Evaluation of mRNA Expression Levels of cyp51A and mdr1, Candidate Genes for Voriconazole Resistance in Aspergillus flavus

Invasive aspergillosis (IA) is a severe condition mainly caused by Aspergillus fumigatus, although other species of the genus, such as section Nigri members, can also be involved. Voriconazole (VRC) is the recommended treatment for IA; however, the prevalence of azole-resistant Aspergillus isolates has alarmingly increased in recent years, and the underlying resistance mechanisms in non-fumigatus species remain unclear. We have determined the in vitro susceptibility of 36 strains from section Nigri to VRC, posaconazole (POS), and itraconazole (ITC), and we have explored the role of Cyp51A and Cyp51B, both targets of azoles, in azole resistance. The three drugs were highly active; POS displayed the best in vitro activity, while ITC and VRC showed MICs above the established epidemiological cutoff values in 9 and 16% of the strains, respectively. Furthermore, expression studies of cyp51A and cyp51B in control condition and after VRC exposure were performed in 14 strains with different VRC susceptibility. We found higher transcription of cyp51A, which was upregulated upon VRC exposure, but no correlation between MICs and cyp51 transcription levels was observed. In addition, cyp51A sequence analyses revealed nonsynonymous mutations present in both, wild-type and non-wild-type strains of A. niger and A. tubingensis. Nevertheless, a few mutations were exclusively present in non-wild-type A. tubingensis strains. Altogether, our results suggest that azole resistance in section Nigri is not clearly explained by Cyp51A protein alteration or by cyp51 gene upregulation, which indicates that other mechanisms might be involved.

Section: Discussionmentioning

confidence: 91%

New Insights into the Cyp51 Contribution to Azole Resistance in Aspergillus Section Nigri

Pérez-Cantero

López-Fernández

Guarro

et al. 2019

“…Azole resistance mechanisms in clinical A. fumigatus isolates have been investigated extensively, and the main mechanism is the presence of point mutations in the target gene cyp51A (4,5,22,23). In contrast to A. fumigatus, the resistance mechanisms of A. flavus have been examined in a limited number of clinical strains (15,20,21,24). Liu et al (25) suggested that a T788G (S240A) missense mutation in cyp51C conferred voriconazole resistance on A. flavus isolates.…”

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confidence: 99%

“…To date, three studies investigated the overexpression of target or efflux pump genes in clinical isolates of A. flavus (20,21,24). In a study, four of five voriconazole non-WT isolates had cyp51A and MDR1 overexpression (24), while in the other study, one of three non-WT isolates exhibited overexpression of target genes (cyp51A, cyp51B, and cyp51C) and efflux pump genes (MDR1, MDR2, atrF, and mfs1) (20). In a study by Paul et al (21), two non-WT isolates showed voriconazole-induced overexpression of Cdr1, suggesting a possible role of multidrug efflux pumps.…”

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confidence: 99%

Microsatellite Typing and Resistance Mechanism Analysis of Voriconazole-Resistant Aspergillus flavus Isolates in South Korean Hospitals

Choi

Won

Joo

et al. 2019

A recent surveillance study in South Korea revealed that 14% (7/50) of Aspergillus flavus clinical isolates had a voriconazole minimum inhibitory concentration of ≥4 μg/ml. Of seven non-wild-type (non-WT) isolates, six ear isolates from four hospitals shared the same microsatellite genotype. None of the non-WT isolates showed cyp51 mutations associated with azole resistance. However, the mean expression levels of efflux pump (MDR2, atrF, and mfs1) and target (cyp51A) genes exhibited significant differences between non-WT and other isolates.

“…Consequently, in the last few years molecular mechanisms underlying azole resistance in A. fumigatus strains have been thoroughly investigated with regard to either point mutation(s) or overexpression of the target gene of azoles (cyp51A) and non-cyp51-mediated resistance (11). In contrast to the case with A. fumigatus, in the last 5 years only a few sporadic reports documenting VRC-resistant A. flavus (VRC-RAfla) isolates have been recorded (13)(14)(15)(16)(17). Also, the true burden of azole resistance in A.…”

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confidence: 99%

“…Unlike for other Aspergillus species, which possess two cyp51 proteins, i.e., cyp51A, and cyp51B, in A. flavus, three cyp51 proteins are found: cyp51A, cyp51B, and cyp51C. So far, limited studies (Ͻ10) analyzing the genetic changes and/or overexpression of azole target genes, i.e., cyp51A and cyp51C, and efflux pump (EP) genes causing decreases in intracellular drug concentrations in laboratory-generated or clinical isolates of azole-resistant A. flavus have been described (13)(14)(15)(16)(17)(18)(19). Krishnan-Natesan et al (19) demonstrated the role of cyp51A mutations in VRC-resistant laboratory-generated A. flavus strains, whereas in two recent studies, mutations in the cyp51C gene in clinical azole-resistant A. flavus strains were reported (13,15).…”

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confidence: 99%

Investigation of Multiple Resistance Mechanisms in Voriconazole-Resistant Aspergillus flavus Clinical Isolates from a Chest Hospital Surveillance in Delhi, India

Sharma

Kumar

et al. 2018

Invasive and allergic infections by are more common in tropical and subtropical countries. The emergence of voriconazole (VRC) resistance in impacts the management of aspergillosis, as azoles are used as the first-line and empirical therapy. We screened 120 molecularly confirmed isolates obtained from respiratory and sinonasal specimens in a chest hospital in Delhi, India, for azole resistance using the CLSI broth microdilution (CLSI-BMD) method. Overall, 2.5% ( = 3/120) of isolates had VRC MICs above epidemiological cutoff values (>1 μg/ml). The whole-genome sequence analysis of three non-wild-type (WT) isolates with high VRC MICs showed polymorphisms in azole target genes (, , and). Further, four novel substitutions (S196F, A324P, N423D, and V465M) encoded in the gene were found in a single non-WT isolate which also exhibited overexpression of (, -, and -) genes and transporter genes, namely, ,, , and The homology model of the non-WT isolate suggests that substitutions S196F and N423D exhibited major structural and functional effects 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 isolates, including the one which had a pan-azole resistance 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.