RESUMEN

BACKGROUND: Panton-Valentine leucocidin-positive meticillin-resistant Staphylococcus aureus (PVL-MRSA) has become a globally common cause of community-acquired infections. AIM: We report an outbreak of PVL-MRSA in a regional neonatal unit in the UK involving three babies and three staff members. METHODS: Quinolone susceptibility was helpful in identifying potential PVL-MRSA but toxin gene profiling and sequence-based typing were required to distinguish between two PVL-MRSA strains present in the unit. FINDINGS: All three symptomatic babies and two staff carriers, one of whom was symptomatic, were found to be carrying the South West Pacific (SWP) clone of PVL-MRSA (ST30). One of the staff carriers had recently visited the Philippines and was thought to be the source of the outbreak. Control was established using standard infection control procedures but one baby with relapsing MRSA colonization has required more than 100 days in isolation. CONCLUSION: This is the first reported neonatal outbreak associated with the SWP clone in the UK. Our study highlights the potential risk of further introductions of this organism by healthcare staff or patients epidemiologically linked with the Philippines.

Asunto(s)

Toxinas Bacterianas/genética , Brotes de Enfermedades , Exotoxinas/genética , Leucocidinas/genética , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Tipificación Molecular , Infecciones Estafilocócicas/epidemiología , Adulto , Antibacterianos/farmacología , Portador Sano/epidemiología , Portador Sano/microbiología , Portador Sano/transmisión , Transmisión de Enfermedad Infecciosa/prevención & control , Femenino , Personal de Salud , Humanos , Lactante , Recién Nacido , Control de Infecciones/métodos , Unidades de Cuidado Intensivo Neonatal , Masculino , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Filipinas , Quinolonas/farmacología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/transmisión , Viaje , Reino Unido/epidemiología , Factores de Virulencia/genética

RESUMEN

We present a case of a confirmed Candida albicans endogenous endophthalmitis in a 35-year-old diabetic white female patient with a long standing history of severe chronic vaginal C. albicans infection. The patient had recently undergone ureteric stenting and received intravenous broad-spectrum antibiotics for renal stones complicated by urinary sepsis. Pan-fungal polymerase chain reaction (PCR) analysis of vitreous aspirate confirmed the presence of C. albicans. Samples showed no microbial growth.

RESUMEN

In response to changes in ambient pH the opportunistic pathogen Candida albicans differentially expresses a number of genes. The response to pH affects morphological differentiation and virulence. The pathway controlling the pH response terminates in the zinc-finger containing transcription factor encoded by RIM101/PRR2. By analogy to the pH response pathway of Aspergillus nidulans, PRR1 of C. albicans encodes a protein that is presumably required to convert Rim101p from an inactive to an active form by proteolytic removal of a C-terminal peptide. A prr1Delta mutant is compromised in its ability to differentiate into the filamentous form. Spontaneous phenotypic revertants of a prr1Delta mutant were selected by their ability to form filamentous colonies. These mutants were also found to be defective in pH-dependent gene expression. Each of the eight mutants examined contained a heterozygous dominant mutation at the RIM101 locus. This was demonstrated genetically in all of the mutants, and directly by sequence determination of both alleles in two of the mutants. The mutant alleles conferred the ability to filament to a prr1Delta mutant, thus demonstrating that they were directly responsible for suppressing the filamentation defect. Seven of the mutant alleles contained a 1-bp substitution and one contained two substitutions at adjacent positions. The mutations were clustered within a 90-bp region near the 3'-end of the gene. In all cases the mutation generated a nonsense codon that resulted in premature termination of Rim101p; the mutant proteins were truncated by 75-104 amino acids. The results define a critical region in the C-terminal region of Rim101p and are consistent with the proposed proteolytic activation of Rim101p.

Asunto(s)

Candida albicans/citología , Candida albicans/genética , Codón sin Sentido , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Factores de Transcripción/genética , Alelos , Diferenciación Celular/genética , Concentración de Iones de Hidrógeno , Fenotipo , Transducción de Señal , Supresión Genética

RESUMEN

In this work we cloned CdPHR1 and CdPHR2 from the human fungal pathogen Candida dubliniensis. The two genes are homologues to the pH-regulated genes PHR1 and PHR2 from Candida albicans. The pH-dependent pattern of expression of CdPHR1 and CdPHR2 was conserved in C. dubliniensis. CdPHR1 could be shown to be functionally equivalent to PHR1. The pH-regulated mode of expression was maintained when CdPHR1 was integrated in C. albicans. This indicates a fundamentally similar mode of expressional regulation in the two species. CdPHR1 was furthermore capable of reversing the aberrant phenotype of a Saccharomyces cerevisiae GAS1 deletion mutant. In this species, however, expression of CdPHR1 was no longer under control of the external pH. Expression of CdPHR1 was not detected when it was introduced into Aspergillus nidulans. In conclusion, C. dubliniensis and C. albicans respond to changes in the environmental pH with a change in cell shape and differential gene expression.

Asunto(s)

Apoenzimas/genética , Candida albicans/genética , Candida/genética , Desoxirribodipirimidina Fotoliasa/genética , Proteínas Fúngicas/genética , Glicoproteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Apoenzimas/aislamiento & purificación , Apoenzimas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Secuencia de Bases , Northern Blotting , Southern Blotting , Candida/metabolismo , Candida albicans/metabolismo , Clonación Molecular , Desoxirribodipirimidina Fotoliasa/aislamiento & purificación , Desoxirribodipirimidina Fotoliasa/metabolismo , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Concentración de Iones de Hidrógeno , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Fenotipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN

RESUMEN

Morphological development of the fungal pathogen Candida albicans is profoundly affected by ambient pH. Acidic pH restricts growth to the yeast form, whereas neutral pH permits development of the filamentous form. Superimposed on the pH restriction is a temperature requirement of approximately 37 degrees C for filamentation. The role of pH in development was investigated by selecting revertants of phr2Delta mutants that had gained the ability to grow at acid pH. The extragenic suppressors in two independent revertants were identified as nonsense mutations in the pH response regulator RIM101 (PRR2) that resulted in a carboxy-terminal truncation of the open reading frame. These dominant active alleles conferred the ability to filament at acidic pH, to express PHR1, an alkaline-expressed gene, at acidic pH, and to repress the acid-expressed gene PHR2. It was also observed that both the wild-type and mutant alleles could act as multicopy suppressors of the temperature restriction on filamentation, allowing extensive filamentation at 29 degrees C. The ability of the activated alleles to promote filamentation was dependent upon the developmental regulator EFG1. The results suggest that RIM101 is responsible for the pH dependence of hyphal development.

Asunto(s)

Candida albicans/fisiología , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Genes Dominantes , Glicoproteínas de Membrana , Factores de Transcripción , Apoenzimas/genética , Apoenzimas/metabolismo , División Celular/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Proteínas Fúngicas/metabolismo , Dosificación de Gen , Regulación Fúngica de la Expresión Génica , Heterocigoto , Concentración de Iones de Hidrógeno , Mutación , Supresión Genética

RESUMEN

There is an increasing interest in non-albicans Candida species because of the increasing number of fungal infections they cause. Most of these infections can be found in immunocompromised individuals, especially in those infected with human immunodeficiency virus (HIV). Candida dubliniensis is a recently identified yeast, mostly isolated in HIV-positive individuals with oral candidiasis. Candida dubliniensis is a germ tube- and chlamydospore-form yeast. Thus, it shares diagnostic characteristics with Candida albicans. Probably, Candida dubliniensis has been present in the community for a long time and has been misidentified as Candida albicans. Significant phenotypic characteristics of Candida dubliniensis (difference in the carbohydrate assimilation profile, difference in colony color on CHROMagar Candida, and positive tetrazolium test, etc.) have been found, but none of them seem to be sufficient alone for the definitive identification of the species. Recently, PCR tests were developed to discriminate Candida albicans from Candida dubliniensis. However, these prove difficult in the context of routine mycological diagnostics. Moreover, an increased resistance to antifungal drugs has been described. This shows the importance of identification of Candida dubliniensis. To elucidate the current insight into Candida dubliniensis, the phenotypic and genotypic characteristics as well as the prevalence and the antifungal drug susceptibilities of this species are discussed from a clinical standpoint.

Asunto(s)

Candida/clasificación , Candidiasis Bucal/microbiología , Infecciones por VIH/microbiología , Antifúngicos/farmacología , Candida/patogenicidad , Candidiasis Bucal/complicaciones , Candidiasis Bucal/epidemiología , Farmacorresistencia Microbiana , Fluconazol/farmacología , Genotipo , Infecciones por VIH/complicaciones , Humanos , Fenotipo , Prevalencia

RESUMEN

The functionality of beta-galactosidase encoded by the E. coli lacZ gene as a reporter of gene expression in C. glabrata was investigated. C. glabrata/E. coli shuttle vectors were constructed, containing both a C. glabrata CEN-ARS cassette, to allow regular segregation and episomal replication of the plasmids, and the lacZ coding sequence of E. coli. The functionality of beta-galactosidase in C. glabrata was verified by inserting the promoter and the 5' coding region of the HIS3 gene from C. glabrata directionally upstream of the lacZ gene. By fusing the promoter of the copper-controlled MTII gene to the lacZ reporter, we showed that beta-galactosidase activity can be differentially induced in C. glabrata. beta-galactosidase reporter activities were detected qualitatively by an indirect filter assay and quantitatively from permeabilized cells.

Asunto(s)

Candida/genética , Vectores Genéticos/genética , Operón Lac/genética , beta-Galactosidasa/genética , Centrómero/genética , Sulfato de Cobre/farmacología , ADN de Hongos/genética , ADN Recombinante , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Proteínas Fúngicas/genética , Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Hidroliasas/genética , Metalotioneína/genética , Plásmidos , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Replicón/genética , beta-Galactosidasa/efectos de los fármacos , beta-Galactosidasa/metabolismo

RESUMEN

Candida dubliniensis is an emerging yeast pathogen generally misclassified as Candida albicans by standard diagnostic procedures. This study examined the efficiency of molecular identification, based on a discriminative PCR test, in a prospective study on the prevalence of C. dubliniensis among 103 oropharyngeal isolates from HIV-infected individuals or transplant recipients, and 30 vaginal isolates. All of the isolates had been classified as C. albicans by standard laboratory procedures. The PCR was evaluated in a blinded fashion against classification achieved by sequencing rDNA. Sequencing results corresponded 100% to the results of the discriminative PCR, indicating the validity of this rapid test. Twenty-one C. dubliniensis isolates were identified, all of them from HIV-infected individuals (prevalence 30%). The internal transcribed spacer regions of the C. dubliniensis isolates were sequenced. Phenotypic features of C. dubliniensis, namely abundant chlamydospore formation, atypical color on CHROMagar, growth defect at 45 degrees C, and colony morphology on Staib agar, were evaluated in a blinded fashion with respect to their discriminative potential, facilitating the design of further epidemiological studies. Carbohydrate assimilation patterns were determined for C. dubliniensis with a novel automated system showing that, in contrast to previous reports, C. dubliniensis is able to utilize D-xylose and trehalose. In evaluating these tests we present a rational approach to identification of the new species and characterization of C. dubliniensis isolates.

Asunto(s)

Candida/clasificación , Candida/genética , Candidiasis/microbiología , ADN de Hongos/genética , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Candida/fisiología , Metabolismo de los Hidratos de Carbono , ADN de Hongos/análisis , ADN Espaciador Ribosómico/genética , Genes de ARNr , Humanos , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Ribosómico/genética , Análisis de Secuencia de ADN , Temperatura

RESUMEN

A Taenia solium metacestode cDNA expression library in the lambda ZAPII vector was screened with pooled sera from patients with neurocysticercosis. Sixty primary clones were identified and shown to belong to two classes. The clones NC-3 and NC-9 did not reveal any significant homologies to sequences deposited in the databases and were further characterized. Both recombinant antigens were expressed as glutathione S-transferase fusion proteins and applied for serological diagnosis of human cysticercosis. An enzyme-linked immunosorbent assay was established and evaluated with 27 serum samples of La Réunion and Madagascar patients with cysticercosis. Diagnosis in these patients was established with radiological and serological procedures. For antigen NC-3 a sensitivity of 96.3% and a specificity of 91.5% for the serodiagnosis were achieved. In contrast, the sensitivity of antigen NC-9 was only 33.3%.

Asunto(s)

Cisticercosis/diagnóstico , Cisticercosis/inmunología , Taenia/inmunología , Animales , Antígenos Helmínticos , Células Clonales/inmunología , ADN Complementario/clasificación , Ensayo de Inmunoadsorción Enzimática , Biblioteca de Genes , Humanos , Proteínas Recombinantes , Pruebas Serológicas , Taenia/genética

RESUMEN

The development of a satisfactory means to reliably distinguish between the two closely related species Candida albicans and Candida dubliniensis in the clinical mycology laboratory has proved difficult because these two species are phenotypically so similar. In this study, we have detected homologues of the pH-regulated C. albicans PHR1 and PHR2 genes in C. dubliniensis. Restriction fragment length polymorphism analysis suggests that there are significant sequence differences between the genes of the two species. In order to exploit this apparent difference, oligonucleotide primers based on the coding sequence of the C. albicans PHR1 structural gene were designed and used in PCR experiments. Use of these primers with C. albicans template DNA from 17 strains yielded a predicted 1.6-kb product, while C. dubliniensis template DNA from 19 strains yielded no product. We therefore propose that PCR using these primers is a rapid and reliable means of distinguishing the two germ tube- and chlamydospore-producing species C. albicans and C. dubliniensis.

Asunto(s)

Apoenzimas/genética , Candida albicans/aislamiento & purificación , Candida/aislamiento & purificación , Desoxirribodipirimidina Fotoliasa/genética , Proteínas Fúngicas , Genes Fúngicos , Glicoproteínas de Membrana , Reacción en Cadena de la Polimerasa , Candida/genética , Candida albicans/genética , Cartilla de ADN , Concentración de Iones de Hidrógeno

RESUMEN

Little is known of the biological attributes conferring pathogenicity on the opportunistic fungal pathogen Candida albicans. Infection by this pathogen, as for bacterial pathogens, may rely upon environmental signals within the host niche to regulate the expression of virulence determinants. To determine if C. albicans responds to the pH of the host niche, we tested the virulence of strains with mutations in either of two pH-regulated genes, PHR1 and PHR2. In vitro, PHR1 is expressed when the ambient pH is at 5.5 or higher and deletion of the gene results in growth and morphological defects at neutral to alkaline pHs. Conversely, PHR2 is expressed at an ambient pH below 5.5, and the growth and morphology of the null mutant is compromised below this pH. A PHR1 null mutant was avirulent in a mouse model of systemic infection but uncompromised in its ability to cause vaginal infection in rats. Since systemic pH is near neutrality and vaginal pH is around 4.5, the virulence phenotype paralleled the pH dependence of the in vitro phenotypes. The virulence phenotype of a PHR2 null mutant was the inverse. The mutant was virulent in a systemic-infection model but avirulent in a vaginal-infection model. Heterozygous mutants exhibited partial reductions in their pathogenic potential, suggesting a gene dosage effect. Unexpectedly, deletion of PHR2 did not prevent hyphal development in vaginal tissue, suggesting that it is not essential for hyphal development in this host niche. The results suggest that the pH of the infection site regulates the expression of genes essential to survival within that niche. This implies that the study of environmentally regulated genes may provide a rationale for understanding the pathobiology of C. albicans.

Asunto(s)

Candida albicans/patogenicidad , Desoxirribodipirimidina Fotoliasa/fisiología , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Glicoproteínas de Membrana , Animales , Apoenzimas/análisis , Candida albicans/genética , Candidiasis/etiología , Candidiasis/patología , Desoxirribodipirimidina Fotoliasa/análisis , Femenino , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratas , Ratas Wistar , Vagina/patología , Virulencia

RESUMEN

Deletion of PHR1, a pH-regulated gene of Candida albicans, results in pH-conditional defects in growth, morphogenesis, and virulence evident at neutral to alkaline pH but absent at acidic pH. Consequently, we searched for a functional homolog of PHR1 active at low pH. This resulted in the isolation of a second pH-regulated gene, designated PHR2. The expression of PHR2 was inversely related to that of PHR1, being repressed at pH values above 6 and progressively induced at more acidic pH values. The predicted amino acid sequence of the PHR2 protein, Phr2p, was 54% identical to that of Phr1p. A PHR2 null mutant exhibited pH-conditional defects in growth and morphogenesis analogous to those of PHR1 mutants but manifest at acid rather than alkaline pH values. Engineered expression of PHR1 at acid pH in a PHR2 mutant strain and PHR2 at alkaline pH in a PHR1 mutant strain complemented the defects in the opposing mutant. Deletion of both PHR1 and PHR2 resulted in a strain with pH-independent, constitutive growth and morphological defects. These results indicate that PHR1 and PHR2 represent a novel pH-balanced system of functional homologs required for C. albicans to adapt to environments of diverse pH.

Asunto(s)

Apoenzimas/genética , Candida albicans/genética , Desoxirribodipirimidina Fotoliasa/genética , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica/fisiología , Glicoproteínas de Membrana , Secuencia de Aminoácidos , Apoenzimas/análisis , Candida albicans/citología , Clonación Molecular , Desoxirribodipirimidina Fotoliasa/análisis , Desoxirribodipirimidina Fotoliasa/fisiología , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Fenotipo , ARN de Hongos/análisis , ARN Mensajero/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido