The A756T Mutation of the ERG11 Gene Associated With Resistance to Itraconazole in Candida Krusei Isolated From Mycotic Mastitis of Cows

Abstract

Candida krusei (C. krusei) has been recently recognized as an important pathogen involved in mycotic mastitis of cows. The phenotypic and molecular characteristics of 15 C. krusei clinical isolates collected from cows with clinical mastitis in three herds of Yinchuan, Ningxia, were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry. In addition to sequencing analysis, the ERG11 gene that encodes 14α-demethylases, the expression of the ERG11 gene, and efflux transporters ABC1 and ABC2 in itraconazole-susceptible (S), itraconazole-susceptible dose dependent (SDD), and itraconazole-resistant (R) C. krusei isolates was also quantified by a quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay. Sequencing analysis revealed three synonymous codon substitutions of the ERG11 gene including T939C, A756T, and T642C in these C. krusei clinical isolates. Among them, T642C and T939C mutations were detected in itraconazole-resistant and -susceptible C. krusei isolates, but the A756T substitution was found only in itraconazole-resistant isolates. Importantly, the expression of the ERG11 gene in itraconazole-resistant isolates was significantly higher compared with itraconazole-SDD and itraconazole-susceptible isolates (p = 0.052 and p = 0.012, respectively), as determined by the qRT-PCR assay. Interestingly, the expression of the ABC2 gene was also significantly higher in itraconazole-resistant isolates relative to the itraconazole-SDD and itraconazole-susceptible strains. Notably, the expression of ERG11 was positively associated with resistance to itraconazole (p = 0.4177 in SDD compared with S, p = 0.0107 in SDD with R, and p = 0.0035 in S with R, respectively). These data demonstrated that mutations of the ERG11 gene were involved in drug resistance in C. krusei. The A756T synonymous codon substitution of the ERG11 gene was correlated with an increased expression of drug-resistant genes including ERG11 and ABC2 in itraconazole-resistant C. krusei isolates examined in this study.

K143R Amino Acid Substitution in 14-α-Demethylase (Erg11p) Changes Plasma Membrane and Cell Wall Structure of Candida albicans

“…In the case of K143R substitution, it is highly unlikely that TF has an influence on the increased expression of ERG11, as UPC2 binds the ERG11 promoter sequence [28]. Nevertheless, synonymous codon substitutions in ERG11 were shown to lead to the increased expression of the ERG11 gene in C. krusei [29]. Thus, a similar situation can occur in the case of K143R substitutions in Erg11p.…”

Isolation and Whole Genome Sequence Analysis of Cow Mastitis Candida tropicalis

“…In dairy cows, Candida infections may be due to the use and abuse of antimicrobial drugs, contamination of antibiotic solutions and syringes and treatment with ducts or other materials in contact with the mammary gland leading to yeast colonization of the bovine dairy [13,15,16] . Although C. albicans infection is considered to be the most common cause of mastitis, cases of bovine mastitis caused by infections of non-albicans Candida spp., such as C. krusei, C. parapsilosis, C. glabrata, and C. tropicalis, infections are becoming more frequent [17] . C. tropicalis, as an emerging pathogen of mastitis in dairy cows, has signi cantly lower antifungal susceptibility and produces more serious antimicrobial resistance, which seriously affects the development of dairy farming.…”