In Vitro Antifungal Activity of Ibrexafungerp (SCY-078) Against Contemporary Blood Isolates From Medically Relevant Species of Candida: A European Study

In Vitro Antifungal Activity of Ibrexafungerp (SCY-078) Against Contemporary Blood Isolates From Medically Relevant Species of Candida: A European Study Guillermo Quindo ´s 1 * § , Katherine Miranda-Cadena 1§ ,RosarioSan-Milla ´n 1†§ , Katyna Borroto-Esoda 2 ,EmiliaCanto ´n 3 ,Marı ´ aJose ´Linares-Sicilia 4 , Axel Hamprecht 5 , Isabel Montesinos 6‡ , Anna Maria Tortorano 7 , Anna Prigitano 7 , Matxalen Vidal-Garcı ´ a 8‡ , Cristina Marcos-Arias 1 , Andrea Guridi 1 , Ferran Sanchez-Reus 9 ,Jesu ´s Machuca-Ba ´rcena 10 , Manuel Antonio Rodrı ´ guez-Iglesias 10 ,EstrellaMartı ´ n-Mazuelos 11 , Carmen Castro-Me ´ndez 11 , Leyre Lo ´pez-Soria 12 , Alba Ruiz-Gaita ´n 3 , Marcelo Fernandez-Rivero 3 , Damaris Lorenzo 13 , Javier Capilla 13 ,AntonioRezusta 8 , Javier Pema ´n 3 , Josep Guarro 13 ,JoanaPereira 14 , Ce ´lia Pais 14 ,OrazioRomeo 15 , Guillermo Ezpeleta 16 , Nerea Jauregizar 17 , David Angulo 2 and Elena Eraso 1 1 Laboratorio de Micologı ´aMe ´dica, UFI 11/25, Departamento de Inmunologı ´a, Microbiologı ´a y Parasitologı ´a, Facultad de Medicina y Enfermerı ´a, Universidad del Paı ´s Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), Bilbao, Spain, 2 SCYNEXIS, Inc., Jersey City, NJ, United States, 3 Instituto de Investigacio ´n Sanitaria, Hospital Universitario y Polite ´cnico La Fe, Valencia, Spain, 4 Research Group GC24, Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Department of Microbiology, Facultad de Medicina y Enfermerı ´a, Universidad de Co ´rdoba, Co ´rdoba, Spain, 5 University Hospital Cologne, Cologne and Institute for Medical Microbiology and Virology, University of Oldenburg, Oldenburg, Germany, 6 Microbiology Department, LHUB-ULB, Ho ˆpital Erasme, Brussels, Belgium, 7 Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano, Milan, Italy, 8 Servicio de Microbiologı ´a, Hospital Universitario Miguel Servet, Zaragoza, Spain, 9 Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 10 A ´ rea de Microbiologı ´a, Departamento de Biomedicina, Biotecnologı ´a y Salud Pu ´blica, Facultad de Medicina, Universidad de Ca ´diz, Ca ´diz, Spain, 11 Servicio de Microbiologı ´a, Hospital Universitario de Valme, Sevilla, Spain, 12 Servicio de Microbiologı ´a, Hospital Universitario de Cruces and BioCruces Bizkaia, Barakaldo, Spain, 13 Microbiology Unit, Medical School, Universitat Rovira i Virgili, Reus, Spain, 14 Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal, 15 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy, 16 Servicio de Microbiologı ´a, Complejo Hospitalario de Navarra, Pamplona and Departamento de Medicina Preventiva y Salud Pu ´blica, Facultad de Medicina y Enfermerı ´a, Universidad del Paı ´s Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), Bilbao, Spain, 17 Departamento de Farmacologı ´a, Facultad de Medicina y Enfermerı ´a, Universidad del Paı ´s Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), Bilbao, Spain Background: Ibrexafungerp (SCY-078) is the newest oral and intravenous antifungal drug with broad activity, currently undergoing clinical trials for invasive candidiasis. Objective: The aim of this study was to assess the in vitro activity of ibrexafungerp and comparators against a collection of 434 European blood isolates of Candida. Methods: Ibrexafungerp, caspofungin, fluconazole, and micafungin minimum inhibitory concentrations (MICs) were collected from 12 European laboratories for 434 blood isolates, including 163 Candida albicans, 108 Candida parapsilosis,60Candida glabrata,40Candida tropicalis,29Candida krusei,20Candida orthopsilosis,6 Candida guilliermondii,2Candida famata,2Candida lusitaniae, and 1 isolate each of Frontiers in Cellular and Infection Microbiology | www.frontiersin.org May 2022 | Volume 12 | Article 9065631 Edited by: Jose L. Lopez-Ribot, University of Texas at San Antonio, United States Reviewed by: Hamid Badali, The University of Texas Health Science Center at San Antonio, United States Marı ´a Guembe, Gregorio Maraño ´n Hospital, Spain *Correspondence: Guillermo Quindo ´s [email protected] † Deceased ‡ Present addresses: Isabel Montesinos, Laboratoire/Biologie Clinique, CHU UCL Namur, Universite ´catholique de Louvain, Godinne, Belgium Matxalen Vidal-Garcı ´a, Servicio de Microbiologı ´aClı ´nica y Control de Infeccio ´n, Hospital Universitario Basurto & BioCruces Bizkaia, Bilbao, Spain § These authors have contributed equally to this work and share first authorship Specialty section: This article was submitted to Fungal Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 28 March 2022 Accepted: 19 April 2022 Published: 16 May 2022 ORIGINAL RESEARCH published: 16 May 2022 doi: 10.3389/fcimb.2022.906563 Candida bracarensis, Candida catenulata,Candida dubliniensis, and Candida kefyr. MICs were determined by the EUCAST broth microdilution method, and isolates were classified according to recommended clinical breakpoints and epidemiological cutoffs. Additionally, 22 Candida auris from different clinical specimens were evaluated. Results: Ibrexafungerp MICs ranged from 0.016 to ≥8 mg/L. The lowest ibrexafungerp MICs were observed for C. albicans (geometric MIC 0.062 mg/L, MIC range 0.016–0.5 mg/L) and the highest ibrexafungerp MICs were observed for C. tropicalis (geometric MIC 0.517 mg/L, MIC range 0.06–≥8 mg/L). Modal MICs/ MIC 50 s (mg/L) against Candida spp. were 0.125/0.06 for C. albicans, 0.5/0.5 for C. parapsilosis, 0.25/0.25 for C. glabrata, 0.5/0.5 for C. tropicalis, 1/1 for C. krusei, 4/2 for C. orthopsilosis, and 0.5/0.5 for C. auris. Ibrexafungerp showed activity against fluconazoleand echinocandin-resistant isolates. If adopting wild-type upper limits, a non-wild-type phenotype for ibrexafungerp was only observed for 16/434 (3.7%) isolates: 11 (4.6%) C. parapsilosis, 4 (5%) C. glabrata, and 1 (2.5%) C. tropicalis. Conclusion: Ibrexafungerp showed a potent in vitro activity against Candida. Keywords: antifungal testing, antifungal resistance, Candida,ibrexafungerp,SCY-078,EUCAST, caspofungin, micafungin INTRODUCTION Invasive candidiasis (IC) is the most common healthcareassociated invasive mycosis, being a major cause of human morbidity and mortality. Candida albicans is the most prevalent etiology, but other species, such as Candida glabrata, Candida parapsilosis,Candida krusei (Pichia kudriavzevii), and, more recently, Candida auris, are increasing causes of IC. These emergent species are usually less susceptible to current antifungal drugs. Although Candida isolates displaying antifungal resistance are still uncommon, they are increasingly reported worldwide. Therapy of IC is an unsolved clinical challenge and, for this reason, monitoring antifungal susceptibility patterns and resistance mechanisms is of utmost importance. Moreover, new antifungal drugs are needed as the number of available antifungal drug classes, and particularly those for oral administration, is limited (Arendrup and Patterson, 2017;Quindos et al., 2018; Fuller et al., 2019;Pfaller et al., 2019). Ibrexafungerp (formerly SCY-078) is a semisynthetic triterpenoid glycoside derived from enfumafungin, which is structurally different from echinocandins and form a new class of antifungal drugs called “fungerps”that strongly inhibit fungal 1,3-b-glucan synthase (Davis et al., 2020). Even ibrexafungerp and echinocandins share similar mechanisms of action, and their binding sites to the target enzyme is not the same, resulting in very limited cross-resistance (Jimenez-Ortigosa et al., 2017; Pfaller et al., 2017). Ibrexafungerp displays significant in vitro and in vivo activities against azoleand echinocandin-resistant isolates of Candida species, including biofilm-forming strains (Jimenez-Ortigosa et al., 2014;Pfaller et al., 2017;Schell et al., 2017;Gamal et al., 2021). Ibrexafungerp aims to be the first orally and intravenously available glucan synthase inhibitor useful in the treatment of lifethreatening fungal infections (Davis et al., 2020) as well as superficial ones, such as vulvovaginal candidiasis (Schwebke et al., 2021;Sobel et al., 2022). Currently, there are 13 listed clinical trials for ibrexafungerp, eight of which have been completed (https://ClinicalTrials.gov/; accessed on March 8, 2022). In the current study, we have determined the anti-Candida in vitro activity of ibrexafungerp, caspofungin, fluconazole, and micafungin against 434 European Candida blood isolates analyzed in 12 European laboratories. MATERIALS AND METHODS Microorganisms In vitro susceptibility of a collection of 434 Candida blood isolates (2016–2018) from 434 patients was determined at 12 laboratories from Belgium, Germany, Italy, Portugal, and Spain. Abbreviations: ATCC,AmericanTypeCultureCollection;CBP,clinicalbreakpoints; DMSO,dimethylsulfoxide;ECV,epidemiologicalcutoffvalues;EUCAST,European Committee on Antimicrobial Susceptibility Testing; GM, geometric mean; IC, invasive candidiasis; ITS, internal transcribed spacer; MALDI-TOF, matrix-assisted laser desorption/ionization–time-of-flight; MM, modal MIC; MIC, minimum inhibitory concentration; MOPS, morpholinopropane sulphate; NWT, non-wild type; PK/PD, pharmacokinetics and pharmacodynamics; QC, quality control; UPV/EHU, Universidad del Paı  s Vasco/Euskal Herriko Unibertsitatea; WT, wild type; WTUL, wild-type upper limits. Quindo ´s et al. Anti-Candida Activity of Ibrexafungerp Frontiers in Cellular and Infection Microbiology | www.frontiersin.org May 2022 | Volume 12 | Article 9065632 Each laboratory studied its own clinical isolates. The collection included 163 C. albicans, 108 C. parapsilosis,60C. glabrata,40 C. tropicalis,29C. krusei,20Candida orthopsilosis,6Candida guilliermondii (Meyerozyma guilliermondii), 2 Candida famata (Debaryomyces hansenii), 2 Candida lusitaniae (Clavispora lusitaniae), and 1 isolate each of Candida bracarensis, Candida catenulata (Diutina catenulata), Candida dubliniensis,and Candida kefyr (Kluyveromyces marxianus). Additionally, 22 C. auris from different clinical specimens were evaluated: Eight isolates were from blood, seven from oral specimens, and 7 from urine (Ruiz-Gaitan et al., 2017;Ruiz-Gaitan et al., 2018). Isolates were identified by phenotypic methods, MALDI-TOF (proteomic method), and, when needed, internal transcribed spacer (ITS) sequencing (genotypic method) (Miranda-Zapico et al., 2011). C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were included as quality control (QC) strains. Reference strain C. albicans ATCC 64550 was also included as recommended by EUCAST for detecting variation in echinocandin activities. Prior to testing, each isolate was cultured onto Sabouraud dextrose agar and/or CHROMagar Candida medium (Becton Dickinson, Sparks, MD, USA) to ensure purity and viability. QC and reference strain were included every testing day and all their results were within published ranges. Antifungal Drugs and Susceptibility Testing All isolates were tested for in vitro susceptibility to ibrexafungerp, caspofungin, fluconazole, and micafungin using the EUCAST broth microdilution method (Arendrup et al., 2020a;Arendrup et al., 2020b;EUCAST, 2020;EUCAST, 2021). The reference powder of ibrexafungerp was obtained from the manufacturer (SCYNEXIS Inc., Jersey City, NJ, USA). Stock solutions were prepared in dimethyl sulfoxide (DMSO), and the final range of ibrexafungerp, caspofungin, and micafungin concentrations tested was 0.008 to 8 mg/L except for fluconazole, which ranged from 0.125 to 128 mg/L. The EUCAST method was performed by using RPMI 1640 with 2% glucose and buffered to pH 7.0 with 0.165 M morpholinopropane sulphate (MOPS). Panels were inoculated with a final standardized cell concentration of 0.5–2.5 × 10 5 cells/ml. Panels were read spectrophotometrically at 450 nm after 24 h of incubation at 36 ± 1°C. Although caspofungin presents excessive inter-laboratory variation in minimum inhibitory concentration (MIC) results by the EUCAST broth microdilution method (Espinel-Ingroff et al., 2013), MICs were calculated for comparison purposes. Data Analysis MIC values were determined as the lowest concentration of antifungal drug that inhibited 50% of growth compared to that of the growth control. For individual species of Candida for which ten or more isolates were tested, summary descriptive statistics, including MIC ranges, modal MIC (MM) (the most reported MIC), geometric mean MIC (GM), MIC at which 50% of the isolates was inhibited (MIC 50 ), and MIC at which 90% of the isolates was inhibited (MIC 90 ), were calculated for each species and drug. High off-scale MIC results were converted to the next highest concentration, and low off-scale MIC results were left unchanged. Epidemiological cutoff values (ECVs) were used to differentiate wild-type (WT) (without acquired resistance mechanisms) from non-wild-type (NWT) (may harbor acquired resistance mechanisms) isolates. For antifungal drugs, such as ibrexafungerp and caspofungin, or/and species, such as C. auris and C. orthopsilosis, without EUCAST ECVs, WT upper limits (WTUL) were used as the susceptibility cutoff values and MICs > 2 dilution steps above the MM were regarded as NWT (Table 1). Comparison of efficacy among antifungal drugs was based on MIC 90 differences: Those antifungal drugs with a difference of one double dilution were considered to have a similar activity (Wiederhold, 2021). This comparison should not be considered absolute, as other pharmacokinetics and pharmacodynamics (PK/PD) parameters should be taken into account. The study was approved by the Ethics Committee of the Universidad del Paı  s Vasco/Euskal Herriko Unibertsitatea (UPV/EHU, Bilbao, Spain, CEIAB Ethics reference number M30_2015_248). RESULTS MICs of ibrexafungerp against all species were tested in vitro ranging from 0.016 to ≥8mg/L(MIC 90 2 mg/L). These values were comparable to those of caspofungin and micafungin (MIC 90 2 mg/L). The lowest ibrexafungerp MICs were observed for C. albicans (GM 0.062 mg/L, MIC range 0.016–0.5 mg/L, MIC 90 0.125 mg/L) and the highest ibrexafungerp MICs were observed for C. tropicalis (GM 0.517 mg/L, MIC range 0.06–≥8mg/L,MIC 90 2mg/L)(Table 2). Echinocandins MIC values for C. albicans ranged from ≤0.008 mg/L to 2 mg/L for micafungin (MM 0.008 mg/L, GM 0.001 mg/L, MIC 90 0.016 mg/L) and from 0.016 mg/L to 2 mg/L for caspofungin (MM 0.125 mg/L, GM 0.104 mg/L, MIC 90 0.125 mg/L). For the three isolates with elevated caspofungin or/and micafungin MICs, ibrexafungerp MIC range was 0.06 mg/L to 0.25 mg/L. In the current report, ibrexafungerp MICs ranged from 0.016 mg/Lto8mg/Lfor108C. parapsilosis isolates (MM 0.5 mg/L, GM 0.660 mg/L, MIC 90 4mg/L)(Table 2). If we consider ibrexafungerp WTULs(>2mg/L)obtainedinthisstudy,11C. parapsilosis isolates were NWT (4.6%). These NWT isolates were inhibited by ≤1mg/L of fluconazole and by ≤4 mg/L of caspofungin or micafungin. We also observed that for six C. parapsilosis NWT (5.6%) and two resistant isolates to fluconazole (0.9%), the MIC range of ibrexafungerp was 0.5–2 mg/L. According to MIC90s, ibrexafungerp showed comparable values with caspofungin (MM 1 mg/L, GM 0.846 mg/L, MIC 90 2 mg/L) and micafungin (MM 2 mg/L, GM 0.933 mg/L, MIC 90 2 mg/L). Moreover, two isolates resistant to micafungin were inhibited by 2 mg/L of ibrexafungerp. Ibrexafungerp also displayed potent in vitro activity against 60 C. glabrata isolates (MIC range 0.016–8 mg/L, MM 0.25 mg/ L, GM 0.322 mg/L, MIC 90 1 mg/L) (Table 2). Sixteen C. glabrata isolates resistant to fluconazole were inhibited by ≤1 mg/L of ibrexafungerp. Among 9 C. glabrata isolates with elevated MICs to caspofungin or/and micafungin, the MIC range for Quindo ´s et al. Anti-Candida Activity of Ibrexafungerp Frontiers in Cellular and Infection Microbiology | www.frontiersin.org May 2022 | Volume 12 | Article 9065633 ibrexafungerp was 0.25 mg/L to 4 mg/L with a MIC 50 of 1 mg/L. It was notable that whereas the increase in MM between C. glabrata WT and NWT isolates was 4-fold for caspofungin and 63-fold for micafungin, MM values for ibrexafungerp did not increase (Table 3). In vitro activity of ibrexafungerp was also observed against 40 C. tropicalis blood isolates and MICs ranged from 0.06 mg/L to ≥8 mg/L (MM 0.5 mg/L, GM 0.517 mg/L, MIC 90 2 mg/L) (Table 2). Against 14 C. tropicalis resistant to fluconazole, ibrexafungerp MIC range was 0.25–2 mg/L (MIC 90 1mg/L). Moreover, ibrexafungerp showed high in vitro activity against 29 C. krusei (MIC range 0.125–1 mg/L, MM 1 mg/L, GM 0.666 mg/L, MIC 90 were 1 mg/L). MIC values of micafungin ranged from 0.03 mg/L to 0.25 mg/L (MM 0.125 mg/L, GM 0.122 mg/L, MIC 90 0.125 mg/L) and from 0.25 mg/L to 1 mg/L for caspofungin (MM 0.5 mg/L, GM 0.465 mg/L, MIC 90 1 mg/L). Additionally, all C. auris isolates were resistant in vitro to fluconazole (MIC ≥128 mg/L) while ibrexafungerp showed activity (MIC range 0.5 mg/L to 8 mg/L, MM 0.5 mg/L, GM 0.753 mg/L, MIC 90 2 mg/L) (Table 4). C. auris urinary isolates showed higher MICs (data not shown). Against this species, the activity of ibrexafungerp was similar to the activity of micafungin (MIC range 0.125–>8 mg/L, MM 0.125 mg/L, GM 0.377 mg/L, MIC 90 4 mg/L) and 8-fold more active than caspofungin (MIC range 0.25–>8 mg/L, MM 0.25 mg/L, GM 0.585 mg/L, MIC 90 >8 mg/L). Among six isolates with elevated caspofungin or/and micafungin MICs, ibrexafungerp MICs ranged from 0.5 mg/L to 8 mg/L (MIC 50 0.5 mg/L). MICs of ibrexafungerp were ≤1 mg/L for the 2 C. famata isolates and 1 isolate each of C. bracarensis,C. catenulata,C. dubliniensis, and C. kefyr (Table 5). Ibrexafungerp MIC 50 was 2 mg/L for the 6 C. guilliermondii blood isolates with a MIC range from 2 to 4 mg/L, being twofold less active than caspofungin or micafungin. DISCUSSION The present study aimed to evaluate the in vitro activity of ibrexafungerp against a collection of 434 European blood isolates TABLE 1 | EUCAST clinical breakpoints (CBPs) and epidemiological cutoff values (ECVs). Species Antifungal drug CBPs (mg/L) ECVs (mg/L) Susceptible Resistant Wild-type Non Wild-type Candida albicans (n= 163) Ibrexafungerp ––≤0.5 >0.5 Caspofungin ––≤0.5 >0.5 Micafungin ≤0.016 >0.016 ≤0.016 >0.016 Fluconazole ≤2>4≤0.5 >0.5 Candida auris (n= 22) Ibrexafungerp ––≤2>2 Caspofungin ––≤1>1 Micafungin ––≤0.5 >0.5 Fluconazole ≤2>4≤64 >64 Candida glabrata (n= 60) Ibrexafungerp ––≤1>1 Caspofungin ––≤1>1 Micafungin ≤0.03 >0.03 ≤0.03 >0.03 Fluconazole ≤0.001 >16 ≤16 >16 Candida krusei (n= 29) Ibrexafungerp ––≤4>4 Caspofungin ––≤2>2 Micafungin ––≤0.25 >0.25 Fluconazole ––≤128 >128 Candida parapsilosis (n= 108) Ibrexafungerp ––≤2>2 Caspofungin ––≤8>8 Micafungin ≤2>2≤2>2 Fluconazole ≤2>4≤2>2 Candida orthopsilosis (n= 20) Ibrexafungerp ––≤8>8 Caspofungin ––≤8>8 Micafungin ––≤4>4 Fluconazole ≤2>4≤2>2 Candida tropicalis (n= 40) Ibrexafungerp ––≤2>2 Caspofungin ––≤1>1 Micafungin ––≤0.06 >0.06 Fluconazole ≤2>4≤1>1 Gray shaded area: Potentially non-wild-type MICs > 2 dilution steps above the modal MIC. Quindo ´s et al. Anti-Candida Activity of Ibrexafungerp Frontiers in Cellular and Infection Microbiology | www.frontiersin.org May 2022 | Volume 12 | Article 9065634 TABLE 2 | Summary of ibrexafungerp and comparators’in vitro antifungal activities against blood isolates from medically relevant species of Candida. Species/Antifungal drugs No. of isolates at MIC (mg/L) (Cumulative %) Total MIC (mg/L) ≤0.008 0.016 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 ≥128 Range Mode GM 50 90 Candida albicans (n= 163) Ibrexafungerp 0 7 (4.3) 50 (35) 48 (64.4) 50 (95.1) 6 (98.8) 2 (100) 0 0 0 0 0 –– –163 0.016-0.5 0.125 0.062 0.06 0.125 Caspofungin 0 3 (1.9) 7 (6.1) 38 (29.4) 100 (90.8) 8 (95.7) 4 (98.2) 1 (98.7) 1 (100) 0 0 0 –– –163 0.016-2 0.125 0.104 0.125 0.125 Micafungin 82 (50.3) 79 (98.8) 1 (99.4) 0 0 0 0 0 1 (100) 0 0 0 –– –163 ≤0.008–2 0.008 0.001 0.008 0.016 Fluconazole – ––– 56 (34.4) 90 (89.6) 14 (98.2) 1 (98.8) 0 0 01 (99.4) 0 0 1 (100) 163 0.125- ≥128 0.25 0.226 0.25 0.5 Candida parapsilosis (n= 108) Ibrexafungerp 0 6 (5.6) 0 1 (6.5) 3 (9.3) 10 (18.5) 38 (53.7) 24 (75.9) 15 (89.8) 6 (95.4) 5 (100) 0–– –108 0.016-8 0.5 0.660 0.5 4 Caspofungin 0 0 0 1 (0.9) 5 (5.6) 3 (8.3) 26 (32.4) 51 (79.6) 19 (97.2) 3 (100) 0 0 –– –108 0.06–4 1 0.846 1 2 Micafungin 3 (3.3) 2 (4.6) 1 (5.6) 0 0 5 (10.2) 15 (24.1) 32 (53.7) 48 (98.1) 2 (100) 00 –– –108 ≤0.008–4 2 0.933 1 2 Fluconazole – ––– 0 7 (6.5) 68 (69.4) 21 (88.9) 6 (94.4) 4 (98.1) 00 1 (99.1) 0 1 (100) 108 0.25- ≥128 0.5 0.698 0.5 2 Candida glabrata (n= 60) Ibrexafungerp 0 2 (3.3) 0 1 (5) 10 (21.7) 22 (58.3) 16 (85) 5 (93.3) 1 (95) 2 (98.3) 1 (100) 0–– –60 0.016-8 0.25 0.322 0.25 1 Caspofungin 0 0 0 3 (5) 17 (33.3) 19 (65) 15 (90) 4 (96.7) 0 1 (98.3) 01 (100) –– –60 0.06->8 0.25 0.280 0.25 0.5 Micafungin 18 (30) 30 (80) 3 (85) 1 (86.7) 1 (86.7) 0 1 (90) 4 (96.7) 2 (100) 0 0 0 –– –60 ≤0.008-2 0.016 0.023 0.016 0.5 Fluconazole – ––– 0 2 (3.3) 4 (10) 1 (11.7) 11 (30) 6 (40) 12 (60) 5 (68.3) 2 (71.7) 6 (81.7) 11 (100) 60 0.25- ≥128 8 9.514 8 ≥128 Candida tropicalis (n= 40) Ibrexafungerp 0 0 0 2 (5) 3 (12.5) 9 (35) 12 (65) 8 (85) 5 (97.5) 0 0 1 (100) –––40 0.06->8 0.5 0.517 0.5 2 Caspofungin 0 0 0 1 (2.5) 15 (40) 19 (87.5) 4 (97.5) 0 0 0 0 1 (100) –––40 0.06->8 0.25 0.221 0.25 0.5 Micafungin 1 (2.5) 7 (20) 24 (80) 7 (97.5) 0 0 0 1 (100) 0 0 0 0 –––40 ≤0.008-1 0.03 0.032 0.03 0.06 Fluconazole – –––1 (2.5) 8 (22.5) 9 (45) 6 (60) 1 (62.5) 1 (65) 0 0 2 (70) 12 (100) 040 0.125-64 64 3.364 2 64 Candida krusei (n= 29) Ibrexafungerp 0 0 0 0 1 (3.4) 2 (10.3) 10 (44.8) 16 100) 0 0 0 0 –––29 0.125-1 1 0.666 1 1 Caspofungin 0 0 0 0 0 7 (24.1) 18 (86.2) 4 (100) 0 0 0 0 –––29 0.25 –1 0.5 0.465 0.5 1 Micafungin 0 0 1 (3.4) 1 (6.9) 25 (93.1) 2 (100) 0 0 0 0 0 0 –––29 0.03-0.25 0.125 0.122 0.125 0.125 Fluconazole – ––– 0 0000004 (13.8) 18 (75.9) 5 (93.1) 2 (100) 29 16-≥128 32 37.828 32 64 Candida orthopsilosis (n= 20) (Continued) Quindo ´s et al. Anti-Candida Activity of Ibrexafungerp Frontiers in Cellular and Infection Microbiology | www.frontiersin.org May 2022 | Volume 12 | Article 9065635 of Candida. Ibrexafungerp showed a potent in vitro activity against Candida, with MICs ranging from 0.016 to 16 mg/L. The lowest ibrexafungerp MICs were observed against C. albicans and the highest ibrexafungerp MICs were observed against C. tropicalis Moreover, ibrexafungerp also displayed remarkable activity against fluconazoleand echinocandinresistant isolates. Five species of Candida (C. albicans,C. parapsilosis,C. glabrata, C. tropicalis,andC. krusei) cause more than 90% of IC (Quindos, 2014;Arendrup and Patterson, 2017;Quindosetal.,2018). C. albicans remains the predominant cause, but there is an evident shift in the etiology, and IC caused by other species less susceptible to current antifungal drugs is becoming more frequent (Fuller et al., 2019;Pfaller et al., 2019). Echinocandins, such as anidulafungin, caspofungin, and micafungin, are considered first-line therapy for IC because they possess fungicidal activity against Candida (GilAlonso et al., 2015a;Gil-Alonso et al., 2015b). However, emergence of azoleand echinocandin-resistant [multidrug-resistant (MDR)] isolates has been reported for C. auris,C. glabrata,C. guilliermondii,C. lusitaniae,andC. parapsilosis (Lortholary et al., 2011;Peman et al., 2012;Pfaller et al., 2012;Alexander et al., 2013; Pham et al., 2014;Dudiuk et al., 2017;Ruiz-Gaitan et al., 2019; Tortorano et al., 2021). MDR Candida isolates complicate clinical decision-making and are associated with treatment failure and high mortality rates (Lortholary et al., 2011;Alexander et al., 2013; Shields et al., 2013;Quindos et al., 2018;Tortorano et al., 2021). Ibrexafungerp is a novel orally bioavailable semi-synthetic derivative of the terpenoid enfumafungin that inhibits glucan synthase, decreasing 1,3-b-D-glucan polymers and weakening fungal cell wall (Jimenez-Ortigosa et al., 2014;Pfaller et al., 2017; Schell et al., 2017;Davis et al., 2020). The current study that has evaluated European blood isolates confirms and extends the observations published in previous studies (Pfaller et al., 2013; Jimenez-Ortigosa et al., 2014;Berkow et al., 2017;Larkin et al., 2017;Marcos-Zambrano et al., 2017;Pfaller et al., 2017;Schell et al., 2017;Nunnally et al., 2019;Gamal et al., 2021). However, these previous studies have mostly tested American isolates. Our study with European isolates is in line with the conclusion that ibrexafungerp displays potent in vitro activity against the most clinically relevant species of Candida. Ibrexafungerp showed an excellent activity against the C. albicans isolates included in the present study (MICs range 0.016–0.5 mg/L). These results confirm previous findings by other authors, such as Jimenez-Ortigosa et al. (2014);Schell et al. (2017),andMesquida et al. (2021), demonstrating that ibrexafungerp showed activity against most FKS-mediated echinocandin-resistant C. albicans and against azole-resistant C. albicans. Although there are no clinical breakpoints (CBPs) or ECVs available for ibrexafungerp, the study by Mesquida et al. (2022) as well as the present work have proposed WTULs. There are no differences in the proposed limits for C. glabrata (1 mg/L). However, our study suggests higher WTUL for C. albicans (0.5 mg/L vs. 0.25 mg/L by Mesquida et al.), for C. parapsilosis and C. tropicalis (2 mg/L vs. 1 mg/L by Mesquida et al.), and for C. krusei (4 mg/L vs. 2 mg/L by Mesquida et al.). TABLE 2 | Continued Species/Antifungal drugs No. of isolates at MIC (mg/L) (Cumulative %) Total MIC (mg/L) ≤0.008 0.016 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 ≥128 Range Mode GM 50 90 Ibrexafungerp 0 0 0 1 (5) 1 (10) 0 3 (25) 2 (35) 3 (50) 10 (100) 00 –––20 0.06-4 4 1.566 2 4 Caspofungin 0 0 0 0 0 1 (5) 6 (35) 1 (40) 8 (80) 4 (100) 0 0 –––20 0.25-4 2 1.320 2 4 Micafungin 0 0 0 1 (5) 0 1 (10) 5 (35) 10 (85) 3 (100) 0 0 0 –––20 0.06-2 1 0.756 1 1 Fluconazole – –––1 (5) 0 12 (65) 3 (80) 2 (90) 1 (95) 01 (100) 00020 0.125-16 0.5 0.783 0.5 2 Resistant isolates according to EUCAST criteria for fluconazole and micafungin are highlighted in bold type. Accepted and potentially non-wild-type MIC ranges are shaded for blood isolates with MICs > 2 dilution steps above the modal MIC. Quindo ´s et al. Anti-Candida Activity of Ibrexafungerp Frontiers in Cellular and Infection Microbiology | www.frontiersin.org May 2022 | Volume 12 | Article 9065636 C. parapsilosis is the first or second etiology of IC in China, Japan, Latin America, and the Mediterranean countries of Africa, Asia, and Europe, such as Italy, Portugal, and Spain (Quindos et al., 2018). In the current report, ibrexafungerp MICs ranged from 0.016 mg/L to 8 mg/L. Our results are in accordance to those by Marcos-Zambrano et al. (2017). In both studies, ibrexafungerp displayed remarkably lower MICs than micafungin against C. parapsilosis. Although Mesquida et al. (2022) did not differentiate among C. parapsilosis species complex, they also reported lower MICs for ibrexafungerp than for echinocandins. These high MIC values reported for echinocandins against C. parapsilosis have been associated with substitutions in the hs1 region of FKS1 (Garcia-Effron et al., 2008;Dudiuk et al., 2017;Marcos-Zambrano et al., 2017; Mesquida et al., 2022). Schell et al. (2017) also reported lower ibrexafungerp MIC 90 values for 19 C. parapsilosis blood isolates (0.25 mg/L) compared with echinocandins, suggesting that changes in FKS1 may not affect the capacity of ibrexafungerp to inhibit glucan synthase in this species. C. glabrata has increased its etiological importance in IC in Australia, Canada, the USA, and countries in Central and Northern Europe, such as Belgium and Germany (Quindos, 2014;Trouve et al., 2017;Quindos et al., 2018). In our study, ibrexafungerp displayed potent activity (MIC range 0.016–8 mg/L) also against fluconazole-resistant isolates. No cross-resistance has been found between ibrexafungerp and fluconazole, as previously noted by Marcos-Zambrano et al. (2017). The incidence of echinocandin resistance in C. glabrata is generally considered low, approximately 3%–4%, but can be as high as 30% in specific institutions (Alexander et al., 2013;Pham et al., 2014;Arendrup and Patterson, 2017). In the current study, ibrexafungerp MIC was ≤4 mg/L against four C. glabrata resistant to both fluconazole and micafungin. Pfaller et al. (2013) found similar results to ours but, in their report, ibrexafungerp was 8-fold more active than caspofungin against C. glabrata. Furthermore, ibrexafungerp showed activity against 31 C.glabratastrains with mutations in the hs of FKS1 or FKS2 (MIC ≤2 mg/L) and against 14 strains resistant to both caspofungin and fluconazole. In a later study by the same authors (Pfaller et al., 2017), 20 out of 25 FKS mutant C. glabrata isolates (80%) were NWT to one or more echinocandins, but only six (24%) were NWT to ibrexafungerp. Isolates of C. glabrata for which the ibrexafungerp MIC was > 2 mg/L (NWT) all were NWT and either intermediate or resistant to anidulafungin, caspofungin, and micafungin. In our study, 3 out of 4 isolates potentially NWT for ibrexafungerp were inhibited by ≤1 mg/L of caspofungin and one by 0.03 mg/L of micafungin. However, three of these four ibrexafungerp NWT isolates were resistant to micafungin. Schell et al. (2017) evaluated 34 echinocandin-resistant C. glabrata isolates along with 34 paired control C. glabrata isolates, observing that ibrexafungerp MICs for individual C. glabrata isolates tended to be three to five dilutions higher than those for the echinocandins. However, ibrexafungerp MICs trended in agreement with those for the echinocandins. These authors detected that C. glabrata isolates with FKS1 or FKS2 mutations or echinocandin resistance were inhibited by ≤4mg/Lof ibrexafungerp. Nunnally et al. (2019) also reported good ibrexafungerp activity against 89 C. glabrata isolates with FKS1 or FKS2 mutations that conferred resistance to at least one echinocandin. Ibrexafungerp MIC values ranged from <0.03 mg/L to 4 mg/L while caspofungin and micafungin MICs ranged from 0.03 to >16 mg/L and 0.008 to >16 mg/L, respectively. In the study by Mesquida et al. (2022), an isolate of C. glabrata that displayed an ibrexafungerp MIC of 2 mg/L and echinocandin NWT phenotype harbored a mutation at FKS2. The spectrum of resistance mutations found in C. glabrata suggested a partially overlapping but independent binding site for ibrexafungerp relative to echinocandins on glucan synthase as these drugs are structurally dissimilar and interact differently with the target (Jimenez-Ortigosa et al., 2014). Consequently, this potent in vitro activity for ibrexafungerp has been reported against C. glabrata isolates harboring FKS1 and FKS2 point mutations that cause echinocandin resistance (Marcos-Zambrano et al., 2017;Pfaller et al., 2017;Schell et al., 2017;Nunnally et al., 2019;Mesquida et al., 2022). Moreover, TABLE 3 | MICs distribution of ibrexafungerp and comparator antifungal drugs against echinocandin wild-type (WT) and non-wild-type/resistant (NWTR) Candida spp. Isolates. Species Phenotype (no. of isolates) Modal MIC (MIC range) [mg/L] Ibrexafungerp Caspofungin Micafungin Fluconazole Candida albicans WT (160) 0.125 (0.016–2) 0.125 (0.016–0.5) 0.016 (≤0.008–0.016) 0.25 (0.125–≥128) NWTR (3) −(0.03–0.25) −(0.125–2) −(0.03–>8) 0.25 (0.25) Candida parapsilosis WT (106) 0.5 (0.016–8) 1 (0.06–4) 2 (≤0.008–2) 0.25 (0.25–≥128) NWTR (2) 2 (2) −(1–2) 4 (4) −(0.25–4) Candida glabrata WT (51) 0.25 (0.016–8) 0.25 (0.06–0.5) 0.016 (≤0.008–0.03) 8 (0.25–≥128) NWTR (9) 0.25 (0.25–4) 1 (0.5–>8) 1 (0.06–2) 128 (0.5–128) Candida tropicalis WT (39) 0.25 (0.03–>8) 0.25 (0.03–>8) 0.5 (0.03–>8) 0.25 (0.03–16) NWTR (1) >8 (>8) >8 (>8) 0.5 (0.5) 0.25 (0.25) Candida orthopsilosis WT (16) 4 (0.06–4) 2 (0.25–2) 1 (0.06–2) 0.5 (0.125–16) NWTR (4) 4 (2–4) 4 (4) 1 (1–2) 0.5 (0.5–1) Candida auris WT (16) 0.5 (0.5–4) 0.25 (0.25–2) 0.25 (0.25–1) ≥128 (≥128) NWTR (6) 0.5 (0.5–8) >8 (2–>8) 1 (1–>8) ≥128 (≥128) Candida guilliermondii WT (5) 2 (2–4) 0.5 (0.5–1) 0.5 (0.5–1) 0.5 (0.5–64) NWTR (1) 4 (4) 8 (8) 0.008 (0.008) 8 (8) For antifungals without established epidemiological cutoff points, the points established in Table 1 (shaded area) have been taken into account. Quindo ´s et al. Anti-Candida Activity of Ibrexafungerp Frontiers in Cellular and Infection Microbiology | www.frontiersin.org May 2022 | Volume 12 | Article 9065637 the in vitro efficacy of ibrexafungerp has been supported by successful treatments in murine IC caused by C. glabrata resistant to echinocandins (Lepak et al., 2015;Wiederhold et al., 2018). In a murine model of IC caused by C. albicans,C. parapsilosis,orC. glabrata using an oral therapy with ibrexafungerp, Lepak et al. (2015) demonstrated that the AUC/ MIC was the best pharmacodynamics parameter predicting clinical response. A MIC ≤1 mg/L obtained by CLSI would predict a clinical response using oral ibrexafungerp (MarcosZambrano et al., 2017). In Asia, C. tropicalis was the second etiological agent of IC in many hospitals from China, India, Singapore, Thailand, and Taiwan (Quindos et al., 2018). We observed that ibrexafungerp MICs ranged from 0.06 mg/L to ≥8 mg/L for 40 C. tropicalis blood isolates, which confirms the consistently low ibrexafungerp MICs for azole-resistant C. tropicalis reported by Schell et al. (2017).Mesquida et al. (2022) found mutations in the FKS1 gene of two NWT isolates of C. tropicalis that resulted, in both cases, in ibrexafungerp MICs between 0.5 and 1 mg/L. We observed high activity of ibrexafungerp against 29 C. krusei (MIC range 0.125–1 mg/L). Schell et al. (2017) reported ibrexafungerp MICs for six isolates of C. krusei (MIC range from 0.5 mg/L to 4 mg/L), which were higher than those for the other Candida species. These authors suggested that a naturally occurring unidentified substitution may be responsible for the reduced activity of ibrexafungerp on glucan synthase in C. krusei. However, it is unknown if this translates into clinical failure. In a recent Spanish nationwide study on candidemia, C. orthopsilosis was the fifth most frequently isolated species, preceding C. krusei (Peman et al., 2012). In the present study, ibrexafungerp showed good activity against 20 C. orthopsilosis (MIC range 0.06–4 mg/L). To our knowledge, this is the first study on in vitro activity of ibrexafungerp against C. orthopsilosis. The lack of previous reports in this regard precludes comparison. Differences in ibrexafungerp activity against the closely related species C. orthopsilosis and C. parapsilosis highlights the importance of a correct identification of the Candida species involved in invasive infections. C. auris is an emerging pathogen that has been identified in many countries associated with high mortality and a marked ability to develop resistance to multiple commonly used antifungal agents and to withstand standard infection control practices (Larkin et al., 2017;Ruiz-Gaitan et al., 2017;RuizGaitan et al., 2018;Ruiz-Gaitan et al., 2019). Data from Larkin et al. (2017) show that the C. auris isolates exhibited multidrug resistance against fluconazole and amphotericin B. Moreover, some isolates also exhibited high MIC values for voriconazole and itraconazole. In the present report, all C. auris isolates were resistant to fluconazole while ibrexafungerp showed notable activity (MIC range 0.5 mg/L to 8 mg/L). The in vitro activity of ibrexafungerp against European C. auris isolates from our study was similar to that described by Berkow et al. (2017) who studied 100 isolates of this species from Asia, Africa, and America. Of note, in the current study, MM values between C. auris WT and NWT isolates increased 64-fold for caspofungin and 4-fold for micafungin, whereas MM values for ibrexafungerp did not change. Interestingly, ibrexafungerp has been shown to TABLE 4 | Summary of ibrexafungerp and comparators’in vitro antifungal activities against 22 clinical isolates of Candida auris. Antifungal drugs No. of blood/any origin isolates at MIC (mg/L) (Cumulative %) Total MIC ≤0.008 0.016 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 ≥128 Range Mode GM 50 90 Ibrexafungerp 0 0 0 0 0 0 5/16 (62.5/ 72.7) ½ (75/ 81.8) ½ (87.5/ 90.9) 0/1 (87.5/ 95.5) 1/1 (100) 0 –– – 8/22 0.5-8 0.5 0.753 0.5 2 Caspofungin 0 0 0 0 0 7/16 (87.5/ 72.7) 0 0 0/1 (87.5/ 77.3) 0 0 1/5 (100) –– – 8/22 0.25- >8 0.25 0.585 0.25 >8 Micafungin 0 0 0 0 6/13 (75/ 59.1) 0/1 (75/ 63.6) 0 ¼ (87.5/ 81.8) 0/1 (87.5/ 86.4) 0/1 (87.5/ 90.9) 0/1 (87.5/ 95.5) 1/1 (100) –– – 8/22 0.125- >8 0.125 0.377 0.125 4 Fluconazole 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8/22 (100) 8/22 ≥128 ≥128 211.905 ≥128 ≥128 Potentially non-wild-type MIC ranges are shaded for blood isolates with MICs > 2 dilution steps above the modal MIC. Quindo ´s et al. Anti-Candida Activity of Ibrexafungerp Frontiers in Cellular and Infection Microbiology | www.frontiersin.org May 2022 | Volume 12 | Article 9065638 be effective in vivo in the treatment of experimental infections with C. auris (Wiederhold et al., 2021). Ibrexafungerp was twofold less active than caspofungin or micafungin against six C. guilliermondii blood isolates. Accordingly, Mesquida et al. (2022) also found higher ibrexafungerp MICs for 12 C.guilliermondiiisolates, from 0.125 to 8 mg/L. Naturally occurring high echinocandins MICs against C. guilliermondii have been recognized since these antifungal agents were first introduced probably in association to substitutions in the hs1 region of FKS1 (Dudiuk et al., 2017; Schell et al., 2017). In addition, Schell et al. (2017) reported that ibrexafungerp MIC ranges for three C. lusitaniae blood isolates were 1 to 2 mg/L and ibrexafungerp MICs for C. lusitaniae were three to five dilutions higher than those for the other species of Candida. Ibrexafungerp MICs for the two isolates of C. lusitaniae (MICs 4 and 8 mg/L) tested in the current study were higher than those observed for the other Candida species. Aside from emerging resistance, an important limitation of echinocandins is that they must be administered daily by intravenous infusion, potentially prolonging hospital stays for patients undergoing echinocandin therapy and limiting them to inpatient settings in most instances. Of note, Wring et al. (2018) reported that the risk for interactions of ibrexafungerp with drugs metabolized via the cytochrome P450 family of enzymes is low. Ibrexafungerp exhibited concentrationand timedependent fungicidal activity against C. albicans,C. glabrata, C. krusei,C. parapsilosis, and C. tropicalis in time-kill curve studies (Scorneaux et al., 2017). Moreover, ibrexafungerp has demonstrated efficacy for IC in phase 2 and 3 clinical studies (Spec et al., 2019). The current study demonstrates that ibrexafungerp shows potent in vitro activity against Candida blood isolates and its activity is comparable to that of micafungin. Ibrexafungerp even exhibits good activity against fluconazole-resistant Candida isolates. Moreover, echinocandin-resistant isolates exhibit ibrexafungerp MICs consistent with those of echinocandinsusceptible isolates. However, direct comparisons of ibrexafungerp MICs with those of other antifungal drugs should be interpreted with caution, as different drugs may produce diverse ranges of MICs and yet have equivalent clinical efficacy because of their differences in bioavailability and in PK/PD properties. Although Pfaller et al. (2013) reported >90% essential agreement between both methods, CLSI and EUCAST, the comparison between our results and those of American authors should consider that the EUCAST method tends to yield higher MICs than CLSI, regardless of the studied species. Although the most clinically relevant species of Candida have been included in the current study, it would be interesting to assess the activity of ibrexafungerp against additional species as well as more NWT isolates. In conclusion, we demonstrated that ibrexafungerp, a potent inhibitor of glucan synthase, could be an important acquisition to the antifungal toolbox for the therapy of patients suffering from IC caused by MDR species, such as C. auris,C. glabrata,or C. krusei. Considering the excellent pharmacokinetic properties of ibrexafungerp (oral availability and excellent tissue distribution and concentrations) as well as the potent activity observed against the main species causing candidiasis, ibrexafungerp should be regarded as a potential candidate for the therapy of these important diseases. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. AUTHOR CONTRIBUTIONS GQ,KM-C,andRS-Mdesignedtheresearchandparticipatedin manuscriptwriting.KB-E,EC,MJL-S,AH,IM,AT,AP,MV-G, CM-A, AG, FS-R, JM-B, MR-I, EM-M, CC-M, LL-S, AR-G, MF-R, DL,JC,AR,JaP,JG,JoP,CP,OR,GE,NJ,DA,andEEconducted sampling and clinical measures, carried out the experiments, TABLE 5 | Activities of ibrexafungerp and comparator antifungal drugs against other species of Candida blood isolates. Species Isolate reference MIC (mg/L) Ibrexafungerp Caspofungin Micafungin Fluconazole Candida bracarensis 18-3133Br 0.25 0.5 0.016 4 Candida catenulata 17-011 0.5 0.5 2 1 Candida dubliniensis 18-10677-2C 0.5 0.25 0.008 0.25 Candida famata 17-012 0.5 1 2 0.5 Candida famata 10-10647-1C 1 1 0.125 1 Candida guilliermondii 10-606-1C 2 1 1 1 Candida guilliermondii 17-23377-2C 2 0.5 0.5 0.5 Candida guilliermondii 17-25047C 2 0.5 0.5 0.5 Candida guilliermondii 17-341 2 1 1 64 Candida guilliermondii 17-014 4 0.5 0.5 0.5 Candida guilliermondii 17-324 4 8 0.008 8 Candida kefyr 18-001 0.25 0.5 0.125 0.25 Candida lusitaniae 17-018 8 0.5 0.125 0.125 Candida lusitaniae 11-423-1C 4 0.5 0.06 32 Quindo ´s et al. Anti-Candida Activity of Ibrexafungerp Frontiers in Cellular and Infection Microbiology | www.frontiersin.org May 2022 | Volume 12 | Article 9065639