Response to Oxidative Stress in Eight Pathogenic Yeast Species of the Genus <Emphasis Type="Italic">Candida</Emphasis>

In the course of an infection, the formation of reactive oxygen species by phagocytes and the antioxidant defense mechanisms of microorganisms play a crucial role in pathogenesis. In this study, isolates representing 8 pathogenic Candida species—Candida albicans, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis and Candida tropicalis—were compared with regard to their resistance to oxidative stress in vitro. We evaluated degree of resistance, induction of oxidative damage, capacity to adapt, and induction of antioxidant enzymes. The species showed variable sensitivity to oxidative attack. C. albicans, C. glabrata, and C. krusei were more resistant to oxidative stress under the conditions tested; C. parapsilosis and C. tropicalis presented medium resistance; and C. dubliniensis, C. famata, and C. guilliermondii were more sensitive. The overall greater resistance to oxidative stress of C. albicans and C. glabrata may provide an advantage to these species, which are the major causative agents of candidiasis.     

Molecular Mechanisms of Resistance to 5-Fluorocytosine in Laboratory Mutants of <Emphasis Type="Italic">Candida glabrata</Emphasis>

Resistance to 5-fluorocytosine (5-FC) has been poorly investigated in the yeast Candida glabrata. This study was conducted on laboratory mutants obtained by exposure of a wild-type isolate to 5-FC. Based on their susceptibility to 5-fluorouracil (5-FU), two of these mutants were selected for further analysis of the molecular mechanisms of 5-FC resistance. One mutant, resistant to both compounds, exhibited a missense mutation in the gene coding the cytosine deaminase and a decrease in the expression level of the gene coding the uridine monophosphate pyrophosphorylase. The other mutant that showed a reduced susceptibility to 5-FC and 5-FU exhibited an overexpression of the genes coding the thymidylate synthase and a cytosine permease, associated with a missense mutation in the last gene. Thus, beside mutations in the FUR1 gene which represent the most common cause of resistance to 5-FC, other mechanisms may also occur in C. glabrata.     

Evaluation of the Commercial Rapid Trehalose Test (GLABRATA RTT) for the Point of Isolation Identification of <Emphasis Type="Italic">Candida glabrata</Emphasis> Isolates in Primary Cultures

Candidaemias account for 10–20% of nosocomial bloodstream infections depending on the study. Whilst Candida albicans remains the most frequently isolated species, Candida glabrata may be responsible for as many as 10–25% of all candidaemias. Moreover, C. glabrata is generally less susceptible to the azole antifungals than the majority of other pathogenic yeast species. Thus, a rapid test for the specific identification of isolates of C. glabrata would be useful for patient management if it could be performed at point of isolation, on primary cultures grown on standard mycological media directly from patient specimens. Under certain conditions, C. glabrata rapidly hydrolyses trehalose into glucose. The GLABRATA RTT kit allows detection of the preformed enzyme responsible for this action. This study has assessed GLABRATA RTT as an identification tool specifically at point of isolation. Sixty test isolates were evaluated: 39 clinical isolates of C. glabrata identified at the UK Mycology Reference Laboratory, examples of the recently described genetic relatives of C. glabrata, Candida nivariensis (n = 6) and Candida bracarensis (n = 1), and a selection of other common pathogenic yeast species (n = 14). The test provided results within 30 min. Although 77% (30/39) of confirmed C. glabrata isolates were correctly identified by GLABRATA RTT (positive trehalase test), 23% (9/39) of isolates gave negative or equivocal results. All other yeast species gave negative results. The performance of GLABRATA RTT in this study is compared to previous evaluations of the test which employed isolates pre-cultured on specialised media and to other existing conventional identification methodologies.     

Effect of antifungals on itraconazole resistant <Emphasis Type="Italic">Candida glabrata</Emphasis>

In the last decade, infections caused by Candida glabrata have become more serious, particularly due to its decreased susceptibility to azole derivatives and its ability to form biofilm. Here we studied the resistance profile of 42 C. glabrata clinical isolates to different azoles, amphotericin B and echinocandins. This work was also focused on the ability to form biofilm which plays a role in the development of antifungal resistance. The minimal inhibitory concentration testing to antifungal agents was performed according to the CLSI (Clinical and Laboratory Standards Institute) M27-A3 protocol. Quantification of biofilm was done by XTT reduction assay. All C. glabrata clinical isolates were resistant to itraconazole and sixteen also showed resistance to fluconazole. All isolates remained susceptible to voriconazole. Amphotericin B was efficient in a concentration range of 0.125–1 mg/L. The most effective antifungal agents were micafungin and caspofungin with the MIC₁₀₀ values of ≤0.0313–0.125 mg/L. Low concentrations of these agents reduced biofilm formation as well. Our results show that resistance of different C. glabrata strains is azole specific and therefore a single azole resistance cannot be assumed to indicate general azole resistance. Echinocandins proved to have very high efficacy against clinical C. glabrata strains including those with ability to form biofilm.     

Environmental pH modulates biofilm formation and matrix composition in Candida albicans and Candida glabrata

Abstract

Candida species are fungal opportunistic pathogens capable of colonizing and infecting various human anatomical sites, where they have to adapt to distinct niche-specific pH conditions. The aim of this study was to analyse the features of Candida albicans and Candida glabrata biofilms developed under neutral and vaginal acidic (pH 4) conditions. C. albicans produced thicker and more filamentous biofilms under neutral than under acidic conditions. On the other hand, the formation of biofilms by C. glabrata was potentiated by the acidic conditions suggesting the high adaptability of this species to the vaginal environment. In general, both species developed biofilms containing higher amounts of matrix components (protein and carbohydrate) under neutral than acidic conditions, although the opposite result was found for one C. glabrata strain. Overall, this study contributes to a better understanding of the modulation of C. albicans and C. glabrata virulence by specific pH conditions.     

Wild-Type MIC Distributions and Epidemiologic Cutoff Values for Fluconazole and <Emphasis Type="Italic">Candida</Emphasis>: Time for New Clinical Breakpoints?

Antifungal susceptibility testing of Candida against fluconazole has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Both CLSI and EUCAST have developed clinical breakpoint (CBP) criteria for fluconazole, but these differ in both magnitude and target species. Studies using the EUCAST method have also defined wild-type minimum inhibitory concentration (MIC) distributions and epidemiologic cutoff values (ECVs or ECOFFs) for the common species of Candida. The ECVs serve as a sensitive means of discriminating wild-type strains from those with acquired resistance mechanisms and include MICs of 1 μg/mL for C. albicans, 2 μg/mL for C. tropicalis and C. parapsilosis, 32 μg/mL for C. glabrata, and 128 μg/mL for C. krusei. Because the CLSI CBPs may be too insensitive to detect emerging resistance among strains of C. albicans, C. tropicalis, and C. parapsilosis, and bisect the WT MIC distribution of C. glabrata, we sought to establish the wild-type MIC distribution and ECVs for fluconazole and Candida spp. The establishment of the wild-type MIC distributions and ECVs for fluconazole using CLSI methods will be useful in resistance surveillance and may prove to be an important step in the development of species-specific CBPs for this important antifungal agent.     

The multidrug resistance transporters CgTpo1_1 and CgTpo1_2 play a role in virulence and biofilm formation in the human pathogen Candida glabrata

The mechanisms of persistence and virulence associated with Candida glabrata infections are poorly understood, limiting the ability to fight this fungal pathogen. In this study, the multidrug resistance transporters CgTpo1_1 and CgTpo1_2 are shown to play a role in C. glabrata virulence. The survival of the infection model Galleria mellonella, infected with C. glabrata, was found to increase upon the deletion of either CgTPO1_1 or CgTPO1_2. The underlying mechanisms were further explored. In the case of CgTpo1_1, this phenotype was found to be consistent with the observation that it confers resistance to antimicrobial peptides (AMP), such as the human AMP histatin‐5. The deletion of CgTPO1_2, on the other hand, was found to limit the survival of C. glabrata cells when exposed to phagocytosis and impair biofilm formation. Interestingly, CgTPO1_2 expression was found to be up‐regulated during biofilm formation, but and its deletion leads to a decreased expression of adhesin‐encoding genes during biofilm formation, which is consistent with a role in biofilm formation. CgTPO1_2 expression was further seen to decrease plasma membrane potential and affect ergosterol and fatty acid content. Altogether, CgTpo1_1 and CgTpo1_2 appear to play an important role in the virulence of C. glabrata infections, being at the cross‐road between multidrug resistance and pathogenesis.     

Variations in yeast plasma‐membrane lipid composition affect killing activity of three families of insect antifungal peptides

Naturally occurring antimicrobial peptides and their synthetic analogues are promising candidates for new antifungal drugs. We focused on three groups of peptides isolated from the venom of bees and their synthetic analogues (lasioglossins, halictines and hylanines), which all rapidly permeabilised the plasma membrane. We compared peptides' potency against six pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei and C. dubliniensis) and the non‐pathogenic model yeast Saccharomyces cerevisiae. Their activity was independent of the presence of the multidrug‐resistant pumps of C. glabrata but was influenced by the lipid composition of cell plasma membranes. Although the direct interaction of the peptides with ergosterol was negligible in comparison with amphotericin B, the diminished ergosterol content after terbinafine pretreatment resulted in an increased resistance of C. glabrata to the peptides. The tested peptides strongly interacted with phosphatidylglycerol, phosphatidic acid and cardiolipin and partly with phosphatidylinositol and phosphatidylethanolamine. The interactions between predominantly anionic phospholipids and cationic peptides indicated a mainly electrostatic binding of peptides to the membranes. The results obtained also pointed to a considerable role of the components of lipid rafts (composed from sphingolipids and ergosterol) in the interaction of yeast cells with the peptides.     

Silver nanoparticles,a promising treatment against clinically important fluconazole-resistant Candida glabrata

Resistance to azole antifungal agents is a challenging limitation in Candida glabrata treatment. It is associated with decreased intracellular concentrations of antifungal agents as a result of overexpression of efflux pumps on the cellular plasma membranes. This work evaluates the potential of silver nanoparticles (AgNPs) to reverse the resistance of fungal cells to fluconazole. Silver nanoparticles were prepared using wet chemical method and characterised by UV-Vis spectrophotometry, dynamic light scattering, and zeta potential. Broth microdilution and pour plates methods were used to study the anticandidal activity using two C. glabrata fluconazole-resistant strains (DSY565 and CBS138) known to overexpress active efflux pumps, and a standard fluconazole sensitive strain ATCC 22553. Silver nanoparticles–fluconazole combinations decreased concentrations of fluconazole substantially without compromising the activity. These findings suggest that AgNPs enhance the efficacy of fluconazole and offer a promising application in therapy of C. glabrata infections.     

Molecular typing of clinical Candida strains using random amplified polymorphic DNA and contour‐clamped homogenous electric fields electrophoresis

Aims: This report describes an investigation into the genetic profiles of 38 Candida albicans and 19 Candida glabrata strains collected from a dental hospital of Monastir (Tunisia) and the Laboratory of Parasitology, Farhat Hached Hospital of Sousse (Tunisia), using two typing methods: random amplified polymorphic DNA (RAPD) and contour‐clamped homogenous electric fields (CHEF). Methods and Results: The two methods (RAPD and CHEF electrophoresis) were able to identify clonal‐related isolates from different patients. RAPD method using two primers (CA1 and CA2) exhibited the highest discriminatory power by discriminating 22 genotypes for C. albicans with CA1 oligonucleotides and 19 genotypes with CA2 primer. For C. glabrata, 17 genotypes were obtained when both primers CA1 and CA2 were combined. The CHEF karyotyping of C. albicans has discriminated only 17 different karyotypes. Conclusion: The genotype of each isolate and genotypic difference among C. albicans and C. glabrata isolates were patient specific and not associated with the site of infection, geographic origin or date of isolation. Significance and Impact of the Study: Identification of relatedness between Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for interruption of transmission of this yeast.     

Multilocus Sequence Type Analysis Reveals both Clonality and Recombination in Populations of Candida glabrata Bloodstream Isolates from U.S. Surveillance Studies

The human commensal yeast Candida glabrata is becoming increasingly important as an agent of nosocomial bloodstream infection. However, relatively little is known concerning the genetics and population structure of this species. We have analyzed 230 incident bloodstream isolates from previous and current population-based surveillance studies by using multilocus sequence typing (MLST). Our results show that in the U.S. cities of Atlanta, GA; Baltimore, MD; and San Francisco, CA during three time periods spanning 1992 to 2009, five populations of C. glabrata bloodstream isolates are defined by a relatively small number of sequence types. There is little genetic differentiation in the different C. glabrata populations. We also show that there has been a significant temporal shift in the prevalence of one major subtype in Atlanta. Our results support the concept that both recombination and clonality play a role in the population structure of this species.In the most recently available survey of nosocomial bloodstream infections, Candida species were the fourth most common organism, surpassed only by Staphylococcus and Enterococcus species (24). Although Candida albicans remains the most commonly isolated Candida species worldwide, the incidence of Candida glabrata infection has been increasing steadily so that it is now the second most common cause of Candida infection in the United States (14). C. glabrata is considered a normal component of the human epithelial flora but is capable of causing serious systemic infections in susceptible hosts. This increase in the relative proportion of infections due to C. glabrata has come during the period of the introduction and prophylactic use of azole antifungal drugs (21) and may be a reflection of the decreased susceptibility of C. glabrata to these azole antifungal drugs (7, 15). Many questions regarding the epidemiology of C. glabrata infections have a direct impact on public health and still remain unanswered. Is the decreased susceptibility due to a small number of clones expanding in a population, or are all isolates capable of developing resistance to azole drugs? Are some isolates more virulent than others and therefore more prevalent in a population? Can we monitor the expansion of clonal isolates that may be more virulent or have increased drug resistance? A better understanding of the population genetics of C. glabrata may allow us to answer some of these questions.Many DNA fingerprinting methods have been developed for the investigation of the population genetics of Candida species (19). Two of the most important aspects of a typing system are reproducibility between laboratories and the ability to archive strain types. Multilocus sequence typing (MLST) has been developed as a typing system which allows highly reproducible strain discrimination as well as the development of genotypic strain archives that can be stored digitally for both prospective and retrospective analysis of isolates (13, 22). An MLST system which utilizes six housekeeping genes on six separate chromosomes was developed for C. glabrata (4), and an online archive of sequence types (STs) was established (http://cglabrata.mlst.net). Several studies utilizing this typing system have described the molecular population structure of both regional and worldwide collections of C. glabrata isolates (4, 5, 11, 12).During the past 2 decades, the Centers for Disease Control and Prevention (CDC) and our partners have undertaken three active, population-based surveillance studies in order to determine the incidence of candidemia, the distribution of species causing bloodstream infection, and the prevalence of antifungal drug resistance (8, 10). In each case, two major metropolitan areas were included: San Francisco, CA, and Atlanta, GA (1992 to 1993); Baltimore, MD, and the state of Connecticut (1998 to 2000); and Atlanta, GA, and Baltimore, MD (2008 to 2010). Population-based surveillance is unique in that it includes the total population of a particular geographic area and avoids the biases associated with single or select institutional studies. During each of the surveillance studies, incident bloodstream isolates from all hospitals within each defined geographic area were collected and identified to the species level. While C. glabrata isolates comprised a smaller percentage of the isolates in the 1992-to-1993 and 1998-to-2000 surveillance studies (8, 10), they represent almost a third of the isolates collected during the current surveillance (N. Iqbal and S. Lockhart, unpublished observations).In the present work, we have characterized by MLST analysis 230 isolates of C. glabrata from five populations (excluding Connecticut) separated both geographically and temporally. This unique collection of isolates allowed an analysis of the changing population genetics of this organism. We identified 31 unique STs and showed the maintenance of a major ST both geographically and temporally that is unique to the United States. An analysis of the relatedness of specific C. glabrata populations and a strong indication for recombination within and between populations are provided.     

Two unlike cousins: Candida albicans and C. glabrata infection strategies

Candida albicans and C. glabrata are the two most common pathogenic yeasts of humans, yet they are phylogenetically, genetically and phenotypically very different. In this review, we compare and contrast the strategies of C. albicans and C. glabrata to attach to and invade into the host, obtain nutrients and evade the host immune response. Although their strategies share some basic concepts, they differ greatly in their outcome. While C. albicans follows an aggressive strategy to subvert the host response and to obtain nutrients for its survival, C. glabrata seems to have evolved a strategy which is based on stealth, evasion and persistence, without causing severe damage in murine models. However, both fungi are successful as commensals and as pathogens of humans. Understanding these strategies will help in finding novel ways to fight Candida, and fungal infections in general.     

Iron-depletion promotes mitophagy to maintain mitochondrial integrity in pathogenic yeast Candida glabrata

Candida glabrata, a haploid budding yeast, is the cause of severe systemic infections in immune-compromised hosts. The amount of free iron supplied to C. glabrata cells during systemic infections is severely limited by iron-chelating proteins such as transferrin. Thus, the iron-deficiency response in C. glabrata cells is thought to play important roles in their survival inside the host's body. In this study, we found that mitophagy was induced under iron-depleted conditions, and that the disruption of a gene homologous to ATG32, which is responsible for mitophagy in Saccharomyces cerevisiae, blocked mitophagy in C. glabrata. The mitophagic activity in C. glabrata cells was not detected on short-period exposure to nitrogen-starved conditions, which is a mitophagy-inducing condition used in S. cerevisiae. The mitophagy-deficient atg32Δ mutant of C. glabrata also exhibited decreased longevity under iron-deficient conditions. The mitochondrial membrane potential in Cgatg32Δ cells was significantly lower than that in wild-type cells under iron-depleted conditions. In a mouse model of disseminated infection, the Cgatg32Δ strain resulted in significantly decreased kidney and spleen fungal burdens compared with the wild-type strain. These results indicate that mitophagy in C. glabrata occurs in an iron-poor host tissue environment, and it may contribute to the longevity of cells, mitochondrial quality control, and pathogenesis.     

Dual antifungal properties of cationic antimicrobial peptides polybia-MPI: Membrane integrity disruption and inhibition of biofilm formation

With the increasing emergence of resistant fungi, the discovery and development of novel antifungal therapeutics were urgently needed. Compared with conventional antibiotics, the limited propensity of AMPs to induce resistance in pathogens has attracted great interest. In the present study, the antifungal activity and its mechanism-of-action of polybia-MPI, a cationic peptide from the venom of Social wasp Polybia Paulista was investigated. We demonstrated that polybia-MPI could potently inhibit the growth of Candida albicans (C. albicans) and Candida glabrata (C. glabrata). The 50% inhibitory concentrations (IC₅₀) of Polybia-MPI against cancer cells were much higher than the MICs against the tested C. albicans and C. glabrata cells, indicating that polybia-MPI had high selectivity between the fungal and mammalian cells. Our results also indicated that membrane disturbance mechanism was involved in the antifungal activity. Furthermore, polybia-MPI could inhibit the bio film forming of C. glabrata, which was frequently associated with clinically significant biofilm. These results suggest that polybia-MPI has great advantages in the development of antifungal agents.     

Fluconazole-Resistant Candida: Mechanisms and Risk Factor Identification

The extensive use of fluconazole in the previous two decades has fostered the emergence of azole-resistant strains, including non-albicans species such as C. glabrata. The main mechanisms of fluconazole resistance described involve alteration of the quality or quantity of 14-alpha-demethylase enzyme or reduced access of the drug to the target enzyme by upregulation of efflux pumps. Fluconazole prophylaxis has been used successfully to prevent fungal infections in high-risk neonates, neutropenic patients, individuals with HIV infection, recipients of solid organ (eg, liver) transplants and bone marrow transplants, and patients in intensive care units, with a small increase in the risk of colonization, but not infection, with fluconazole-resistant Candida isolates. Among other risk factors, recent fluconazole exposure is associated with fluconazole-resistant candidemia.