P059 The overexpression of efflux pump gene cdr1B resulting in voriconazole- and isavuconazole- resistance in Aspergillus fumigatus recovered from a patient with chronic pulmonary aspergillosis in China
Authors: Liang Tianyu , Wang Qiqi , Pan Shiyang , Ni Fang , Li Ruoyu , Wan Zhe , Liu Wei 
Published: September 2022

Abstract

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM

Objectives
Triazole resistance in the pathogenic Aspergillus fumigatus has been increasing worldwide, posing a growing therapeutic challenge. To date, triazole resistance in clinical isolates of A. fumigatus causing pulmonary aspergillosis has been mainly attributed to the mutations in the cyp51A gene or its promoter, followed by mutations in cyp51B and hmg1 gene encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. From chronic pulmonary aspergillosis (CPA) patient, we isolated a strain of A. fumigatus (BMU10672) with resistance to voriconazole (VRC) and isavuconazole (ISZ), which was caused by overexpression of efflux pump Cdr1B.

Methods
Antifungal susceptibility testing of the isolate of A. fumigatus BMU10672 was performed using the broth microdilution method (CLSI M38-A3), E-test and disk diffusion method. The promoter region and open reading frame of the cyp51A, cyp51B, and hmg1 gene were amplified and sequenced. Then, the expression levels of cyp51A, cyp51B, and efflux pump gene cdr1B with or without being exposed to VRC or ISZ were quantified using real-time PCR, compared with triazole-susceptible A. fumigatus Af293. And the function of efflux pump Cdr1B was tested by efflux pump substrate (Nile red) accumulation assay and efflux pump inhibitor (FK520) assay.

Results
The minimum inhibitory concentration (MIC) of itraconazole (ITC), VRC, posaconazole (POS), ISZ and amphotericin B (AMB), and the minimal effective concentration (MEC) of caspofungin (CAS) against A. fumigatus BMU10672 was 1 μg/ml, 2 μg/mL, 0.5 μg/mL, 2 μg/mL, 1 μg/mL and 0.125 μg/mL, respectively. The results of E-test and disk diffusion assay were consistent with those of the broth microdilution method (Figs. 1a and b). Together, these results indicate that A. fumigatus BMU10672 is resistant to VRC and ISZ, while being susceptible to ITC, POS, AMB, and CAS. Sequencing of the cyp51A, cyp51B and hmg1 gene of A. fumigatus BMU10672 were all intact. The basal and VRC- or ISZ- induced expression levels of efflux pumps gene cdr1B in A. fumigatus BMU10672 were all higher (&gt; 4-fold) than those in triazole-susceptible A. fumigatus Af293. However, no differences in basal and VRC- or ISZ- induced expression levels of cyp51A gene and cyp51B gene were observed between A. fumigatus BMU10672 and Af293. The efflux pump substrate Nile red accumulation assay showed the A. fumigatus BMU10672 accumulated less Nile red than Af293, confirming that Cdr1B was active at exporting Nile red, while efflux pumps inhibitor FK520 can increase the accumulation of the Nile red in A. fumigatus BMU10672 (Fig. 1c). Inhibition of efflux pumps activity by inhibitor FK520 resulted in a MIC reduction of 4-fold in VRC and ISZ MICs, and 2-fold in ITC and POS, against A. fumigatus BMU10672 (Figs. 1a and b).

Conclusion
Overexpression of efflux pumps gene cdr1B resulting in VRC- and ISZ- resistance in the clinical isolate of A. fumigatus BMU10672.