Candidiasis by Candida glabrata, Candida nivariensis and Candida bracarensis in Galleria mellonella: Virulence and Therapeutic Responses to Echinocandins

Candida albicans is the major etiological agent of invasive candidiasis but the increasing prevalence of emerging species of Candida, such as Candida glabrata and phylogenetically closely related species, Candida nivariensis and Candida bracarensis, requires special attention. Differences in virulence among these species and their therapeutic responses using in vivo non-mammalian models are scarcely analysed. The aim of this study was analyse the survival of G. mellonella and host-pathogen interactions during infection by C. glabrata, C. nivariensis and C. bracarensis. Moreover, therapeutic responses to echinocandins were also assessed in the G. mellonella model of candidiasis. These three species produced lethal infection in G. mellonella; C. glabrata was the most virulent species and C. bracarensis the less. Haemocytes of G. mellonella phagocytised C. bracarensis cells more effectively than those of the other two species. Treatment with caspofungin and micafungin was most effective to protect larvae during C. glabrata and C. nivariensis infections while anidulafungin was during C. bracarensis infection. The model of candidiasis in G. mellonella is simple and appropriate to assess the virulence and therapeutic response of these emerging Candida species. Moreover, it successfully allows for detecting differences in the immune system of the host depending on the virulence of pathogens.This research was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) [SAF2017-86188-P and PID2020-117983RB-I00] and from the Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza [GIC15/78 IT-990-16]. Ainara Hernando-Ortiz was funded by Ph.D. grants from the University of the Basque Country (PIF 16/39)

Antimicrobial Peptides with Anti-Candida Activity

[EN] Mycoses are accountable for millions of infections yearly worldwide. Invasive candidiasis is the most usual, presenting a high morbidity and mortality. Candida albicans remains the prevalent etiologic agent, but the incidence of other species such as Candida parapsilosis, Candida glabrata and Candida auris keeps increasing. These pathogens frequently show a reduced susceptibility to commonly used antifungal drugs, including polyenes, triazoles and echinocandins, and the incidence of emerging multi-drug-resistant strains of these species continues to increase. Therefore, the need to search for new molecules that target these pathogenic species in a different manner is now more urgent than ever. Nature is an almost endless source of interesting new molecules that could meet this need. Among these molecules, antimicrobial peptides, present in different sources in nature, possess some advantages over conventional antifungal agents, even with their own drawbacks, and are considered as a promising pharmacological option against a wide range of microbial infections. In this review, we describe 20 antimicrobial peptides from different origins that possess an activity against Candida.This research was funded by the Spanish Ministry of Science and Innovation (PID2020-117983RB-I00) and by the Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza (IT1607-22). A.P.-R. was funded by a Ph.D. grant from the University of the Basque Country (PIF17/167)

In Vitro Interaction and Killing-Kinetics of Amphotericin B Combined with Anidulafungin or Caspofungin against Candida auris

Treatment of invasive infections caused by Candida auris is challenging due to the limited therapeutic options. The combination of antifungal drugs may be an interesting and feasible approach to be investigated. The aim of this study was to examine the in vitro activity of amphotericin B in combination with anidulafungin or caspofungin against C. auris. In vitro static time–kill curve experiments were conducted for 48 h with different combinations of amphotericin B with anidulafungin or caspofungin against six blood isolates of C. auris. The antifungal activity of 0.5 mg/L of amphotericin B was limited against the six isolates of C. auris. Similarly, echinocandins alone had a negligible effect, even at the highest tested concentrations. By contrast, 1 mg/L of amphotericin B showed fungistatic activity. Synergy was rapidly achieved (8 h) with 0.5 mg/L of amphotericin B plus 2 mg/L of anidulafungin or caspofungin. These combinations lead to a sustained fungistatic effect, and the fungicidal endpoint was reached against some C. auris isolates. Additionally, ≥0.5 mg/L of either of the two echinocandins with 1 mg/L of amphotericin B resulted in fungicidal effect against all C. auris isolates. In conclusion, combinations of amphotericin B with anidulafungin or caspofungin provided greater killing with a lower dose requirement for amphotericin B compared to monotherapy, with synergistic and/or fungicidal outcomes.This research was funded by Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza, GIC15/78 IT-990-16 and by FIS, Spain, PI17/01538. U.C. was funded by a Ph.D. grant from the University of the Basque Country, PIF 17/266

Assessing pH-dependent activities of virulence factors secreted by Candida albicans

Candida albicans is an opportunistic pathogen that can thrive under adverse conditions including suboptimal pH, nutrient scarcity, and low levels of oxygen. Its pathogenicity is associated with the production of virulence factors such as extracellular hydrolytic enzymes and toxins. This study was aimed at determining the effect of external pH, substrate nature, and strain origin on protease, lipase, and hemolysin production. To achieve this objective, agar plate assays were performed at pH 5.0, 6.5, and 7.5 with substrates suitable for the detection of each family of enzymes. Moreover, the study was conducted with 20 clinical C. albicans isolates from blood, oral cavity, skin, urine, and vagina. The hydrolytic zones formed around the colonies were further measured to calculate the Ez (enzymatic zone) indexes. We found that detection of proteases in skim milk agar plates was possible for most isolates only at pH 5 (80%) and pH 6.5 (75%), whereas BSA plates could confer protease detection exclusively at pH 5 (80%). Similarly, the percentage of isolates possessing lipolytic activities was higher at pH 5 (90%) than at pH 6.5 (70%) and pH 7.5 (35%). In contrast, hemolytic activities were detected in all isolates at pH 6.5 and 7.5 but not at pH 5. Further analysis revealed that some differences in the detected activities could potentially be attributed to the anatomical origin of these isolates. Collectively, these findings suggest that the pH of the site of infection might be critical for mimicking the microenvironment employed to experimentally discover the key virulence factors.The work was supported by IKERBASQUE (Basque Foundation for Science). Elena Eraso, Elena Sevillano, and Guillermo Quindós have received grant support from Consejería de Educación, Universidades e Investigación del Gobierno Vasco (GIC15/78 IT-990-16/IT1607-22), Spanish Ministry of Science and Innovation (PID2020-117983RB-I00), and UPV/EHU (COLAB19/11)

In Vitro and In Vivo Activity of Citral in Combination with Amphotericin B, Anidulafungin and Fluconazole against Candida auris Isolates

Candida auris is an emerging fungal pathogen responsible for hospital outbreaks of invasive candidiasis associated with high mortality. The treatment of these mycoses is a clinical challenge due to the high resistance levels of this species to current antifungal drugs, and alternative therapeutic strategies are needed. In this study, we evaluated the in vitro and in vivo activities of combinations of citral with anidulafungin, amphotericin B or fluconazole against 19 C. auris isolates. The antifungal effect of citral was in most cases similar to the effect of the antifungal drugs in monotherapy. The best combination results were obtained with anidulafungin, with synergistic and additive interactions against 7 and 11 of the 19 isolates, respectively. The combination of 0.06 μg/mL anidulafungin and 64 μg/mL citral showed the best results, with a survival rate of 63.2% in Caenorhabditis elegans infected with C. auris UPV 17-279. The combination of fluconazole with citral reduced the MIC of fluconazole from > 64 to 1–4 μg/mL against 12 isolates, and a combination of 2 μg/mL fluconazole and 64 μg/mL citral was also effective in reducing mortality in C. elegans. Amphotericin B combined with citral, although effective in vitro, did not improve the activity of each compound in vivo.The research group was funded by the Consejería de Educación, Universidades e Investigación (GIC IT-1607-22) of Gobierno Vasco-Eusko Jaurlaritza and by the Spanish Ministry of Science and Innovation (PID2020-117983RB-I00)

In Vitro Pharmacokinetic/Pharmacodynamic Modelling and Simulation of Amphotericin B against Candida auris

The aims of this study were to characterize the antifungal activity of amphotericin B against Candida auris in a static in vitro system and to evaluate different dosing schedules and MIC scenarios by means of semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) modelling and simulation. A two-compartment model consisting of a drug-susceptible and a drug-resistant subpopulation successfully characterized the time-kill data and a modified Emax sigmoidal model best described the effect of the drug. The model incorporated growth rate constants for both subpopulations, a death rate constant and a transfer constant between both compartments. Additionally, the model included a parameter to account for the delay in growth in the absence or presence of the drug. Amphotericin B displayed a concentration-dependent fungicidal activity. The developed PK/PD model was able to characterize properly the antifungal activity of amphotericin B against C. auris. Finally, simulation analysis revealed that none of the simulated standard dosing scenarios of 0.6, 1 and 1.5 mg/kg/day over a week treatment showed successful activity against C. auris infection. Simulations also pointed out that an MIC of 1 mg/L would be linked to treatment failure for C. auris invasive infections and therefore, the resistance rate to amphotericin B may be higher than previously reported.This research was funded by Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza, GIC15/78 IT-990-16 and by FIS, Spain, PI17/01538. U.C. was funded by a Ph.D. grant from the University of the Basque Country, PIF 17/266

In Vitro Synergistic Interactions of Isavuconazole and Echinocandins against Candida auris

Candida auris is an emergent fungal pathogen that causes severe infectious outbreaks globally. The public health concern when dealing with this pathogen is mainly due to reduced susceptibility to current antifungal drugs. A valuable alternative to overcome this problem is to investigate the efficacy of combination therapy. The aim of this study was to determine the in vitro interactions of isavuconazole with echinocandins against C. auris. Interactions were determined using a checkerboard method, and absorbance data were analyzed with different approaches: the fractional inhibitory concentration index (FICI), Greco universal response surface approach, and Bliss interaction model. All models were in accordance and showed that combinations of isavuconazole with echinocandins resulted in an overall synergistic interaction. A wide range of concentrations within the therapeutic range were selected to perform time-kill curves. These confirmed that isavuconazole–echinocandin combinations were more effective than monotherapy regimens. Synergism and fungistatic activity were achieved with combinations that included isavuconazole in low concentrations (≥0.125 mg/L) and ≥1 mg/L of echinocandin. Time-kill curves revealed that once synergy was achieved, combinations of higher drug concentrations did not improve the antifungal activity. This work launches promising results regarding the combination of isavuconazole with echinocandins for the treatment of C. auris infections.This research was funded by Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza, GIC15/78 IT-990-16 and by FIS, Spain, PI17/01538. UC was funded by a Ph.D. grant from the University of the Basque Country UPV/EHU, PIF 17/266

In vitro activities of carvacrol, cinnamaldehyde and thymol against Candida biofilms

[EN]Oral candidiasis is frequently associated with Candida biofilms. Biofilms are microbial communities related to persistent, recalcitrant and difficult to-treat infections. Conventional treatments are not sufficient to overcome biofilm-associated candidiasis; thus, the search of new antifungal compounds is necessary. In the current study, we have evaluated the effect of three phytocompounds, carvacrol, cinnamaldehyde and thymol, against Candida planktonic and sessile cells. Reduction in biofilm biomass and metabolic activity was assessed during adhesion and mature biofilm phases. Candida albicans was the most biofilm-producing Candida species. All phytocompounds tested were fungicidal against Candida planktonic cells. Cinnamaldehyde was the most active in inhibiting biofilm adhesion, but carvacrol and thymol significantly reduced both mature biofilm biomass and metabolic activity. These results highlight the role of cinnamaldehyde, carvacrol and thymol as promising alternatives for the treatment of candidiasis due to their antibiofilm capacities, and stress the necessity to continue studies on their safety, toxicity and pharmacodynamics and pharmacokinetics.This work was supported by Gobierno Vasco-Eusko Jaurlaritza, Spain [grant number GIC15/78 IT-990-16, 2016] and Fundacion ONCE "Oportunidad al Talento", Spain and Fondo Social Europeo, Spain [CMA, 2018]

Virulence of Candida Auris from Different Clinical Origins in Caenorhabditis Elegans and Galleria Mellonella Host Models

Candida auris is an emerging multidrug-resistant fungal pathogen responsible for nosocomial outbreaks of invasive candidiasis. Although several studies on the pathogenicity of this species have been reported, the knowledge on C. auris virulence is still limited. This study aims to analyze the pathogenicity of C. auris, using one aggregating isolate and eleven non-aggregating isolates from different clinical origins (blood, urine and oropharyngeal specimens) in two alternative host models of candidiasis: Caenorhabditis elegans and Galleria mellonella. Furthermore, possible associations between virulence, aggregation, biofilm-forming capacity, and clinical origin were assessed. The aggregating phenotype isolate was less virulent in both in vivo invertebrate infection models than non-aggregating isolates but showed higher capacity to form biofilms. Blood isolates were significantly more virulent than those isolated from urine and respiratory specimens in the G. mellonella model of candidiasis. We conclude that both models of candidiasis present pros and cons but prove useful to evaluate the virulence of C. auris in vivo. Both models also evidence the heterogeneity in virulence that this species can develop, which may be influenced by the aggregative phenotype and clinical origin.This work was supported by the Euskal Herriko Unibertsitatea [PIF 16/39]; Euskal Herriko Unibertsitatea [PIF17/167]; Eusko Jaurlaritza [GIC15/78 IT-990-16]; Ministerio de Economia y Competitividad [SAF2017-86188-P]

In vitro and in vivo anti-Candida activity of citral in combination with fluconazole

[EN] Background The ability of Candida to develop biofilms on inert surfaces or living tissues favors recalcitrant and chronic candidiasis associated, in many instances, with resistance to current antifungal therapy. Aim The aim of this study was to evaluate the antifungal activity of citral, a phytocompound present in lemongrass essential oil, in monotherapy and combined with fluconazole against azole-resistant Candida planktonic cells and biofilms. The effect of citral combined with fluconazole was also analysed with regard to the expression of fluconazole resistance-associated genes in Candida albicans and the effectiveness of the combination therapy in a Caenorhabditis elegans model of candidiasis. Results Citral reduced biofilm formation at initial stages and the metabolic activity of the mature biofilm. The combination of citral with fluconazole was synergistic, with a significant increase in the survival of C. elegans infected with Candida. RNA analysis revealed a reduction of the expression of the efflux pump encoded by MDR1, leading to a greater effect of fluconazole. Conclusion Citral in monotherapy and in combination with fluconazole could represent an interesting therapy to treat recalcitrant Candida infections associated to biofilms.This research was supported by Gobierno Vasco-Eusko Jaurlaritza [Eusko Jaurlaritza GIC15/78 IT-990-16] and by Fundacion ONCE "Oportunidad al Talento" and Fondo Social Europeo CM-A [C.M.-A.]; Ministerio de Economia, Industria y Competitividad, Gobierno de Espan [PID2020-117983RB-I00]