Molecular typing of Candida albicans isolates from patients and health care workers in a neonatal intensive care unit.

AIMS: The aim of this study was to investigate the genetic relatedness between Candida albicans isolates and to assess their nosocomial origin and the likeliness of cross-transmission between health care workers (HCWs) and hospitalized neonates in a neonatal intensive care unit (NICU). METHODS: We retrospectively analysed 82 isolates obtained from 40 neonates and seven isolates from onychomycosis of the fingers of five HCWs in a Tunisian NICU by using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis with CA1 and CA2 as primers. RESULTS: In RAPD analysis, the discriminatory power (DP) of CA1 and CA2 primers was 0·86 and 0·81, respectively. A higher DP was achieved by combining patterns generated by both primers (0·92), while PFGE karyotyping exhibited the lowest DP (0·62). The RAPD-CA1/CA2 analysis revealed that 65·8% of isolates obtained from neonates derived from a limited number (6) of groups of genetically identical strains, that five temporal clusterings occurred during the study period and that three HCWs' isolates and 11 isolates obtained from six neonates were identical. CONCLUSIONS: These findings argue for the nosocomial transmission of C. albicans in our NICU and for the transfer of strains from HCWs to patients. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of relatedness between Candida species obtained from neonates and health care workers by using molecular techniques with high discriminatory power is essential for setting up specific control measures in order to reduce the incidence of nosocomial candidiasis.

Identification of pathogenic yeast species by polymerase chain reaction amplification of the RPS0 gene intron fragment.

AIMS: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. METHODS AND RESULTS: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species-specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (10(7)-10(5) cells). CONCLUSION: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.

Genes involved in the regulation of invertase production in Saccharomyces cerevisiae.

The expression of Saccharomyces cerevisiae SUC genes is exclusively regulated by catabolic repression, mediated by glucose. Genes involved in this process have been defined by means of mutants either unable to express invertase or with constitutive phenotype, although none of the genes is specific for invertase regulation. The affected genes in mutants unable to produce invertase are designated SNFX. These genes can be assorted into two groups considering either their function in regulation of gene expression or their epistatic relationships. Mutants with constitutive phenotype have been selected either by resistance to 2-deoxyglucose or by suppression of snf mutations. Among the different genes previously outlined, some of which code for transcription factors, only the MIG1 product, a "zinc finger" protein, shows a clear capacity of binding DNA in vitro. Besides the ON/OFF switch mechanism of the expression of SUC genes, some genes seem to play a role in modulating invertase expression, either hindering or stimulating transcription. A model to define the relationship between the different gene products involved in the regulation of transcription of the SUC genes is proposed.

Differential expression of SUC genes: a question of bases.

Non-coding nucleotide sequences located 5' upstream of the transcriptional start site play an essential role in gene expression as they contain binding sites for transcription and regulatory factors. The yeast SUC gene family is a useful model to study the influence that nucleotide exchanges within the promoter regions have on their expression, since (i) these genes, regulated by glucose repression, are differentially transcribed (invertase activity produced by distinct SUC genes may show variations of about 10-fold); and (ii) promoter sequences of SUC3, SUC4, SUC5 and SUC7 are more than 99% homologous, showing only six base exchanges among all of them. Comparison of these nucleotide exchanges with the expression of each SUC gene (located either on chromosomes or on multicopy and centromeric plasmids) points out that naturally occurring base exchanges as few as one nucleotide modification (G to A transition at position -497 relative to the translational start site, C to T transition at position -460 and insertion/deletion of a T at positions -590, -586 and -435) may have a strong effect on gene expression.

Cloning and characterization of the SEC18 gene from Candida albicans.

The SEC18 gene product is required for protein transport at different stages in the Saccharomyces cerevisiae secretory pathway. The homologous SEC18 gene from Candida albicans has been cloned by complementation of a sec18-1 S. cerevisiae thermosensitive mutant using a C. albicans genomic library in YRp7. Sequence analysis of the gene revealed a 2382-bp open reading frame which coded for a protein of 88,926 kDa. By an in vitro transcription-translation coupled reaction of the C. albicans SEC18 gene, a protein of approximately 85 kDa was obtained. Hydrophobicity analysis of the protein did not show any predicted signal sequence nor transmembrane anchor domain. These results and the fact that glycosylation was absent in the protein indicated that C. albicans Sec18p did not enter in the secretory pathway. The alignment of the amino acid sequence revealed that the SEC18 gene from C. albicans was homologous to the SEC18 from S. cerevisiae (50% amino acid identity) and to the gene that coded the N-ethylmaleimide-sensitive factor (NSF) protein (43% amino acid identity). Moreover, the C. albicans Sec18p also showed the putative ATP binding site present in S. cerevisiae Sec18p and in NSF.

A method for taxonomic determination of Candida albicans with DNA probes.

Determination of Candida species represents an important problem derived from the clinical implications of the species belonging to this genus. DNA probes have already been used for the epidemiology of Candida albicans, as well as for taxonomic analysis of Candida and other genera, although these probes are based on non-species-specific DNA sequences. In this work we carried out a 48-h assay, allowing the identification of C. albicans from clinical isolates, using DNA probes based on C. albicans LEU2 and URA3 genes. Another probe related to C. albicans SEC18 gene was shown not to be C. albicans specific.

Differential expression of the invertase-encoding SUC genes in Saccharomyces cerevisiae.

Invertase (INV) is encoded in Saccharomyces cerevisiae by a family of genes, comprising SUC1-SUC5 and SUC7. Production of INV is highly variable, dependent on the strain and SUC gene present in the cell. The differences in INV production derive from the structure of the genes or are dependent on the genetic background of the strain. Centromeric plasmids (based on YCp50) carrying one of the SUC genes (except SUC7) were introduced into a strain (SEY2101) lacking SUC genes. The INV produced by the transformants was dependent on the individual SUC genes, and correlated with INV mRNA levels. Plasmids in which SUC2 had been placed under control of promoters from the other SUC genes, were used to transform SEY2101 cells. The amounts of INV produced by cells carrying hybrid SUC genes were in agreement with the levels expected if the promoter controlled the expression of the SUC2 structural region. It is suggested that the differences in expression are a function of the transcription efficiency of the different SUC gene promoters, based on the divergence of 5' sequences.

Phenotype traits associated with different alleles at the RPS5 locus in Saccharomyces cerevisiae.

The RPS5 gene has been characterised through its ability to reduce invertase production by the SUC5 gene. In this paper we show that RPS5 acts by maintaining low levels of SUC5 mRNA. We also show that RPS5 acts on the SUC1 and SUC4 genes but not on SUC2 and SUC3, which are members of the SUC family. RPS5 also shows a pleiotropic effect on the amount of mitochondrial cytochromes.

Novel genetic components controlling invertase production in Saccharomyces cerevisiae.

Low levels of invertase (EC 3.2.1.26) activity were observed in most diploid strains of S. cerevisiae used in this work. There was no effect of mating type on invertase levels, and cell surface was not a limiting factor, because an increase in ploidy did not cause further decrease in specific invertase activity. Finally, some diploids showed the activity expected from the additive effects of different SUC genes, and haploid strains possessing two SUC genes expressed very variable invertase activities depending on the strain. This suggested the existence of one or more additional genes which control the levels of invertase. Genetic analysis of SUC5 strains provided evidence of the existence of a new gene, RPS5, which drastically reduced the specific invertase activity in strains possessing active SUC alleles. The recessive allele of this gene (rps5) allows expression of higher levels of invertase. We suggest that genes similar RPS5 are responsible for the low levels of invertase activity observed in diploid strains of S. cerevisiae.