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Title: Azole resistance in clinical isolates of Aspergillus fumigatus
Author: Albarrag, Ahmed
ISNI:       0000 0001 3409 922X
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2008
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Aspergillus fil1nigatus is the most common aetiological agent of aspergillosis. Invasive aspergillosis is a major cause of death in leukaemic and organ transplant patients. The azoles are the largest and the most widely used class of antifungals. Antifungal drug resistance has been observed in many fungi. In many organisms, acquiring resistance to a drug can confer a biological fitness cost that is expressed for example as decreased growth rate or virulence. A total number of 21 A. fill11igatus clinical isolates were used in this project. Sixteen of these isolates were recovered serially from four patients at different times during the treatment period with azoles, including three cases of acquired azole resistance. The antifungal susceptibilities of isolates were determined. The genetic relatedness of the isolates was confirmed by microsatellite length polymorphism typing. Various measures of fitness were used to determine variation between susceptible and resistant isolates. Growth rate (colony radial growth rate and specific growth rate), germination time and conidial yields were determined on various media. Although some resistant isolates had a reduced growth rate compared to their susceptible match, there was no clear evidence of fitness cost of resistance has been found. The mechanisms of drug resistance in these isolates were investigated. Sequencing of the cyp5JA gene was carried out. Novel and previously described mutations in cyp5JA were identified. Three new mutations were detected in isolates recovered from patient D which were shown to have the same genotype. These mutations were G138C (in six isolates), Y431C (in one isolate) and G434C (in one isolate). These isolates have decreased susceptibility to itraconazole (>8.0 mg/l), voriconazole (4-8.0 mg/l), ravuconazole (4-8.0 mg/l), and posaconazole 0-4.0 mg/l). Azole cross-resistance observed in these isolates was confirmed to be caused by two of these mutations, G138C and Y431C, by expression of the mutated cyp5JA alleles in the yeast S. cerevisiae. The relative levels of expression of cyp5JA, cyp5JB and five efflux transporters, AfuMDRJ, AfilMDR2, AfiIMDR3, AfuMDR4 and atrF, genes were analysed using real-time RT-PCR. Gene expression analysis revealed that isolates from patient D had their cyp5JA gene up-regulated by 7.2- to 13.4-fold. Generally, susceptible and resistant isolates had the same level of expression of all five efflux transporters examined. Interestingly, a type II transposon insertion (1882 bp) in the region upstream of the start codon of cyp5JA, at position -317, was detected in one isolate from patient D, which exhibited the highest level of cyp5JA expression and so transposon insertion was associated with elevation of cyp5JA expression. In conclusion, this thesis describes novel mutations in the cyp5JA gene of clinical isolates that confer and azole cross-resistance. It also identified up-regulation of the cyp5JA gene as an additional azole resistance mechanism in some isolates. An active transposon was identified and found to be associated with resistance by elevating gene expression, an observation not previously made in A. fill11igatus for any phenotype. Up-regulation of 5 transporters and mutations in the cyp5JB gene were not related to resistance.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available