Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Jan 29, 2005
After the process of DNA barcoding has become well advanced in a group of organisms, as it has in... more After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base pairs long), uniquely distinctive oligonucleotide 'microcodes' of less than 25 bp can be found that allow rapid identification of circa 100-200 species on various array-like platforms. Microarrays can in principle fulfill the function of microcode-based species identification but, because of their high cost and low level of reusability, they tend to be less cost-effective. Two alternative platforms in current use in fungal identification are reusable nylon-based macroarrays and the Luminex system of specific, colour-coded DNA detection beads analysed by means of a flow cytome...
Diagnoses of opportunistic mycotic infections constitute an increasing clinical problem. Conventi... more Diagnoses of opportunistic mycotic infections constitute an increasing clinical problem. Conventional diagnostic tests are time consuming and lack specificity and sensitivity for accurate and timely prognoses. This unit provides a comprehensive description of a fungal detection method that combines nucleic acid signatures with flow cytometry. The multiplexed assay, which uses xMAP technology, consists of unique fluorescent microspheres covalently bound to species-specific fungal oligonucleotide probes. In the presence of the complementary target sequence, the probe hybridizes to its biotinylated target. Quantification of the reaction is based on the fluorescence signal of the reporter molecule that binds to the biotin moieties of the target. The assay can be expanded to include other microorganisms and has the capability to simultaneously test 100 different fungal probes per tube/well. The speed, flexibility in design, and high-throughput capability makes this assay an attractive diagnostic tool for fungal infections and other related maladies.
Yeasts of the genus Malassezia have been associated with a variety of dermatological disorders in... more Yeasts of the genus Malassezia have been associated with a variety of dermatological disorders in humans and domestic animals. With the recent recognition of new members of the genus, new questions are emerging with regard to the pathogenesis and epidemiology of the new species. As new species are recognized, a precise and comprehensive identification system is needed. Herein is described a bead suspension culture-based array that combines the specificity and reliability of nucleic acid hybridization analysis with the speed and sensitivity of the Luminex analyser. The developed 16-plex array consisted of species- and group-specific capture probes that acted as ‘microcodes' for species identification. The probes, which were designed from sequence analysis in the D1/D2 region of rRNA and internal transcribed spacer (ITS) regions, were covalently bound to unique sets of fluorescent beads. Upon hybridization, the biotinylated amplicon was detected by the addition of a fluorochrome c...
A Luminex suspension array, which had been developed for identification of Cryptococcus neoforman... more A Luminex suspension array, which had been developed for identification of Cryptococcus neoformans and Cryptococcus gattii isolates, was tested by genotyping a set of 58 mostly clinical isolates. All genotypes of C. neoformans and C. gattii were included. In addition, cerebrospinal fluid (CSF) obtained from patients with cryptococcal meningitis was used to investigate the feasibility of the technique for identification of the infecting strain. The suspension array correctly identified haploid isolates in all cases. Furthermore, hybrid isolates possessing two alleles of the Luminex probe region could be identified as hybrids. In CSF specimens, the genotype of the cryptococcal strains responsible for infection could be identified after optimization of the PCR conditions. However, further optimization of the DNA extraction protocol is needed to enhance the usability of the method in clinical practice.
Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immun... more Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immunocompromised patients. The disease, cryptococcosis, is mostly acquired by inhalation and can result in a chronic meningoencephalitis, which can be fatal. Here, we describe a molecular method to identify the varieties and genotypic groups within the C. neoformans species complex from culture-based assays. The method employs a novel flow cytometer with a dual laser system that allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes that are covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, which hybridized to their complementary probe sequences, are quantified by the addition of the conjugate, streptavidin R-phycoerythrin. In this study we developed and validated eight probes derived from sequen...
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2000
Species of the genus Mrakia are currently classified as synonyms based on molecular sequence anal... more Species of the genus Mrakia are currently classified as synonyms based on molecular sequence analyses of the large sub-unit ribosomal DNA (LrDNA). Physiological and protein electrophoretic studies, however, reveal possible species differences. To clarify this discrepancy, we undertook molecular sequence analyses of the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of rDNA from the four psychrophilic Mrakia species
Great Bahama Bank (GBB) is the principal location of the formation and accumulation of ooids (con... more Great Bahama Bank (GBB) is the principal location of the formation and accumulation of ooids (concentrically coated, sand-size carbonate grains) in the world today, and as such has been the focus of studies on all aspects of ooids for more than half a century. Our view from a close look at this vast body of literature coupled with our continuing interests stresses that biological mechanisms (microbially mediated organomineralization) are very important in the formation of ooids, whereas the controlling factor for the distribution and size of ooid sand bodies is the physical energy. Mapping and coring studies of the modern ooid sand bodies on GBB provide insight into the rock record from different perspectives. An important consequence of the dual influence of ooid formation and distribution is that the geochemical signature of ooids is not in equilibrium with the seawater in which ooids form; therefore, extracting the paleophysical energy record from oolitic deposits is potentially ...
... J. Syst. Bacteriol. 28: 618. 1978, nom. rej. prop. Holotypus: ex cerebral spinal fluid, from ... more ... J. Syst. Bacteriol. 28: 618. 1978, nom. rej. prop. Holotypus: ex cerebral spinal fluid, from a patient from the San Fernando Valley, Cali-fornia, USA in the Veteran's Administration hospital, Los Angeles, isolated by Milton Huppert prior to 1971 ([lyophilised culture] ATCC No. ...
Microorganisms are abundant and ubiquotous components of carbonate sediments where their metaboli... more Microorganisms are abundant and ubiquotous components of carbonate sediments where their metabolisms can modify the geochemical conditions of their surroundings. The consortia of microorganisms along with their vast metabolic diversity allow these communities to efficiently cycle major elements and mediate the degree of carbonate saturation (González-Muñoz 2010; Dittrich and Sibler 2005; Baskar et al 2006) Heterotrophic and autotrophic bacteria are known to play a role in carbonate precipitation (Dupraz et al 2009). These microbes contribute to the formation of carbonates deposits that potentially form reservoir rocks, such as stromatolites (Dupraz and Visccher 2005), dolomites; (Wright and Wacey 2005) and ooid shoals (Kahle 2007). The primary role of bacteria in the precipitation process has been attributed to their metabolic activity (sulfate reduction, denitrification, ammonification, anaerobic sulphide oxidation, and photosynthesis) creating an alkaline environment that induces ...
The present study characterized bacterial communities associated with oolitic car- bonate sedimen... more The present study characterized bacterial communities associated with oolitic car- bonate sediments from the Bahamas Archipelago, ranging from high-energy ‘active’ to lower energy ‘non-active’ and ‘mat-stabilized’ environments. Bacterial communities were analyzed using terminal restriction fragment length polymorphisms (TRFLP), clone analyses of the 16S rRNA gene, confocal laser scanning microscopy (CLSM) and the quantitative phenol-sulfuric acid assay for extracellular polymeric substances (EPS). Confocal imaging of oolitic grains stained with cyanine dye-conjugated lectin and EPS quantification demonstrated that all 3 environments har- bored attached biofilm communities, but densities increased from the active to the mat-stabilized environment. Bacterial communities associated with all 3 settings were highly diverse and domi- nated by Proteobacteria (50 to 61%). Analysis of similarity (ANOSIM) and similarity percentages (SIMPER) revealed significant differences among the 3 environ...
The identity and concentrations of intracellular organic solutes were determined by nuclear magne... more The identity and concentrations of intracellular organic solutes were determined by nuclear magnetic resonance spectroscopy for two strains of aerobic, gram-negative bacteria isolated from Mono Lake, Calif., an alkaline, moderately hypersaline lake. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was the major endogenous solute in both organisms. Concentrations of ectoine varied with external NaCl levels in strain ML-D but not in strain ML-G, where the level was high but invariant from 1.5 to 3.0 M NaCl. Hydroxyectoine also occurred in strain ML-D, especially at elevated NaCl concentrations (2.5 and 3.0 M), but at levels lower than those of ectoine. Exogenous organic solutes that might occur in Mono Lake were examined for their effects on the de novo synthesis of ectoine. Dimethylsulfoniopropionate (DMSP) (0.1 or 1 mM) did not significantly lower ectoine levels in either isolate, and only strain ML-G showed any capacity for DMSP accumulation. With nitrogen limitati...
Amplified fragment length polymorphism (AFLP) genotyping of isolates of the pathogenic fungus Cry... more Amplified fragment length polymorphism (AFLP) genotyping of isolates of the pathogenic fungus Cryptococcus neoformans suggested a considerable genetic divergence between the varieties C. neoformans var. neoformans and C. neoformans var. grubii on the one hand versus C. neoformans var. gattii on the other. This divergence is supported by additional phenotypic, biochemical, clinical and molecular differences. Therefore, the authors propose the existence of two species, C. neoformans (Sanfelice) Vuillemin and C. bacillisporus Kwon-Chung, which differ in geographical distribution, serotypes and ecological origin. Within each species three AFLP genotypes occur, which differ in geographical distribution and serotypes. Differences in ecological origin (AIDS patients, non-AIDS patients, animals or the environment) were found to be statistically not significant. In C. neoformans as well as in C. bacillisporus one of the genotypes represented a hybrid. The occurrence of hybridization has cons...
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Jan 29, 2005
After the process of DNA barcoding has become well advanced in a group of organisms, as it has in... more After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base pairs long), uniquely distinctive oligonucleotide 'microcodes' of less than 25 bp can be found that allow rapid identification of circa 100-200 species on various array-like platforms. Microarrays can in principle fulfill the function of microcode-based species identification but, because of their high cost and low level of reusability, they tend to be less cost-effective. Two alternative platforms in current use in fungal identification are reusable nylon-based macroarrays and the Luminex system of specific, colour-coded DNA detection beads analysed by means of a flow cytome...
Diagnoses of opportunistic mycotic infections constitute an increasing clinical problem. Conventi... more Diagnoses of opportunistic mycotic infections constitute an increasing clinical problem. Conventional diagnostic tests are time consuming and lack specificity and sensitivity for accurate and timely prognoses. This unit provides a comprehensive description of a fungal detection method that combines nucleic acid signatures with flow cytometry. The multiplexed assay, which uses xMAP technology, consists of unique fluorescent microspheres covalently bound to species-specific fungal oligonucleotide probes. In the presence of the complementary target sequence, the probe hybridizes to its biotinylated target. Quantification of the reaction is based on the fluorescence signal of the reporter molecule that binds to the biotin moieties of the target. The assay can be expanded to include other microorganisms and has the capability to simultaneously test 100 different fungal probes per tube/well. The speed, flexibility in design, and high-throughput capability makes this assay an attractive diagnostic tool for fungal infections and other related maladies.
Yeasts of the genus Malassezia have been associated with a variety of dermatological disorders in... more Yeasts of the genus Malassezia have been associated with a variety of dermatological disorders in humans and domestic animals. With the recent recognition of new members of the genus, new questions are emerging with regard to the pathogenesis and epidemiology of the new species. As new species are recognized, a precise and comprehensive identification system is needed. Herein is described a bead suspension culture-based array that combines the specificity and reliability of nucleic acid hybridization analysis with the speed and sensitivity of the Luminex analyser. The developed 16-plex array consisted of species- and group-specific capture probes that acted as ‘microcodes' for species identification. The probes, which were designed from sequence analysis in the D1/D2 region of rRNA and internal transcribed spacer (ITS) regions, were covalently bound to unique sets of fluorescent beads. Upon hybridization, the biotinylated amplicon was detected by the addition of a fluorochrome c...
A Luminex suspension array, which had been developed for identification of Cryptococcus neoforman... more A Luminex suspension array, which had been developed for identification of Cryptococcus neoformans and Cryptococcus gattii isolates, was tested by genotyping a set of 58 mostly clinical isolates. All genotypes of C. neoformans and C. gattii were included. In addition, cerebrospinal fluid (CSF) obtained from patients with cryptococcal meningitis was used to investigate the feasibility of the technique for identification of the infecting strain. The suspension array correctly identified haploid isolates in all cases. Furthermore, hybrid isolates possessing two alleles of the Luminex probe region could be identified as hybrids. In CSF specimens, the genotype of the cryptococcal strains responsible for infection could be identified after optimization of the PCR conditions. However, further optimization of the DNA extraction protocol is needed to enhance the usability of the method in clinical practice.
Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immun... more Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immunocompromised patients. The disease, cryptococcosis, is mostly acquired by inhalation and can result in a chronic meningoencephalitis, which can be fatal. Here, we describe a molecular method to identify the varieties and genotypic groups within the C. neoformans species complex from culture-based assays. The method employs a novel flow cytometer with a dual laser system that allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes that are covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, which hybridized to their complementary probe sequences, are quantified by the addition of the conjugate, streptavidin R-phycoerythrin. In this study we developed and validated eight probes derived from sequen...
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2000
Species of the genus Mrakia are currently classified as synonyms based on molecular sequence anal... more Species of the genus Mrakia are currently classified as synonyms based on molecular sequence analyses of the large sub-unit ribosomal DNA (LrDNA). Physiological and protein electrophoretic studies, however, reveal possible species differences. To clarify this discrepancy, we undertook molecular sequence analyses of the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of rDNA from the four psychrophilic Mrakia species
Great Bahama Bank (GBB) is the principal location of the formation and accumulation of ooids (con... more Great Bahama Bank (GBB) is the principal location of the formation and accumulation of ooids (concentrically coated, sand-size carbonate grains) in the world today, and as such has been the focus of studies on all aspects of ooids for more than half a century. Our view from a close look at this vast body of literature coupled with our continuing interests stresses that biological mechanisms (microbially mediated organomineralization) are very important in the formation of ooids, whereas the controlling factor for the distribution and size of ooid sand bodies is the physical energy. Mapping and coring studies of the modern ooid sand bodies on GBB provide insight into the rock record from different perspectives. An important consequence of the dual influence of ooid formation and distribution is that the geochemical signature of ooids is not in equilibrium with the seawater in which ooids form; therefore, extracting the paleophysical energy record from oolitic deposits is potentially ...
... J. Syst. Bacteriol. 28: 618. 1978, nom. rej. prop. Holotypus: ex cerebral spinal fluid, from ... more ... J. Syst. Bacteriol. 28: 618. 1978, nom. rej. prop. Holotypus: ex cerebral spinal fluid, from a patient from the San Fernando Valley, Cali-fornia, USA in the Veteran's Administration hospital, Los Angeles, isolated by Milton Huppert prior to 1971 ([lyophilised culture] ATCC No. ...
Microorganisms are abundant and ubiquotous components of carbonate sediments where their metaboli... more Microorganisms are abundant and ubiquotous components of carbonate sediments where their metabolisms can modify the geochemical conditions of their surroundings. The consortia of microorganisms along with their vast metabolic diversity allow these communities to efficiently cycle major elements and mediate the degree of carbonate saturation (González-Muñoz 2010; Dittrich and Sibler 2005; Baskar et al 2006) Heterotrophic and autotrophic bacteria are known to play a role in carbonate precipitation (Dupraz et al 2009). These microbes contribute to the formation of carbonates deposits that potentially form reservoir rocks, such as stromatolites (Dupraz and Visccher 2005), dolomites; (Wright and Wacey 2005) and ooid shoals (Kahle 2007). The primary role of bacteria in the precipitation process has been attributed to their metabolic activity (sulfate reduction, denitrification, ammonification, anaerobic sulphide oxidation, and photosynthesis) creating an alkaline environment that induces ...
The present study characterized bacterial communities associated with oolitic car- bonate sedimen... more The present study characterized bacterial communities associated with oolitic car- bonate sediments from the Bahamas Archipelago, ranging from high-energy ‘active’ to lower energy ‘non-active’ and ‘mat-stabilized’ environments. Bacterial communities were analyzed using terminal restriction fragment length polymorphisms (TRFLP), clone analyses of the 16S rRNA gene, confocal laser scanning microscopy (CLSM) and the quantitative phenol-sulfuric acid assay for extracellular polymeric substances (EPS). Confocal imaging of oolitic grains stained with cyanine dye-conjugated lectin and EPS quantification demonstrated that all 3 environments har- bored attached biofilm communities, but densities increased from the active to the mat-stabilized environment. Bacterial communities associated with all 3 settings were highly diverse and domi- nated by Proteobacteria (50 to 61%). Analysis of similarity (ANOSIM) and similarity percentages (SIMPER) revealed significant differences among the 3 environ...
The identity and concentrations of intracellular organic solutes were determined by nuclear magne... more The identity and concentrations of intracellular organic solutes were determined by nuclear magnetic resonance spectroscopy for two strains of aerobic, gram-negative bacteria isolated from Mono Lake, Calif., an alkaline, moderately hypersaline lake. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was the major endogenous solute in both organisms. Concentrations of ectoine varied with external NaCl levels in strain ML-D but not in strain ML-G, where the level was high but invariant from 1.5 to 3.0 M NaCl. Hydroxyectoine also occurred in strain ML-D, especially at elevated NaCl concentrations (2.5 and 3.0 M), but at levels lower than those of ectoine. Exogenous organic solutes that might occur in Mono Lake were examined for their effects on the de novo synthesis of ectoine. Dimethylsulfoniopropionate (DMSP) (0.1 or 1 mM) did not significantly lower ectoine levels in either isolate, and only strain ML-G showed any capacity for DMSP accumulation. With nitrogen limitati...
Amplified fragment length polymorphism (AFLP) genotyping of isolates of the pathogenic fungus Cry... more Amplified fragment length polymorphism (AFLP) genotyping of isolates of the pathogenic fungus Cryptococcus neoformans suggested a considerable genetic divergence between the varieties C. neoformans var. neoformans and C. neoformans var. grubii on the one hand versus C. neoformans var. gattii on the other. This divergence is supported by additional phenotypic, biochemical, clinical and molecular differences. Therefore, the authors propose the existence of two species, C. neoformans (Sanfelice) Vuillemin and C. bacillisporus Kwon-Chung, which differ in geographical distribution, serotypes and ecological origin. Within each species three AFLP genotypes occur, which differ in geographical distribution and serotypes. Differences in ecological origin (AIDS patients, non-AIDS patients, animals or the environment) were found to be statistically not significant. In C. neoformans as well as in C. bacillisporus one of the genotypes represented a hybrid. The occurrence of hybridization has cons...
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