Investigation of multiple resistance mechanisms in voriconazole resistant Aspergillus flavus clinical isolates from a chest hospital surveillance in Delhi, India [PublishAheadOfPrint]

Invasive and allergic infections by Aspergillus flavus are more common in tropical and subtropical countries. The emergence of voriconazole (VRC) resistance in A. flavus impacts the management of aspergillosis as azoles are used as the first line and empiric therapy. We screened molecularly confirmed 120 A. flavus isolates obtained from respiratory and sino-nasal specimens in a chest hospital in Delhi for azole resistance using CLSI BMD method. Overall, 2.5% (n=3/120) of A. flavus isolates had VRC MICs above epidemiological cutoff values (>1μg/ml). The whole genome sequence analysis of three non-wildtype (WT) A. flavus isolates with high VRC MICs showed polymorphisms in azole target genes (cyp51A, cyp51B and cyp51C). Further, four novel substitutions (S196F, A324P, N423D and V465M) in cyp51C gene were found in a single non-WT isolate which also exhibited over expression of cyp51 (A, B and C) genes and transporter genes namely MDR1, MDR2, atrF and mfs1. The homology model of the non-WT isolate suggests that substitutions S196F and N423D exhibited major structural and functional effect on cyp51C drug binding. The substrate (drug) may not be able to bind to binding pocket due to changes in the pocket size or closing down or narrowing of cavities in drug entry channels. Notably, the remaining two VRC resistant A. flavus isolates including the one which had pan azole phenotype (itraconazole and posaconazole) did not show upregulation of any of the analyzed target genes. These results suggest that multiple target genes and mechanisms could simultaneously contribute to azole resistance in A. flavus.

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