Morphological characterization of in-vitro human hair keratinolysis,produced by identified wild strains of Chrysosporium species

Chrysosporium species were isolated from soil and keratinized material. Primary isolation was performed following the general method of hair baiting on modified Czapek-agar media with washed, defated and sterilized human hair fragments added. Strains were maintained in test tubes of potato dextrose agar at 29 °C and cultivated on phytone yeast extract agar at 28 °C for 14 days for identification. Isolates were characterized using Van Oorschot's key. Keratinolytic activity was expressed following a subjective scale representing degree/severity of attack upon hair surface and presence of fungal structures observed in substrate. Culture results and characterization methods were effective for soil Chrysosporium strain isolation. A new hair attack mode is described. Of 71 keratinolytic fungal isolates, eight (12%) Chrysosporium species were identified. One keratinolytic Chrysosporium sp. isolate is yet to be identified.This revised version was published online in October 2005 with corrections to the Cover Date.     

Fungi in the sea fan <Emphasis Type="Italic">Gorgonia ventalina</Emphasis>: diversity and sampling strategies

Fungal communities from gorgonians have been poorly documented and most studies of these communities have lacked defined sampling strategies. The objectives of this study were: (1) to estimate fungal diversity in Gorgonia ventalina; (2) to compare two sampling and tissue processing strategies: tissue fragments of different sizes vs. homogenized tissue. A total of seven genera and fourteen species of fungi were isolated on culture medium and identified by sequencing the nrITS. All but one species were new reports. In both treatments Aspergillus and Penicillium were the most common genera isolated. Most species isolated from fragments were not observed from homogenized tissue and vice versa. Reducing the size fragment increased significantly the number of species isolated per fragment. To better estimate fungal diversity in sea fans a strategy is proposed that combines sampling of small tissue fragments with homogenized tissue, since each technique yielded fungal species not detected by the other.     

Comparison of PCR-RFLP with 21-plex PCR and rDNA Sequencing for Identification of Clinical Yeast Isolates

Non-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which are time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR. One hundred and seventy-three yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method. The results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=?C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR-RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR. The application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

     

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

Aims: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (10⁷–10⁵ cells). Conclusion: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.     

An electrophoretic karyotype for Candida albicans reveals large chromosomes in multiples

Summary Using field-inversion gel electrophoresis we defined an electrophoretic karyotype for the yeast, Candida albicans. The karyotype is distinct from other species of Candida and is species specific. A total of five distinct chromosomal mobility groups were observed, at least four of which are composed of a minimum of two fragments each. From the apparent sizes of these fragments relative to the large chromosomes of the morphologically related yeast Saccharomyces cerevisiae, together with the known genome size of this organism, we conclude that the karyotype is the result of the migration of intact chromosomes.     

High‐resolution DNA melt‐curve analysis for cost‐effective mass screening of pairwise species interactions

Ecological studies of pairwise interactions are constrained by the methods available for rapid species identification of the interacting organisms. The resolution of data required to characterize species interaction networks at multiple spatio‐temporal scales can be intensive, and therefore laborious and costly to collect. We explore the utility of high‐resolution DNA melt‐curve analysis (HRM) as a rapid species identification method. An approach was developed to identify organisms at the pairwise interaction level, with particular application to cryptic species interactions that are traditionally difficult to study. Here, we selected a challenging application; to identify the presence/absence of pathogenic fungi (Sporothrix inflata, Ophiostoma nigrocarpum and Ophiostoma galeiforme) transported by bark beetle vectors (Hylastes ater and Hylurgus ligniperda). The technique was able to distinguish between different species of DNA within a single, pooled sample. In test applications, HRM was effective in the mass screening and identification of pathogenic fungal species carried by many individual bark beetle vectors (n = 455 beetles screened) across large geographic scales. For two of the fungal species, there was no difference in the frequency of association with either of their vectors, but for the third fungal species there was a shift in vector–pathogen associations across locations. This technique allows rapid, mass screening and characterization of species interactions at a fraction of the time and cost of traditional methods. It is anticipated that this method can be readily applied to explore other cryptic species interactions, or other studies requiring rapid generation of large data sets and/or high‐throughput efficiency.     

Fungi Vectored by the Bark Beetle Ips typographus Following Hibernation Under the Bark of Standing Trees and in the Forest Litter

The bark beetle Ips typographus has different hibernation environments, under the bark of standing trees or in the forest litter, which is likely to affect the beetle-associated fungal flora. We isolated fungi from beetles, standing I. typographus-attacked trees, and forest litter below the attacked trees. Fungal identification was done using cultural and molecular methods. The results of the two methods in detecting fungal species were compared. Fungal communities associated with I. typographus differed considerably depending on the hibernation environment. In addition to seven taxa of known ophiostomoid I. typographus-associated fungi, we detected 18 ascomycetes and anamorphic fungi, five wood-decaying basidomycetes, 11 yeasts, and four zygomycetes. Of those, 14 fungal taxa were detected exclusively from beetles that hibernated under bark, and six taxa were detected exclusively from beetles hibernating in forest litter. The spruce pathogen, Ceratocystis polonica, was detected occasionally in bark, while another spruce pathogen, Grosmannia europhioides, was detected more often from beetles hibernating under the bark as compared to litter. The identification method had a significant impact on which taxa were detected. Rapidly growing fungal taxa, e.g. Penicillium, Trichoderma, and Ophiostoma, dominated pure culture isolations; while yeasts dominated the communities detected using molecular methods. The study also demonstrated low frequencies of tree pathogenic fungi carried by I. typographus during its outbreaks and that the beetle does not require them to successfully attack and kill trees.     

Rapid identification of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> within the section <Emphasis Type="Italic">Fumigati</Emphasis>

Background  

New fungal species that are morphologically similar to Aspergillus fumigatus were recently described and included in section Fumigati. Misidentification of such fungal species, particularly of the human pathogens, Aspergillus lentulus, Neosartorya fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri and Neosartorya udagawae, has been increasingly reported by numerous clinical labs. Nevertheless, A. fumigatus still accounts for more than 90% of all invasive aspergillosis cases. The purpose of the present study was to develop a rapid method for the molecular identification of A. fumigatus to distinguish it from other species within the section Fumigati.     

The dynamics of the yeast community of the Tagus river estuary: testing the hypothesis of the multiple origins of estuarine yeasts

Yeasts are common inhabitants of different types of aquatic habitats, including marine and estuarine waters and rivers. Although numerous studies have surveyed yeast occurrence in these habitats, the identification of autochthonous populations has been problematic because several yeast species seem to be very versatile and therefore mere presence is not sufficient to establish an ecological association. In the present study we investigated the dynamics of the yeast community in the Tagus river estuary (Portugal) by combining a microbiological study involving isolation, quantification, and molecular identification of dominant yeast populations with the analysis of hydrological and hydrographical data. We set out to test the hypothesis of the multiple origins of estuarine yeast populations in a transect of the Tagus estuary and we postulate four possible sources: open sea, terrestrial, gastrointestinal and the estuary itself in the case of populations that have become resident. Candida parapsilosis and Pichia guilliermondii were correlated with Escherichia coli, which indicated an intestinal origin. Other cream-colored yeasts like Debaryomyces hansenii and Candida zeylanoides had similar dynamics, but no association with E. coli and quite distinct ecological preferences. They might represent a group of resident estuarine populations whose primary origin is diverse and can include marine, terrestrial, and gastrointestinal habitats. Another major yeast population was represented by Rhodotorula mucilaginosa. The cosmopolitan nature of that species and its moderate association with E. coli point to terrestrial sources as primary habitats.     

辽宁碱蓬根际土壤真菌多样性的季节变化及其耐盐性

采用稀释平板法和形态学鉴定法进行了辽宁碱蓬(Suaeda liaotungensis)根际土壤真菌的分离鉴定及季节、盐度对真菌多样性的影响分析。结果表明:春、夏、秋、冬四季辽宁碱蓬根际土壤中的真菌菌落数分别为6410、4180、5730和3340,种类分别为6属13种、9属16种、11属31种、6属12种,共分离鉴定出13属42种真菌;其根际土壤真菌的多样性指数、丰富度指数和均匀度指数,从大到小的次序均为秋夏春冬,3种指数均在秋季达到峰值;多样性指数和丰富度指数差异较大,均匀度指数波动较小,春季和冬季的均匀度指数相近。辽宁碱蓬根际土壤真菌种类明显多于无植被土壤,是无植被土壤的7倍,多样性指数和均匀度指数分别是无植被土壤的4—8倍和1—1.2倍;其根际土壤真菌优势种群包括青霉属(Penicillium)、葡萄穗霉属(Stachybotrys)、枝孢属(Cladosporium)、木霉属(Trichoderma)、曲霉属(Aspergillus)和镰孢属(Fusarium);4个季节的优势菌不尽相同,但均有一个共同的优势菌属青霉属。以Na Cl浓度梯度法制造盐胁迫生境,检测辽宁碱蓬根际土壤真菌对盐胁迫的响应,结果表明:60%左右的菌种能耐受5%以下盐度、15%左右的菌种能耐10%—20%盐度;筛选出了6株高度耐盐菌:细交链孢霉、草酸青霉、产黄青霉、烟曲霉、细极链格孢和赭曲霉,其中最高耐盐菌种赭曲霉可耐20%盐度。创新之处在于以盐度和季节为变量检测辽宁碱蓬根际土壤真菌的种群构成,为其深入研究奠定了理论基础。     

Molecular characterization of carotenogenic yeasts from aquatic environments in Patagonia, Argentina

Fifteen aquatic environments (lakes, lagoons and rivers) of glacial origin in the northern Andean Patagonia (Argentina) were surveyed for the occurrence of red yeasts. Subsurface water samples were filtered and used for colony counting and yeast isolation. A preliminary quantitative analysis indicated that total yeast counts ranged between 0 and 250 cells l⁻¹. A polyphasic approach including physiological and molecular methods was used for the identification of 64 carotenogenic yeast strains. The molecular characterisation of the isolates was based on the mini/microsatellite-primed PCR technique (MSP-PCR) employing the (GTG)₅ and the M13 primers. Comparison of representative fingerprints of each group with those of the type strains of pigmented yeasts allowed the expeditious identification of 87.5% isolates. The sequence analysis of the D1/D2 domains of the 26S rDNA was employed to confirm identifications and in the characterization of the unidentified MSP-PCR groups. Teleomorphic yeast species were detected by performing sexual compatibility assays. The isolates corresponded to 6 genera and 15 yeast species, including four new yeast species of the genera Cryptococcus (1), Rhodotorula (1) and Sporobolomyces (2). Rhodotorula mucilaginosa was found in the majority of the samples and represented ca. 50% of the total number of isolates. However, this yeast was not detected in aquatic environments with very low anthropic influence. Other frequent yeast isolates were teleomorphic yeast species of Rhodosporidium babjevae, R. kratochvilovae and Sporidiobolus salmonicolor. This study represents the first report on red yeast occurrence and biodiversity in northwestern Patagonia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hypertonic Sabouraud Dextrose Agar as a Substrate for Differentiation of Candida dubliniensis

In this study, we aimed to detect the proportion of Candida dubliniensis among yeast strains previously identified as C. albicans by using several phenotypic methods and PCR. For this purpose, we screened 300 strains by using phenotypic tests suggested for the identification of C. dubliniensis in the literature, but we detected high proportion of false-positive reactions. Only two strains (0.6%) were detected as true C. dubliniensis by PCR and API ID 32C methods. Moreover, these two strains gave the expected results with all the phenotypic tests, including modified salt tolerance test for C. dubliniensis. In conclusion, none of the phenotypic methods, except for the modified salt tolerance test, revealed 100% successful results in discrimination of C. albicans and C. dubliniensis species. However, in the tobacco agar test, the rate of false positivity was as low as 0.6%. We suggest that in the case of absence of PCR and other automatized identification systems, these two phenotypic tests can be used in routine laboratories to obtain a presumptive result.     

Characterization of three endophytic, indole-3-acetic acid-producing yeasts occurring in Populus trees

Three endophytic yeast, one isolated from stems of wild cottonwood (Populus trichocarpa), two from stems of hybrid poplar (P. trichocarpa × Populus deltoides), were characterized by analyzing three ribosomal genes, the small subunit (18S), internal transcribed spacer (ITS), and D1/D2 region of the large subunit (26S). Phenotypic characteristics of the yeast isolates were also obtained using a commercial yeast identification kit and used for assisting the species identification. The isolate from wild cottonwood was identified to be closest to species Rhodotorula graminis. The two isolates from hybrid poplar were identified to be species Rhodotorula mucilaginosa. In addition, the three yeast isolates were observed to be able to produce indole-3-acetic acid (IAA), a phytohormone which can promote plant growth, when incubated with l-tryptophan. To our knowledge, the yeast strains presented in this study were the first endophytic yeast strains isolated from species of Populus.     

Limitations of the RAPD technique in phylogeny reconstruction in Drosophila

In this study the limitations of the RAPD technique for phylogenetic analysis of very closely related and less related species of Drosophila are examined. In addition, assumptions of positional homology of amplified fragments in different species are examined by cross-hybridization of RAPD fragments. It is demonstrated that in Drosophila the use of RAPD markers is very efficient in identification of species. For assessment of phylogenetic relationships, however, the method is limited to sibling species, and reliable measures for genetic distances cannot be obtained. Hybridization experiments demonstrate that fragments of similar length amplified from different species are not always derived from corresponding loci, and that not all RAPD fragments within the same amplification pattern are independent.     

Evaluation of the Commercial Rapid Trehalose Test (GLABRATA RTT) for the Point of Isolation Identification of <Emphasis Type="Italic">Candida glabrata</Emphasis> Isolates in Primary Cultures

Candidaemias account for 10–20% of nosocomial bloodstream infections depending on the study. Whilst Candida albicans remains the most frequently isolated species, Candida glabrata may be responsible for as many as 10–25% of all candidaemias. Moreover, C. glabrata is generally less susceptible to the azole antifungals than the majority of other pathogenic yeast species. Thus, a rapid test for the specific identification of isolates of C. glabrata would be useful for patient management if it could be performed at point of isolation, on primary cultures grown on standard mycological media directly from patient specimens. Under certain conditions, C. glabrata rapidly hydrolyses trehalose into glucose. The GLABRATA RTT kit allows detection of the preformed enzyme responsible for this action. This study has assessed GLABRATA RTT as an identification tool specifically at point of isolation. Sixty test isolates were evaluated: 39 clinical isolates of C. glabrata identified at the UK Mycology Reference Laboratory, examples of the recently described genetic relatives of C. glabrata, Candida nivariensis (n = 6) and Candida bracarensis (n = 1), and a selection of other common pathogenic yeast species (n = 14). The test provided results within 30 min. Although 77% (30/39) of confirmed C. glabrata isolates were correctly identified by GLABRATA RTT (positive trehalase test), 23% (9/39) of isolates gave negative or equivocal results. All other yeast species gave negative results. The performance of GLABRATA RTT in this study is compared to previous evaluations of the test which employed isolates pre-cultured on specialised media and to other existing conventional identification methodologies.     

Molecular divergence of the soil yeasts <Emphasis Type="Italic">Williopsis</Emphasis> sensu stricto

Fifty-three strains of Saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA comprising the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, and Komagataea pratensis and the Williopsis sensu stricto complex. The minisatellite primer M13 was proposed for differentiation between sibling species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of Saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 768–776.Original Russian Text Copyright © 2004 by Naumova, Gazdiev, Naumov.     

Molecular identification of dolphinfish species (genus Coryphaena) using multiplex haplotype-specific PCR of mitochondrial DNA

A rapid and reliable method of dolphinfish species identification was designed based on PCR amplification of diagnostic DNA fragments from the mitochondrial cytochrome b gene. It consisted in a tetraplex reaction producing a positive control amplicon and species-specific fragments in Coryphaena hippurus and C. equiselis. It was successfully tested in specimens of known identity and in nominal C. hippurus samples among which two C. equiselis were discovered. This approach has significant advantages over other molecular species identification methods and may help in determining species composition of mixed catches, and in forensic and food control applications of dolphinfish specimens or products.     

Comparing Methods for Identifying Bacillus Strains Capable of Producing the Antifungal Lipopeptide Iturin A

Lipopeptides represent a unique class of bioactive microbial secondary metabolites, and iturin A shows attractive antibiotic properties among them. This study compares three methods, such as yeast/fungal growth inhibition assay, quantitative high-performance liquid chromatography (HPLC) and polymerase chain reaction (PCR) for identifying a number of Bacillus species that produce iturin A. We examined the feasibility of screening iturin A-producing Bacillus strains by PCR using specific primers for ituD and lpa-14 amplification. Twenty standard strains and 120 field-collected Bacillus spp. isolates were tested in this study. Four B. subtilis and one B. circulan strains from ATCC, and B. amyloliquefaciens B128, a known iturin A producer, exhibited positive results. Of the 120 field-collected isolates, 42 strains were positive. The potential of producing iturin A by these PCR-positive strains were then confirmed by conventional methods such as fungal growth inhibition assay and HPLC analysis. The consistency between results of PCR, HPLC, and fungal growth inhibition assay suggests that the PCR method could be used as an alternative tool for fast screening of iturin A-producing Bacillus strains from the environment. This is the first report of detecting iturin A production from B. circulans.     

Recent Taxonomic Developments with <Emphasis Type="Italic">Candida</Emphasis> and Other Opportunistic Yeasts

Increases in susceptible patient populations and advances in identification methods have resulted in the continued recognition of novel yeasts as agents of human infection. Most of these agents are members of the well-recognized genera Candida, Cryptococcus, Trichosporon, and Rhodotorula. Some of these agents are “cryptic species,” members of species complexes, and may not be detectable using classical carbohydrate assimilation-based methods of yeast identification. Such species require DNA- or MALDI-based methods for correct identification, although sporadic isolates may not routinely require delineation to the individual species level. The coming end of the fungal taxonomy rules requiring separate names for sexual and asexual forms of the same fungus will hopefully allow greater clarity, as names for medically important yeast can now be based on the needs of the medical mycology community and the common goal of better communication between laboratory and clinician.