Study On Phenotype And Genome-wide Expression Profile Of Candida Albicans Clinical Isolates Associated With Stepwise Acquisition Of Azole Resistance

Chapter I Study on phenotypic changes of Candida albicans during the stepwise acquisition of azole resistance in vivoObjectives To investigate the phenotypic changes of Candida albicans during the stepwise acquisition of azole resistance in vivo. Methods A series of six isolates (Ca1、 2、5、8、14、17, Cal was isolated before drug treatment) from the same HIV patient showing increasing azole resistance after long-time use of fluconazole were selected. Optical microscope、transmission electron microscope 、hyphal formation test、cell surface hydrophobicity test (CSH) and flocculation test were performed to explore the changes of morphological structure、hyphal formation、 flocculation and adhesion during the stepwise acquisition of azole resistance in vivo. Results No significant changes in morphological structure、ultrastructure and intercellular adhesive ability were observed in these isolates. Except Ca17, the ratio of hyphal formation and value of CSH in other posttreatment isolates(Ca2、5、8、14) were significantly higher than those of the sensitive isolate Cal. Conclusions This set of isolates with gradually decreased azole susceptibility showed difference in the hyphal formation and adhesive capacity. Further study is needed to explore the mechanism and significance of these changes.Chapter II Study on the ability to form biofilm and susceptibility to antifungal agents of Candida albicans during the stepwise acquisition of azole resistance in vivo Section I Study on the ability to form biofilm of Candida albicansObjectives To study the development and structure characteristics of Candida albicans biofilm in vitro and compare the differences of ability to form biofilm among the tested strains. Methods Candida albicans biofilms were constructed in 24-well microculture plates and observed under inverted microscope at the time points 2、4、8、 12、24h. XTT assay was used to evaluate the metabolic activity of biofilm at various time points during biofilm formation. Results The ability to form biofilm differs in tested strains. We divided them into two groups, namely weak ability to form biofilm group (Ca1、17) and strong ability to form biofilm group (Ca2、5、8、14). Conclusions The ability of Candida albicans to form biofilm tends to change during the stepwise acquisition of azole resistance, which plays an important role under selective pressure from antifungal agents.Section II Study on susceptibility of antifungal drugs against planktonic or biofilm cells of Candida albicans.Objectives To compare the difference in susceptibility of antifungal drugs between planktonic or biofilm cells of Candida albicans. Methods Susceptibilities of strains in planktonic state to fluconazole、itraconazole、 voriconazole 、amphotericin B and caspofungin were tested by a modified M27-A3 method of CLSI in 96-well microculture plates. Besides, biofilms in 96-well microculture plates were constructed, and XTT reduction assay was used to evaluate the effect of antifungal agents on biofilms. Results All the tested strains were susceptive to amphotericin B and caspofungin in planktonic state and gradually resistant to azole drugs. Biofilms displayed higher levels of resistance to azole drugs, especially to fluconazole. Amphotericin B was proved effective in reducing viability of cells within the biofilms, but only at high concentrations. Caspofungin showed strongest activity against C. albicans biofilm. Conclusions During the stepwise acquisition of drug resistance, susceptibilities of strains to antifungal agents are gradually decreased; Compared with planktonic cells of C.albicans, biofilms are more resistant to antifungal agents, especially to azoles. Though Amphotericin B can reduce viability of cells within the biofilms, its clinical application was limited due to its side effects. Caspofungin showed strongest activity against C. albicans biofilm and is thus promising for the treatment of biofilm-related infections.Chapter DI Using RNA-seq to determine the transcriptional landscape of the tested strainsObjectives To study the changes of gene expression in C.albicans during the acquisition of azole resistance. Methods RNA-seq was used to examine the genes which have significantly changed related to an increase in MIC and the differential gene expression was analyzed in fluconazole-susceptible(Cal) and-resistant isolates (Ca17); the functional enrichment analysis and biological pathway analysis of differential genes were also perfomed. Results A total of 55 genes expressed differently betweenCal and Ca17, including 49 up-regulated genes and 6 down-regulated genes. The up-regulation of MDR1 was the greatest, followed by CDR1 and CDR2. We also identified some genes whose differential expression was for the first time associated with this resistant phenotype such as HWP1、GLX3、GEF2、HSL1、MED9、OYE32、RPS19A、 HST6、NAD4、NAD5、NAD4L、PGA31、GST3 and so on. The up-regulated genes are often related with resistance and oxidative stress response. There were 655 genes related with an increase in MIC, including 303 genes with significant relevance. The annotation of GO function and biological pathway revealed genes with different expression were mainly associated with metabolism. Conclusions A variety of genes contribute to acquired drug resistance of Candida albicans through different mechanisms at different periods. Among these mechanisms, the increased mRNA levels of efflux pump genes is crucial for the development of severe resistance. Drug could induce strains to change metabolic pathways, which may play an important role in the formation of resistance.