Azole resistance in Aspergillus flavus and associated fitness cost.

BACKGROUND: The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals - CYP 51 A, B and C - are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. OBJECTIVES: This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. METHODS: The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. RESULTS: In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. CONCLUSIONS: These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus.

In vitro efficacy of essential oils against various Candida species.

Candida species are responsible for the most common fungal infections worldwide. We studied the in vitro antifungal activity of a large panel of essential oils (EOs) against various Candida species. The EOs activity against Candida spp. was tested using a gradient microdilution assay ranging from 4% to 0.008% (v/v). After a preliminary screening including 31 EOs, seven selected EOs were tested against 13 clinical isolates and four reference strains belonging to six Candida species. Cinnamomum zeylanicum and Cymbopogon giganteus EOs exhibited the best antifungal activity against all clinical and reference strains, with MIC ranges of 0.015%-0.25% (v/v). EOs from Litsea citrata, Backhousia citriodora and Ocimum sanctum presented MIC ranges of 0.03%-0.5% (v/v). The antifungal efficacy of EOs was independent of the susceptibility of Candida strains to usual antifungal agents. These EOs could have a promising antifungal action.

The Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Aspergillus Section Flavi from Three French Hospitals.

(1) Background: Aspergillus flavus is a cosmopolitan mold with medical, veterinary, and agronomic concerns. Its morphological similarity to other cryptic species of the Flavi section requires molecular identification techniques that are not routinely performed. For clinical isolates of Aspergillus section Flavi, we present the molecular identification, susceptibility to six antifungal agents, and clinical context of source patients. (2) Methods: One hundred forty fungal clinical isolates were included in the study. These isolates, recovered over a 15-year period (2001-2015), were identified based on their morphological characteristics as belonging to section Flavi. After the subculture, sequencing of a part of the ß-tubulin and calmodulin genes was performed, and resistance to azole antifungals was screened on agar plates containing itraconazole and voriconazole. Minimum inhibitory concentrations were determined for 120 isolates by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. (3) Results: Partial ß-tubulin and calmodulin sequences analysis showed that 138/140 isolates were A. flavus sensu stricto, 1 isolate was A. parasiticus/sojae, and 1 was A. nomiae. Many of the isolates came from samples collected in the context of respiratory tract colonization. Among probable or proven aspergillosis, respiratory infections were the most frequent, followed by ENT infections. Antifungal susceptibility testing was available for isolates (n = 120, all A. flavus ss) from one hospital. The MIC range (geometric mean MIC) in mg/L was 0.5-8 (0.77), 0.5-8 (1.03), 0.125-2 (0.25), 0.03-2 (0.22), 0.25-8 (1.91), and 0.03-0.125 (0.061) for voriconazole, isavuconazole, itraconazole, posaconazole, amphotericin B, and caspofungin, respectively. Two (1.67%) isolates showed resistance to isavuconazole according to current EUCAST breakpoints with MICs at 8 mg/L for isavuconazole and voriconazole. One of these two isolates was also resistant to itraconazole with MIC at 2 mg/L. (4) Conclusions: The present characterization of a large collection of Aspergillus belonging to the Flavi section confirmed that A. flavus ss is the predominant species. It is mainly implicated in respiratory and ENT infections. The emergence of resistance highlights the need to perform susceptibility tests on section Flavi isolates.

In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates.

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.

Colistin and Isavuconazole Interact Synergistically In Vitro against Aspergillus nidulans and Aspergillus niger.

The in vitro interactions of isavuconazole in combination with colistin were evaluated against 55 clinical Aspergillus species isolates belonging to the five most important species (Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) responsible for human aspergillosis by a microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method for antifungal susceptibility testing. Selected isolates (A. nidulans, n = 10; A. niger, n = 15) were additionally evaluated by an agar diffusion assay using isavuconazole gradient concentration strips with or without colistin incorporated Roswell Parc Memorial Institute (RPMI) agar. Interpretation of the checkerboard results was done by the fractional inhibitory concentration index. Using the checkerboard method, combination isavuconazole-colistin was synergistic for 100% of the 15 A. nidulans isolates and for 60% of the 20 A. niger isolates. No interactions were found for any of the other isolates. By agar diffusion assay, minimal inhibitory concentrations (MICs) in combination decreased compared to isavuconazole alone for 92% of the isolates. No interactions were found for any A. nidulans isolates, but synergy was observed for 40% of the A. niger isolates. A poor essential agreement of EUCAST and gradient concentration strip MICs at ± 2 log2 dilutions with 0% was obtained. Antagonistic interactions were never observed regardless of the technique used.