Molecular expression of itraconazole resistance in clinical isolates of Candida krusei

Abstract

Objective To investigate the main molecular expressions responsible for itraconazole resistance in clinical isolates of Candida krusei (C.krusei). Methods The ERG11 gene in the 16 C. krusei clinical isolates was amplified by polymerase chain reaction (PCR). Point mutations were determined by nucleotide sequence and compared among itraconazole-resistant (R, n=5), itraconazole-susceptible dose dependent (SDD, n=8) and itraconazole-susceptible (S, n=3) C. krusei. Meanwhile, ERG11 and efflux transporter genes (ABC1 and ABC2) were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in 3 groups at the mRNA level. All variables were transformed to obey normal distribution and variance homogenity and compared by two sided t test between two groups. Results Seven point mutations in ERG11 gene of all the C. krusei clinical isolates were detected, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in all three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones (P=0.015 and P=0.002, respectively). ABC2 gene mRNA level in itraconazole-resistant group was significantly higher than the other two groups (P=0.016 and P<0.01, respectively), and the level of its expression in susceptible dose-dependent group was higher than dose susceptible group (P=0.007). ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in one C. krusei (CK10) strain resistant to both itraconazole and voriconazole were highest in all the itraconazole-resistant isolates. Conclusions There are ERG11 gene polymorphisms in clinical isolates of C. krusei. ERG11 gene mutations are not found to be involved in the development of itraconazole resistance in C. krusei. ERG11 and ABC2 upregulation might be responsible for the acquired itraconazole resistance of these clinical isolates. Key words: Candida krusei; Itraconazole; Drug resistance, multiple; Polymerase chain reaction; Reverse transcriptase polymerase chain reaction