Créteil University (UPEC, EnvA), France
3
Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi,
Delhi, India
Running heading: Colistin-echinocandins combination against Candida auris Word count: Abstract: 227; Text: 2952 Key words: Candida auris, antifungals, combination, caspofungin, micafungin, colistin, synergy, checkerboard
*Corresponding author: Eric DANNAOUI, MD, PhD, FECMM Hôpital Européen Georges Pompidou, Unité de Parasitologie-Mycologie, Service de Microbiologie, Paris, France Phone: +33 1 56 09 39 48 Fax:
+33 1 56 09 24 46
Email: [email protected]
Abstract Antifungal combination is an interesting approach for the treatment of several fungal infections but there is currently little evidence to support combined therapy in C. auris infections. Recently, it has been shown that the antibacterial colistin could interact synergistically with antifungals against Candida spp., including azole-resistant isolates. In this study, we evaluated the in vitro interaction between colistin and either caspofungin or micafungin against C. auris isolates. Interaction between colistin and either caspofungin or micafungin was evaluated against 15 C. auris isolates by a checkerboard methodology based on the EUCAST reference method. Results were analysed by two approaches: calculation of the fractional inhibitory concentration index (FICI) and response surface analysis based on the Bliss model. The MIC range (Gmean) of caspofungin and micafungin were 0.25 to 1 µg/mL (0.691 µg/mL) and 0.03 to 0.125 µg/mL (0.114 µg/mL), respectively. No activity of colistin alone was observed with MIC of >64 µg/mL for all the isolates. When colistin was combined with caspofungin, synergistic interactions were observed for all strains with FICI values of 0.08 to 0.14. In contrast, indifferent interactions were observed for the combination of colistin with micafungin with FICI values of 0.51 to 1.01. Synergy was also demonstrated by using the Bliss model against all isolates for the colistin-caspofungin combination and in 60% of the isolates for the colistin-micafungin combination. Antagonism was never observed, for any combination.
Introduction Candida auris is an emerging, multi-resistant pathogen responsible for invasive hospitalacquired infections [1–3]. Isolated for the first time from an ear sample in Japan [4] and initially described as a rare pathogen, this species was increasingly detected worldwide in less than a decade after its first isolation. The first known C. auris infections were documented in South Korea in 1996 [5]. Infections due to C. auris have been reported in several countries across all four continents [6–18]. Initially, C. auris was probably underestimated due to misidentification of C. auris as C. haemulonii, Candida famata, Candida sake, Saccharomyces cerevisiae or Rhodotorula glutinis by commercial identification techniques in clinical laboratories [19–22]. To enable correct identification, MALDI-TOF MS is required [19]. This method performs well when a sufficient number of Candida auris reference spectra from different geographical origins are included in the database. The in vitro susceptibility profile of C. auris isolates has been explored by using several antifungal susceptibility testing methods, including commercial kits. Nevertheless, to correctly interpret the results, confirmation by the reference dilution method is necessary [19,20]. The resistance of C. auris to antifungals is worrying for the treatment of infections caused by this yeast. Although few C. auris strains exhibit elevated MIC for all three major classes of antifungal drugs i.e. azoles, polyenes and echinocandins [23,24], resistance can evolve quite rapidly in this species especially to echinocandins [25]. Therefore, continuous monitoring of the emergence of resistance is recommended in patients infected or colonized with C. auris. Echinocandins are the first line of treatment for C. auris infections [26] and amphotericin B is an alternative choice, depending on the in vitro susceptibility of the isolate [2,3]. In the face of this multi-resistance and the lack of evidence to support combined therapies, it seems
interesting to explore antifungal interactions against C. auris. In a previous study, it has been shown that combination of flucytosine with amphotericin B, voriconazole or micafungin didn’t show any synergism but could be nevertheless of interest due to the absence of antagonism [27]. Another combination study showed that the interaction between micafungin and voriconazole was synergistic against Candida auris [28]. Because the treatment of fungal infections is limited by the existence of resistance and the low number of antifungal families, drug repurposing could be a good alternative for managing difficult-to-treat infections [29]. This strategy has already allowed to identify off-patent compounds against Candida species including C. auris [30,31]. Another interesting possibility is the combination of polymyxin B, an antibiotic of the family of polymyxins, with an antifungal of the family of echinocandins, as these compounds have different cellular targets. The first evaluation of colistin against yeasts was published in 1970 and showed the in vitro fungicidal activity of colistin against C. tropicalis [32]. Combinations of polymyxin B or colistin with caspofungin have been shown to be synergistic against several Candida spp. including azole-resistant isolates [33–35]. The purpose of the present study was to evaluate the in vitro interaction between colistin, and either caspofungin or micafungin, against C. auris isolates.
Materials and Methods Isolates Fifteen strains previously identified as C. auris by DNA sequencing [4,6,36,37] were used. Strains were from different geographical origin (India (n=12), Korea (n=2), Japan (n=1)), and included the type strain of the species (CBS 10913). Isolates were retrieved from frozen stock on Sabouraud dextrose agar supplemented with chloramphenicol and gentamicin to ensure purity. The two reference strains Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality controls. Drugs and medium RPMI 1640 medium with l-glutamine but without sodium bicarbonate (Sigma-Aldrich, Saint Quentin Fallavier, France) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (VWR, Fontenay-sous-Bois, France) and supplemented with 2% of glucose was used as the test medium. The drug tested included caspofungin (Sigma-Aldrich), micafungin (Astellas Pharma, Tokyo, Japan), and colistin (Acros Organics BVBA, Geel, Belgium). Stock solutions were prepared at 1600 µg/mL in DMSO for both echinocandins and at 12800 µg/mL in water for colistin. Stock solutions were kept at -80°C until used. Microplate preparation for checkerboard The interactions of colistin with either caspofungin or micafungin were investigated by using the guidelines of the Antifungal Susceptibility Testing subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST) reference technique, modified for a broth microdilution checkerboard procedure [38]. For that, 96-well flat-bottom microtiter plates (VWR) were used. Drugs dilutions were prepared at four times the final concentration by following the drug dilution scheme recommended by EUCAST [39]. Final concentrations ranged from 1 to 64 µg/mL for colistin, 0.004 to 2 µg/mL for caspofungin and micafungin. For two-dimensional microplate preparation, 50 µL of each concentration of
colistin was added into wells 1 to 11 of each column, and then 50 µL of the echinocandin was added into wells A to H of each line. The wells of column 11 and the wells of line H contained colistin and the echinocandin alone, respectively. Colum 12 served as the growth control and contained only the RPMI medium with the solvent but without antifungal drugs. Microplates were kept at −20°C until the day of testing. Inoculum and incubation Isolates were grown on Sabouraud dextrose agar for 24h at 37°C and yeast cell suspension were prepared in sterile water and adjusted to 0.5 McFarland. After a 1/10 dilution step in water, each well of the plate was inoculated with 100 µl of the yeast cell suspension resulting in a final inoculum size of 0.5-2.5x105 CFU/ml. Microplates were incubated at 37°C and read spectrophotometrically after 24h of incubation. The experiments were performed in triplicate. For each combination, an uninoculated blank plate (100µl of sterile water was added to each well) was incubated in the same conditions. Reading Spectrophotometric
reading
was
performed
with
an
automated
Dynex
MRX
spectrophotometer (Dynex Technology, Chantilly, VA, USA) at 550 nm. After subtraction of the blank optical density values of the uninoculated plate, the percentage of growth for each well was calculated by comparison with the growth for the drug-free control wells. A growth inhibition endpoint of 50% was used both for the drugs tested alone and in combination. Analysis of results High off-scale MICs were converted to the next highest concentration. Two different methods were used to analyse the drug interactions: one based on the Loewe additivity model (by calculation of the fractional inhibitory concentration index (FICI)) and one based on the Bliss independence model (by response surface modelling).
FICI. For calculation of the FICI, the FIC of each drug was first calculated as follow. In all wells corresponding to an MIC, the FICA was obtained by dividing the concentration of drug A when used in combination (CA) by the MIC of the drug when used alone (MICA). Similarly, FICB was obtained by dividing the concentration of drug B when used in combination (C B) by the MIC of the drug when used alone (MICB). FICI was then calculated by the following equation: FICI = FICA + FICB = (CA / MICA) + (CB / MICB) Interactions were interpreted as follows: synergy for FICI ≤ 0,5, indifference for FICI >0.5 to ≤ 4,0, and antagonism for FICI >4 [40]. Response surface modelling. There are several problems with the FICI approach. First, not all the data generated by the checkerboard experiments are used (only those data corresponding to an MIC are used for the calculation of the FICI). Second, the MIC determination is dependent on the breakpoint used (complete or partial inhibition) and therefore the final results may be different based on the choice of the breakpoint. Therefore, a response surface approach, independent of an endpoint and using all the data, was also used. This kind of approach has been previously used for testing drug interactions against yeasts [41]. Briefly, from the experimental data expressed as percentage of growth for each well of the microplate, the dose-response curve of each drug alone is fitted to a Hill equation [42]. From the two dose-response curves, a theoretical response surface of the combination corresponding to an indifferent interaction is calculated based on the Bliss model. The experimental response surface is then compared to the modelled response surface to calculate the synergy distribution. For visualization, the synergy levels can be mapped on the experimental combination dose-response surface. To summarize the synergy distribution, the SUM-SYN-ANT metric was used. This metric represent the sum of synergy and antagonism observed in the concentration space. For interpreting the SUM-SYN-ANT metric, an
experimental plate of a combination of an antifungal with itself (micafungin combined with micafungin) was performed. The SUM-SYN-ANT of this experimental plate was 24.5%. Synergy and antagonism were assumed when the SUM-SYN-ANT was >24.5% and <-24.5%, respectively. Between -24.5 and 24.5%, a no-interaction was concluded. All the calculations were performed with the Combenefit software [42,43].
Results The results of one of the three replicates, for the tested drugs alone and in combination against C. auris isolates are summarized in Tables 1 and 2. The MIC ranges (and GM) of drugs alone against the strains were 0.25 to 1 µg/mL (0.691 µg/mL) and 0.03 to 0.125 µg/mL (0.114 µg/mL) for caspofungin and micafungin, respectively. There was no activity of colistin alone with MIC of >64 µg/mL for all the isolates. Similar results were obtained for the three replicates: MIC values for drugs alone were within +/-2log2 dilutions in 100% of the cases. Analysis of interactions was evaluated by two different approaches: calculation of the FIC indices based on Loewe additivity and by a response surface method based on Bliss independence. By FIC indices calculation, when caspofungin was combined with colistin, the MIC ranges decreased to 0.015 to 0.125 µg/mL and 1 to 8 µg/mL for caspofungin and colistin, respectively. A strong synergy was observed for all the isolates with FICI ranging from 0.08 to 0.14. When micafungin was combined with colistin the MIC ranges of micafungin remained unchanged from 0.03 to 0.125 µg/mL while colistin decreased to 1 µg/mL. FICI ranged from 0.51 to 1.01 which was indicative of no-interaction against all Candida auris isolates. When the data were analysed with the response surface approach based on the Bliss model, similar results were obtained. For the combination of colistin with caspofungin the SUMSYN-ANT metric ranged from 92.8 to 185.07 indicative of strong synergy for all the strains (Table 1). An example of the synergy mapped on the dose-response surface, along with the matrix of synergy for the strain CBS 10913 is shown in Figure 1. When micafungin was combined with colistin, the SUM-SYN-ANT metric ranged from 8.82 to 40.87. With a significant threshold at 24.5%, synergy was observed for 9 isolates out of 15 (60%). Among these 9 isolates, 8/9 belongs to the Indian clade and the last belongs to the east Asian clade.
The dose-response surface, along with the matrix of interaction for the strain CBS 10913 is shown in Figure 2. Antagonism was never observed for any isolates, whatever the method of analysis.
Discussion C. auris is a multi-drug resistant organism that emerged across the five continents [3]. Therapeutic options for C. auris are limited. According to the recommendations, echinocandins are the best choice for initial treatment [44]. Nevertheless, the mortality remains high and echinocandins resistance has been reported [45,46]. Therefore, new therapeutic options are needed. In this context, combination therapy may be of interest. Currently, few antifungal combinations have been evaluated against C. auris. Interactions between flucytosine and amphotericin B, voriconazole, or micafungin have been recently tested against a collection of 15 clinical isolates of C. auris and showed the absence of antagonism. Several other combinations between an azole and an echinocandins have been evaluated against 10 C. auris isolates [28]. Synergy was observed for the combination of micafungin with voriconazole against all the isolates tested while indifferent interactions were seen for the combinations micafungin + fluconazole, caspofungin + fluconazole, and caspofungin + voriconazole. Combinations of antifungals with non-antifungal drugs have also been tested in the context of drug repurposing. Indeed, since a few years, the drug repurposing of “off-patent” molecules is an alternative for the treatment of invasive infections [29]. The advantages of this approach are a lower cost and a shorter development timeline compared to the development of new molecules. In a study of 1280 molecules belonging to the Prestwick Chemical library, two drugs (ebselen, an anti-inflammatory and suloctidil, a vasodilator) showed synergistic interactions with anidulafungin and voriconazole (FICI <0.44 and <0.5, respectively) on C. auris [29,30]. These new drugs should be evaluated as therapeutic alternative to treat C. auris infections. In another study of 10 isolates of C. auris it was shown that the combination of sulfamethoxazole with voriconazole restored the fungistatic activity of voriconazole against voriconazole-resistant isolates. The same results were found for the combination of
sulfamethoxazole with itraconazole. But in the study, combinations were effective according to the underlying mechanism of resistance to azoles. Combinations were effective if the mechanism was an overproduction of or decreased affinity for the azole target (ERG 11). Combinations were ineffective when the mechanism of azole resistance was due to efflux pump overexpression [47]. In the present study, we tested the combination of colistin with two antifungals belonging to the family of the echinocandins (caspofungin and micafungin). When colistin was combined with caspofungin, FICI was <0.15 indicative of a strong synergy for all the strains. For the combination of colistin with micafungin, FICI was >0.5 and no antagonism was observed. Synergy was nevertheless observed in 60% of the isolates when the checkerboard results were analysed by the response surface approach. The differential effect between caspofungin and micafungin when there are tested in combination has been previously reported [28,48,49]. The explanation of the differential behaviour between this two echinocandins remains unclear. With these encouraging results of combination of colistin with caspofungin, it would be interesting to further explore combinations of colistin with other classes of antifungals. To our knowledge, this is the first attempt to use colistin in combination against C. auris and our results are in agreement with previous studies that evaluate combinations including colistin as a drug partner against Candida spp. As for colistin, it belongs to the family of polymyxins, from the group of polymyxins E. This class of antibiotic was rejected in the 1980s because of its neuro and nephrotoxicity. Currently, it is being re-used as an option for treating multidrug-resistant gram negative bacteria [50]. It targets the external membrane of the bacteria and more precisely the lipid A of lipopolysaccharide. There is a displacement of the Ca2+ and Mg2+ who belong to the lipid A, which results in alteration of the external membrane and then an increase in membrane permeability leading to cell death. Colistin could favour the penetration of other antibiotics or
antifungals as suggested in the study of Yousfi and al. that showed synergistic interaction between azoles and colistin [50]. In this same study, the combination of colistin with amphotericin B was evaluated against 11 multi-drug resistant yeasts. The permeabilization of the fungal membrane induced by amphotericin B plus the damage of the membrane induced by colistin would explain the synergistic effect of the two combined [50]. Combination of colistin with caspofungin has also been evaluated in several studies. Notably one from 2013, highlighting a synergy of this combination against Candida albicans both in vitro and in vivo in a Galleria mellonella model [33]. Another study focused on the synergy between colistin and caspofungin. It was suggested that the alteration of the cell wall by the echinocandin could facilitate the access of colistin to the fungal membrane. In addition, the study showed that for the association to be synergistic, the strains had to be previously susceptible to echinocandins, otherwise the combination was ineffective [34]. In present study, we used the checkerboard method to generate drug interaction data and the results were primarily analysed by the FICI calculation (based on the Loewe additivity model). Nevertheless, it is noteworthy that this kind of analysis may be difficult, particularly for the choice of the most suitable inhibition endpoint which could be different for testing the drug alone and the drugs in combinations. For this reason, we also employed an alternative method, namely the response-surface modelling (based on the Bliss independence model). This approach is not dependent of an MIC endpoint and all the concentrations tested in combination can be used for the calculations. Moreover, with this method, the synergy can be visualized on the three-dimensional response surface. In the present study, there are two main limits. The first and most important is the resistance profile of our strains. Indeed, none of the strains were resistant to echinocandins and only 5/15 had a resistance to voriconazole (if we consider a breakpoint 2 µg/mL) and all strains were susceptible to other antifungals. Combination of colistin with caspofungin should be tested on
echinocandin-resistant strains and multi-resistant strains. The second is the representativeness of our study cohort, which has only two clades out of the existing four. It is known that there is a strong phylogeographic structure within the same clade. It would be interesting to test isolates belonging to the other clades. It has been shown that the inter-laboratory reproducibility for caspofungin susceptibility testing by reference methods was not optimal [51]. This may be a problem for categorization of strains as susceptible or resistant but certainly not in assessing the interaction between caspofungin and another antimicrobial. In summary we demonstrated that colistin in combination with caspofungin is synergistic against C. auris and that there is no antagonism when colistin is combined with micafungin. The development of new antifungal drugs being active against C. auris will be essential to eradicate multidrug-resistant isolates. Before new drugs are available on the market, the emergence of this species and its disturbing resistance to antifungals encourage the study of new therapeutic strategies, including the combination of antibiotics and antifungals.
Declarations Funding: No specific funding has been received for this study. Competing Interests: During the past 5 years, Eric Dannaoui has received research grants from MSD and Gilead; travel grants from Gilead, MSD, Pfizer, and Astellas, and speaker’s fee from Gilead, MSD, and Astellas. Françoise Botterel has received research grants from MSD; travel grants from Gilead, MSD, Pfizer, and speaker’s fee from Gilead, MSD, and Pfizer. Elie Djenontin: no conflict; Anne-Laure Bidaud: no conflict; Anuradha Chowdhary: no conflict Ethical Approval: Not required
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Table 1: In vitro interaction of caspofungin with colistin against 15 isolates of Candida auris
Isolate
MIC (µg/mL) of drug alone
MIC (µg/mL) of the drugs in combination
CAS CST CAS CST CBS 12372 0.25 128 0.015 8 CBS 12373 0.50 128 0.03 4 CBS 12766 1 128 0.06 2 CBS 12767 1 128 0.06 2 CBS 12768 1 128 0.06 4 CBS 12769 1 128 0.06 2 CBS 12770 1 128 0.125 2 CBS 12771 1 128 0.125 1 CBS 12772 1 128 0.125 1 CBS 12773 1 128 0.06 4 CBS 12774 0.50 128 0.06 2 CBS 12775 0.50 128 0.06 1 CBS 12776 0.50 128 0.06 2 CBS 12777 1 128 0.06 2 CBS 10913 0.25 128 0.015 4 CAS Caspofungin; CST Colistin; N: No interaction; S: Synergy
Lowest FICI for the combination CAS/CST 0.125 0.09 0.08 0.08 0.09 0.08 0.14 0.13 0.13 0.09 0.14 0.13 0.14 0.08 0.09
Response surface analysis
Interpretation SUM-SYN-ANT Interpretation S 169.11 S S 158.51 S S 109.25 S S 123.68 S S 112.08 S S 92.80 S S 103.89 S S 108.37 S S 121.19 S S 124.16 S S 96.04 S S 102.39 S S 130.20 S S 97.76 S S 185.07 S
Table 2: In vitro interaction of micafungin with colistin against 15 isolates of Candida auris
Isolate
MIC (µg/mL) of drug alone MICA
CST
MIC (µg/mL) of the drugs in combination MICA
CST
CBS 12372 0.125 128 0.06 1 CBS 12373 0.125 128 0.125 1 CBS 12766 0.125 128 0.125 1 CBS 12767 0.125 128 0.125 1 CBS 12768 0.125 128 0.125 1 CBS 12769 0.125 128 0.125 1 CBS 12770 0.125 128 0.06 1 CBS 12771 0.125 128 0.125 1 CBS 12772 0.125 128 0.125 1 CBS 12773 0.125 128 0.125 1 CBS 12774 0.125 128 0.125 1 CBS 12775 0.125 128 0.06 1 CBS 12776 0.125 128 0.125 1 CBS 12777 0.125 128 0.06 1 CBS 10913 0.03 128 0.03 1 MICA Micafungin; CST Colistin; N: No interaction; S: Synergy
Figure legends:
Lowest FIC index for the combination MICA/CST
Interpretation
0.51 1.01 1.01 1.01 1.01 1.01 0.51 1.01 1.01 1.01 1.01 0.51 1.01 0.51 1.01
N N N N N N N N N N N N N N N
Response surface analysis SUM-SYN- Interpretation ANT 20.53 N 21.06 N 34.17 S 34.33 S 34.14 S 26.39 S 20.34 N 8.82 N 40.87 S 23.48 N 17.19 N 33.61 S 30.99 S 25.15 S 28.34 S
Figure 1: Combination of caspofungin with colistin against C. auris CBS 10913 analysed by response surface modelling based on the Bliss model. (A) Synergy mapped on the experimental response surface, (B) Matrix of the synergy distribution, (C, D) Dose-response curve of each drug alone. Combined results from three independent experiments were used for analysis.
Figure 2: Combination of micafungin with colistin against C. auris CBS 10913 analysed by response surface modelling based on the Bliss model. (A) Synergy mapped on the experimental response surface, (B) Matrix of the synergy distribution, (C, D) Dose-response curve of each drug alone. Combined results from three independent experiments were used for analysis.