Gerhard Haase

Multicenter evaluation of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization method for simultaneous dual-color identification of C. albicans and C. glabrata directly from blood culture bottles

ABSTRACT We evaluated the performance of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method, a rapid two-color assay for detection of C. albicans and C. glabrata, in a multicenter study. The assay is designed for use directly from positive blood culture bottles in a FISH format. Intact, fixed cells are labeled fluorescent green (C. albicans) or fluorescent red (C. glabrata) by rRNA hybridization of fluorophore-labeled PNA probes. Results are available <3 h after cultures signal positive. An evaluation of 197 routine blood culture bottles newly positive for yeast by Gram staining was performed at five hospitals. The sensitivities of detection for C. albicans, and C. glabrata were 98.7% (78/79) and 100% (37/37), respectively, and the specificity for both components of the assay was 100% (82/82). The assay was also evaluated with 70 fungal reference strains and was challenged in the BacT/ALERT microbiological detection system with spiked blood culture bottles. These results support the use of the assay for rapid, simultaneous identification of C. albicans and C. glabrata in positive blood culture bottles. This rapid assay may aid in the selection of initial antifungal drugs, leading to improved patient outcomes.

Species Diversity and Polymorphism in the Exophiala spinifera Clade Containing Opportunistic Black Yeast-Like Fungi

ABSTRACT A monophyletic group of black yeast-like fungi containing opportunistic pathogens around Exophiala spinifera is analyzed using sequences of the small-subunit (SSU) and internal transcribed spacer (ITS) domains of ribosomal DNA. The group contains yeast-like and annellidic species (anamorph genus Exophiala) in addition to sympodial taxa (anamorph genera Ramichloridium and Rhinocladiella). The new species Exophiala oligosperma, Ramichloridium basitonum, and Rhinocladiella similis are introduced and compared with their morphologically similar counterparts at larger phylogenetic distances outside the E. spinifera clade. Exophiala jeanselmei is redefined. New combinations are proposed in Exophiala: Exophiala exophialae for Phaeococcomyces exophialae and Exophiala heteromorpha for E. jeanselmei var. heteromorpha.

High prevalence of the neurotrope Exophiala dermatitidis and related oligotrophic black yeasts in sauna facilities

Summary. The black yeast Exophiala dermatitidis, an agent of fatal brain infections in East Asia, is common in European steam baths. The related fungi Sarcinomyces phaeomuriformis and Exophiala mesophila were isolated from locations in these complexes with lower ambient temperature and/or moisture. The latter two species had dry, rather than slimy, colonies and lower maximum growth temperatures (38 °C, 32 °C) than E. dermatitidis (42 °C). Exophiala dermatitidis produces abundant extracellular polysaccharide (EPS). The only E. dermatitidis strains lacking EPS were found outside the steam baths. Therefore it is likely that the extracellular polysaccharides commonly produced by E. dermatitidis are significant to survival under hot and moist conditions. Substrates sampled as controls, such as fruit surfaces and human faeces, yielded Exophiala dermatitidis at very low frequency.

Molecular Cloning and Characterization of WdPKS1, a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in Wangiella (Exophiala) dermatitidis

ABSTRACT 1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resultingwdpks1Δ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intactWdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a β-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Δ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in awdpks1Δ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.

Differentiation of Candida albicans and Candida dubliniensis by Fluorescent In Situ Hybridization with Peptide Nucleic Acid Probes

ABSTRACT The recent discovery of Candida dubliniensis as a separate species that traditionally has been identified asCandida albicans has led to the development of a variety of biochemical and molecular methods for the differentiation of these two pathogenic yeasts. rRNA sequences are well-established phylogenetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection and identification of microorganisms. Peptide nucleic acid (PNA) is a DNA mimic with improved hybridization characteristics, and the neutral backbone of PNA probes offers significant advantages in whole-cell in situ hybridization assays. In this study, we developed PNA probes targeting the rRNAs ofC. albicans and C. dubliniensis and applied them to a fluorescence in situ hybridization method (PNA FISH) for differentiation between C. albicans and C. dubliniensis. Liquid cultures were smeared onto microscope slides, heat fixed, and then hybridized for 30 min. Unhybridized PNA probe was removed by washing, and smears were examined by fluorescence microscopy. Evaluation of the PNA FISH method using smears of 79C. dubliniensis and 70 C. albicans strains showed 100% sensitivity and 100% specificity for both PNA probes. We concluded that PNA FISH is a powerful tool for the differentiation ofC. albicans and C. dubliniensis.

Direct Identification of Staphylococcus aureus from Positive Blood Culture Bottles

ABSTRACT Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes targeting Staphylococcus aureus 16S rRNA is a novel method for direct identification of S. aureus from positive blood culture bottles. The test (S. aureus PNA FISH) is performed on smears made directly from positive blood culture bottles with gram-positive cocci in clusters (GPCC) and provides results within 2.5 h. A blinded comparison of S. aureus PNA FISH with standard identification methods was performed in collaboration with eight clinical microbiology laboratories. A total of 564 routine blood culture bottles positive for GPCC recovered from both aerobic and anaerobic media from three different manufacturers (ESP, BACTEC, and BacT/Alert) were included in the study. The sensitivity and specificity of S. aureus PNA FISH were 100% (57 of 57) and 99.2% (116 of 117), respectively, with 174 GPCC-positive ESP blood culture bottles, 98.5% (67 of 68) and 98.5% (129 of 131), respectively, with 200 GPCC-positive BACTEC blood culture bottles, and 100% (74 of 74) and 99.1% (115 of 116), respectively, with 190 GPCC-positive BacT/Alert blood culture bottles. It is concluded that S. aureus PNA FISH performs well with commonly used continuously monitoring blood culture systems.

Proposed nomenclature for Pseudallescheria, Scedosporium and related genera

As a result of fundamental changes in the International Code of Nomenclature on the use of separate names for sexual and asexual stages of fungi, generic names of many groups should be reconsidered. Members of the ECMM/ISHAM working group on Pseudallescheria/Scedosporium infections herein advocate a novel nomenclature for genera and species in Pseudallescheria, Scedosporium and allied taxa. The generic names Parascedosporium, Lomentospora, Petriella, Petriellopsis, and Scedosporium are proposed for a lineage within Microascaceae with mostly Scedosporium anamorphs producing slimy, annellidic conidia. Considering that Scedosporium has priority over Pseudallescheria and that Scedosporium prolificans is phylogenetically distinct from the other Scedosporium species, some name changes are proposed. Pseudallescheria minutispora and Petriellidium desertorum are renamed as Scedosporium minutisporum and S. desertorum, respectively. Scedosporium prolificans is renamed as Lomentospora prolificans.

Phylogenetic analysis of ten black yeast species using nuclear small subunit rRNA gene sequences

The nuclear small subunit rRNA genes of authentic strains of the black yeastsExophiala dermatitidis, Wangiella dermatitidis, Sarcinomyces phaeomuriformis, Capronia mansonii, Nadsoniella nigra var.hesuelica, Phaeoannellomyces elegans, Phaeococcomyces exophialae, Exophiala jeanselmei var.jeanselmei andE. castellanii were amplified by PCR and directly sequenced. A putative secondary structure of the nuclear small subunit rRNA ofExophiala dermatitidis was predicted from the sequence data. Alignment with corresponding sequences fromNeurospora crassa andAureobasidium pullulans was performed and a phylogenetic tree was constructed using the neighbor-joining method. The obtained topology of the tree was confirmed by bootstrap analysis. Based upon this analysis all fungi studied formed a well-supported monophyletic group clustering as a sister group to one group of the Plectomycetes (Trichocomaceae and Onygenales). The analysis confirmed the close relationship postulated betweenExophiala dermatitidis, Wangiella dermatitidis andSarcinomyces phaeomuriformis. This monophyletic clade also contains the teleomorph speciesCapronia mansonii thus confirming the concept of a teleomorph connection of the genusExophiala to a member of the Herpotrichiellaceae. However,Exophiala castellanii did not belong to this clade. Therefore, this species is not the anamorph ofCapronia mansonii as it was postulated.

A co-stimulatory signal through ICAM-β 2 integrin-bindingpotentiates neutrophil phagocytosis

The β2 integrin LFA–1 (lymphocyte function associated antigen; CD11a/CD18) is the common ligand for the intercellular adhesion molecules (ICAMs). Integrins support cell function by providing co-stimulatory second signals that are a precondition for full cell activation first described for ICAM–1–binding to LFA–1 in lymphocytes. Integrins can also serve to activate functions associated with distinct subunits of other integrins. In addition to LFA–1, neutrophils express the β2 integrin Mac–1 (CD11b/CD18; CR3) that apparently contains multiple sites that bind invading microbes directly or through surface–fixed C3 (ref. 6), resulting in activation of the phagocyte function. Expression of the LFA–1 counter–receptor ICAM–1 on endothelial cells occurs only at the site of inflammation. Therefore, in neutrophils, ICAM–1 ligand binding could, as with lymphocytes, also play a part as a co–stimulatory signal to induce full phagocytotic function. We show that in neutrophils, the LFA–1 ligand interaction is the stimulatory signal to express full phagocytotic activation. This is best demonstrated by the rapid association of Streptococcus pyogenes with neutrophils, followed by ingestion, strong oxidative–burst induction and enhanced killing of these bacteria, which are well–known for their resistance to human neutrophil defense. These findings may contribute to the development of therapeutic strategies targeting the modulation of ICAM–1–leukocyte interaction.

Coniosporium perforans and C-apollinis, two new rock-inhabiting fungi isolated from marble in the Sanctuary of Delos (Cyclades, Greece)

Coniosporium perforans and C. apollinis, orginating from marble in the Mediterranean basin, are described as new species of rock inhabiting microcolonial fungi. The morphologically similar species Monodictys castaneae (Wallr.) S. Hughes, Phaeosclera dematioides Sigler et al., and a Coniosporium-like strain are compared using 18S rDNA phylogeny and Restriction Length Fragment Polymorphism analysis of Internal Transcribed Spacer regions. Sarcinomyces crustaceus Lindner is additionally compared on the basis of 18S rDNA sequencing data. Phylogenetic analysis suggests that Phaeosclera dematioides is related to the ascomycetous order Dothideales and Monodictys castaneae to the Pleosporales, whereas the three Coniosporium species studied are a sister group to the Herpotrichiellaceae (Chaetothyriales). A similar affinity was suggested previously for the recently described meristematic rock-fungus Sarcinomyces petricola Wollenzien & de Hoog. Sarcinomyces crustaceus appears unrelated to this group, and hence the present new taxa cannot be described in this genus.