Dynamics of Mixed- Candida Species Biofilms in Response to Antifungals.

Oral infections caused by Candida species, the most commonly isolated human fungal pathogen, are frequently associated with biofilms. Although Candida albicans is the predominant organism found in patients with oral thrush, a biofilm infection, there is an increasing incidence of oral colonization and infections caused by non- albicans Candida species, including C. glabrata, C. dubliniensis, and C. tropicalis, which are frequently more resistant to antifungal treatment. While single-species Candida biofilms have been well studied, considerably less is known about the dynamics of mixed- Candida species biofilms and how these dynamics are altered by antifungal treatment. To address these questions, we developed a quantitative polymerase chain reaction-based approach to determine the precise species composition of mixed- Candida species biofilms formed by clinical isolates and laboratory strains in the presence and absence of clinically relevant concentrations of 3 commonly used antifungals: fluconazole, caspofungin, and amphotericin B. In monospecies biofilms, fluconazole exposure favored growth of C. glabrata and C. tropicalis, while caspofungin generally favored significant growth of all species to a varying degree. Fluconazole was not effective against preformed mixed- Candida species biofilms while amphotericin B was potent. As a general trend, in mixed- Candida species biofilms, C. albicans lost dominance in the presence of antifungals. Interestingly, presence in mixed versus monospecies biofilms reduced susceptibility to amphotericin B for C. tropicalis and C. glabrata. Overall, our data suggest that antifungal treatment favors the growth of specific non- albicans Candida species in mixed- Candida species biofilms.

Meningoencephalitis in a dog due to Trichosporon montevideense.

Meningoencephalitis due to infection with Trichosporon montevideense was diagnosed in a 4-year-old dog with a brief clinical history of rapidly progressing neurological signs that culminated in a comatose state. No significant gross lesions were found at post-mortem examination. Microscopically, a few scattered areas of pyogranulomatous inflammation with a few small, non-pigmented fungal hyphae were found within the cerebrum surrounding the lateral ventricles. A Trichosporon sp. was identified through culture of the brain and species was determined via sequence analysis of the internal transcribed spacer region of the Trichosporon rRNA gene. DNA in-situ hybridization confirmed the diagnosis. This is the first reported case of Trichosporon-associated meningoencephalitis in a dog.

A universal DNA extraction and PCR amplification method for fungal rDNA sequence-based identification.

Accurate identification of fungal pathogens using a sequence-based approach requires an extraction method that yields template DNA pure enough for polymerase chain reaction (PCR) or other types of amplification. Therefore, the objective of this study was to develop and standardise a rapid, inexpensive DNA extraction protocol applicable to the major fungal phyla, which would yield sufficient template DNA pure enough for PCR and sequencing. A total of 519 clinical and culture collection strains, comprised of both yeast and filamentous fungi, were prepared using our extraction method to determine its applicability for PCR, which targeted the ITS and D1/D2 regions in a single PCR amplicon. All templates were successfully amplified and found to yield the correct strain identification when sequenced. This protocol could be completed in approximately 30 min and utilised a combination of physical and chemical extraction methods but did not require organic solvents nor ethanol precipitation. The method reduces the number of tube manipulations and yielded suitable template DNA for PCR amplification from all phyla that were tested.

Isolation and characterisation of the fungus Spiromastix asexualis sp. nov. from discospondylitis in a German Shepherd dog, and review of Spiromastix with the proposal of the new order Spiromastixales (Ascomycota).

The genus Spiromastix consists of several fungal species that have been isolated from soil and animal dung in various parts of the world. However, these species are considered to be of low pathogenic potential, as no cases of infections caused by these fungi have been reported. Here, we describe the clinical course of discospondylitis in a dog from which a fungus was cultured from a biopsy and identified as a Spiromastix species by morphologic characteristics and sequencing. Phylogenetic analysis determined this to be a new species, Spiromastix asexualis, which is described, and a new order, Spiromastixales, is proposed.

Phylogeny of chrysosporia infecting reptiles: proposal of the new family Nannizziopsiaceae and five new species.

We have performed a phenotypic and phylogenetic study of a set of fungi, mostly of veterinary origin, morphologically similar to the Chrysosporium asexual morph of Nannizziopsis vriesii (Onygenales, Eurotiomycetidae, Eurotiomycetes, Ascomycota). The analysis of sequences of the D1-D2 domains of the 28S rDNA, including representatives of the different families of the Onygenales, revealed that N. vriesii and relatives form a distinct lineage within that order, which is proposed as the new family Nannizziopsiaceae. The members of this family show the particular characteristic of causing skin infections in reptiles and producing hyaline, thin- and smooth-walled, small, mostly sessile 1-celled conidia and colonies with a pungent skunk-like odour. The phenotypic and multigene study results, based on ribosomal ITS region, actin and ß-tubulin sequences, demonstrated that some of the fungi included in this study were different from the known species of Nannizziopsis and Chrysosporium and are described here as new. They are N. chlamydospora, N. draconii, N. arthrosporioides, N. pluriseptata and Chrysosporium longisporum. Nannizziopsis chlamydospora is distinguished by producing chlamydospores and by its ability to grow at 5 °C. Nannizziopsis draconii is able to grow on bromocresol purple-milk solids-glucose (BCP-MS-G) agar alkalinizing the medium, is resistant to 0.2 % cycloheximide but does not grow on Sabouraud dextrose agar (SDA) with 3 % NaCl. Nannizziopsis arthrosporioides is characterised by the production of very long arthroconidia. Nannizziopsis pluriseptata produces 1- to 5-celled sessile conidia, alkalinizes the BCP-MS-G agar and grows on SDA supplemented with 5 % NaCl. Chrysosporium longisporum shows long sessile conidia (up to 13 µm) and does not produce lipase.

Isolation and characterization of a new fungal genus and species, Aphanoascella galapagosensis, from carapace keratitis of a Galapagos tortoise (Chelonoidis nigra microphyes).

A new fungal genus and species, Aphanoascella galapagosensis, recovered from carapace keratitis in a Galapagos tortoise residing in a south Texas zoological collection, is characterized and described. The presence of a pale peridium composed of textura epidermoidea surrounded by scarce Hülle cell-like chlamydospores, and the characteristic reticulate ascospores with an equatorial rim separates it from other genera within the Onygenales. The phylogenetic tree inferred from the analysis of D1/D2 sequences demonstrates that this fungus represents a new lineage within that order. As D1/D2 and ITS sequence data also shows a further separation of Aphanoascus spp. into two monophyletic groups, we propose to retain the generic name Keratinophyton for species whose ascospores are pitted and display a conspicuous equatorial rim, and thereby propose new combinations in this genus for four Aphanoascus species.

Disseminated Burkholderia gladioli infection in a lung transplant recipient with underlying hypocomplementemic urticarial vasculitis.

Burkholderia gladioli is difficult to definitively identify within the laboratory using phenotypic testing alone. We describe a case of recurrent B. gladioli infection in a lung transplant recipient with underlying hypocomplementemic urticarial vasculitis syndrome, discuss the difficulties encountered with laboratory identification, provide a review of the methodology required for definitive identification, and discuss potential pathophysiologic mechanisms in this patient responsible for the difficulty in treatment.

Insertional mutagenesis combined with an inducible filamentation phenotype reveals a conserved STE50 homologue in Cryptococcus neoformans that is required for monokaryotic fruiting and sexual reproduction.

Cryptococcus neoformans typically grows in a yeast-like morphology; however, under specific conditions the fungus can produce hyphae that are either dikaryotic or monokaryotic. In this study, we developed a simple method for inducing robust monokaryotic fruiting and combined the assay with Agrobacterium tumefaciens insertional mutagenesis to screen for hyphal mutants. A C. neoformans homologue of the Saccharomyces cerevisiae STE50 gene was identified and characterized. STE50 was found to be required for sexual reproduction and monokaryotic fruiting. Ste50p has conserved SAM and RA domains, as well as two SH3 domains specific to basidiomycetous Ste50 proteins. Analysis of protein-protein interaction showed that Ste50p can interact with Ste11p and Ste20p, and epistasis experiments placed STE50 between STE20 and STE11. Genetic analysis of the role of STE50 in sexual reproduction showed that it was required for all steps, from response to pheromone to production of hyphae. Analysis of the effect of individual Ste50p domains on sexual reproduction and monokaryotic fruiting revealed domain-specific effects for both processes. This study revealed that the C. neoformans STE50 gene has both conserved and novel functions during sexual reproduction and monokaryotic fruiting, and these functions are domain-dependent.

Disseminated Oxyporus corticola infection in a German shepherd dog.

The filamentous basidiomycetous fungus, Oxyporus corticola, has not previously been reported in the human or veterinary medical literature. Identification of this organism as the etiologic agent of fungal osteomyelitis and multiorgan dissemination in a German shepherd dog was confirmed by comparison of ITS and D1/D2 sequences with known isolates.

Strain-dependent variation in 18S ribosomal DNA Copy numbers in Aspergillus fumigatus.

Enumerating Aspergillus fumigatus CFU can be challenging since CFU determination by plate count can be difficult. CFU determination by quantitative real-time PCR (qPCR), however, is becoming increasingly common and usually relies on detecting one of the subunits of the multicopy rRNA genes. This study was undertaken to determine if ribosomal DNA (rDNA) copy number was constant or variable among different A. fumigatus isolates. FKS1 was used as a single-copy control gene and was validated against single-copy (pyrG and ARG4) and multicopy (arsC) controls. The copy numbers of the 18S rDNA subunit were then determined for a variety of isolates and were found to vary with the strain, from 38 to 91 copies per genome. Investigation of the stability of the 18S rDNA copy number after exposure to a number of different environmental and growth conditions revealed that the copy number was stable, varying less than one copy across all conditions, including in isolates recovered from an animal model. These results suggest that while the ribosomal genes are excellent targets for enumeration by qPCR, the copy number should be determined prior to using them as targets for quantitative analysis.

Interlaboratory reproducibility of a single-locus sequence-based method for strain typing of Aspergillus fumigatus.

Seven international laboratories tested the recently proposed single-locus typing strategy for Aspergillus fumigatus subtyping for interlaboratory reproducibility. Comparative sequence analyses of portions of the locus AFUA_3G08990, encoding a putative cell surface protein (denoted CSP), was performed with a panel of Aspergillus isolates. Each laboratory followed very different protocols for extraction of DNA, PCR, and sequencing. Results revealed that the CSP typing method was a reproducible and portable strain typing method.

Isolation and characterization of a new fungal species, Chrysosporium ophiodiicola, from a mycotic granuloma of a black rat snake (Elaphe obsoleta obsoleta).

Isolation and characterization of the new species Chrysosporium ophiodiicola from a mycotic granuloma of a black rat snake (Elaphe obsoleta obsoleta) are reported. Analysis of the sequences of different fragments of the ribosomal genes demonstrated that this species belongs to the Onygenales and that this species is genetically different from other morphologically similar species of Chrysosporium. This new species is unique in having both narrow and cylindrical-to-slightly clavate conidia and a strong, pungent odor.

Pulmonary Phialemonium curvatum phaeohyphomycosis in a Standard Poodle dog.

Phialemonium curvatum, frequently misidentified as an Acremonium species, is reported here as a new agent of pulmonary phaeohyphomycosis in a Standard Poodle dog, and added as a new species in the genus to cause mycoses in canines. In vitro susceptibility data, for both human and animal isolates, suggests resistance to amphotericin B and susceptibility to the triazole agents itraconazole, voriconazole, and posaconazole.

Split marker transformation increases homologous integration frequency in Cryptococcus neoformans.

Gene disruption in Cryptococcus neoformans can be problematic due to high frequencies of ectopic integration and telomerization. To improve the frequency of homologous integration, a transformation strategy was employed called split marker, which utilizes a mixture of DNAs comprised of overlapping truncations of the selectable marker. Five genes were compared for homologous integration frequencies using various constructs. Homologous integration was highest when the split marker approach was used, with rates as high as 60% depending on target gene. A second factor that contributed to an increased homologous integration frequency was strain background, which was highest when a double auxotroph was used as a host. The split marker strategy was combined with an ura-blaster construct, which has been used in other fungi to recycle ura5 or ura3 mutations. When a hisG-URA5-hisG cassette was successfully integrated at the target locus, the URA5 gene could be easily evicted by plating onto 5-FOA agar. The cassette was then successfully used for a second cycle of transformation-eviction. The effectiveness of the split marker disruption strategy suggests that continued investigation and modification of traditional molecular techniques could increase the efficiency of C. neoformans molecular manipulation.

Genotyping of Turkish environmental Cryptococcus neoformans var. neoformans isolates by pulsed field gel electrophoresis and mating type.

A total of 26 environmental Cryptococcus neoformans var. neoformans strains isolated from 634 samples of pigeon droppings collected from 54 different provinces of Turkey in 1996 and 1997 were included in this study. The results of pulsed-field gel electrophoresis (PFGE) showed that the 26 strains could be separated into 24 different PFGE patterns. In a mating-type study, of 26 strains, 20 were MATalpha, four were MATa, one was MATa/alpha and one was non-typable by STE20 specific primers. By the polymerase chain reaction typing, all the isolates were serotype A. The extensive heterogeneity among these isolates suggests that a single clonal population may not be present in Turkey. Additionally, the presence of an AMATa/DMATalpha hybrid may indicate the existence of strains that are AMATa mating type in Turkish environment.

Molecular epidemiology of clinical Cryptococcus neoformans strains from India.

Little is known about the molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans in India, a country now in the midst of an epidemic of AIDS-related cryptococcosis. We studied 57 clinical isolates from several regions in India, of which 51 were C. neoformans var. grubii, 1 was C. neoformans var. neoformans, and 5 were C. neoformans var. gattii. This strain set included 18 additional sequential isolates from 14 patients. Strains were characterized phenotypically by measuring the polysaccharide capsule and by determining the MICs of standard antifungals. Molecular typing was performed by a PCR-based method using the minisatellite-specific core sequence (M13), by electrophoretic karyotyping, by restriction fragment length polymorphisms with the C. neoformans transposon 1 (TCN-1), and by URA5 DNA sequence analysis. Overall, Indian isolates were less heterogeneous than isolates from other regions and included a subset that clustered into one group based on URA5 DNA sequence analysis. In summary, our results demonstrate (i) differences in genetic diversity of C. neoformans isolates from India compared to isolates from other regions in the world; (ii) that DNA typing with the TCN-1 probe can adequately distinguish C. neoformans var. grubii strains; (iii) that TCN-1 sequences are absent in many C. neoformans var. gattii strains, supporting previous studies indicating that these strains have a limited geographical dispersal; and (iv) that human cryptococcal infection can be associated with microevolution of the infecting strain and by simultaneous coinfection with two distinct C. neoformans strains.

Comparative analysis of pathogenicity of Cryptococcus neoformans serotypes A, D and AD in murine cryptococcosis.

OBJECTIVES: To characterize the pathogenicity of 15 strains of Cryptococcus neoformans belonging to several serotype/mating type allele patterns (Dalpha, Da, A(alpha), A(a), A(alpha)/D(a) and D(alpha)/A(a)) in experimental models of murine cryptococcosis. METHODS: CD1-infected mice were examined for survival and fungal loads in either brain or lung during the course of infection. RESULTS: All strains, with the exception of one Da strain, produced melanin in vitro. Similarly, all strains were encapsulated and produced phospholipase. When CD1 mice were challenged intravenously (i.v.) with 5x10(5)CFU/mouse and observed for 60 days post-infection, a significant variation of mortality rate was observed among mice infected with different strains. A(alpha) and A(alpha)/D(a) strains all produced 100% mortality within the study period with mean survivals significantly shorter than those of mice infected with strains belonging to any other allele type (P<0.0001). A wide range of pathogenicity was shown by haploid and diploid strains presenting D(alpha) allele. This finding was confirmed by an intranasal model of challenge. To investigate the progression of infection, the mice were challenged i.v. with 5x10(4)CFU/mouse and tissue burden experiments (brain and lung) were performed on days 6 and 12 post-infection. Only the mice infected with A(alpha) and A(alpha)/D(a) strains showed a >1 log(10) increase of CFU/g in both tissues throughout the study period. CONCLUSIONS: Our results suggest that the presence of the A(alpha) mating type allele in either haploid or diploid strains is correlated with virulence, while the presence of the A(a) or D(a) allele in haploid strains is associated with moderate or no virulence. Finally, either haploid or diploid strains presenting D(alpha) allele vary in virulence.

Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans.

The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.

Simple chemical extraction method for DNA isolation from Aspergillus fumigatus and other Aspergillus species.

DNA was successfully isolated from numerous Aspergillus spp. by use of a commercial kit. DNA that was easily digested and yielded PCR products up to 8.5 kb in size was recovered from broth or agar cultures. The ease and speed of this protocol provide an alternative to physical methods of DNA isolation.