Mechanisms Of Fluconazole Resistance In Clinical Isolates Of Candida Glabrata

In recent years,invasive fungal infections(IFIs) have increased dramatically, largely because of the increasing size of the immunocompromised population (especially AID patients).Candida albicans remains the most common conditional pathogenic fungi isolated from clinical samples,however,there has been a significant increase in the frequency of non-albicans Candida species isolation in recent years.C. glabrata is now emerging as an important agent in both mucosal and systemic infections.C.glabrata currently ranks as the second or third most frequently isolated Candida species from all reported cases of candidiasis in some areas,second only to C.albicans.C.glabrata infections are of particular importance because of their natural increased resistance to antifungal agents(specifically the azoles) and consequently a high mortality rate in compromised,at-risk hospitalised patients. Several studies have revealed that a growing percentage of C.glabrata clinical isolates are resistant to fluconazole.The rise in the number of C.glabrata systemic infections deserves a great deal of concern due to the propensity of this microorganism to rapidly develop resistance to azole antifungal.Many researches have focused on the diagnosis and treatment of C.glabrata infections,particularly mechanisms of drug resistance of C.glabrata.SectionⅠRetrospective study on invasive fungal infections among people with acquired immune deficiency syndromeInvasive fungal infections have become a major cause of morbidity and mortality among people with AIDS,however,little is known about the clinical features and prognosis of IFI in AIDS in China.This study aimed to characterize the clinical features and prognosis of IFI in AIDS patients in China.We retrospectively reviewed the records of all HIV-infected patients at a Chinese university hospital between December 2004 and May 2006.We identified 35 patients with IFI.44.4%of IFIs occurred in the digestive tract.57.4%of fungal pathogens isolated were C.albicans, non-albicans Candida species accounted for 22.3%.39.5%of C.albicans isolates were resistant to fluconazole,41.7%of non-albicans Candida isolates were resistant to fluconazole.71.8%of IFIs occurred in patients with CD4~+T lymphocyte counts＜100 cells per cubic millimeter.All the patients received both antiretroviral and antifungal therapy;27 patients were cured and 8 died.The study shows that IFI is one of the most common opportunistic infections in AIDS patients in China.IFIs mainly occur in patients with low CD4~+T lymphocyte counts.The majority of IFIs occur in the digestive tract.The most common pathogen causing IFI is C.albicans, non-albicans Candida species are increasingly seen in the HIV-infected patients in recent years.The fungal resistance rate of Candida isolates from AIDS patients is high.The mortality rate remains high though antiretroviral therapy and many newer antifungals are available in China.SectionⅡSusceptibility of non-albicans Candida isolates to common antifungal agentsIn order to investigate the prevalence of antifungal resistance of non-albicans Candida isolates,we studied the frequency of resistance profile to amphotericin B, flucytosine,fluconazole and itraconazole of 116 isolates of non-albicans Candida isolates from clinical samples from patients at Shanghai Public Health Clinical Center affiliated to Fudan University,using Neo-Sensitabs tablet assay.All but two C. glabrata isolates were susceptible to amphotericin B.86.4%of C.tropicalis and 80% of C.parapsilosis were susceptible to fluconazole.The resistant rates of C.glabrata isolates to amphotericin B,fluconazole and itraconazole were 3.6%,16.1%and 10.7%, respectively.All C.krusei isolates were resistant to fluconazole and flucytosine.We conclude that most non-albicans Candida are susuceptible to amphotericin B,and that C.krusei exhibits low susceptibilities to the triazole agents and flucytosine.This investigation reveals that C.glabrata is less susceptible to the antifungal agents (particularly the triazole agents) than are most other species of Candida and exhibits cross-resistance to the triazole agents.SectionⅢAnalysis of ERG11 gene mutations in fluconazole-resistant Candida glabrata strainsTo elucidate if the mutation in ERG11 gene encoding P450 lanosterol 14-α demethylase could also be implicated in fluconazole resistance in C.glabrata,ERG11 genes of nine fluconazole-resistant and 10 fluconazole-sensitive C.glabrata isolates were cloned and sequenced,the sequences were compared to the published sequence. We found 10 point mutations which did not lead to amino acid changes.There existed neither missense mutationss or frame-shifting mutations.Amomg the 10 point mutations,five existed in both fluconazole-resistant and fluconazole-sensitive C. glabrata isolates,three were only present in fluconazole-resistant isolates,two only existed in fluconazole-sensitive ones.The majority of the point mutations were located between 1320-2200 base pair of ERG11 gene.The results indicate that there is sequence polymorphism at DNA level in clinical strains of C.glabrata which are resistant to fluconazole.ERG11 gene mutations are not found to be involved in the development of fluconazole resistance in C.glabrata in this study.SectionⅣRelationship between the expression of ERG11,CDR1 and CDR2 genes and fluconazole resistance in clinical isolates of Candida glabrataTo determine if changes in the levels of expression of ERG11,CDR1 and CDR2 genes could be associated with resistance phenotype in clinical isolates of C.glabrata, we used quantitative RT-PCR analysis to evaluate the expression of the ERG11, CDR1 and CDR2 genes in clinical isolates including nine fluconazole-resistant,nine fluconazole-S-DD(susceptible dose dependent) and 10 fluconazole-sensitive C. glabrata isolates.Quantitative RT-PCR analyses revealed that the fluconazole-resistant isolates expressed ERG11 at higher levels than fluconazole-sensitive isolates.CDR1 expression was significantly higher in the fluconazole-resistant isolates compared to the fluconazole-sensitive isolates,as well as in the S-DD isolates compared to the fluconazole-sensitive isolates.CDR2 upregulation was observed in the fluconazole-resistant isolates compared to the susceptible isolates.With the decrease in susceptibilities to fluconazole,the levels of expression of ERG11,CDR1 and CDR2 genes in the isolates appeared to be increased. These results provide evidence that the overexpression of ERG11,CDR1 and CDR2 genes is associated with fluconazole resistance in clinical isolates of C.glabrata. ERG11,CDR1 and CDR2 upregulation is a major molecular mechanism of fluconazole resistance in clinical isolates of C.glabrata. SectionⅤComparative proteomic analysis of fluconazole-resistant and fluconazole-sensitive Candida glabrata isolatesThe known molecular mechanisms of fluconazole resisitance in C glabrata are not sufficient to explain the resistance in clinical isolates of C.glabrata,new resistance-associated genes and other possible underlying resistance mechanisms deserve a great deal of concern and investigation.We used proteomics-associated techniques to identify changes in the proteome of fluconazole-resistant isolates of C. glabrata compared with fluconazole-sensitive ones in order to identify proteins that are differentially expressed in associated with fluconazole resistance.Eight proteins were found to be more abundantly represented,and four proteins were found to be less abundantly represented,in fluconazole-resistant strains compared with fluconazole-sensitive ones.These differentially expressed proteins involved in energy metabolism,stress response and macromolecule synthesis.These results indicate that proteins involved in energy metabolism,stress response and macromolecule synthesis may play a role in the development of fluconazole resistance in clinical isolates of C. glabrata.The study provides further evidence that many different mechanisms are involved in the development of fluconazole resistance in C.glabrata.These findings provide scientific basis for discovering new genes and mechanisms associated with fluconazole resistance in C.glabrata.