RESUMO
While Candida albicans is the most significant fungal pathogen for humans, Candida glabrata accounts for an increasing number of infections. Little is known about how C. glabrata interacts with the innate immune system, the first line of defense against such organisms. The C. glabrata adhesin Epa1p was previously shown to bind mammalian epithelial cells. We hypothesized that Epa1p mediates unique, nonopsonic binding to macrophages, leading to induction of immune responses. We found that Epa1p mediated adhesion by both C. glabrata (Cg) and transformed Saccharomyces cerevisiae (Sc(EPA1) ) to human macrophage-like cells, including Thp1 and U937 lines, and donor PBMCs. Adhesion was distinct from described mechanisms such as Dectin-1. Epa1p expression was necessary and sufficient for S. cerevisiae binding and phagocytosis, the latter of which was actin-mediated. Sc(EPA1) induced inflammatory cytokine production (IL-8 and TNF-α) by human PBMC-derived macrophages. Despite expressing Epa1p and binding to macrophages, Cg avoided phagocytosis and cytokine induction. In contrast to human results, in murine cell models (RAW264.7, J774A.1, and C57BL/6-derived cells), Epa1p-mediated binding was only revealed after blocking the Dectin-1 system. Recognition of Epa1p represents a novel mechanism by which human innate immune cells bind fungi, and for Sc(EPA1) results in phagocytosis and subsequent cytokine production.
Assuntos
Candida glabrata/imunologia , Adesão Celular , Citocinas/metabolismo , Proteínas Fúngicas/imunologia , Lectinas/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Fagocitose , Animais , Células Cultivadas , Expressão Gênica , Humanos , Camundongos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologia , Transformação GenéticaRESUMO
INTRODUCTION: Ubiquinol (reduced coenzyme Q10) is essential for adequate aerobic metabolism. The objective of this trial was to determine whether ubiquinol administration in patients resuscitated from cardiac arrest could increase physiological coenzyme Q10 levels, improve oxygen consumption, and reduce neurological biomarkers of injury. MATERIALS AND METHODS: This was a randomized, double-blind, placebo-controlled trial in patients successfully resuscitated from cardiac arrest. Patients were randomized to receive enteral ubiquinol (300â¯mg) or placebo every 12â¯h for up to 7 days. The primary endpoint was total coenzyme Q10 plasma levels at 24â¯h after enrollment. Secondary endpoints included neuron specific enolase, S100B, lactate, cellular and global oxygen consumption, neurological status, and in-hospital mortality. RESULTS: Forty-three patients were included in the modified intention-to-treat analysis. Median coenzyme Q10 levels were significantly higher in the ubiquinol group as compared to the placebo group at 24â¯h (441 [IQR, 215-510] ηg/mL vs. 113 [IQR, 94-208] ηg/mL, Pâ¯<â¯0.001). Similar results were observed at 48 and 72â¯h. There were no differences between the two groups in any of the secondary endpoints. Median neuron specific enolase levels were not different between the two groups at 24â¯h (16.8 [IQR, 9.5-19.8] ηg/mL vs. 8.2 [IQR, 4.3-19.1] ηg/mL, Pâ¯=â¯0.61). CONCLUSIONS: Administration of enteral ubiquinol increased plasma coenzyme Q10 levels in post-cardiac arrest patients as compared to placebo. There were no differences in neurological biomarkers and oxygen consumption between the two groups.
Assuntos
Parada Cardíaca , Ubiquinona , Biomarcadores , Método Duplo-Cego , Parada Cardíaca/tratamento farmacológico , Humanos , Ubiquinona/análogos & derivadosRESUMO
Intravascular catheter infections are a major cause of morbidity and mortality in hospitalized patients, accounting for the majority of the 200,000 nosocomial bloodstream infections occurring in the US annually. Of the intravenous lines that are culture-positive for Candida, 40% actually represent fungemia, which generally necessitates systemic treatment and line removal to affect cure. Until recently, the reason for the need for device removal was unclear. However, our research group and others have demonstrated a near-total resistance to antifungals by biofilm-associated Candida. Similar to bacterial species, Candida biofilm formation proceeds through early, intermediate and maturation phases. This process is associated with the generation of a polysaccharide extracellular matrix (ECM). Mature C. albicans biofilms have a heterogeneous architecture, in terms of distribution of fungal cells and ECM, and exhibit broad antimicrobial resistance. The mechanisms causing such profound antifungal resistance are beginning to be understood. Recent data indicate that resistance is phase-specific and multifactorial, involving efflux pumps and sterol synthesis (at early and mature biofilm phases, respectively). Neither metabolic quiescence nor the ECM appear to contribute substantially. Susceptibility testing and confocal scanning laser microscopy demonstrated that azoles failed to exert activity against mature Candida biofilms. However, sub-inhibitory concentrations of voriconazole impaired biofilm formation and caused cell morphological aberrations. In contrast, lipid-formulation amphotericins and the echinocandins uniquely exhibited activity against mature biofilms. The mechanisms underlying this ability are unknown. The role of other pharmacological (eg, catheter coatings, antimicrobial peptides and antibiotic locks) and non-pharmacological methods in the prevention and treatment of device-related biofilms is discussed in this review.
Assuntos
Biofilmes/crescimento & desenvolvimento , Candida/crescimento & desenvolvimento , Candidíase/tratamento farmacológico , Farmacorresistência Fúngica/fisiologia , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , HumanosRESUMO
OBJECTIVE: To evaluate clinical data for cold-stunned Kemp's ridley turtles (Lepidochelys kempii) with Enterococcus spp infections during rehabilitation. DESIGN: Retrospective case series. ANIMALS: 50 stranded cold-stunned Kemp's ridley turtles hospitalized between 2006 and 2012. PROCEDURES: Medical records for turtles from which Enterococcus spp were isolated were reviewed retrospectively, and clinical data, including morphometric data, body temperature at admission, physical examination findings, antimicrobial medication history, history of medications administered IV, environmental data, day of diagnosis, clinical signs at diagnosis, microbiological testing results, sources of positive culture results, hematologic and plasma biochemical data, cytologic and histopathologic results, radiographic findings, antimicrobial treatments, time to first negative culture result, treatment duration, results of subsequent cultures, and case outcome, were collated and analyzed. RESULTS: Enterococcus spp were isolated from bacteriologic cultures of blood, bone, joint, and respiratory tract samples and a skin lesion, with supporting evidence of infection provided by histopathologic, cytologic, and radiographic data. Positive culture results were associated with clinical problems such as lethargy, anorexia, and lameness. Most (34/43 [79%]) turtles for which an antemortem diagnosis was made survived with treatment and were released into the wild. CONCLUSIONS AND CLINICAL RELEVANCE: Cold-stunned Kemp's ridley turtles may be affected by serious Enterococcus spp infections during rehabilitation. Recognition and treatment of these infections are important for successful rehabilitation.
Assuntos
Temperatura Baixa/efeitos adversos , Enterococcus/isolamento & purificação , Infecções por Bactérias Gram-Positivas/veterinária , Tartarugas , Animais , Antibacterianos/uso terapêutico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/mortalidade , Estudos Retrospectivos , Fatores de RiscoRESUMO
Candida albicans biofilms are formed through three distinct developmental phases and are associated with high fluconazole (FLU) resistance. In the present study, we used a set of isogenic Candida strains lacking one or more of the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p to determine their role in FLU resistance of biofilms. Additionally, variation in sterol profile as a possible mechanism of drug resistance was investigated. Our results indicate that parent and mutant strains formed similar biofilms. However, biofilms formed by double and triple mutants were more susceptible to FLU at 6 h (MIC = 64 and 16 microg/ml, respectively) than the wild-type strain (MIC > 256 microg/ml). At later time points (12 and 48 h), all the strains became resistant to this azole (MIC > or = 256 microg/ml), indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation. Northern blot analyses revealed that Candida biofilms expressed CDR and MDR1 genes in all the developmental phases, while planktonic cells expressed these genes only at the 12- and 48-h time points. Functionality of efflux pumps was assayed by rhodamine (Rh123) efflux assays, which revealed significant differences in Rh123 retention between biofilm and planktonic cells at the early phase (P = 0.0006) but not at later stages (12 and 48 h). Sterol analyses showed that ergosterol levels were significantly decreased (P < 0.001) at intermediate and mature phases, compared to those in early-phase biofilms. These studies suggest that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Fluconazol/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Candida albicans/genética , Candida albicans/fisiologia , DNA Fúngico/genética , Farmacorresistência Fúngica/genética , Corantes Fluorescentes/farmacocinética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Rodamina 123/farmacocinética , Esteróis/metabolismoRESUMO
Candida is the fourth most common organism responsible for bloodstream infections in many intensive care units, with Candida albicans being the most predominant species isolated in such cases. It has previously been shown that candidal phospholipase B, encoded by the PLB1 gene, is an important virulence factor for C. albicans pathogenesis. In this study, the effects of environmental factors (carbohydrate source and pH) and physiological conditions (serum, phospholipids and temperature) on the expression of PLB1 by C. albicans cells grown in rich [Sabouraud dextrose broth (SB) or yeast extract/peptone/dextrose] or chemically defined [Lee's, RPMI-1640 or yeast nitrogen base (YNB)] media were investigated. Northern blot analyses revealed that PLB1 mRNA was expressed in C. albicans cells grown in rich media at 30 degrees C but not at 37 degrees C. However, the protein Plb1p was detected in fungal cells growing at 37 degrees C in SB, as determined by Western blot analysis, indicating that although the mRNA for this gene was not detected, the actual gene product was present at this temperature. Expression of PLB1 was detected in cells grown in YNB/glucose at 30 degrees C but not at 37 degrees C. However, growth of C. albicans in YNB/glucose supplemented with serum and phospholipids resulted in expression of PLB1 at 37 degrees C also. Additionally, acidic pH induced higher levels of PLB1 mRNA expression compared to neutral pH, while the morphological form of C. albicans did not have any influence on the expression of this gene. The studies described here show that the expression of PLB1 is regulated by nutritional supplementation, environmental factors and the growth phase of the C. albicans cells, as well as by physiological conditions. The differential expression of PLB1 in response to environmental factors may be correlated to host-specific components available to C. albicans during infection.
Assuntos
Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Lisofosfolipase/metabolismo , Candida albicans/genética , Meios de Cultura/química , Proteínas Fúngicas/genética , Humanos , Concentração de Íons de Hidrogênio , Lisofosfolipase/genética , TemperaturaRESUMO
Candida parapsilosis is an important non-albicans species which infects hospitalized patients. No studies have correlated outbreak infections of C. parapsilosis with multiple virulence factors. We used DNA fingerprinting to determine genetic variability among isolates from a C. parapsilosis outbreak and from our clinical database. We compared phenotypic markers of pathogenesis, including adherence, biofilm formation, and protein secretion (secretory aspartic protease [SAP] and phospholipase). Adherence was measured as colony counts on silicone elastomer disks immersed in agar. Biofilms formed on disks were quantified by dry weight. SAP expression was measured by hydrolysis of bovine albumin; a colorimetric assay was used to quantitate phospholipase. DNA fingerprinting indicated that the outbreak isolates were clonal and genetically distinct from our database. Biofilm expression by the outbreak clone was greater than that of sporadic isolates (p < or =0.0005). Adherence and protein secretion did not correlate with strain pathogenicity. These results suggest that biofilm production plays a role in C. parapsilosis outbreaks.