Elisabete Ricardo

Ibuprofen reverts antifungal resistance on Candida albicans showing overexpression of CDR genes

Several mechanisms may be associated with Candida albicans resistance to azoles. Ibuprofen was described as being able to revert resistance related to efflux activity in Candida. The aim of this study was to uncover the molecular base of antifungal resistance in C. albicans clinical strains that could be reverted by ibuprofen. Sixty-two clinical isolates and five control strains of C. albicans were studied: the azole susceptibility phenotype was determined according to the Clinical Laboratory for Standards Institute, M27-A2 protocol and minimal inhibitory concentration values were recalculated with ibuprofen (100 microg mL(-1)); synergistic studies between fluconazole and FK506, a Cdr1p inhibitor, were performed using an agar disk diffusion assay and were compared with ibuprofen results. Gene expression was quantified by real-time PCR, with and without ibuprofen, regarding CDR1, CDR2, MDR1, encoding for efflux pumps, and ERG11, encoding for azole target protein. A correlation between susceptibility phenotype and resistance gene expression profiles was determined. Ibuprofen and FK506 showed a clear synergistic effect when combined with fluconazole. Resistant isolates reverting to susceptible after incubation with ibuprofen showed CDR1 and CDR2 overexpression especially of the latter. Conversely, strains that did not revert displayed a remarkable increase in ERG11 expression along with CDR genes. Ibuprofen did not alter resistance gene expression significantly (P>0.05), probably acting as a Cdrp blocker.

In vivo and in vitro acquisition of resistance to voriconazole by Candida krusei

ABSTRACT Candida krusei is an important agent of opportunistic infections that often displays resistance to several antifungals. We describe here the in vivo acquisition of resistance to voriconazole (VRC) by C. krusei isolates recovered from a leukemia patient during a long period of VRC therapy. In order to mimic the in vivo development of VRC resistance, a susceptible C. krusei isolate was exposed daily to 1 μg/ml of VRC in vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 μg/ml was achieved; this value remained constant after 25 additional days of treatment with VRC and also after 30 consecutive days of incubation in VRC-free medium. Our objective was to determine the associated molecular resistance mechanisms, such as expression of efflux pump genes and ERG11 gene mutations, among the resistant strains. Synergistic effects between the efflux blocker tacrolimus (FK506) and VRC were found in all of the resistant strains. Moreover, ABC1 gene expression increased over time in both the in vivo- and in vitro-induced resistant strains, in contrast to the ABC2 and ERG11 genes, whose expression was invariably lower and constant. ERG11 gene sequencing showed two different types of mutations, i.e., heterozygosity at T1389T/C, corresponding to synonymous mutations, in C. krusei strains and a missense mutation at position T418C, resulting in a change from Tyr to His, among resistant C. krusei clinical isolates. This study highlights the relevance of ATP-dependent efflux pump (namely, Abc1p) activity in VRC resistance and describes new mutations in the ERG11 gene among resistant C. krusei clinical isolates.

Erratum to: Species distribution and in vitro antifungal susceptibility profiles of yeast isolates from invasive infections during a Portuguese multicenter survey.

This is the first Portuguese multicenter observational and descriptive study that provides insights on the species distribution and susceptibility profiles of yeast isolates from fungemia episodes. Ten district hospitals across Portugal contributed by collecting yeast isolates from blood cultures and answering questionnaires concerning patients’ data during a 12-month period. Molecular identification of cryptic species of Candida parapsilosis and C. glabrata complex was performed. The susceptibility profile of each isolate, considering eight of the most often used antifungals, was determined. Both Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocols were applied. The incidence of 240 episodes of fungemia was 0.88/1,000 admissions. Fifteen different species were found, with C. albicans (40 %) being the most prevalent, followed by C. parapsilosis (23 %) and C. glabrata (13 %). Most isolates were recovered from patients admitted to surgical wards or intensive care units, with 57 % being males and 32 % aged between 41 and 60 years. For both the CLSI and EUCAST protocols, the overall susceptibility rates ranged from 74 to 97 % for echinocandins and from 84 to 98 % for azoles. Important resistance rate discrepancies between protocols were observed in C. albicans and C. glabrata for echinocandins and in C. parapsilosis and C. tropicalis for azoles. Death associated with fungemia occurred in 25 % of the cases, with more than half of C. glabrata infections being fatal. The great number of Candida non-albicans is noteworthy despite a relatively low antifungal resistance rate. Studies like this are essential in order to improve empirical treatment guidelines.

The effect of antibacterial and non-antibacterial compounds alone or associated with antifugals upon fungi

During the last 30 years the incidence of fungal infections has increased dramatically. While the antifungal therapeutic options available are somewhat reduced, most pathogenic microorganisms have an incredible capacity to mutate and acquire resistance. In addition, multiple drugs are often required concomitantly to manage clinically complex disorders. The combination of antibiotics or other compounds with antifungal drugs, simultaneously or sequentially, is commonly adopted in clinical practice, although without a full knowledge of the consequences. Thus, the role of combined therapy and the effect of antibiotics upon fungal growth promotion need to be critically evaluated and understood in order to avoid undesirable drug interactions. With this review we intend discuss the studies that report about antibiotics inhibiting fungal growth, as well as studies describing the synergistic effect of the combined therapy, i.e., associations between antibiotics or other compounds with antifungal drugs. Alternative therapeutic protocols for fungal disease could be designed, taking advantage of such drug combinations. Critical revision of previously published data is crucial in order to define future research strategies.

Genital candidosis in heterosexual couples

Background  Evidence suggests that Candida can be sexually transmitted; however, the contribution of sexual transmission to the pathogenesis of genital candidosis needs further elucidation.

Candida krusei reservoir in a neutropaenia unit: molecular evidence of a foe?

Candida krusei has been documented as an emerging pathogen causing nosocomial outbreaks. The consecutive isolation of C. krusei strains in three patients admitted to the same hospital department within 2 months lead us to consider the possibility of an outbreak. Additionally, C. krusei isolates were collected from the room surfaces, whereas another isolate had been recovered from the blood of one patient 2 years before. HinfI DNA restriction endonuclease-based analysis of all C. krusei isolates was performed and restriction profiles were compared. Surprisingly, isolates from different patients were unrelated, whereas isolates from biological products of the same patient showed indistinguishable HinfI restriction patterns and were similar to those obtained from the surrounding environment of the respective patients. The study approach revealed the endogenous origin of the C. krusei infectious episodes observed and demonstrated that, subsequent to colonizing a patient, C. krusei can be involved in infectious episodes distant in time. The hypothesis of an outbreak was excluded, although we believe that the methodology employed in the present study represents a valuable tool for diagnostic and epidemiological surveys.

GRG5/AES Interacts with T-Cell Factor 4 (TCF4) and Downregulates Wnt Signaling in Human Cells and Zebrafish Embryos

Transcriptional control by TCF/LEF proteins is crucial in key developmental processes such as embryo polarity, tissue architecture and cell fate determination. TCFs associate with β-catenin to activate transcription in the presence of Wnt signaling, but in its absence act as repressors together with Groucho-family proteins (GRGs). TCF4 is critical in vertebrate intestinal epithelium, where TCF4-β-catenin complexes are necessary for the maintenance of a proliferative compartment, and their abnormal formation initiates tumorigenesis. However, the extent of TCF4-GRG complexes’ roles in development and the mechanisms by which they repress transcription are not completely understood. Here we characterize the interaction between TCF4 and GRG5/AES, a Groucho family member whose functional relationship with TCFs has been controversial. We map the core GRG interaction region in TCF4 to a 111-amino acid fragment and show that, in contrast to other GRGs, GRG5/AES-binding specifically depends on a 4-amino acid motif (LVPQ) present only in TCF3 and some TCF4 isoforms. We further demonstrate that GRG5/AES represses Wnt-mediated transcription both in human cells and zebrafish embryos. Importantly, we provide the first evidence of an inherent repressive function of GRG5/AES in dorsal-ventral patterning during early zebrafish embryogenesis. These results improve our understanding of TCF-GRG interactions, have significant implications for models of transcriptional repression by TCF-GRG complexes, and lay the groundwork for in depth direct assessment of the potential role of Groucho-family proteins in both normal and abnormal development.

Rapid flow cytometry test for identification of different carbapenemases in Enterobacteriaceae

ABSTRACT A flow cytometry test was developed to identify carbapenemase production by Enterobacteriaceae and to discriminate between the different types of carbapenemases (classes A, B, and D). It is based on the detection of meropenem activity against bacteria, coupled with different carbapenemase inhibitors, which is assessed by flow cytometry. It represents a convenient, fast, and reliable approach (100% sensitivity and 100% specificity) for the detection and characterization of different carbapenemases.

New insights regarding yeast survival following exposure to liposomal amphotericin B

ABSTRACT In vitro resistance to amphotericin B is an extremely rare event among pathogenic yeasts. However, in vivo response is sometimes reduced, resulting in an unfavorable outcome. Such adverse outcomes might be related to subfungicidal plasma concentrations. We aimed to clarify the mechanisms of liposomal amphotericin B (AMB-L; AmBisome)-induced lesions and the mechanisms responsible for yeast cell recovery following exposure at plasma concentrations. The physiological statuses developing following exposure to AMB-L at simulated plasma concentrations (20 to 0.1 mg/liter) and at a constant concentration (3 mg/liter) were assessed in a 24-h time course assay. Time-kill experiments also were carried out under the same AMB-L treatment conditions. Our results suggest that yeast cells develop compensatory responses related to membrane polarization, metabolic activity, and reactive oxygen species (ROS) production after exposure to high plasma concentrations (20 to 5 mg/liter) during the first 6 h; in the remaining 18 h, when exposed to lower concentrations, cells reveal almost full recovery with no evidence of fungicidal activity. In contrast, whenever cells are exposed to a constant concentration above the MIC, despite initially exhibiting compensatory stress responses, soon afterwards they exhibit membrane depolarization, a decrease of metabolic activity, increasing ROS production, and lastly, programmed cell death and necrosis, resulting in succumbing to AMB-L fungicidal effects. This study may represent a step forward in the support of AMB-L use for clinical treatment of invasive fungal infections, since it demonstrates the importance of maintaining levels of AMB-L above the MIC in plasma and tissues to ensure it produces its fungicidal effects.

Unveiling the Synergistic Interaction Between Liposomal Amphotericin B and Colistin

Patients with multiple comorbidities are often administered simultaneously or sequentially antifungals and antibacterial agents, without full knowledge of the consequences of drug interactions. Considering the clinical relevance of liposomal amphotericin B (L-AMB), the association between L-AMB and six antibacterial agents was evaluated against four clinical isolates and one type strain of Candida spp. and two clinical isolates and one type strain of Aspergillus fumigatus. In order to evaluate such combined effects, the minimal inhibitory concentration (MIC) of L-AMB was determined in the presence of 0.5-, 1-, 2-, and 4-fold peak plasma concentrations of each of the antibacterial drugs. Since the L-AMB/colistin (CST) association was the most synergic, viability assays were performed and the physiological status induced by this association was characterized. In addition, computational molecular dynamics studies were also performed in order to clarify the molecular interaction. The maximum synergistic effect with all antibacterial agents, except CST, was reached at fourfold the usual peak plasma concentrations, resulting in 2-to 8-fold L-AMB MIC reduction for Candida and 2-to 16-fold for Aspergillus. For CST, the greatest synergism was registered at peak plasma concentration (3 mg/L), with 4-to 8-fold L-AMB MIC reduction for Candida and 16-to 32-fold for Aspergillus. L-AMB at subinhibitory concentration (0.125 mg/L) combined with CST 3 mg/L resulted in: a decrease of fungal cell viability; an increase of cell membrane permeability; an increase of cellular metabolic activity soon after 1 h of exposure, which decreased until 24 h; and an increase of ROS production up to 24 h. From the molecular dynamics studies, AMB and CST molecules shown a propensity to form a stable molecular complex in solution, conferring a recognition and binding added value for membrane intercalation. Our results demonstrate that CST interacts synergistically with L-AMB, forming a stable complex, which promotes the fungicidal activity of L-AMB at low concentration.