David Patrick Overy

Spoilage fungi and their mycotoxins in commercially marketed chestnuts

A nationwide survey was carried out to assess mould spoilage of Castanea sativa nuts sold in Canadian grocery stores in 1998-99. Morphological and cultural characters, along with secondary metabolite profiles derived from thin-layer chromatography, were used to sort and identify fungi cultured from nut tissue. Three mycotoxigenic fungi dominated (Penicillium crustosum, Penicillium glabrum/spinulosum and Penicillium discolor) and were isolated at frequencies of 67.1%, 18.6% and 17.7%, respectively, from a total sample size of 350 nuts. Another mycotoxin producer, Aspergillus ochraceus was also isolated, but at a much lower frequency. HPLC and diode array detection were used to confirm the suspected presence of the mycotoxins penitrem A, chaetoglobosin A and C, emodin and ochratoxin A in extracts prepared from naturally infected nut tissue. To the best of our knowledge, this is the first time emodin has been found in a naturally contaminated food source.

New penicillin-producing Penicillium species and an overview of section Chrysogena

Species classified in Penicillium sect. Chrysogena are primary soil-borne and the most well-known members are P. chrysogenum and P. nalgiovense. Penicillium chrysogenum has received much attention because of its role in the production on penicillin and as a contaminant of indoor environments and various food and feedstuffs. Another biotechnologically important species is P. nalgiovense, which is used as a fungal starter culture for the production of fermented meat products. Previous taxonomic studies often had conflicting species circumscriptions. Here, we present a multigene analysis, combined with phenotypic characters and extrolite data, demonstrating that sect. Chrysogena consists of 18 species. Six of these are newly described here (P. allii-sativi, P. desertorum, P. goetzii, P. halotolerans, P. tardochrysogenum, P. vanluykii) and P. lanoscoeruleum was found to be an older name for P. aethiopicum. Each species produces a unique extrolite profile. The species share phenotypic characters, such as good growth on CYA supplemented with 5 % NaCl, ter- or quarterverticillate branched conidiophores and short, ampulliform phialides (< 9 μm). Conidial colours, production of ascomata and ascospores, shape and ornamentation of conidia and growth rates on other agar media are valuable for species identification. Eight species (P. allii-sativi, P. chrysogenum, P. dipodomyis, P. flavigenum, P. nalgiovense, P. rubens, P. tardochrysogenum and P. vanluykii) produce penicillin in culture.

New Penicillium species associated with bulbs and root vegetables.

Taxa of the Penicillium series Corymbifera are known for their strongly fasciculate growth and association with the rhizosphere of vegetables and flower bulbs. Using micromorphology, colony characteristics on various media and chemotaxonomic profiling, P. albocoremium sensu stricto and two new species, P. radicicola and P. tulipae, are redescribed during a taxonomic survey of P. albocoremium isolates contained within the IBT culture collection. Although these novel taxa are micromorphologically quite similar, their unique secondary metabolite profiles individually distinguish them from isolates of P. albocoremium. Moreover, the following metabolites produced by these species are known mycotoxins: citrinin, penicillic acid and terrestric acid by P. radicicola and terrestric acid and penitrem A by P. tulipae.

Andrastin A and barceloneic acid metabolites, protein farnesyl transferase inhibitors from Penicillium albocoremium : chemotaxonomic significance and pathological implications

A survey of Penicillium albocoremium was undertaken to identify potential taxonomic metabolite markers. One major and four minor metabolites were consistently produced by the 19 strains surveyed on three different media. Following purification and spectral studies, the metabolites were identified as the known protein farnesyl transferase inhibitors andrastin A (1) and barceloneic acid A (2) along with barceloneic acid B (3), barceloneic lactone (4), and methyl barceloneate (5). These compounds are significant taxonomic markers for P. albocoremium; moreover this is the first report of a methyl ester of a barceloneic acid being produced as a secondary metabolite. Tissue extracts created following pathogenicity trials involving P. albocoremium and Allium cepa confirmed the production of these five metabolites in planta. Barceloneic acid B was found to be biologically active against a P388 murine leukemia cell line.

Cryptococcus gattii VGIIb-like variant in white-tailed deer, Nova Scotia, Canada

To the Editor: Cryptococcus gattii is a fungal pathogen that is emerging in the Pacific Northwest of North America. In Nova Scotia, Canada, previously not recognized as a C. gattii–endemic area, a variant strain similar to VGIIb caused cryptococcosis with nasopulmonary, lymph node and central nervous system involvement in a free-ranging, yearling white-tailed deer (Odocoileus virginianus). The deer was found in the village of Greenwood (latitude 44.9717246; longitude −64.9341295) on July 14, 2014. The deer exhibited behavioral and neurologic abnormalities, including loss of fear of humans, ataxia, circling, high-stepping gait, torticollis, and a fixed stare. Additional clinical signs were ptyalism with frothing from the mouth and dyspnea with gurgling respiration. The animal was euthanized, and the head, lungs, heart, gastrointestinal tract, liver, and kidneys were submitted for pathologic examination. n nGross examination revealed multifocal, soft, round, expansile, pale tan masses of variable sizes that had replaced or effaced the normal architecture of the tracheobronchial lymph nodes and pulmonary parenchyma. The center of the largest lymph node mass was necrotic and filled with viscous yellow material. Similar yellow gelatinous material obliterated the right ethmoturbinates rostral to the cribriform plate. In the brain, cerebellar coning was prominent. Several small, pitted lesions with dark rims were noted in the neuropil of the thalami, superior colliculi, and hippocampus. Gross lesions were absent in the liver, kidney, and gastrointestinal tract (Technical Appendix). n nMicroscopically, the nasal cavity, lung, tracheobronchial lymph node, and brain lesions were similar, consisting of variably sized, cystic spaces supported by various thicknesses of well-differentiated fibrovascular septa or remaining normal parenchyma. The cystic spaces, immediately adjacent tissues, meninges, and ependyma contained variable numbers of yeast associated with a granulomatous inflammatory response or a pleocellular population of lymphocytes, plasma cells, macrophages, and neutrophils (Figure). The yeast were round to oval, 15–33 μm in total diameter, with poorly staining central portions (5–12 μm in diameter) surrounded by a pale acidophilic or basophilic capsule (5–21 μm thick), which stained positively with a mucicarmine stain. Some yeast were dematiaceous, and Fontana-Masson staining was consistent with presence of melanin. Very rarely, narrow-based budding was observed in the yeast. All aforementioned morphologic characteristics, staining affinities, and lesion distributions are consistent with an infection with fungi in the genus Cryptococcus (1,2). n n n nFigure n nTissue from white-tailed deer (Odocoileus virginianus), showing microscopic lesions caused by a unique Cryptococcus gattii VGIIb-like variant strain most similar to that of the VGIIb genotype; etiology was confirmed by molecular sequencing. A) Photomicrograph ... n n n nOne species of Cryptococcus was isolated from a tracheobronchial lymph node aspirate. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting of the isolate (Biotyper RTC software; Bruker Daltonics Ltd, Bremen, Germany) yielded 8 diagnostic signals consistent with C. gattii VGIIb and VGIIc; therefore, the isolate was further classified by multilocus sequence typing based on 7 genetic loci, following the International Society for Human and Animal Mycology consensus multilocus sequence typing scheme for the C. neoformans/C. gattii species complex (3). Further discrimination based on allele congruence with established C. gattii VGII genotypes (4) classified the isolate as being most similar to genotype C. gattii VGIIb (CAP59 allele no. 2, GPD1 allele no. 6, LAC1 allele no. 4, PLB1 allele no. 2, URA5 allele no. 2, IGS1 allele no. 10). However, because of a slight difference in the SOD1 allele (99.5% similarity with allele no. 15), this strain is considered to be a unique variant strain, most similar to that of the VGIIb genotype. Whole-genotyping studies have provided evidence of multiple distinct introductions of the VGIIb genotype to North America (5). Because of the observed difference in the SOD1 allele, the VGIIb-like variant strain may represent a fourth introduction or a different VGII genotype altogether. n nThe white-tailed deer represents a new host species for C. gattii in North America. Because white-tailed deer are nonmigratory, generally exhibiting only minor seasonal movements (6), this infection was considered to be autochthonous, indicating endemicity of the C. gattii VGIIb-like variant in Nova Scotia and highlighting the value of nonmigratory animals as sentinels for emerging diseases (7). Incidence for this disease is highest in the Pacific Northwest, where the primary agents are C. gattii VGII genotypes (2,4). A pertinent literature review and consultation with regional public and veterinary health authorities determined that Quebec was the most eastern province in Canada where crytococcosis associated with C. gattii VGII has caused clinical disease that was not potentially travel related in humans (Phillippe Dufresne, pers. comm.). In eastern North America, the C. gattii VGIIb genotype is reported to have caused disseminated cryptococcosis in a human in Florida, USA (8,9). Because C. gattii is potentially pervasive in the environment, the Nova Scotia Department of Health has alerted provincial infectious disease specialists and the provincial public health laboratory to ensure availability of the diagnostic capacity to test for the fungus. n nThe C. gattii VGIIb genotype causes substantial, life-threatening disease in otherwise healthy hosts (2), and a unique VGIIb-like variant is endemic to Atlantic Canada. Therefore, continued surveillance by physicians and veterinarians in the region is warranted. n nTechnical Appendix: nGross pathologic findings in white-tailed deer infected with Cryptococcus gattii VGIIb-like variant, Nova Scotia, Canada. n nClick here to view.(418K, pdf)

Dating the Cryptococcus gattii Dispersal to the North American Pacific Northwest

The recent emergence of the pathogenic fungus Cryptococcus gattii in the Pacific Northwest (PNW) resulted in numerous investigations into the epidemiological and enzootic impacts, as well as multiple genomic explorations of the three primary molecular subtypes of the fungus that were discovered. These studies lead to the general conclusion that the subtypes identified likely emerged out of Brazil. Here, we conducted genomic dating analyses to determine the ages of the various lineages seen in the PNW and propose hypothetical causes for the dispersal events. Bayesian evolutionary analysis strongly suggests that these independent fungal populations in the PNW are all 60 to 100 years old, providing a timing that is subsequent to the opening of the Panama Canal, which allowed for more direct shipping between Brazil and the western North American coastline, a possible driving event for these fungal translocation events. ABSTRACT The emergence of Cryptococcus gattii, previously regarded as a predominantly tropical pathogen, in the temperate climate of the North American Pacific Northwest (PNW) in 1999 prompted several questions. The most prevalent among these was the timing of the introduction of this pathogen to this novel environment. Here, we infer tip-dated timing estimates for the three clonal C. gattii populations observed in the PNW, VGIIa, VGIIb, and VGIIc, based on whole-genome sequencing of 134 C. gattii isolates and using Bayesian evolutionary analysis by sampling trees (BEAST). We estimated the nucleotide substitution rate for each lineage (1.59 × 10−8, 1.59 × 10−8, and 2.70 × 10−8, respectively) to be an order of magnitude higher than common neutral fungal mutation rates (2.0 × 10−9), indicating a microevolutionary rate (e.g., successive clonal generations in a laboratory) in comparison to a species’ slower, macroevolutionary rate (e.g., when using fossil records). The clonal nature of the PNW C. gattii emergence over a narrow number of years would therefore possibly explain our higher mutation rates. Our results suggest that the mean time to most recent common ancestor for all three sublineages occurred within the last 60 to 100 years. While the cause of C. gattii dispersal to the PNW is still unclear, our research estimates that the arrival is neither ancient nor very recent (i.e., <25 years ago), making a strong case for an anthropogenic introduction. IMPORTANCE The recent emergence of the pathogenic fungus Cryptococcus gattii in the Pacific Northwest (PNW) resulted in numerous investigations into the epidemiological and enzootic impacts, as well as multiple genomic explorations of the three primary molecular subtypes of the fungus that were discovered. These studies lead to the general conclusion that the subtypes identified likely emerged out of Brazil. Here, we conducted genomic dating analyses to determine the ages of the various lineages seen in the PNW and propose hypothetical causes for the dispersal events. Bayesian evolutionary analysis strongly suggests that these independent fungal populations in the PNW are all 60 to 100 years old, providing a timing that is subsequent to the opening of the Panama Canal, which allowed for more direct shipping between Brazil and the western North American coastline, a possible driving event for these fungal translocation events.