Australian medical mycology culture collections: fundamental resources for mycological diagnosis and research

Currently in Australia, there are four major medical mycology culture collections that form a close collaborative network. They provide fundamental resources for diagnosis and research and are part of the World Federation of Culture Collections.

Mycological cultures have been deliberately cultured, stored and maintained for centuries, mainly as starter cultures in fermenting food and drinks, including in the beer and wine production process. At least since the discovery of penicillin, fungal cultures have been preserved as resources of biological, pharmacological and industrial substances. At the turn of the century living fungal cultures started to be stored as an important resource for research and teaching, with the first fungal cultures having been made commercially available from the German Technical University in Prague at the end of the nineteenth century by Professor Frantisek Karl¹. Since then many countries have established government funded microbial/mycology culture collections, with the oldest being the Centraalbureau voor Schimmelcultures (now Westerdijk Institute for Fungal Biodiversity Institute) in Baarn in 2004 and now situated in Utrecht, The Netherlands¹.

In the absence of a national mycological culture collection a number of diagnostic and medical mycology research related collections have been established throughout Australia. There are currently four major medical mycology culture collections in Australia, holding together over 30 000 fungal cultures, which are briefly described below. They form a close collaborative network and are part of the World Federation of Culture Collections (WFCC; http://www.wfcc.info).

WFCC registration number: AMMRL-42; Curator: Ms Kerry Weeks; Contact: Tel: +61 2 9926 4319, Email: Kerry.Weeks@health.nsw.gov.au

The medical mycology collection of the Australian National Reference Laboratory in Medical Mycology (AMMRL) was established in 1949 at the Royal North Shore Hospital (RNSH) in Sydney, NSW, when a small number of live cultures from overseas were received, which were first stored in the Department of Bacteriology. In 1954 with the appointment of a full-time research employee, medical mycology became an officially acknowledged unit within the framework of the Department of Bacteriology. With assistance from the Institute of Medical Research, the mycology unit and its associated culture collection grew steadily. In 1964, Dr Dorothea Frey was appointed Principal Mycologist until she retired in 1982. In 1965 the Department of Medical Mycology was designated the National Reference Laboratory in Medical Mycology by the National Health and Medical Research Council (NHMRC). It was maintained by the Board of the Hospital and the Council of the Institute of Medical Research. From 1954 to 1965 it was supported by grants from the NHMRC. In 1983, the mycology laboratory was incorporated into the Microbiology Department at RNSH and is now under the mantle of NSW Health Pathology. From 1975 until 1999 Mr. David Muir was the principle Mycologist. On his retirement, Ms. Kerry Weeks was appointed as curator. The collection is part of the Atlas of Living Australia (ALA; http://www.ala.org.au).

The collection currently holds approximately 2200 clinical, veterinary and environmental strains, including 670 yeast strains, representing 84 species, 1372 filamentous fungal strains, representing 350 species, and 165 aerobic actinomycetes, which were obtained from routine clinical diagnostics, outside referred and published reference sources, both nationally and internationally. The cultures are mainly identified using classical morphological/phenotypic characteristics and some are further identified by ITS1/2 sequencing and MALDI-TOF. A genomics program is currently being developed. Isolates are stored in 5 mL sterile water vials at room temperature. Associated metadata are stored as a Microsoft Word document (currently being converted to MS Excel format) and in a Laboratory Information System (LIS). Cultures are open access and are available commercially on a fee basis and freely for collaborative teaching/research studies.

The collection has been involved in many publications involving first time reports of fungal pathogens. Recently it formed the basis for a study of the Metarhizium anisopliae species complex implemented in human disease².

WFCC registration number: ACH-47; Curator: Dr Sarah Kidd; Contact: Tel: +61 8 8222 3544, Email: Sarah Kidd@sa.gov.au

The Adelaide Children’s Hospital Mycology Culture Collection (ACH) was established by Ms. Geraldine Kaminski (née Brown) in the early 1960s in Adelaide, SA, collecting fungal cultures stored under oil and/or in sterile water. A special teaching collection of 100 cultures was maintained via monthly sub-culturing. Professor David Ellis took over the collection in 1983 and after his retirement in 2011, Dr Sarah Kidd took over the curation.

While the Adelaide Children’s Hospital became the Women’s and Children’s Hospital in 1989, and the Mycology Unit moved to the Institute of Medical and Veterinary Sciences (IMVS) in 2013 and became the National Mycology Reference Centre, under the banner of SA Pathology. The laboratory offers molecular identification and genotyping of human and animal pathogenic fungi as well as antifungal susceptibility testing. Cultures remain available to non-profit organisations for use in teaching and collaborative research. Many of the earlier isolates belong to the Cryptococcus gattii species complex and formed the basis of the fundamental studies by Professor Ellis on the environmental niche of C. gattii³. The collection has also been used to screen for multi-azole resistant Aspergillus fumigatus isolates, the first confirmed isolates in Australia⁴.

The collection now houses ~22 700 fungal cultures comprising local and referred clinical, veterinary and environmental isolates of around 80 yeast species (∼12 300 isolates) and 340 filamentous fungal species (∼10 400 isolates). The isolates were initially stored at −80°C, but new isolates are now largely stored as water cultures. Early cultures were identified by morphological/phenotypic means but are now confirmed by MALDI-TOF and ITS1/2, D1/D2 rDNA gene cluster, and/or β-tubulin sequencing as required. Electronically stored metadata include antifungal minimum inhibitory concentrations, which have been used in a number of international collaborative studies to assess susceptibility patterns and set interpretive criteria for yeast and fungal species⁵.

Curator: Mr Ian Arthur; Contact: Tel: +61 8 6383 4531, Email: Ian.Arthur@health.wa.gov.au

The medical mycology culture collection at the Department of Microbiology at Path West at the Queen Elizabeth II Medical Centre (DMFMC), Nedlands, WA was established in 1966 by Ms. Rose McAleer for reference and clinical fungal strains obtained by the Mycological Laboratory in Perth., at what was at that time ‘The State Health Laboratory Services (SHLS), WA’. It has since been transferred across to institutions that have superseded SHLS, now being Path West. It continues to accumulate clinical and quality-controlled reference strains as deemed important to store by the curator, Ian Arthur. The collection is part of the Atlas of Living Australia (ALA; http://www.ala.org.au).

The PathWest QEII culture collection currently contains 3500 human and animal pathogenic fungal strains, predominantly fungi but also Nocardia spp., which have been obtained through affiliated institutions state-wide. Early cultures were identified solely by morphological criteria, but now strains have MALDI-TOF or relevant sequence data. The cultures are stored in 15 mL sterile water suspensions in glass bottles at room temperature and as 2 mL glycerol stocks in cryogenic bottles stored at –80°C. Associated metadata are stored in an access database. Cultures are available for collaborative studies. The collection offers molecular identification and genotyping of human and animal pathogenic fungi. The laboratory provides diagnostic and referred identification services including fungal pathogens and aerobic actinomycetes.

The collection has formed the basis for a number of national studies of mycoses including the investigation of Australian Sporothrix isolates by whole genome sequence analysis⁶.

WFCC registration number: WM-1205; Curator: Professor Wieland Meyer; Contact: Tel: +61 2 8627 3430, Email: wieland.meyer@sydney.edu.au

With the establishment of the Centre for Infectious Diseases and Microbiology (CIDM) at Westmead Hospital in 1980 the first collection of fungal cultures was established by Professor Tania Sorrell forming the basis of the WM culture collection, which was formally established by Professor Wieland Meyer at the Molecular Mycology Research Laboratory (MMRL) at CIDM at the Faculty of Medicine and Health of the University of Sydney in 1995 and is currently housed at the Westmead Institute for Medical Research. The collection is partially funded by grants from the NHMRC and the Western Sydney Local Health District. The collection is part of the Atlas of Living Australia (ALA; http://www.ala.org.au).

Currently the WM culture collection holds 11 137 strains, representing 580 human and animal pathogenic fungal species, isolated from clinical, veterinary and environmental sources from 63 countries. The majority of strains were collected as part of national and international clinical, molecular epidemiological and basic science projects. It maintains 174 type cultures and the reference strains of the major molecular types of the Cryptococcus neoformans (VNI, VNII, VNB, VNIII and VNIV) and C. gattii (VGI, VGII, VGIII and VGIV) species complexes⁷. Among the 11 137 fungal cultures 9242 are yeast strains and 1671 are filamentous fungal strains. Among the 9242 yeast isolates, 5135 are C. neoformans and C. gattii species complex isolates, representing 49 out of 623 C. neoformans and 88 out of 551 C. gattii globally identified MultiLocus Sequence (MLST) Types (ST). The second largest proportion of yeasts in the collection are from Candida spp. with 2798 isolates, representing 117 species.

Strains are initially identified phenotypically and biochemically followed by either sequencing of the ITS1/2 region (primary fungal DNA barcode⁸) or the translocation elongation factor 1α (TEF1α) (secondary fungal DNA barcode⁹), and LSU D1/D2 region of the rDNA gene cluster for yeasts, or MALDI-TOF analysis. After identification, strains are subcultured onto Sabouraud dextrose agar for 48h at 30°C for yeast and 20°C for filamentous fungi to prepare long-term storage cultures. The strains are stored as lyophilised and glycerol cultures at –80°C, with a small number being also stored as living cultures at 14°C. The strain associated metadata are stored electronically using the BioloMICS software package (http://www.bio-aware.com, Hannut, Belgium). Strains are freely available for collaborative research projects or as reference strains for diagnostics and molecular typing studies. The collection offers molecular identification and genotyping of human and animal pathogenic fungi in outbreak settings.

The collection formed the basis for global molecular typing studies, especially using Multilocus Sequence Typing (MLST) (e.g. Meyer et al.⁷) and whole genome sequencing based SNP analysis for the C. neoformans and C. gattii species complexes (e.g. Firacative et al.¹⁰), Scedosporium spp.¹¹ and Pneumocystis jirovecii at http://mlst.mycologylab.org. It was also the primary resource for the development of molecular identification methods for human pathogenic fungi, especially in the development of the Dual DNA barcoding scheme, combining the ITS1/2 region of the rDNA gene cluster (primary fungal DNA barcode⁸) and the TEF1α (secondary fungal DNA barcode⁹), and it’s associated quality controlled reference sequence database (http://its.mycologylab.org or http://www.isham.org) for which more than 1000 strains are reference strains^12,13.

The main purpose of the four major Australian Medical Mycology Culture Collections is to preserve and provide the mycological research community with fungal strains and their associated metadata, representing Australian and global clinical biodiversity of human and animal pathogenic fungi and related environmental fungal isolates. In addition, these collections preserve quality controlled, well documented reference strains for validation and interlaboratory comparisons of diagnostics tests. They maintain a bank of cultures used in research studies, which provide the basis for reproducibility studies of the obtained research findings and for the inclusion of those isolates into future national and international research studies.

The authors declare no conflicts of interest.