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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.

SUBMITTER: Du J

PROVIDER: S-EPMC8385537 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveTo study the main molecular mechanisms responsible for itraconazole resistance in clinical isolates of Candida krusei.MethodsThe 14α-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction (PCR), and their nucleotide sequences were determined to detect point mutations. Meanwhile, ERG11 and efflux transporters (ABC1 and ABC2) genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for their expression in itraconazole-resistant (R), itraconazole-susceptible dose dependent (SDD) and itraconazole-susceptible (S) C.krusei at the mRNA level.ResultsWe found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in the 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 levels in itraconazole-resistant group was significantly higher than the other two groups, and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole (P = 0.007 in SDD compared with S, P = 0.016 in SDD with R, and P<0.001 in S with R respectively). While ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in a C.krusei (CK10) resistant to both itraconazole and voriconazole were expressed highest in all the itraconazole-resistant isolates.ConclusionsThere are ERG11 gene polymorphisms in clinical isolates of C.krusei. ERG11 gene mutations may not be involved in the development of itraconazole resistance in C.krusei. ERG11 and ABC2 overexpression might be responsible for the acquired itraconazole resistance of these clinical isolates.

Project description:Abstract Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objective To determine the profile of fluconazole-resistant Candida isolates (FRCS) and the proportion of erg11 gene mutation in fluconazole-resistant C. albicans (FRCA) isolates Methods After getting approval from ethics the study was conducted on 150 isolates of FRCS obtained from patients admitted to our tertiary care hospital from September 2019 to December 2021. All the isolates were revived onto Sabouraud dextrose agar (SDA) and identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Fluconazole-resistance was detected by VITEK 2 automated system. Deoxyribonucleic acid (DNA) of FRCA isolates were extracted by QIAamp DNA Mini kit (Qiagen-51 304, Hilden, Germany) as per manufacturer's instruction. A single primer pair targeting the nucleotide sequence from 1067 to 1576 bp region of erg11 gene was used for amplification as per Xu et al. with minor modification and 500 bp polymerase chain reaction (PCR) products were observed by gel electrophoresis. A positive clinical isolate of FRCA was used as a positive control. The PCR products were purified and subjected to Sanger sequencing with an ABI PRISM DNA analyzer (Applied Biosystems) and mutations were detected by using Mega (version 7) software by comparing with the published GenBank sequence AF153844.1 of C. albicans strain ATCC 28516. Categorical variables were expressed in percentages and continuous variables were expressed in mean (average). Results Out of the total of 150 FRCS, the most common fluconazole-resistant agent of candidiasis was C. auris, which was isolated from 60 (40%) cases followed by 24 (16%) isolates of C. glabrata, 19 (12.7%) isolates of C. krusei, 18 (12%) isolates of C. parapsilosis, 17 (11.3%) isolates of C. albicans, 10 (6.7%) isolates of C. tropicalis, and only two (1.3%) isolates of C. guilliermondii. Among these 17 FRCA isolates, 9 isolates were obtained from genital swabs, 5 from blood, and a single isolate each from the perianal swab, ear swab, and sputum. A total of 12 (70.6%) out of 17 FRCA isolates yielded 500 bp region in erg11 gene. Gel electrophoresis image of PCR products of erg11 gene is depicted in Figure 1. Sequencing of these products detected a single missense mutation A1309G (V437I) in one FRCA isolate. Most of the FRCA isolates had an average of 4 silent mutations (observed in 6 isolates) and a single isolate had 6 silent mutations. A total of 8 different silent mutations were observed among FRCA isolates T1296C (83.3%), C1203T (66.7%), A1440G (66.7%), C1302T (50%), T1470C (50%), T1140C (25%), T1110C (16.7%), and T1284C (8.3%). Conclusion C. auris was the most common fluconazole resistant isolate observed, followed by C. glabrata, C. krusei, C. parapsilosis, C. albicans, C. tropicalis, and C. guilliermondii. There were many silent mutations observed in erg11 gene of FRCA isolates and detected only a single missense mutation (V437I). Fluconazole-resistant in FRCA isolates may be due to mechanisms other than the studied one in our region.

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

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