Salma Abbes

Interactions between copy number and expression level of genes involved in fluconazole resistance in Candida glabrata

Objectives: This study aimed to elucidate the relative involvement of drug resistance gene copy number and overexpression in fluconazole resistance in clinical C. glabrata isolates using a population-based approach. Methods: Fluconazole resistance levels were quantified using the minimal inhibitory concentration (MIC) via Etest method. Both gene expression levels and gene copy number of CgCDR1, CgPDH1, CgERG11, and CgSNQ2 were assessed via quantitative real-time PCR. The influence of the main effects and first-level interactions of both the expression level and copy number of these genes on fluconazole resistance levels were analyzed using a multivariate statistical model. Results: Forty-three C. glabrata isolates were collected from 30 patients during in a hospital survey. In the multivariate analysis, C. glabrata fluconazole MICs were independently increased by CgSNQ2 overexpression (p < 10−4) and the interaction between CgPDH1 gene copy number and CgPDH1 expression level (p = 0.038). In contrast, both CgPDH1 overexpression (p = 0.049) and the interaction between CgSNQ2 and CgERG11 expression (p = 0.003) led to a significant decrease in fluconazole MICs. Conclusion: Fluconazole resistance in C. glabrata involves complex interactions between drug resistance gene expression and/or copy number. The population-based multivariate analysis highlighted the involvement of the CgSNQ2 gene in fluconazole resistance and the complex effect of the other genes such as PDH1 for which overexpression was associated with reduced fluconazole resistance levels, while the interaction between PDH1 overexpression and copy number was associated with increased resistance levels.

Microsatellite analysis of Candida isolates from recurrent vulvovaginal candidiasis.

Candida albicans and Candida glabrata are the most common causative agents of both vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). Studying the population structure and genotype differentiation of Candida species that cause RVVC may lead to a significant improvement in clinical management. A total of 106 isolates were collected from 55 patients who were subdivided into three groups. Group I comprised 15 patients with RVVC (n=50 isolates); group II comprised 16 patients, who had a history of at least two episodes of VVC in the last year (n=32 isolates, two from each patient); and group III comprised 24 patients (n=24 isolates) who had experienced a single episode of VVC in the previous 1 year period. C. albicans microsatellite markers CAI, CAIII and CAIV and C. glabrata RPM2, MTI and ERG3 microsatellites were amplified in a multiplex PCR. All isolates were subjected to population genetic analysis, which provided evidence that there is a predominantly clonal population structure of C. albicans in each group. However, recombination was detected to some degree in C. albicans isolates in group III. A genetic homogeneity between the different C. albicans groups was observed. Although, C. glabrata isolates showed an important genetic differentiation between group I and group III (F(ST)=0.207). Genotype analysis revealed that the dominant genotypes of C. glabrata and C. albicans strains were more prevalent in patients with RVVC. The frequent scenario for cases of recurrent infection in our study was strain replacement (53.3%). In conclusion, the identification of recurrence-associated genotypes and a specific C. glabrata population structure in the RVVC group could be a significant marker for further investigations of virulence factors and RVVC management.

Analysis of virulence factors and in vivo biofilm-forming capacity of Yarrowia lipolytica isolated from patients with fungemia

Abstract Yarrowia lipolytica is ubiquitous in the environment, opportunistic, and might be considered as one of the causative agents of catheter‐related candidemia. Our work aimed to study some virulence factors of Y. lipolytica such as hydrolases production and biofilm formation with comparison to the most frequent Candida specie in human disease. In sum, 58 clinical isolates of Y. lipolytica, 16 C. glabrata, and 12 C. albicans were collected from Intensive care unit (ICU). All were tested for enzymatic production and biofilm formation. All tested isolates of C. albicans and C. glabrata were able to degrade casein, and 98.2% of Y. lipolytica showed caseinase activity but no gelatinase activity was detected in all isolates. Y. lipolytica strains showed significantly lower (3.4%) in vitro phospholipase activity than C. albicans and C. glabrata (P < .05). No significant differences of the hemolytic activity were detected between the three species (P > .05). Concerning biofilm formation, and unlike the results obtained on polystyrene plate, the number of adhered and biofilm cultivable cells obtained by Y. lipolytica after 168 hours of catheter subcutaneous implantation is significantly greater and tends to be more compact and structured hyphal layer. Although C. albicans remains the most pathogenic yeast, development of selective ability of Y. lipolytica to adhere, to form a biofilm on catheter medical devices, and to produce phospholipase and hemolytic enzyme is of particular interest, and it is strongly recommended to be vigilant in the use of medical implanted medical devices, particularly in ICU.

Changes in Genotype and Fluconazole Susceptibility of Isolates from Patients with Candida glabrata in Tunisia

UNLABELLED Candida glabrata has emerged as an opportunistic pathogen of considerable importance in invasive and superficial infections. AIMS To analyze the development of fluconazole resistance in patients under treatment through epidemiological survey in our hospital. PATIENTS AND METHODS Twenty two patients (89 clinical strains) were collected. Molecular typing of isolates was performed by polymorphic markers. Analysis of gene expression was realized by reverse transcriptase-real time polymerase chain reactions (RT-qPCR). RESULTS Genetic analysis showed that 63% persists with apparently unchanged strains (n=14). Among them, four showed fluconazole resistance development. A strain replacement was observed in 6 patients and two patients selected more resistant isolates during the course of treatment. An analysis of Candida glabrata cerebellar degeneration-related protein 1 (CgCDR1), Candida glabrata cerebellar degeneration-related protein 2 (CgCDR2) and Candida glabrata sterol 14 alpha-demetylase Erg 11 (CgERG11) expression revealed an over-expression in 10 resistant isolates. CONCLUSION This study demonstrated that C. glabrata strain undergo frequent changes in vivo. The increase in CgCDR1 and CgCDR2 expression was the most mechanism associated with fluconazole resistance.