Clonal and spontaneous origins of fluconazole resistance in Candida albicans

The genotypes and susceptibilities to fluconazole of 78 strains of the human pathogenic yeast Candida albicans were compared. The strains comprised two sets of samples from Durham, N.C.: one from patients infected with the human immunodeficiency virus (HIV) and the other from healthy volunteers. For each strain, the MIC of fluconazole was determined by the standard National Committee for Clinical Laboratory Standards protocol. Genotypes were determined by PCR fingerprinting with five separate primers. The analysis revealed little evidence for genotypic clustering according to HIV status or body site. However, a small group of fluconazole-resistant strains isolated from patients infected with HIV formed a distinct cluster. In addition, two fluconazole-resistant strains were isolated from individuals who never took fluconazole, one from a patient infected with HIV and the other from a healthy person. The results suggest both clonal and spontaneous origins of fluconazole resistance in C. albicans.     

Establishing surrogate markers for fluconazole resistance in Candida albicans

Azole-resistant Candida can be a confounding factor for clinical management of opportunistic infections in immunocompromised patients, but rapid identification of such resistant organisms can improve patient outcome. New target-based molecular diagnostic strategies have the potential to identify resistant organisms faster than current culture-based assays. It was the objective of this study to determine whether target site mutations and/or drug pump over-expression are suitable surrogate markers of drug resistance that could aid new molecular-based diagnostic assays. A collection of 59 clinical isolates displaying a range of azole susceptibilities were assayed for mutations within the target gene Erg 11 and for over-expression of drug-efflux pumps Cdr 1, Cdr 2, Flu 1, and Mdr 1, as well as drug target gene Erg 11 by quantitative real-time PCR with molecular beacons. A fluconazole-resistant (MIC>or=64 microg/ml) phenotype was closely associated with over-expression of Cdr 1 (p=0.005), Cdr 2 (p=0.01), and Mdr 1 (p=0.03) along with four mutations in Erg 11 (T 229 A, Y 132 F, S 405 F, G 464 S). Changes in expression levels for Erg 11 and Flu 1 were not statistically correlated with resistance (p=0.27 and p=0.86, respectively). Overall, these findings provide a statistical basis to establish Erg 11 mutations and drug pump over-expression as surrogate markers for phenotypic fluconazole resistance.     

Environmental pH influences Candida albicans biofilms regarding its structure,virulence and susceptibility to fluconazole

Candida albicans colonizes sites with different environmental pH. However, it is unclear how these conditions can interfere on biofilms. This study aimed to evaluate the influence of environmental pH on behavior of C. albicans regarding its structure, virulence and susceptibility to fluconazole (FLZ). Minimal inhibitory concentration, minimal fungicidal concentration and time kill were used to evaluate the susceptibility to FLZ in planktonic cells under three pH values (4.0, 5.5, 7.0). These pH values were used for biofilms analysis. C. albicans ATCC 90028 was developed on poly(methlymethacrylate) resin for 48 h. Then, 2.56 μg/mL of FLZ was added to experimental groups for 24 h, and biofilms were analyzed by cell quantification, bioactivity, secretion of proteinases and phospholipases and structure. All data were analyzed by two-way ANOVA, followed by Tukey's test (α = 0.05). For planktonic cells, changes in environmental pH decreased the susceptibility to FLZ. C. albicans biofilms developed at pH 5.5 showed higher cell counts, bioactivity, bio-volume, average thickness and roughness coefficient (p < 0.05). In contrast, the presence of FLZ at pH 4.0 did not influence the structural parameters (p > 0.05), but increased secretion of proteinase and phospholipase (p < 0.05). Within the conditions studied, it was shown that environmental pH modulates the structure, virulence and susceptibility of C. albicans to FLZ.     

Molecular characterization of fluconazole resistance in a case of Candida albicans ocular infection

Ocular yeast infections in diabetics are a therapeutic challenge. Drug resistance and reduced azole susceptibility are major concerns. The case we describe characterizes a Candida albicans strain from a vitrectomy specimen that was susceptible to fluconazole by in vitro testing but recalcitrant to therapy. Molecular studies revealed transient overexpression of CDR1 and ERG11 mRNA in the presence of fluconazole that may have contributed to poor clinical response in this patient.     

Lipids of Candida albicans and their role in multidrug resistance

Over the years, lipids of non-pathogenic yeast such as Saccharomyces cerevisiae have been characterized to some details; however, a comparable situation does not exist for the human pathogenic fungi. This review is an attempt to bring in recent advances made in lipid research by employing high throughput lipidomic methods in terms of lipid analysis of pathogenic yeasts. Several pathogenic fungi exhibit multidrug resistance (MDR) which they acquire during the course of a treatment. Among the several causal factors, lipids by far have emerged as one of the critical contributors in the MDR acquisition in human pathogenic Candida. In this article, we have particularly focused on the role of lipids involved in cross talks between different cellular circuits that impact the acquisition of MDR in Candida.     

Activity of coumarin against Candida albicans biofilms

Objective

The aim of this study was to investigate the antibiofilm activity of coumarin against Candida albicans.

药物流出泵抑制剂联合咪康唑清除白假丝酵母菌生物被膜的研究

目的 观察咪康唑分别与两种药物流出泵抑制剂联用清除耐药株(persister)的效果.方法 白假丝酵母菌参考株YEM30,在96孔板中形成生物被膜(biofilm),CDR1抑制剂Enniatin B、CDR1/CDR2抑制剂Milbemycins α25单独或联合与咪康唑作用后,采用菌落形成单位(CFU)计数的方法统计耐药株的数量.采用SAS8.0统计软件包对数据进行q检验.结果 咪康唑分别联合两种药物流出泵抑制剂清除生物被膜耐药株的效果明显优于咪康唑单独使用(P<0.001),其中咪康唑与Enniatin B联用效果更佳.结论 咪康唑与药物流出泵抑制剂联合应用具有清除生物被膜中表现型耐药株的作用,这为提高抗真菌治疗的效果提供了一条新途径和新思路.     

Candida albicans genotyping in studies with patients with AIDS developing resistance to fluconazole.

We characterized Candida albicans strains responsible for recurrent oropharyngeal candidosis (OPC) in four patients with AIDS who developed clinical and mycological resistance to fluconazole (FCZ). Karyotype and restriction fragment length polymorphism analyses were performed on the clonal populations to differentiate relapse from reinfection, and the results were assessed with those of serotype and FCZ MICs. Despite the polymorphism in chromosomal bands larger than 2.2 Mbp related to an intraclonal variation, karyotype analysis showed a single strain type attributable to each patient. On the other hand, EcoRI and HinfI restriction fragments revealed a polymorphism for one patient between the first sample and the subsequent ones, relevant to the acquisition of a new strain causing the following episodes of OPC. This result coincided with switching of the serotype and with the acquisition of a resistance to FCZ. For the other three patients, the similarity of the DNA electrophoretic patterns and the serotype of the samples suggested that recurrence can be due to the initial strain that generates FCZ resistance. Although useful for epidemiological studies, molecular typing methods seem to be inadequate to detect the acquisition of FCZ resistance.     

Mixed species biofilms of Candida albicans and Staphylococcus epidermidis

A simple catheter disk model system was used to study the development in vitro of mixed species biofilms of Candida albicans and Staphylococcus epidermidis, two organisms commonly found in catheter-associated infections. Two strains of S. epidermidis were used: a slime-producing wild type (strain RP62A) and a slime-negative mutant (strain M7). In mixed fungal-bacterial biofilms, both staphylococcal strains showed extensive interactions with C. albicans. The susceptibility of 48-h biofilms to fluconazole, vancomycin and mixtures of the drugs was determined colorimetrically. The results indicated that the extracellular polymer produced by S. epidermidis RP62A could inhibit fluconazole penetration in mixed fungal-bacterial biofilms. Conversely, the presence of C. albicans in a biofilm appeared to protect the slime-negative staphylococcus against vancomycin. Overall, the findings suggest that fungal cells can modulate the action of antibiotics, and that bacteria can affect antifungal activity in mixed fungal-bacterial biofilms.     

Effect of fluconazole on agar invasion by Candida albicans

Subinhibitory concentrations of azoles are known to inhibit hyphal branching of Candida albicans in liquid media. This study showed that subinhibitory concentrations of fluconazole also inhibit agar invasion by C. albicans in YPD solid medium. Agar invasion was markedly inhibited in two C. albicans strains that were resistant to fluconazole. This suggests that the degree to which fluconazole inhibits agar invasion by C. albicans hyphae could serve as a marker of susceptibility to azoles.     

Role of dimorphism in the development of Candida albicans biofilms.

Two model biofilm systems, involving growth on disks of catheter material or on cylindrical cellulose filters, were used to investigate the structure of Candida albicans biofilms. To assess the importance of dimorphism in biofilm development, biofilms produced by two wild-type strains were compared with those formed by two morphological mutants, incapable of yeast and hyphal growth, respectively. Scanning electron microscopy and thin sections of biofilms examined by light microscopy revealed that biofilms of the wild-type strains formed on catheter disks consisted of two distinct layers: a thin, basal yeast layer and a thicker, but more open, hyphal layer. The hypha- mutant produced only the basal layer, whereas the yeast- mutant formed a thicker, hyphal biofilm equivalent to the outer zone of the wild-type structures. Biofilms of the yeast- mutant were more easily detached from the catheter surface than the others, suggesting that the basal yeast layer has an important role in anchoring the biofilm to the surface. Biofilms formed on cylindrical cellulose filters were quite different in appearance. The hypha- mutant and both wild types produced exclusively yeast-form biofilms whereas the yeast- mutant generated a dense hyphal mat on the top of the filter. All these biofilms, irrespective of morphological form, were resistant to the antifungal agent, amphotericin B. Overall, these results indicate that the structure of a C. albicans biofilm depends on the nature of the contact surface, but that some surfaces produce biofilms with a layered architecture resembling to that described for bacterial systems.     

Rapid identification of fluconazole resistance using Chromagar Candida

Although extremely rare 10 years ago, antifungal drug resistance is becoming a major problem in certain populations, especially in those infected with HIV. This study was undertaken to study the resistance of Candida species isolated in our hospital to Fluconazole using Chrom agar Candida. The Candida strains which were routinely isolated from clinical specimens like blood, urine, sputum, pus, fluid and homograft isolates were included in the study. 142 Candida isolates were tested by using Chrom agar Candida incorporated with fluconazole. 16 strains were found to be resistant to Fluconazole and 126 strains sensitive to Fluconazole. Nine were C tropicalis, 3 C krusei, 2 C guillermondii, 1 Geotrichum candidum and one was an unidentified strain of Candida. The MIC of the 16 strains were done using RPMI 1640 medium by macro broth dilution method. MIC of 9 strains was 64 & > 64 micrograms/ml of 6 strains 32 micrograms/ml and 1 strain 16 micrograms/ml.     

Influence of doxorubicin on fluconazole susceptibility and efflux pump gene expression of Candida dubliniensis

The effect of doxorubicin (DOX) on the fluconazole (FLU) susceptibility of C. dubliniensis was investigated. Isolates were exposed to DOX and FLU in a chequerboard assay and resistance gene expressions were analysed after DOX exposure. The susceptibility of the yeast to FLU was decreased in the presence of DOX in the chequerboard assay with FIC indices suggesting an antagonistic effect. Gene expression analyses showed an overexpression of CdCDR2. Hence, DOX was found to have an impact on resistance mechanisms in C. dubliniensis isolates.     

Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria

Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed.     

Rapid identification of fluconazole resistance using Chrom agar Candida

All though extremely rare 10 years ago, antifungal drug resistance is becoming a major problem in certain populations, especially in those infected with HIV. This study was undertaken to study the resistance of Candida species isolated in our hospital to Fluconazole using Chrom agar Candida. The Candida strains which were routinely isolated from clinical specimens like blood, urine, sputum, pus, fluid and homograft isolates were included in the study. 142 Candida isolates were tested by using Chrom agar Candida incorporated with Fluconazole. 16 strains were found to be resistant to Fluconazole and 126 strains sensitive to Fluconazole. Nine were C. tropicalis, 3 C. krusei, 2 C. guillermondii, 1 Geotrichum candidum and one was an unidentified strain of Candida. The MIC of the 16 strains were done using RPMI 1640 medium by macro broth dilution method. MIC of 9 strains was 64 & > 64 ug/ml of 6 strains 32 ug/ml and 1 strain 16 ug/ml.     

特比萘芬与氟康唑或伊曲康唑联合对氟康唑诱导耐药稳定白念珠菌的影响

目的探讨特比萘芬与氟康唑或伊曲康唑联合抗氟康唑诱导产生的耐药稳定白念珠菌的作用.方法采用多步诱导法,在YEPD培养基中,利用氟康唑诱导白念珠菌敏感株产生耐药稳定菌株.根据美国国家临床实验标准委员会（NCCLS）制定的标准,采用棋盘微量稀释法测定特比萘芬与氟康唑或伊曲康唑对耐药稳定菌株的联合药敏试验,并对诱导耐药稳定菌株ERG11基因的编码区序列进行DNA测序.结果临床敏感菌株和标准敏感菌株能被诱导形成耐药菌株,但大部分不稳定,诱导耐药稳定株ERG11基因的编码区DNA测序有突变点存在,特比萘芬与氟康唑或伊曲康唑联用对诱导耐药稳定株可产生协同作用.结论特比萘芬与氟康唑或伊曲康唑联合应用对基因突变产生的耐药株有协同作用,可阻止或延迟氟康唑诱导的耐药性白念珠菌菌株的产生.