Novel Polymorphic Multilocus Microsatellite Markers to Distinguish Candida tropicalis Isolates.
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ABSTRACT:
SUBMITTER: Fan X
PROVIDER: S-EPMC5098789 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundCandida tropicalis is considered to be the leading pathogen causing nosocomial fungemia and hepatosplenic fungal infections in patients with cancer, particularly those with leukemia. Microsatellite-based typing methods using sets of genetic markers have been developed and reported for population structure analysis of C. albicans, C. glabrata, and C. parapsilosis, but no studies have been published for genetic analysis of C. tropicalis. The objective of this study was to develop new microsatellite loci that have the ability to distinguish among C. tropicalis isolates.ResultsDNA sequences containing over 10 bi- or tri-nucleotide repeats were selected from the C. tropicalis genome database. Thirty PCR primers sets specific for the microsatellite loci were designed and tested using eight clinically independent isolates. According to the amplification efficiency, specificity, and observed polymorphisms, eight markers were selected for further population structure analysis and molecular typing. Sixty-five independent C. tropicalis isolates were genotyped using these 8 markers. Based on these analyses, six microsatellite loci were confirmed, although two loci were found to be with unstable flanking areas. The six polymorphic loci displayed 4-22 alleles and 7-27 genotypes. The discriminatory power of the six loci ranged from 0.70 to 0.95. Genotyping results obtained by microsatellite analysis were compared to PCR-fingerprinting and multi-locus sequence typing (MLST). The comparisons showed that microsatellite analysis and MLST had the similar discriminatory power for C. tropicalis, which were more powerful than PCR-fingerprinting.ConclusionsThis is the first attempt to develop new microsatellite loci for C. tropicalis. These newly developed markers will be a valuable resource for the differentiation of C. tropicalis isolates. More C. tropicalis isolates will need to be sequenced and analyzed in order to fully show the potential of these newly developed microsatellite markers.
| S-EPMC4247128 | biostudies-literature
Project description:Candida parapsilosis, a pathogenic yeast, is composed of three newly designated genomic species that are physiologically and morphologically indistinguishable. Nosocomial infections caused by group I C. parapsilosis are often associated with the breakdown of infection control practices and the contamination of medical devices, solutions, and indwelling catheters. Due to the low levels of nucleotide sequence variation that are observed, an investigation of the size polymorphisms in loci harboring microsatellite repeat sequences was applied for the typing of C. parapsilosis group I isolates. PCR primer sets that flank the microsatellite repeats for seven loci were designed. Following amplification by PCR, the size of each amplification product was determined automatically by capillary electrophoresis. A total of 42 C. parapsilosis group I isolates were typed by microsatellite analysis, and their profiles were compared to the hybridization profiles obtained by use of the Cp3-13 DNA probe. A high degree of discrimination (discriminatory power = 0.971) was observed by microsatellite analysis. The number of different alleles per locus ranged from 14 for locus B to 5 for locus C. Microsatellite analysis detected 30 different microsatellite genotypes, with 24 genotypes represented by a single isolate. Comparison of the genotypes obtained by microsatellite analysis and those obtained by analysis of the Cp3-13 hybridization profiles showed that they were similar, and these methods were able to identify related and unrelated isolates. Some discrepancies were observed between the methods and may be due to higher mutation rates and/or homoplasy by microsatellite markers. Identical results were observed between microsatellite analysis and Cp3-13 DNA hybridization profile analysis for C. parapsilosis isolates obtained from two patients, demonstrating the reproducibilities of the methods in vivo. Identical microsatellite profiles were observed for isolates displaying different phenotypic switching morphologies. Indistinguishable Cp3-13 DNA hybridization profiles were observed for six epidemiologically related isolates; however, only three of six primary isolates had identical microsatellite profiles. Size variation at a single locus was observed for three of six isolates obtained either after the outbreak period or from a different body site, suggesting the potential of the method to detect microevolutionary events. Interestingly, for most loci a single allele per strain was observed; in contrast, two alleles per locus were observed for some strains, and consistent with the findings for natural isolates, some isolates may be aneuploid. Due to the potential for high throughput, reproducibility, and discrimination, microsatellite analysis may provide a robust and efficient method for the genotyping of large numbers of C. parapsilosis group I isolates.
| S-EPMC1393075 | biostudies-literature
Project description:Penicillium marneffei is an emerging opportunistic dimorphic fungal pathogen that is endemic in Southeast Asia. A typing method based on the analysis of size polymorphisms in microsatellite loci was investigated. Three loci available from the GenBank database were identified to harbor microsatellites. PCR primers flanking the microsatellite repeats were designed with one primer in the set fluorescently labeled. PCR products were then sized by automated capillary electrophoresis. As expected for a haploid fungus, a single band was observed for each microsatellite locus for all isolates. Polymorphic microsatellite marker (PMM) analysis detected a total of 22 different allelic types for 35 isolates of P. marneffei with a high discriminatory power (D = 0.956). Microsatellites I, II, and III detected 14, 10, and 7 alleles, respectively. The reproducibility of length polymorphisms was confirmed by using different DNA preparations from the same isolate or by repeated runs from the same DNA preparation. PMM profiles for eight isolates passaged in vitro for 7 to 8 weeks were identical to the original culture, demonstrating short-term stability and reproducibility. PCR products were not observed for other dimorphic fungi or human DNA. Comparison of allelic frequencies in isolates obtained from China and Thailand identified distinct allele combinations, suggesting the potential geographic isolation of populations. Due to the high discriminatory power, reproducibility, and potential for high throughput, PMM analysis may provide a good typing method for epidemiologic and surveillance investigations of P. marneffei.
| S-EPMC387604 | biostudies-literature
Project description:The infection proportion of Candida orthopsilosis, a member of the C. parapsilosis complex, has increased globally in recent years, and nosocomial outbreaks have been reported in several countries. This study aimed to establish microsatellite loci-based typing method that was able to effectively distinguish among C. orthopsilosis isolates. Three reference C. orthopsilosis genome sequences were analyzed to identify repeat loci. DNA sequences containing over eight bi- or more nucleotide repeats were selected. A total of 51 loci were initially identified, and locus-specific primers were designed and tested with 20 epidemiologically unrelated isolates. Four loci with excellent reproducibility, specificity, and resolution for molecular typing purposes were identified, and the combined discriminatory power (DP, based on 20 epidemiologically unrelated isolates) of these four loci was 1.0. Reproducibility was demonstrated by consistently testing three strains each in triplicate, and stability, demonstrated by testing 10 successive passages. Then, we collected 48 C. orthopsilosis non-duplicate clinical isolates from the China Hospital Invasive Fungal Surveillance Net study to compare the DP of the microsatellite-based typing with internal transcribed spacer (ITS) and amplified fragment length polymorphism (AFLP) typing analyses, using ATCC 96139 as a reference strain. These 49 isolates were subdivided into 12 microsatellite types (COMT1-12), six AFLP types, and three ITS types, while all the isolates with the same COMT belonged to consistent AFLP and ITS type, demonstrating the high DP of our microsatellite-type method. According to our results, COMT12 was found to be the predominant type in China, and COMT5 was the second largest and responsible for causing a nosocomial outbreak. This microsatellite-type method is a valuable tool for the differentiation of C. orthopsilosis and could be vital for epidemiological studies to determine strain relatedness and monitor transmission.
| S-EPMC10662339 | biostudies-literature
Project description:Molecular typing studies have shown that the predominant form of reproduction of Candida albicans is clonal and that, in a majority of situations, persistent or recurrent infections are due to a unique strain. Characterization of distinct subpopulations and correlation with clinical features may thus be important to understanding the pathogenesis of candidiasis. In a clonal model, a unique polymorphic marker may identify populations with different biological properties. We therefore compared 48 bloodstream isolates and 48 nonbloodstream matched strains of C. albicans at the elongation factor 3-encoding gene (CEF3) polymorphic microsatellite locus of C. albicans. Sizing of the alleles was performed by automated capillary electrophoresis. A new, 137-bp allele was characterized, and seven nondescribed combinations were observed, resulting in 15 and 11 distinct CEF3 profiles in bloodstream and control strains, respectively. Genotypes 126-135, 130-136, and 131-131 accounted for 60.4% of both bloodstream and control strains. Four bloodstream isolates but no control strains displayed the 135-135 combination. None of the other genotypes was present at an increased frequency in bloodstream isolates. Bloodstream and nonbloodstream strains of C. albicans thus have a heterogeneous structure at the CEF3 locus, with three major and multiple minor allelic combinations.
| S-EPMC87636 | biostudies-literature
Project description:An important emerging application of high-throughput 454 sequencing is the isolation of molecular markers such as microsatellites from genomic DNA. However, few studies have developed microsatellites from cDNA despite the added potential for targeting candidate genes. Moreover, to develop microsatellites usually requires the evaluation of numerous primer pairs for polymorphism in the focal species. This can be time-consuming and wasteful, particularly for taxa with low genetic diversity where the majority of primers often yield monomorphic polymerase chain reaction (PCR) products. Transcriptome assemblies provide a convenient solution, functional annotation of transcripts allowing markers to be targeted towards candidate genes, while high sequence coverage in principle permits the assessment of variability in silico. Consequently, we evaluated fifty primer pairs designed to amplify microsatellites, primarily residing within transcripts related to immunity and growth, identified from an Antarctic fur seal (Arctocephalus gazella) transcriptome assembly. In silico visualization was used to classify each microsatellite as being either polymorphic or monomorphic and to quantify the number of distinct length variants, each taken to represent a different allele. The majority of loci (n = 36, 76.0%) yielded interpretable PCR products, 23 of which were polymorphic in a sample of 24 fur seal individuals. Loci that appeared variable in silico were significantly more likely to yield polymorphic PCR products, even after controlling for microsatellite length measured in silico. We also found a significant positive relationship between inferred and observed allele number. This study not only demonstrates the feasibility of generating modest panels of microsatellites targeted towards specific classes of gene, but also suggests that in silico microsatellite variability may provide a useful proxy for PCR product polymorphism.
| S-EPMC3154332 | biostudies-literature
Project description:Polymorphic Microsatellite Markers for the Tetrapolar Anther-Smut Fungus Microbotryum saponariae Based on Genome Sequencing.
| PRJEB11435 | ENA
Project description:A system is described for typing isolates of the pathogenic fungus Candida tropicalis, based on sequence polymorphisms in fragments of six genes: ICL1, MDR1, SAPT2, SAPT4, XYR1, and ZWF1a. The system differentiated 87 diploid sequence types (DSTs) among a total of 106 isolates tested or 80 DSTs among 88 isolates from unique sources. Replicate isolates from the same source clustered together with high statistical similarity, with the exception of one isolate. However, a clade of very closely related isolates included replicate isolates from three different patients, as well as single isolates from eight other patients. This clade, provisionally designated clade 1, was one of three clusters of isolates with high statistical similarity. Five of six isolates in one cluster that may acquire clade status were resistant to flucytosine. This study adds C. tropicalis to Candida albicans and Candida glabrata as Candida species for which a multilocus sequence typing (MLST) system has been set up. The C. tropicalis MLST database can be accessed at http://pubmlst.org/ctropicalis/.
| S-EPMC1287820 | biostudies-literature
Project description:BackgroundGlobal reports have highlighted the increasing prevalence of Candida tropicalis infections as well as organism(') s drug resistance. This study aimed at identifying azole resistance markers in clinical isolates of C. tropicalis, which will be a great resource for developing new drugs.MethodsTwo susceptible and resistant isolates of C. tropicalis were recovered from an epidemiological investigation of candidiasis in immunocompromised patients. C. tropicalis ATCC 750 was used as reference strain. Antifungal susceptibility to fluconazole and itraconazole was determined using Clinical and Laboratory Standards Institute (CLSI) method. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology and real-time reverse-transcriptase (RT) PCR were used for identification of potential genes involved in azole resistance of C. tropicalis clinical isolates.ResultsFive genes encoding the following enzymes were identified as superoxide dismutase (SOD) implicated in antioxidant defense, ornithine aminotransferase (OAT), acetyl ornithine aminotransferase (ACOAT), adenosylmethionine-8-amino-7-oxononanoate aminotransferase (DAPA AT), and 4-aminobutyrate aminotransferase (ABAT)-belonging to pyridoxal phosphate (PLP) dependent enzymes and acting in an important physiological role in many fungal-cell cycles. Real-time RT-PCR confirmed mRNA level of the aforementioned genes.ConclusionOur findings showed that factors such as PLP-dependent enzymes and SOD might be implicated in drug resistance in C. tropicalis clinical isolate. Therefore, further studies are required to explore the accurate biological functions of the mentioned genes that would be helpful for effective drug development.
| S-EPMC6807149 | biostudies-literature
Project description:BackgroundCandida tropicalis is a human pathogenic yeast frequently isolated in Latin America and Asian-Pacific regions, although recent studies showed that it is also becoming increasingly widespread throughout several African and south-European countries. Nevertheless, relatively little is known about its global patterns of genetic variation as most of existing multilocus sequence typing (MLST) data come from Asia and there are no genotyped African isolates.ObjectivesWe report detailed genotyping data from a large set of C. tropicalis isolates recovered from different clinical sources in Italy, Egypt and Cameroon in order to expand the allele/genotype library of MLST database (https://pubmlst.org/ctropicalis), and to explore the genetic diversity in this species.MethodsA total of 103 C. tropicalis isolates were genotyped using the MLST scheme developed for this species. All isolates were also tested for in vitro susceptibility to various antifungals to assess whether certain genotypes were associated with drug-resistance.Results and conclusionsA total of 104 different alleles were detected across the MLST-loci investigated. The allelic diversity found at these loci resulted in 51 unique MLST genotypes of which 36 (70.6%) were novel. Global optimal eBURST analysis identified 18 clonal complexes (CCs) and confirm the existence of a specific Italian-cluster (CC36). Three CCs were also statistically associated with fluconazole resistance, which was elevated in Cameroon and Egypt. Our data show high genetic diversity in our isolates suggesting that the global population structure of C. tropicalis is still poorly understood. Moreover, its clinical impact in Italy, Egypt and Cameroon appears to be relevant and should be carefully considered.
| S-EPMC9796097 | biostudies-literature