RESUMEN
The aim of this work was to elucidate the molecular mechanisms of flucytosine (5FC) resistance and 5FC/fluconazole (FLC) cross-resistance in 11 genetically and epidemiologically unrelated clinical isolates of Candida lusitaniae. We first showed that the levels of transcription of the FCY2 gene encoding purine-cytosine permease (PCP) in the isolates were similar to that in the wild-type strain, 6936. Nucleotide sequencing of the FCY2 alleles revealed that 5FC and 5FC/FLC resistance could be correlated with a cytosine-to-thymine substitution at nucleotide 505 in the fcy2 genes of seven clinical isolates, resulting in a nonsense mutation and in a putative nonfunctional truncated PCP of 168 amino acids. Reintroducing a FCY2 wild-type allele at the fcy2 locus of a ura3 auxotrophic strain derived from the clinical isolate CL38 fcy2(C505T) restored levels of susceptibility to antifungals comparable to those of the wild-type strains. In the remaining four isolates, a polymorphic nucleotide was found in FCY1 where the nucleotide substitution T26C resulted in the amino acid replacement M9T in cytosine deaminase. Introducing this mutated allele into a 5FC- and 5FC/FLC-resistant fcy1Delta strain failed to restore antifungal susceptibility, while susceptibility was obtained by introducing a wild-type FCY1 allele. We thus found a correlation between the fcy1 T26C mutation and both 5FC and 5FC/FLC resistances. We demonstrated that only two genetic events occurred in 11 unrelated clinical isolates of C. lusitaniae to support 5FC and 5FC/FLC resistance: either the nonsense mutation C505T in the fcy2 gene or the missense mutation T26C in the fcy1 gene.
Asunto(s)
Antifúngicos/farmacología , Candidiasis/microbiología , Codón sin Sentido/genética , Fluconazol/farmacología , Flucitosina/farmacología , Mutación Missense/genética , Northern Blotting , Southern Blotting , Candida/efectos de los fármacos , Candida/genética , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la PolimerasaRESUMEN
We report on the underlying molecular mechanisms likely responsible for the high-level fluconazole resistance in a Candida lusitaniae clinical isolate. Fluconazole resistance correlated with overexpression of ERG11 and of several efflux pump genes, in particular, the orthologs of the Candida albicans MDR1, PDR16, CDR1, CDR2, and YOR1.
Asunto(s)
Candida/efectos de los fármacos , Candida/genética , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Sistema Enzimático del Citocromo P-450/genética , Cartilla de ADN , Proteínas Fúngicas/genética , Humanos , Proteínas de Transporte de Membrana/genética , Metiltransferasas/genética , Oxidorreductasas/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
In yeast, external signals such as high osmolarity or oxidant conditions activate the high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) cascade pathway, which consists of two upstream branches, i.e. Sho1p and Sln1p and common downstream elements, including the Pbs2p MAPK kinase and the Hog1p MAPK. We recently showed that the Candida lusitaniae SLN1 gene, potentially encoding a histidine kinase receptor, is crucial for oxidative stress adaptation when the fungus grows as budding yeast and during the early steps of pseudohyphal development. In the current study, we characterized the SHO1 gene of this opportunistic fungus. Complete loss of SHO1 function causes profound defects in pseudohyphal differentiation, especially in high osmolarity and oxidative stress conditions, suggesting a crucial role of SHO1 in the pseudohyphae morphogenetic transitions. Moreover, when grown as budding yeast, the sho1Delta mutant revealed a sensitivity to compounds that interfere with the cell wall assembly, pointing to a potential role of Sho1p in cell wall biogenesis. However, the sho1Delta mutant does not display evident cell-wall architecture modifications, such as aggregation phenotypes. Although not hypersusceptible to antifungals of clinical relevance, the sho1Delta mutants are susceptible to the filamentous fungi-specific antifungals dicarboximides and phenylpyrroles. Finally, our findings highlight some significant phenotypic differences when the C. lusitaniae sho1Delta mutant is compared with the corresponding mutants described in Saccharomyces cerevisiae, Candida albicans and Aspergillus fumigatus.
Asunto(s)
Candida/fisiología , Proteínas Fúngicas/fisiología , Proteínas de la Membrana/fisiología , Transducción de Señal , Antifúngicos/farmacología , Aspergillus fumigatus/genética , Candida/genética , Candida/crecimiento & desarrollo , Candida albicans/genética , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Eliminación de Gen , Hifa/crecimiento & desarrollo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Morfogénesis , Presión Osmótica , Estrés Oxidativo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADNRESUMEN
In the present study, we have investigated the role of SSK2, PBS2, and HOG1, encoding modules of the high-osmolarity-glycerol mitogen-activated protein kinase pathway in Candida lusitaniae. Functional analysis of mutants indicated that Ssk2p, Pbs2p, and Hog1p are involved in osmotolerance, drug sensitivity, and heavy metal tolerance but not in oxidant stress adaptation.
Asunto(s)
Candida/enzimología , Candidiasis/microbiología , Proteínas Fúngicas/metabolismo , Glicerol/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal , Candida/genética , Candida/fisiología , Proteínas Fúngicas/genética , Humanos , Proteínas Quinasas Activadas por Mitógenos/genética , Concentración OsmolarRESUMEN
We recently characterized the histidine kinase receptor genes of Candida lusitaniae. For the present study, we have further investigated the role of SSK1 and SKN7, encoding response regulators. The results of functional analysis of mutants indicated that Ssk1p is involved in osmotolerance and pseudohyphal development, whereas Skn7p appears crucial for oxidative stress adaptation.
Asunto(s)
Candida/citología , Candida/metabolismo , Proteínas Fúngicas/fisiología , Candida/efectos de los fármacos , Proteínas Fúngicas/metabolismo , Estrés Oxidativo , Transducción de SeñalRESUMEN
Inactivation of the FCY2 (cytosine permease), FCY1 (cytosine deaminase), and FUR1 (uracil phosphoribosyltransferase) genes in Candida lusitaniae produced two patterns of resistance to flucytosine. Mutant fur1 demonstrated resistance to 5-fluorouracil, whereas mutants fcy1 and fcy2 demonstrated fluconazole resistance in the presence of subinhibitory flucytosine concentrations.
Asunto(s)
Candida/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Flucitosina/farmacología , Fluorouracilo/farmacología , Alelos , Antifúngicos/farmacología , Candida/enzimología , Candida/genética , Clonación Molecular , Citosina Desaminasa/genética , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación , Pentosiltransferasa/genética , Plásmidos/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Current laboratory and radiological methods for diagnosis of invasive aspergillosis (IA) lack sensitivity and specificity. METHODS: We prospectively evaluated the diagnostic value of twice-weekly screening for circulating Aspergillus fumigatus and A. flavus DNA with a polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA). RESULTS: Among the 201 adult patients with hematological malignancies who were included in the study, 55 IA cases were diagnosed. On the basis of the analysis of 1205 serum samples from 167 patients, the sensitivity, specificity, and positive and negative predictive values of the PCR-ELISA for proven and probable IA cases were 63.6%, 89.7%, 63.6%, and 89.7%, respectively, when samples with 2 consecutive positive results were used. The use of a combination of the PCR-ELISA and a galactomannan (GM) assay increased the sensitivity to 83.3%, increased the negative predictive value to 97.6%, and decreased the specificity to 69.8%. In most patients with IA, PCR-ELISA positivity anticipated or was simultaneous with the initiation of antifungal therapy, the abnormalities found by computed tomography, the mycological/histological diagnosis, and the GM positivity. Overall, 56.3% of the patients had at least 1 positive sample, and the false single-positive rate was 44.8%. CONCLUSIONS: In addition to serial screening for GM antigenemia and radiological surveillance, PCR-ELISA may improve the rates of early diagnosis of IA and the management of patients with hematological malignancies.
Asunto(s)
Aspergilosis/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Neoplasias Hematológicas/complicaciones , Técnicas de Diagnóstico Molecular , Reacción en Cadena de la Polimerasa/métodos , Aspergilosis/complicaciones , Aspergillus flavus/genética , Aspergillus flavus/aislamiento & purificación , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , ADN de Hongos/análisis , Diagnóstico Precoz , Galactosa/análogos & derivados , Humanos , Mananos/sangre , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND & OBJECTIVES: A limited number of biotypes, T-types, and emm-types have been found to be associated with invasive isolates of group A streptococci, confirming the involvement of the M protein in virulence and its importance as an epidemiological marker. In this study, the epidemiological markers in the clinical isolates of group A streptococci were compared in invasive and non invasive isolates. METHODS: From 1998 to 2001, 141 invasive and 353 non invasive isolates in France were studied and their biotype, T-type, and emm-type were determined. RESULTS: The invasive isolates were mostly obtained from blood whereas the non invasive isolates were isolated from throat. Most of the isolates were of biotype 1. The invasive isolates were mostly of the T-type 1 associated with emm-type 1. The T-type 4 associated with emm-type 4 and the T-type 28 associated with emm-type 28 were also frequent. Invasive isolates responsible for puerperal sepsis and non invasive isolates were mostly of the T-type 28 associated with emm-type 28. INTERPRETATION & CONCLUSION: This study confirms the high prevalence of isolates of biotype 1, T-type 1, and emm-type 1 among invasive isolates of group A streptococci. The emm-type 28 associated with T-type 28 was frequently observed in non-invasive isolates. A prospective study is being conducted to update the prevalence of the different emm-types in France, which will be of importance for the development of future vaccines.
Asunto(s)
Infecciones Estreptocócicas/epidemiología , Streptococcus pyogenes/aislamiento & purificación , Secuencia de Bases , Cartilla de ADN , Francia/epidemiología , Humanos , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Infecciones Estreptocócicas/microbiologíaRESUMEN
Myiasis is the infestation of vertebrate tissues with fly larvae (Diptera). Most human cases in North America are subcutaneous forms due to Dermatobia hominis imported from Central and South America. Human cases of myiasis acquired in North America are rare and are primarily subdermal or ophthalmologic forms of infestation caused by early stages of Cuterebra larvae. We report an unusual case of tracheopulmonary myiasis, resulting from the in situ development of a mature cuterebrine larva associated with high eosinophilia. Only two other cases of tracheopulmonary cuterebrid myiasis have been reported in humans, and they are reviewed herein. Cuterebra myiasis (cuterebrosis) remains a rare and aberrant cause of tracheopulmonary disease and is a newly described cause of eosinophilia in humans.