Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

A database of microsatellite genotypes for <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A system for genotyping Saccharomyces cerevisiae is described based on a multiplex of ten microsatellite loci and the MAT locus. A database of genotypes has been developed for 246 yeast strains, including a large set of commercial wine yeasts, as well as 35 sequenced natural isolates currently being sequenced. The latter allow us, for the first time, to make direct comparisons of the relationship between DNA sequence data and microsatellite-based genotypes. The genotyping system provides a rapid and valuable system for strain identification as well as studying population genetics of S. cerevisiae.     

The genetic structure of fermentative vineyard-associated <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> populations revealed by microsatellite analysis

From the analysis of six polymorphic microsatellite loci performed in 361 Saccharomyces cerevisiae isolates, 93 alleles were identified, 52 of them being described for the first time. All these isolates have a distinct mtDNA RFLP pattern. They are derived from a pool of 1620 isolates obtained from spontaneous fermentations of grapes collected in three vineyards of the Vinho Verde Region in Portugal, during the 2001–2003 harvest seasons. For all loci analyzed, observed heterozygosity was 3–4 times lower than the expected value supposing a Hardy–Weinberg equilibrium (random mating and no evolutionary mechanisms acting), indicating a clonal structure and strong populational substructuring. Genetic differences among S. cerevisiae populations were apparent mainly from gradations in allele frequencies rather than from distinctive “diagnostic” genotypes, and the accumulation of small allele-frequency differences across six loci allowed the identification of population structures. Genetic differentiation in the same vineyard in consecutive years was of the same order of magnitude as the differences verified among the different vineyards. Correlation of genetic differentiation with the distance between sampling points within a vineyard suggested a pattern of isolation-by-distance, where genetic divergence in a vineyard increased with size. The continuous use of commercial yeasts has a limited influence on the autochthonous fermentative yeast population collected from grapes and may just slightly change populational structures of strains isolated from sites very close to the winery where they have been used. The present work is the first large-scale approach using microsatellite typing allowing a very fine resolution of indigenous S. cerevisiae populations isolated from vineyards.     

Population Structure of <Emphasis Type="Italic">Candida parapsilosis</Emphasis>: No Genetic Difference Between French and Uruguayan Isolates Using Microsatellite Length Polymorphism

Candida parapsilosis is a human commensal yeast, frequently involved in infection worldwide and especially in neonates. It is the second species responsible for bloodstream infections in Uruguay and the third species in France. We were interested in knowing whether the population structure of isolates responsible for candidemia in France and in Uruguay was different. Genotyping methods based on microsatellite length polymorphism (MLP) have been described and are especially used for investigation of local outbreaks. We therefore determined the genotypes of 159 C. parapsilosis isolates recovered from 122 patients (84 French patients from 43 hospitals and 38 Uruguayan patients from 10 hospitals) using three microsatellites markers previously described. Our results confirmed that C. parapsilosis population has a high genetic diversity, clonal inheritance and that majority of patients were infected by a single isolate. But we described recurrent infections due to related or unrelated genotypes resulting from isolates harboring loss or gain of heterozygosity. We also described three cases of coinfections due to unrelated genotypes. We did not uncover geographic specificity but observed two linked genotypes that seem to be associated with voriconazole resistance. Finally, among eight isolates involved in grouped cases, the genotypes were similar in six cases supporting the hypothesis of inter-patient transmission. These results confirmed the usefulness of performing MLP genotyping analysis for grouped cases of C. parapsilosis isolates in order to reinforce preventive hygiene measures.     

Detection of Malaysian methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA) clinical isolates using simplex and duplex real-time PCR

The aim of this study was to develop a methicillin-resistant Staphylococcus aureus (MRSA) detection method based on the melting temperature analysis profiling of S. aureus clinical isolates from three different hospitals in Malaysia. Simplex and duplex real-time PCR assay was used for the simultaneous detection of nuc (species-specific) and mecA (methicillin-resistance) genes in a single SYBR Green I real-time PCR tube assay. Evaluations were based on the melting temperature (T _m) analysis of the amplicons using 23 S. aureus clinical isolates including three ATCC S. aureus standard strains. Real-time PCR amplification products with melting peaks at 78.39 ± 0.4°C and 74.41 ± 0.6°C were detected for nuc and mecA genes, respectively. Each real-time PCR assay was completed within two hours. This rapid genotypic method is useful for the detection of resistant determinant (mecA) and identification of S. aureus (nuc) clinical isolates, thus benefiting patient therapy in hospitals.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

The gene <Emphasis Type="Italic">bap</Emphasis>, involved in biofilm production,is present in <Emphasis Type="Italic">Staphylococcus</Emphasis> spp. strains from nosocomial infections

This study analyzed ten strains of coagulase-negative staphylococci (CNS) involved in nosocomial infections in three Brazilian hospitals. Their antibiotic susceptibility profile showed that most strains exhibited multiple antibiotic resistance and possessed the mecA gene. The ability of these strains to adhere to polystyrene microtiter plates was also tested and nine of them proved to be biofilm producers at least in one of the three conditions tested: growth in TSB, in TSB supplemented with NaCl, or in TSB supplemented with glucose. The presence of the bap gene, which codes for the biofilm-associated protein (Bap), was investigated in all ten strains by PCR. AU strains were bop-positive and DNA sequencing experiments confirmed that the fragments amplified were indeed part of a bap gene. The presence of the icaA gene, one of the genes involved in polysaccharide intercellular adhesin (PIA) formation, was also detected by PCR in eight of the ten strains tested. The two icaA-negative strains were either weak biofilm producer or no biofilm producer, although they were bop-positive. To our knowledge, this is the first report demonstrating the presence of the bap gene in nosocomial isolates of CNS, being also the first report on the presence of this gene in Staphylococcus haemolyticus and S. cohnii. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Polymorphic microsatellite loci for population studies of the razor clam, <Emphasis Type="Italic">Sinonovacula constricta</Emphasis>

The razor clam (Sinonovacula constricta) is an important aquacultured bivalve in China. The natural populations of this species are decreasing quickly. To facilitate studies on genetic diversity and population structure of wild populations, microsatellites were isolated from a CA enriched genomic library. Eight microsatellite loci were polymorphic in 30 individuals from Chongming in Shanghai, China. The number of alleles per polymorphic locus varied from 6 to 13 and the values of observed heterozygosity and expected heterozygosity ranged from 0.350 to 1.000 and from 0.602 to 0.902, respectively. These microsatellites are being used in studying population differentiation and genetic diversity for effective conservation and management genetic resources of S. constricta.     

Development and characterization of microsatellite loci in Chinese medicinal plant Akebia trifoliate ssp. australis and cross-species amplification in closely related taxa

Eleven polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of Akebia trifoliate ssp. australis. The number of allele per locus ranged from 3 to 14. The observed heterozygosity and expected heterozygosity at population level were 0.196–1.000 and 0.522–0.902, respectively. In addition, this set of microsatellites produced robust cross-species amplification in other two related taxa, suggesting these microsatellite markers should provide a useful tool for genetic and conservation studies of the Akebia species.     

Early,non-destructive selection of microspore-derived embryo genotypes in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) by molecular markers and oil quality analysis

In oilseed rape (Brassica napus L.) breeding, microspore culture is frequently applied for the immediate regeneration of homozygous doubled haploid (DH) plants. From the regenerated microspore-derived embryos (MDEs), usually only a smaller subset of around 200 are used for plantlet regeneration and cultivation in the greenhouse until seed harvest, without there being any knowledge about their quality traits and agronomic performance. The random selection of MDEs implies that valuable rare recombinant genotypes may be discarded at an early stage of in vitro culture. We report here on the development of a simple protocol for simultaneously extracting lipids (for oil quality analysis) and the isolation of DNA (for marker-assisted selection) from single cotyledons dissected from MDEs under aseptic conditions, thus keeping the rest of the embryo in vitro for plantlet regeneration. Neither the fatty acid extraction nor the transmethylation with sodium methylate at high pH interfered with subsequent DNA isolation. The feasibility of the protocol was tested using MDEs from a cross segregating for two linked transgenes, fae1 and plsC, affecting the fatty acid composition. Multiplex PCR was performed with specific PCR primers for the plsC gene and with locus-specific primers for a resident single copy fad2 gene. The amplification of the fad2 gene provided a control for the presence of DNA in sufficient quantity and quality, whereas the amplification of the plsC gene showed a 1:1 segregation expected for a single copy transgene in a segregating DH population. The early identification of the 50% MDE genotypes carrying the desired transgenes, along with a high expression of the trait, allows their early selection for plantlet regeneration.     

Microsatellite DNA loci from the typical halophyte <Emphasis Type="Italic">Thellungiella salsuginea</Emphasis> (Brassicaceae)

Thellungiella salsuginea (Brassiaceae) is a typical halophyte which can tolerate extreme cold, drought, and salinity. In order to understand the adaptive evolution of this species in the arid habitats, it is important to know its genetic structure. In this study, 17 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of this species. We further assessed the polymorphisms of each locus in 18 individuals from nine geographically distant populations. The number of alleles per locus ranged from six to fourteen. The observed and expected heterozygosity ranged from 0.17 to 0.28 and 0.32 to 0.45, respectively. These markers have been crossly checked in another congeneric species, T. halophila. These microsatellite markers will be useful for investigating population genetics and adaptive evolution of this species and morphological divergence between and it and the closely related species.     

Characterization of 14 novel microsatellite loci in the endangered <Emphasis Type="Italic">Liriodendron chinense</Emphasis> (Magnoliaceae) and cross-species amplification in closely related taxa

Fourteen polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of Liriodendron chinense (Magnoliaceae). The average allele number of these microsatellites was 5.2 per locus, ranging from three to eight. The observed and expected heterozygosity at population level were 0.07–1.00 and 0.10–0.83, respectively. These sets of microsatellites will be useful for studies of population genetic structure of L. chinense and L. tulipifera as well as to estimate fine-scale gene flow rates.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

Isolation and characterization of microsatellite DNA primers for <Emphasis Type="Italic">Sinadoxa corydalifolia</Emphasis> (Adoxaceae)

Sinadoxa corydalifolia is the only species of Sinadoxa (Adoxaceae) with the aberrant morphology. This species has become extremely endangered in the Qinghai-Tibetan Plateau. To provide a population-level genetic profile for investigation and conservation of genetic diversity of this species, we developed 10 new microsatellite loci for this species by the combining biotin capture method. About 31 microsatellites were screened from the library, 10 of the screened microsatellites are polymorphic. The number of alleles per locus in 18 individuals ranged from 3 to 11, expected heterozygosity and observed heterozygosity ranged from 0.3071 to 0.6243 and from 0.1675 to 0.4357, respectively. We further performed cross-priming tests of these primers in another species of the Adoxaceae: Adoxa moschatellina and found 9 of 10 successfully amplified the targeted sequences. These newly developed loci provide a useful tool to investigate the genetic diversity and design the conversation measures of S. corydalifolia and study the genetic divergence and the initial speciation pattern between it and the related species in the Adoxaceae.     

Use of CHROMagar in the Differentiation of Common Species of <Emphasis Type="Italic">Candida</Emphasis>

CHROMagar has been reported to be useful for the rapid and accurate identification of Candida species. We tested 135 isolates of Candida species isolated from oropharyngeal candidiasis in HIV patients and found that it was useful in the presumptive identification of Candida albicans and Candida krusei. Occasional strains of C. tropicalis produced colonies with a greenish tinge making it difficult to differentiate from C. albicans.     

An efficient method for screening faecal DNA genotypes and detecting new individuals and hybrids in the red wolf (<Emphasis Type="Italic">Canis rufus</Emphasis>) experimental population area

Previously, sequencing of mitochondrial DNA (mtDNA) from non-invasively collected faecal material (scat) has been used to help manage hybridization in the wild red wolf (Canis rufus) population. This method is limited by the maternal inheritance of mtDNA and the inability to obtain individual identification. Here, we optimize the use of nuclear DNA microsatellite markers on red wolf scat DNA to distinguish between individuals and detect hybrids. We develop a data filtering method in which scat genotypes are compared to known blood genotypes to reduce the number of PCR amplifications needed. We apply our data filtering method and the more conservative maximum likelihood ratio method (MLR) of Miller et al. (2002 Genetics 160:357–366) to a scat dataset previously screened for hybrids by sequencing of mtDNA. Using seven microsatellite loci, we obtained genotypes for 105 scats, which were matched to 17 individuals. The PCR amplification success rate was 50% and genotyping error rates ranged from 6.6% to 52.1% per locus. Our data filtering method produced comparable results to the MLR method, and decreased the time and cost of analysis by 25%. Analysis of this dataset using our data filtering method verified that no hybrid individuals were present in the Alligator River National Wildlife Refuge, North Carolina in 2000. Our results demonstrate that nuclear DNA microsatellite analysis of red wolf scats provides an efficient and accurate approach to screen for new individuals and hybrids.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Isolation and characterization of first microsatellite markers for the noble crayfish, <Emphasis Type="Italic">Astacus astacus</Emphasis>

Noble crayfish (Astacus astacus) is listed as vulnerable by the IUCN Red List of Threatened Species in Europe. However, very little is known about genetic diversity and structuring of noble crayfish populations, mainly because of the lack of informative genetic markers. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4,000 recombinant clones. Eight loci revealed polymorphisms in a panel of 172 individuals from seven populations in Northern Europe. Number of alleles per locus ranged from two to 10 (average 4.4) and heterozygosity levels among populations varied from 0 to 0.80 for H _o and from 0 to 0.72 for H _e.     

Development and characterization of microsatellite loci for lotus (Nelumbo nucifera)

This paper reports the development of microsatellite primers for Nelumbo nucifera Gaerten. By screening genomic libraries enriched with 10 kinds of probes, Seventeen polymorphic loci were isolated and primers were designed. Polymorphism of these 17 loci was assessed in 24 individuals. All the 17 loci are polymorphic and the number of alleles ranged from two to seven. Observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.9176 and from 0.2837 to 0.7917 respectively. These microsatellite loci should be useful for studying the genetic diversity of N. nucifera.