No abstract

13The identification of fungal species and determination of their significance in the clinical laboratory are complex practices that help establish or exclude a fungal cause of disease. In the past, the clinical mycologist utilized a limited array of phenotypic measurements for categorizing isolates to the species level. This scenario is shifting in favor of molecular identification strategies largely due to a combination of several factors: (i) the changing landscape of epidemiology of medically important fungi, in which novel organisms never before implicated in human infection are being reported from clinical samples (10, 41); (ii) reports of species-specific differences in antifungal susceptibilities of these newly recognized fungi (4, 10, 41); (iii) numerous studies demonstrating that morphology alone may not be a sufficiently objective method for species determination (7,8,10,23,41); and (iv) a growing scarcity of bench scientists and microbiologists trained in traditional mycology. With the increasing incidence of fungal infections and reports of invasive fungal infections in nontraditional populations, such as patients with critical illnesses, the onus is on the clinical microbiologist/mycologist to return a timely and accurate identification. Molecular methods are rapid with a turnaround time of about 24 h from the time of DNA extraction, yield results that are objective with data portable between labs, and could be more economical in the long run.Few topics are more controversial or evoke such a passionate response as the term "species" to a mycologist. Molecular studies have demonstrated that a strategy where multiple genes (or portions thereof) are sequenced and the resultant data are analyzed by phylogenetic methods is a robust strategy for fungal species recognition. This concept, known as phylogenetic species recognition (PSR) (40), has been used successfully to define species in the genera Fusarium and Aspergillus (8,23,29,31,32). The advent of PSR has greatly clarified the taxonomy of these genera and as such is a powerful tool for fungal species delimitation. However, this methodology is expensive and requires phylogenetic expertise, which may be limiting factors in clinical microbiology laboratories. In reality, once a species has been delimited by PSR using several robust loci, sequence diversity within the species is known, and on the basis of this knowledge, comparative sequence analyses from a single locus can be used for rapid species identification. "Cutoff scores," which are dependent on genetic diversity within and between sibling species, can then be provided.Thus, it is important to clarify that our intent in this editorial is to address the practice of species "identification" as applied to a clinical setting and not species "classification" necessary for taxonomic categorization. Although the two terms can be overlapping, the purpose of an "identification" method in a clinical microbiology laboratory is the ability to provide a specific name or epithet to an organism rapidly and wi...

No 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.A significant portion of the species of black yeasts and their filamentous relatives, anamorphs of members of the order Chaetothyriales, are regularly encountered as causative agents of human mycoses (9). They exhibit a relatively high degree of molecular diversity (10) but seem to possess common factors which enable them to invade the human host, resulting in a bewildering diversity of mycoses, such as chromoblastomycosis, mycetoma, brain infection, and other types of phaeohyphomycosis (9). In harboring a wide array of clinically relevant species, the Chaetothyriales are unique in the fungal kingdom: they are only matched by the Onygenales, the order containing the dermatophytes and the dimorphic pathogens. Understanding the species diversity of the Chaetothyriales and their specific ecology is of considerable medical relevance.This wide species spectrum is only poorly understood, as until recently insufficient markers were available for a reliable distinction of taxa. Morphology is poorly developed in these fungi, and when present, very similar microscopic structures can be expressed in phylogenetically remote species (15). Sequencing studies of the ribosomal operon have shown that this gene can be successfully applied to species delimitation and identification. A large number of new taxa have to be introduced; many of these have a pathogenic potential.In an extended 18S ribosomal DNA (rDNA) sequencing study of black yeasts and their allies, Haase et al. (15) showed that the phylogenetic tree of the Chaetothyriales is poorly resolved, which indicates a radiation of taxa within a relatively short evolutionary period. All anamorph genera concerned proved to be polyphyletic (15); the morphological entities were nevertheless maintained for practical reasons. The single teleomorph genus in the order, Capronia, was found throughout the tree but appeared to have limited clinical relevance.

Beta‐haemolytic streptococci are important human and animal pathogens: their genetic traits that are associated with the ability to infect human hosts remain, however, unclear. The surface protein, Lmb, mediates the adherence of Streptococcus agalactiae to human laminin. For further analysis of the corresponding gene, the adjacent genomic regions were sequenced. Lmb is localized on a putative composite transposon of 16 kb and is flanked by two copies of a novel insertion sequence element (ISSag2). It harbours the genes scpB and lmb, which are 98% identical with the respective genes of Streptococcus pyogenes. Analysis of the distribution of these genes and ISSag2 among 131 streptococcal strains revealed that all of the human isolates, but only 20% (12 of 61) of the animal isolates, contained scpB and lmb or their homologues. To investigate if the putative transposon can be mobilized, an erythromycin resistance marker was incorporated into the lmb gene of S. agalactiae. Screening for mutant strains with a regained susceptibility for erythromycin identified strains with a deletion of scpB, lmb, and one copy of ISSag2. We hypothesize that a horizontal gene transfer caused the exchange of scpB and lmb and that the ability of S. pyogenes, S. agalactiae and group C and G streptococcal strains to colonize or infect human hosts is dependent on their presence.

Candida dubliniensis is often associated with C. albicans in cultures. Easy-to-perform selective isolation procedures for these closely related species do not exist. Therefore, we evaluated previously described discriminatory phenotypic markers forC. dubliniensis. A total of 150 oral rinses from human immunodeficiency virus (HIV)-infected patients were cultured on CHROMagar Candida. Dark green colonies described as being indicative ofC. dubliniensis and other green colonies, 170 in total, were isolated. Chlamydospore formation, intracellular β-d-glucosidase activity, ability to grow at 42°C, carbohydrate assimilation pattern obtained by the API ID 32C, and Fourier transform infrared (FT-IR) spectroscopy were used for phenotypic characterization. Sequencing of the 5′ end of the nuclear large-subunit (26S) ribosomal DNA gene was used for definitive species identification for C. dubliniensis. C. dubliniensis was found in 34% of yeast-colonized HIV-infected patients. The color of the colonies on CHROMagar Candida proved to be insufficient for selecting C. dubliniensis, since only 30 of 53 provenC. dubliniensis isolates showed a dark green color in primary cultures. The described typical chlamydospore formation can give only some indication of C. dubliniensis. The assimilation pattern proved to be insufficient to discriminate C. dubliniensis from C. albicans. All C. dubliniensis strains showed no or highly restricted growth at 42°C and a lack of β-d-glucosidase activity. Unfortunately, atypical C. albicans strains can also exhibit these phenotypic traits. FT-IR spectroscopy combined with hierarchical clustering proved to be as reliable as genotyping for discriminating the two species.

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 resulting wdpks1⌬ 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 intact WdPKS1 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 ␤-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 wdpks1⌬ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1⌬ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.The zoopathogenic fungus Wangiella (Exophiala) dermatitidis is one of many form species of the Fungi Imperfecti, which are darkly pigmented (dematiaceous) owing to the deposition of 1,8-dihydroxynaphthalene (1,8-DHN) melanin in their cell walls (22,45). This fungus has recently become better known as a paradigm for the causative agents of phaeohyphomycosis and other emerging dermatomycoses of humans, because of its increasing detection as a systemic pathogen in both immunocompetent and immunocompromised patients (34, 35). Moreover, because W. dermatitidis has a well-defined polymorphic nature and a well-characterized cell wall chemistry, it serves as an excellent model for the more than 100 other dematiaceous fungal pathogens of humans (14,37,42).Although dark pigments of fungi are often called melanin without regard to mode of enzymatic synthesis or chemical composition, most syntheses of melanin are attributed to either a phenoloxidase, e.g., laccases and tyrosinases, or a polyketide synthase (Pks) of a pentaketide biosynthetic pathway (52). The phenoloxidases have been found mostly among basidiomycete fungi and are usually composed of soluble enzymes with broad substra...

No abstract