Search

Ooid shoals, sedimentary structures consisting of carbonate grains, are often used as proxies to explore changes in seawater chemistry over Earth history. Despite their importance as contributors to the global carbonate budget and as paleo-environmental indicators for water depth, pH, temperature and water energy, the mechanism of formation of these concentric, sub-spherical concretions of tangentially or radially arranged aragonite or calcite crystals is still the subject of intense debate. Unlike oncoids, whose microbial origins are well acknowledged, ooids are traditionally viewed as abiotic grains, mainly produced in suspension by agitated, warm waters that are supersaturated with respect to CaCO3. Yet, there is considerable evidence, including geochemical bio-signatures and detection of viable microbes capable of mineralization and biofilm production and degradation, to suggest otherwise. The emergence of new and sophisticated technologies reveals that the level of microbial diversity of these sedimentary grains exceeds that of some microbialite systems. These findings have refined our understanding of ooid genesis and ooid accretion processes and argue toward microbial mediation both as a direct result of metabolic activities inducing changes in the environment conducive to precipitation and by a passive mechanism, in which mucilaginous extracellular polymeric substances (EPS) act as a template for mineral nucleation. Furthermore, new developments in the field indicate that carbonate mineralization in marine ooids occurs through sequential precipitations that involve an amorphous calcium carbonate phase (ACC), which some argue are of biotic origins. Herein, we discuss the most relevant theories of ooid formation, including some of the early pioneering studies that laid the groundwork for new theories attempting to decode the origins of these controversial grains.

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 more accurate than doing so for the paleochemical record.

Ooid formation remains elusive despite their importance as palaeoclimatic indicators and important contributors to global carbonate budget. Based on stable isotopes, nutrient and elemental analyses on solid components and ooidal leachates, this study supports the notion of microbial involvement in the development of ooids from Great Bahama Bank. Carbon and nitrogen isotopic analyses on organic fractions identified geochemical signatures of microbial activity. The δ13C values for organic carbon in the bulk (−11·9 to −16·9‰); intercrystalline/intracrystalline (−11·9 to 16·7‰); and intracrystalline phases (−12·4 to −17·7‰) were similar and, except for the more enriched values of ooids from Butterfly Beach, were within the range of photosynthesisers. The δ15N values for the bulk (+0·5 to −0·2‰); intercrystalline/intracrystalline (−0·3‰ to −0·7‰) and intracrystalline organic matter (−0·3 to −1·7‰) showed a narrow range consistent with nitrogen fixation. While positive δ15N and δ18O values of the leached from the ooids provided evidence of denitrification, the carbonate associated sulphate δ34SCAS of the bulk sediments (+19·2 to +19·6‰) and δ34S of the leachates (+16·6 to +18·3‰) provided weak indication of sulphate reduction, suggesting either that high concentrations of isotopically enriched S are overriding bio-signatures of sulphate reduction or that microbes are preferentially using as an electron acceptor. In contrast, the elevated sulphate concentrations of the leachates suggest the occurrence of microbial sulphide oxidation within ooids. The high Mg/Ca of the leachates and scanning electron microscope analyses provide putative evidence of amorphous calcium carbonate and a formative role in CaCO3 precipitation. Together, these findings indicate that a redox dependent microbial consortium may influence CaCO3 precipitation in the form of ooid accretion, cementation and micritization. It is also inferred that ooid deposits are not suitable indicators of palaeoclimate because ooids are affected throughout their life by a complex chain of abiotic and biological processes which can lead to large geochemical offsets.

Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made., Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature “C. neoformans species complex” and “C. gattii species complex.” Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

Despite the importance of oolitic depositional systems as indicators of climate and reservoirs of inorganic C, little is known about the microbial functional diversity, structure, composition, and potential metabolic processes leading to precipitation of carbonates. To fill this gap, we assess the metabolic gene carriage and extracellular polymeric substance (EPS) development in microbial communities associated with oolitic carbonate sediments from the Bahamas Archipelago. Oolitic sediments ranging from high-energy 'active' to lower energy 'non-active' and 'microbially stabilized' environments were examined as they represent contrasting depositional settings, mostly influenced by tidal flows and wave-generated currents. Functional gene analysis, which employed a microarray-based gene technology, detected a total of 12,432 of 95,847 distinct gene probes, including a large number of metabolic processes previously linked to mineral precipitation. Among these, gene-encoding enzymes for denitrification, sulfate reduction, ammonification, and oxygenic/anoxygenic photosynthesis were abundant. In addition, a broad diversity of genes was related to organic carbon degradation, and N2 fixation implying these communities has metabolic plasticity that enables survival under oligotrophic conditions. Differences in functional genes were detected among the environments, with higher diversity associated with non-active and microbially stabilized environments in comparison with the active environment. EPS showed a gradient increase from active to microbially stabilized communities, and when combined with functional gene analysis, which revealed genes encoding EPS-degrading enzymes (chitinases, glucoamylase, amylases), supports a putative role of EPS-mediated microbial calcium carbonate precipitation. We propose that carbonate precipitation in marine oolitic biofilms is spatially and temporally controlled by a complex consortium of microbes with diverse physiologies, including photosynthesizers, heterotrophs, denitrifiers, sulfate reducers, and ammonifiers.

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C. neoformans molecular type VNI and C. gattii molecular type VGII.

Aims: Research into the relationship between pathogens, faecal indicator microbes and environmental factors in beach sand has been limited, yet vital to the understanding of the microbial relationship between sand and the water column and to the improvement of criteria for better human health protection at beaches. The objectives of this study were to evaluate the presence and distribution of pathogens in various zones of beach sand (subtidal, intertidal and supratidal) and to assess their relationship with environmental parameters and indicator microbes at a non-point source subtropical marine beach. Methods and Results: In this exploratory study in subtropical Miami (Florida, USA), beach sand samples were collected and analysed over the course of 6 days for several pathogens, microbial source tracking markers and indicator microbes. An inverse correlation between moisture content and most indicator microbes was found. Significant associations were identified between some indicator microbes and pathogens (such as nematode larvae and yeasts in the genus Candida), which are from classes of microbes that are rarely evaluated in the context of recreational beach use. Conclusions: Results indicate that indicator microbes may predict the presence of some of the pathogens, in particular helminthes, yeasts and the bacterial pathogen Staphylococcus aureus including methicillin-resistant forms. Indicator microbes may thus be useful for monitoring beach sand and water quality at non-point source beaches. Significance and Impact of the Study: The presence of both indicator microbes and pathogens in beach sand provides one possible explanation for human health effects reported at non-point sources beaches.

Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid—modified oligonucleotides (LNA) and Mirus Label IT® nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5′ terminal labeling technique, the chemicallabeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

High incidence of cryptococcosis in these patients is in contrast to reports from other countries., To determine the population structure of the cryptococcosis agents in China, we analyzed the genotype of 120 Cryptococcus neoformans and 9 Cryptococcus gattii strains isolated from 1980 through 2006 from cryptococcosis patients residing in 16 provinces of mainland China. A total of 71% (91/129) of the clinical strains isolated from 1985 through 2006 were from patients without any apparent risk factors. Only 8.5% (11/129) were from AIDS patients; the remaining 20.5% (27/129) were from patients with underlying diseases other than HIV infection. One hundred twenty of the 129 isolates were C. neoformans serotype A, mating type MATα strains that exhibited an identical M13-based VNI subtype, which was distinguishable from the reference VNI molecular type. The 9 remaining isolates were serotype B, MATα strains of C. gattii and portrayed a typical VGI molecular type. Data analyzed from multilocus sequences showed no variation and that these Chinese C. neoformans isolates belong to a cluster that has phylogenetically diverged from the VNI reference strain. Our finding that most cryptococcosis patients in China had no apparent risk factor is in stark contrast with reports from other countries.

Research to understand and remediate coastal pollution is moving toward a multitiered approach in which traditional enumeration of fecal indicators is accompanied by molecular analysis of a variety of targets. Technology that rapidly detects multiple microbial contaminants would benefit from such an approach. The Luminex® 100™ system is a suspension array that assays multiple analytes rapidly in a single well of a microtiter plate. The ability of the system to simultaneously detect multiple fecal indicating bacteria in environmental samples was tested. Primer/probe sets were designed to simultaneously detect the following fecal indicators: the Bacteroides fragilis group, Enterococcus spp., Escherichia coli and Shigella spp., Bacteroides distasonis, and Ent. faecalis. Specificity and sensitivity of the Luminex probes was tested against laboratory cultures. In addition, sequencing, culture plate testing, and specificity testing with environmental isolates were steps taken to validate the function of the assay with environmental samples. Luminex response to cultures and to environmental samples was consistent with sequencing results, suggesting that the technology has the potential to simultaneously detect multiple targets for coastal water quality applications, particularly as progress is made to efficiently extract DNA from water and sediment matrices.

Fungal infections, on the rise for several decades, can prove deadly, particularly for immunocompromised patients. Among the most common fungal pathogens are yeasts such as Candida and Cryptococcus neoformans, and filamentous fungi such as Aspergillus and Fusarium spp. In North America, Candida ranks as the fourth most common bloodstream pathogen, accounting for as many as 8–15% of such infections.

Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI + 5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neo- formans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels. � 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The polysaccharide capsule is a unique feature of Cryptococcus neoformans that distinguishes the organism from other medically important yeasts. Various histochemical stains react with cryptococci, including mucin stains such as mucicarmine, alcian blue, and periodic acid-Schiff that stain the capsule and Fontana-Masson stain, which interacts with the cell wall. The sensitivity of cerebrospinal fluid (CSF) antigen detection is highest for diagnosing cryptococcal meningitis in HIV-infected persons and varies based upon factors such as the fungal burden in the CSF and the presence of well-encapsulated organisms. Since the serum cryptococcal antigen is positive in almost all AIDS patients with cryptococcal meningitis, there has been interest in using the test to screen high-risk HIV-infected persons, such as those with fevers, neurological symptoms, and/or low CD4 counts. At the current time, there are four commercially available assays that detect β-Glucan (BG) in serum. Over the past two decades, a myriad of molecular techniques have been explored for the detection of pathogenic fungi. Molecular fingerprinting techniques, e.g., randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis, amplified fragment length polymorphism (AFLP), PCR fingerprinting with minisatellite (M13) or microsatellite primers [e.g., (GACA)4 or (CTG)5], karyotypes, sequencing, multilocus microsatellite typing, mating-type locus, and multilocus sequence typing (MLST), are among the most popular typing techniques that have been applied to discern and characterize the genetic heterogeneity within the species of the C. neoformans species complex.

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 and the psychrophilic yeast, Cryptococcus curiosus. Identical ITS sequences were found between C. curiosus, M. nivalis and M. frigida. Although, M. stokesii and M. gelida displayed identical ITS and IGS sequences, their sequences differed from the other three species by 2.3% and 38%, respectively. The results suggest that M. stokesii is a synonym of M. gelida, whereas M. nivalis is a synonym of M. frigida. Sequence differences (1.9%) observed in the IGS region indicates that C. curiosus is a distinct strain of M. frigida.

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 limitation, however, DMSP (0.1 mM) substituted for ectoine in strain ML-G and became the main organic solute. Glycine betaine (GB) was more effective than DMSP in affecting ectoine levels, principally in strain ML-D. Strain ML-D accumulated GB to 50 or 67% of its organic solute pool at 2.5 M NaCl, at an external level of 0.1 or 1 mM GB, respectively. Strain ML-D also accumulated arsenobetaine. The methylated zwitterionic compounds, probably metabolic products of phytoplankton (DMSP and GB) or brine shrimps (arsenobetaine) in Mono Lake, may function as osmolytes for indigenous bacteria when present at high concentrations or under conditions of nitrogen limitation or salt stress.

Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT(®) nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5' terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

The metabolism of the methylated osmolytes glycine betaine (GB) and dimethylsulfoniopropionate (DMSP) was studied in a bacterium (strain MD 14–50) isolated from a colony of the cyanobacterium Trichodesmium. MD 14–50 when grown on DMSP cleaved dimethylsulfide (DMS) from DMSP and oxidized acrylate. In contrast to DMSP, GB was metabolized by sequential N-demethylations. Low concentrations (100 μM) of DMSP or GB allowed the growth of MD 14–50 on glucose at higher salinities than in their absence. At elevated salinities, DMSP was accumulated intracellularly with less catabolism and DMS production. Thus, DMSP and GB were catabolized by different mechanisms but functioned interchangeably as osmolytes.

PURPOSE. Fungal ocular infections cause significant ocular morbidity, particularly when diagnosis and treatment are delayed. Accurate morphologic identification of Fusarium spp. beyond the genus is time-consuming and insensitive. It was the purpose of this study to examine the usefulness of the nuclear ribosomal RNA (rRNA) internal transcribed spacer regions (ITS1 and -2) to detect and differentiate Fusarium spp. responsible for ocular infections. METHODS. Fifty-eight archived isolates from ocular sources of 52 patients diagnosed with Fusarium keratitis at the Bascom Palmer Eye Institute (Miami, FL) from April 2000 to May 2007 were analyzed. The archived samples, which were initially classified according to morphologic characteristics, were analyzed by DNA sequence data generated from the ITS regions of the rRNA genes. RESULTS. Fifteen distinct sequences were identified among the 58 isolates. Sequence analysis identified the isolates as Fusarium solani (75%), F. oxysporum (16%), F. incarnatum-equiseti (5%), F. dimerum (2%), and one Fusarium sp. (2%) that was not classified within any species complex. Species identification based on sequence data correlated well with the morphologic classification when performed by a mycology reference laboratory, but a higher rate of mismatch was observed based on identification by a nonreference laboratory. CONCLUSIONS. Most of the isolates of Fusarium ocular infections belong to the F. solani or F. oxysporum species complexes. Morphologic classification at the species level yielded inconsistent results at a general microbiology laboratory. In contrast, the sequence variation within the ITS region allowed reliable and faster discrimination of the isolates at both the genus and species level.

This communication describes the consensus multi-locus typing scheme established by the Cryptococcal Working Group I (Genotyping of Cryptococcus neoformans and C. gattii) of the International Society for Human and Animal Mycology (ISHAM) using seven unlinked genetic loci for global strain genotyping. These genetic loci include the housekeeping genes CAP59,GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region. Allele and sequence type information are accessible at http://www.mlst.net/ .

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 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 coupled to a reporter molecule. The hybridized beads were subsequently analysed by flow cytometric techniques. The developed array, which allowed the detection of species in a multiplex and high-throughput format, was accurate and fast, since it allowed precise identification of species and required less than 1 h following PCR amplification. The described protocol, which can integrate uniplex or multiplex PCR reactions, permitted the simultaneous detection of target sequences in a single reaction, and allowed single mismatch discrimination between probe and non-target sequences. The assay has the capability to be expanded to include other medically important pathogenic species in a single or multiplex array format.

Cryptococcus neoformans and Cryptococcus gattii are yeasts that cause meningoencephalitis, but that differ in host range and geographical distribution. Cryptococcus neoformans occurs world-wide and mostly infects immunocompromised patients, whereas C. gattii occurs mainly in (sub)tropical regions and infects healthy individuals. Anomalous C. neoformans strains were isolated from patients. These strains were found to be monokaryotic, and diploid or aneuploid. Amplified Fragment Length Polymorphism (AFLP) and sequence analyses indicated that AFLP genotypes 2 (C. neoformans) and 4 (C. gattii) were present. The strains were serologically BD. Mating- and serotype-specific PCR reactions showed that the strains were MATa-serotype D/MATa-serotype B. This study is the first to describe naturally occurring hybrids between C. neoformans and C. gattii. [KEYWORDS: Cryptococcus neoformans ; Cryptococcus gattii ; BD hybrid ; recurrent meningitis]

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 sequence analysis of the intergenic spacer region of the rRNA gene region. The assay proved to be specific and sensitive, allowed discrimination of a 1-bp mismatch with no apparent cross-reactivity, and detected 10 1 to 10 3 genome copies. The described protocol, which can be used directly with yeast cells or isolated DNA, can be undertaken in less than 1 h following PCR amplification and permits identification of species in a multiplex format. In addition to a multiplex capability, the assay allows the simultaneous detection of target sequences in a single reaction. The accuracy, speed, flexibility, and sensitivity of this technology are a few of the advantages that will make this assay useful for the diagnosis of human cryptococcal infections and other pathogenic diseases.

Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI +5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neoformans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels.

The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon . This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 10 2 genome molecules in PCRs and 10 7 to 10 8 molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.

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 consequences for the reproductive biology of the species, as new genotypes with altered virulence or susceptibility to antifungal drugs may arise through the exchange of genetic material.