S4.3d Mechanism of fluconazole resistance in Candida vulturna, a member of the Candida haemulonii complex of multidrug - resistant yeasts
Authors: Macedo Daiana , Berrio Indira , Scandon Patricia , Gamarra Soledad , Effron Guillermo Garcia 
Published: September 2022

Abstract

Emergent species of the Candida genus, September 22, 2022, 10:30 AM - 12:00 PM

Objetives
Candida vulturna is an emerging yeast firstly isolated in 2016 from flowers and from a blood sample in Malaysia. It is a member of the Candida haemulonii species complex. This complex has 9 species within which C. haemulonii (including the vulnera variety), C. pseudohemulonii, C. duobushaemulonii, C. auris, and now C. vulturna are considered human pathogens. Like the other members of the haemulonii complex, the identification of C. vulturna is difficult. Due to the scarcity of strains to study, the susceptibility pattern of this species is unknown. The objectives of this work were to study C. vulturna strains isolated from clinical samples from different cities of Colombia, to study their susceptibility patterns, and to establish their mechanisms of azole resistance.

Methods
Five C. vulturna strains isolated from urine (n = 1) and blood (n = 4) in three different cities in Colombia were studied. Strains were identified as C. duobushaemulonii (n = 4) and C. haemulonii (n = 1) by means of VITEK2 (software version 9.02) and as C. pseudohaemulonii (n = 5) by using Bruker's MALDI-TOF in Colombia. Upon arrival in Argentina, isolates were molecularly identified by sequencing the ITS regions (rDNA). Antifungal susceptibility testing was performed following the CLSI´s M27 4th ed document. Amphotericin B, anidulafungin, caspofungin, 5-flucytosine, fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole were tested. In addition, ERG11 genes of the five strains were amplified and sequenced.

Results
The sequences of the ITS regions were analyzed and the 5 strains were identified as C. vulturna (100% homology). All the strains presented low echinocandins MICs (0.06-0.12 μg/ml), 5-flucytosine (0.06-0.12 μg/ml) and triazoles with the exception of fluconazole (isavuconazole: &lt;0.015 -0.12 μg/ml, itraconazole: 0.06-0.50 μg/ml, posaconazole: &lt;0.015-0.015 μg/ml and voriconazole: 0.03-0.50 μg/ml). In the case of amphotericin B and fluconazole, 4 of the 5 strains presented high MICs for both antifungals (&gt;8 μg/ml and &gt;64 μg/ml), while the remaining one presented MIC values = 1 μg/ml for the polyene and for the azole (cross amphotericin B and fluconazole high MICs). The strain with low fluconazole and amphotericina B MICs was the one isolated from urine. ERG11 sequences analysis showed that the 4 strains with high fluconazole MICs (blood isolates) harbor five amino acid substitutions: S119A, P135S, D180E, K266S, N326K (using C. albicans numbering). Only the P135 residue is highly conserved among ERG11 from different species (Fig. 1, box shows the substitution). In addition, this substitution is present in the Erg11p of fluconazole-resistant Debaryomyces hansenii and the P135 residue is located near the Erg11p´s Heme group where azole drugs bind (Fig. 2: Saccharomyces cereviciae Erg11p complexed with voriconazole 3D structure. In yellow the region between Y131 and P135 residues. In grey voriconazole binding the Heme group).

Conclusion
This emerging multi-resistant pathogen may be underdiagnosed by the methods commonly used in clinical laboratories. The only method capable of identifying this species is DNA sequencing. The strains studied presented very high MIC values for amphotericin B and fluconazole. The P135S substitution could be responsible for the high fluconazole MIC values observed in 4 of the 5 strains of C. vulturna.