bccm activity report 2009-2012

BCCM Activity report 2009-2012

Foreword by Philippe Courard, State Secretary for Science Policy


BCCM, the Belgian Biological Resource Centre

In 1983 the Council of Ministers decided to bring the microbial resources and the expertise available in different Belgian institutes together in a network of culture collections: with this the consortium of Belgian Co-ordinated Collections of Micro-organisms (BCCM) saw the light of day. Indeed, the Council of Ministers sought to set up an efficient and specialised infrastructure to support the many (academic and industrial) researchers in the fields of biotechnology and life sciences, which are of vital importance to Belgium.

Today, 30 years later, the BCCM consortium has grown to become one of the most important culture collections in the world, both in terms of the size and quality of the collections (bacteria, yeasts, fungi, plasmids, diatoms, DNA libraries) and its expertise. Not only does the consortium keep more than 64,000 quality controlled, characterised and documented units of biological material, but it also offers its expertise through services and partnership projects.

The Organisation for Economic Cooperation and Development (OECD) has recognised the importance of biological diversity and microbial culture collections to the economy and research in the fields of healthcare, agriculture, food, environment, etc. This is because the promise held by the development of biotechnology and environment technology can only be fulfilled with appropriate support from Biological Resource Centres (BRCs). By developing the activities of the BCCM consortium Belgium will be in a position to satisfy the OECD’s recommendations on BRCs.

Under a Belgian Government initiative the World Intellectual Property Organization (WIPO) has recognised the BCCM consortium as an International Depositary under the Budapest Treaty on the International Recognition of Deposit of Microorganisms for Patent Procedure. The BCCM contributes to the innovation process by accepting and storing deposits of the biological materials referred to in patent applications.

Within the framework of the Convention on Biological Diversity the BCCM consortium pioneered the development of a code of conduct for microbial culture

collections covering both access to microbial genetic resources and opportunities to share the benefits arising from their use. This MOSAICC1 code of conduct, which came about with the support of the European Commission, was very highly valued by the Secretariat of the CBD and generally considered as one of the predecessors to the Bonn guidelines. The BCCM carries its proactive approach of the Nagoya protocol’s "access and benefit sharing" clauses forward in the TRUST2 project, in which international experts develop specific guidelines from these clauses for microbial culture collections.

All of the above makes the BCCM consortium a much sought after partner in European partnership projects. And so it goes without saying that the BCCM takes part in the MIRRI3 project, in the framework of the European Strategic Forum for Research Infrastructure (ESFRI). This project lays the foundations for a European, decentralized infrastructure for microbial genetic resources. Close cooperation between the European culture collections will benefit the many users and help stimulate the further development of biotechnology.

I wish the BCCM consortium many more years to come...

Philippe CourardState Secretary for Science Policy

1 MOSAICC: Micro-Organisms Sustainable use and Access regulation International Code of Conduct 2 TRUST: TRansparant User-friendly System of Transfer3 MIRRI: MIcrobial Resources Research Infrastructure

Foreword by Philippe Mettens, President of the Committee of Directors


BCCM, a treasure trove of biological material

It will have escaped no one’s attention that the Federal Science Policy Office is expert in the management of collections. And not just the artistic, natural science and historical collections held by the Federal Science Institutes, but management of the live collections of micro-organisms held by the BCCM consortium. Indeed, this consortium of seven culture collections coordinated and financed by the Federal Science Policy Office looks after more than 64,000 specimens of biological material in its public collections. It is a professional centre that stores and manages microbial diversity by quality methods and offers the material as a reference for further study and valorisation.

Micro-organisms are an important raw material in biotechnology. The properties of bacteria, fungi, yeasts and diatoms are used in countless industrial applications and processes. Consider, for example, fermentation processes and the use of probiotics in foods, the production of antibiotics in medicine, the use of microorganisms as growth promoting elements in agriculture and aids to bioremediation on polluted sites, etc. Not only that, but we do not yet know the properties of every microbial species. Therefore the public collections held by the BCCM truly are a treasure trove of biological material, which can be explored through screening projects, for example.

Since federal funding began in 1983 the BCCM collections have developed from individual, research-based collections to a consortium of public, service-oriented BRCs. The scientific expertise of the BCCM is offered in a variety of forms, from specialised services to extensive scientific collaboration projects. Every year about 700 clients call on services provided by the BCCM. These involve the identification of strains, characterisation of strain genotypes by molecular techniques, microbial counts, screening projects, etc. The BCCM collections have also been involved in more than 40 formal scientific collaboration projects in recent years.

The BCCM’s policy and strategy are set within the consortium and each and every entity plays a part in their implementation. A special role is set aside for the BCCM coordination unit of the Federal Science Policy Office. This unit coordinates the quality control, information technology, external and internal communication, international cooperation and legal aspects of the collections. The BCCM consortium attaches a great deal of importance to the quality of the services it provides. In 2005 the consortium’s quality management system was ISO 9001 certified for the acquisition, control, preservation, storage and distribution of biological material and relevant information within the framework of public depots, safe depots and patent depots. The coordination unit’s coordination, project management and representation processes are also ISO 9001 certified.

This certification was the driver behind the quality control system set up by the Federal Science Policy Office, in which all Federal Science Institutes will be integrated at a later stage.

It is from this perspective that the BCCM aims to continue to meet the needs of its clients and partners. Let this activity report be the first step in developing a continuing dialogue with all stakeholders.

Philippe MettensPresident of the Committee of DirectorsFederal Science Policy Office


BCCM mission and vision....................................................................................8

BCCM structure ...................................................................................................8

BCCM services ....................................................................................................9

The BCCM multi-site quality management system .........................................10

Information Management at BCCM ..................................................................11

International cooperation and legal issues ......................................................12

Biodiversity .......................................................................................................14

Fostering bacterial biodiversity .........................................................................16

Dermatophytes ..................................................................................................17

Polypores ...........................................................................................................18

Fusarium ............................................................................................................19

Exploring yeast diversity....................................................................................20

Diatoms ..............................................................................................................21

Cyanobacteria ....................................................................................................22

Mycobacteria .....................................................................................................23

Culture collections for science and industry .................................................24

Microbial diversity in spontaneous food fermentations ...................................26

Microbial characterization of probiotics ...........................................................27

Plant associated organisms..............................................................................28

Antibiotic resistance (ITM) .................................................................................29

Fusarium as a human pathogen .......................................................................30

Pathogenic genes ..............................................................................................31

Detection of Bacillus contamination in gelatin ................................................32

Challenges in mycorrhizal preservation ...........................................................33

Challenges in preservation: methane-oxidizing bacteria ................................34

Recombinant plasmids as biotechnological tools ...........................................35

MALDI-TOF mass spectrometry: a straightforward tool for identification

and preservation quality control of microbes ...................................................36

BCCM facts & figures........................................................................................38

The BCCM patrimony ........................................................................................39

Distribution of material .....................................................................................42

Other scientific services ....................................................................................42

Research projects ..............................................................................................43

The BCCM budget..............................................................................................43

New taxa described in BCCM ............................................................................44

Bibliography .......................................................................................................46

Contacts .............................................................................................................51

Table of content


BCCM mission and vision

Our vision"BCCM, great at small things"

To be a solution partner providing quality services in microbial and genetic resources for academia and industry

Our mission The BCCM consortium aims to:

• acquire, preserve and distribute microbial and genetic resources,• identify and characterise these biological materials,• offer customer-oriented services,• increase the understanding of microorganisms and their function in ecosystems,• foster the application of biological resources.

BCCM strategy The BCCM consortium brings together 7 decentralised biological resource centres with complementary expertise.

BCCM:• continues to upgrade its service portfolio and scientific expertise;• develops a powerful data management system;• runs a multi-site ISO 9001 quality management system; • fosters (inter)national collaboration, linkage and exchange;• complies with (inter)national regulations regarding the handling of microbial and

genetic resources..

BCCM structure

The BCCM consortium is composed of 7 decentralised culture collections that are coordinated by a central team at the Belgian Science Policy Office. Each collection is part of a binomial with its host laboratory. The close cooperation between these two partners leads to an optimal balance between research and conservation activities.

Moreover, the cooperation between the collections has as its aim the exchange and implementation of best practices for the conservation of microbial genetic resources and the harmonised application of international standards and regulations in microbiology.

In close collaboration with the host institutes and a recurrent funding programme from the Belgian Science Policy Office to support biotechnology and life sciences, about 70 people study and conserve the biodiversity present in the BCCM collections.

BCCM/MUCLEnvironmental and applied Mycology

Université Catholique de Louvain

BCCM/ULCCyanobacteriaUniversity of Liège

BCCM/ITMMycobacteria

Institute of Tropical Medicine, Antwerp

BCCM/DCGDiatomsGhent University

BCCM/IHEMBiomedical Fungi & Yeasts

Scientific Institute of Public Health, Brussels

BCCM/LMBPPlasmidsGhent University

BCCM/LMGBacteria

Ghent University

BCCM/CCCoordination CellBelgian Science Policy Office


BCCM services

Are you looking for biological material?

Go to the BCCM online catalogue: http://bccm.belspo.be

• Subsets of specific interest available: - test and control strains, type strains- isolates from clinical cases, antimicrobial resistant strains, mycobacteria,

medical fungi, etc.- lactic acid bacteria, bifidobacteria, probiotics, brewing yeasts, etc.- degraders- producers of secondary metabolites, additives, aquaculture inoculants, etc. - full genome strains

• Distribution of expression vectors or empty backbone vectors• Custom cloning of expression vectors• Customised subsets for screening under special conditions• Selected sets of strains for educational purposes at reduced prices• Inactivated material available to meet your bio-safety requirements

Need to preserve isolates or plasmids?

BCCM preserves and keeps biological material in quality controlled conditions.

• Free public deposit under a unique access number in online BCCM catalogues• Restricted access deposits:

- safe deposit - patent deposit - storage of third party material

Want to know your cultures?

Make use of the BCCM characterisation expertise.

• Identification: molecular biology – physiology – morphology – taxonomy • Research: biodiversity – taxonomy – functional properties• Bioassays: resistance – inhibition • Tailor-made approaches • Food and agricultural products and processes: quality – safety – monitoring • Plasmid profile analysis• Screenings (e.g. genomic) for properties of interest

Looking for training?

BCCM provides training for groups or individuals.

• On isolation, cultivation and preservation techniques• On identification, polyphasic taxonomy and classification

Require more specific services?

Ask BCCM for consultancy & contract research.

BCCM/CCCoordination CellBelgian Science Policy Office


The BCCM consortium regards it as essential to meet the expectations of its customers and stakeholders and seeks to ensure the quality of its biological material and related information. Hence it was one of the first European culture collections to implement a formal quality management system.

Since July 2005 the BCCM quality management system has been certified as meeting the requirements of the international ISO 9001 standard. The multi-site certificate covers the following core activities of the collections, as well as those of the coordination cell:• Access to, control, preservation, storage and supply of biological material and

related information in public deposits, safe deposits and patent deposits;• Coordination, project management and representation of the BCCM consortium..

The BCCM quality management system is centrally administered and built around a unique quality policy and a single strategic plan with objectives that are shared by all the BCCM entities. It is subject to local as well as central management review and is monitored according to common performance indicators. The unique core documents of the quality management system are complemented by harmonised, specific local documents at each site. Planning of internal audits is monitored centrally. Dealing with complaints, non-conformities, corrective and preventive actions and improvements is also identical for all BCCM entities.

At regular intervals the BCCM consortium organises client satisfaction surveys in order to get feedback from its users on the quality of the services delivered. To anticipate future users' needs, the BCCM consortium strives to have an open and constructive dialogue with its users.

BCCM/IHEM and BCCM/ITM are active in the medical field and have implemented an additional quality management system. At BCCM/IHEM the quality controls to test viability, identity, fungal and bacterial purity are accredited according to the ISO 17025 standard. At BCCM/ITM the viability, identity and mycobacterial purity checks on batches for release are accredited according to the ISO 15189 standard.

The BCCM quality management system will be further developed in the drive for continuous improvement. In this sense, all your input and feedback is greatly appreciated.

The BCCM multi-site quality management system


Since their creation the BCCM collections have managed the information related to the preserved biological material. In the 1980s the digitisation of their culture catalogues in related databases began, and in 1989 a first printed version of the BCCM catalogues was published.

In addition to catalogue data, the BCCM collections also progressively digitised other data for daily management of the collections (stock management, clients/services, operations, equipment...) as well as literature and scientific data (sequences, fatty acids...). BCCM is currently finalising the implementation of a centralised Laboratory Information Management System (LIMS) that will be used by all the BCCM collections for managing, storing and querying their digital contents.

In 1995 BCCM created their website (bccm.belspo.be), where an online version of all the BCCM catalogues is published and where general information, the list of services, the newsletter, detailed information on the Budapest Treaty and patent deposition and on-going projects can be found.

The BCCM website has been updated and will gradually be further improved to include the latest functionalities for online communication and a 'one-stop-shop' that will make it possible for customers to register and track orders of biological material.

Information Management at BCCM

BCCM actively participated in a number of initiatives aimed at creating data standards for microbial information exchange and linking: Microbial Information Network Europe project (MINE), Common Access to Biological Resources and Information (CABRI, www.cabri.org), European Biological Resources Centres Network (EBRCN, www.ebrcn.net) and the OECD Task Force on Biological Resources Centres.

The BCCM collections also exchange their catalogue data in a standardised way with several other international initiatives and biodiversity information infrastructures, such as the Global Biodiversity Information Facility (GBIF, www.gbif.org), the Global Catalogue of Microorganisms (gcm.wfcc.info) and StrainInfo (www.straininfo.net).


The BCCM consortium has developed long term cooperative links with partners worldwide. Collaborations are built both between the BCCM consortium as a whole or between individual BCCM collections and partners abroad.

The objective of BCCM is the mutual enrichment of the holdings and expertise of every partner through capacity building programmes and research projects, seeking ex situ conservation and valorisation of microbial genetic resources.

Development of new international rules has greatly impacted on the operations of culture collections. Since 1983 BCCM has proactively developed legal and technical solutions in order to conform to the evolving legal framework, especially the Convention on Biological Diversity1(CBD) and the Nagoya Protocol on Access and Benefit Sharing to the Convention on Biological Diversity (Nagoya Protocol)2. BCCM combines its approach to these international agreements with other relevant rules, mainly the TRIPs Agreement3 and the Budapest Treaty4. In this context, BCCM is also recognised as an International Depository Authority entitled to accept microbial material for the purposes of patent procedure.

BCCM participates in and initiates collaborations to develop a coherent set of legal and administrative instruments and recommendations facilitating access to microbial genetic resources. Secured access to microbial material and reliable data integration is also a prerequisite for the cumulative research that underpins bioscience. Therefore BCCM is actively seeking balanced cooperation with providers, as well as with users, of microbial genetic resources and related information.

BCCM launched the concerted action ‘MOSAICC’ (Micro-Organisms Sustainable use and Access regulation International Code of Conduct). It translates the principles of the CBD into practical procedures. Funded by the European Commission's Directorate-General for Research, MOSAICC is a voluntary code of conduct. Regularly updated, the MOSAICC project gets input from multiple socio-economic partners. Predating the Nagoya Protocol negotiations, MOSAICC is now revised through the TRUST project to set up a Transparent User-friendly System of Transfer for Science & Technology to enforce the Nagoya Protocol when it enters into force in the near future.

Considering their mission to provide scientifically, technically and also legally fit-for-use material and related information, and to protect the rights of their partners, the BCCM collections were among the first in Europe to draw up their legal policy and set a Material Transfer Agreement as general conditions of distribution of microbial material to customers worldwide. It has also defined its policy of acquisition of microbial resources to meet the legitimate expectation of depositors in terms of scientific or commercial return for their contribution to science with common social benefits.

BCCM collections have contacts worldwide through bilateral and multilateral projects. Collaborations include, for instance, one with partners from the People’s Republic of China, under the terms of a bilateral agreement and according to the principles of article 7 of the CBD, to promote the identification and monitoring of biodiversity. Similarly, under the terms of another bilateral agreement with the Kingdom of Morocco, BCCM has contributed, as an effective implementation of the directives of CBD article 9 on the ex situ conservation of biological resources, to the setting up of the Moroccan Coordinated Collections of Microorganisms.. These are only two examples of many connections on all seven continents. With regard to the general global evolution of biological resource centres, in which bioinformatics is essential, BCCM seeks the development and optimal use of ICT instruments designed to facilitate exchange of microbial genetic resources, related

International cooperation and legal issues

1 CBD (Rio de Janeiro, 5 June 1992). This convention lays down new principles governing, among others, conservation, sustainable use as well as access to genetic resources and fair sharing of benefits arising from the use of these resources.2 Nagoya Protocol Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity.3 Agreement on Trade-Related Aspects of Intellectual Property Rights (Marrakech, 15 April 1994). It develops standards and principles concerning the availability, scope and use of trade-related intellectual property rights, and sets up rules for effective and appropriate enforcement of these rights applying also to the use of biological resources.4Budapest Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure (Budapest, 28 April 1977). It sets practical rules for patent procedure involving micro-organisms.


data and information, technology and know-how. Therefore BCCM has participated in numerous projects aimed at boosting the E-management and study of microbiological resources. It started with the CABRI project (Common Access to Biological Resources and Information) to launch a first common public web-based catalogue at European level, and currently continues with its contribution to the ambitious WFCC Global Catalogue of Microorganisms programme.

In the European Union BCCM has been active in culture collection networking, participating in successive structuring projects. The aim of the latest programme is a permanent European research infrastructure dedicated to microbial resources called MIRRI, or Microbial Resources Research Infrastructure.

Furthermore, BCCM - as a consortium or through its individual collections - is involved in different forums and networks; it participates in international organisations such as the European Culture Collections' Organisation (ECCO). Recognised by its peers for its specific experience in networking management, the BCCM consortium has already seen two of its staff members elected as President of the World Federation for Culture Collections (WFCC) and still contributes to UNESCO Life sciences programmes, including the Microbial Resources Centres Network (MIRCEN).

A strong national base in research institutions and a broad scientific network worldwide increase BCCM's capacity to meet the future bio-economy needs, being a reliable interface between public and private scientists both at home and abroad.


Biodiversity


Biodiversity refers to the wide variety of ecosystems and living organisms, their habitats and their genes.

The greatest diversity of life is found in the world of micro-organisms. An estimated 1 million bacterial species and 5.1 million fungal species exist on this planet, of which as yet fewer than 1% and 5% respectively have been described.

Micro-organisms are found in all imaginable habitats, ranging from sub-freezing temperatures to hot springs, from the high Rocky Mountains to the deepest oceans, and producing a diversity of bioactive compounds, ranging from toxins or antibiotics to industrial enzymes and dyes. They can be harmful or helpful to humans; can exist in symbiotic relationship with humans or simply have no interaction.

Many of them are difficult to isolate and culture, despite evidence of their existence. Even if a strain cannot be cultured, its presence can be confirmed by PCR-technology. Specific genes can be directly cloned from soil and water samples and the entire genome can be reconstructed. However, in the absence of the living material, very little can be known about the properties of the strain as an organism.

The study of microbial life is of paramount importance, as it forms the backbone of biotechnology and life sciences.

The Belgian Co-ordinated Collections of Micro-organisms, BCCM, is a centre of excellence which combines expertise in conservation and preservation of microbial diversity with quality in scientific services for academia and industry and which uses focused research to constantly increase its understanding of microorganisms and their functions in ecosystems.

A huge patrimony of 64,000 of micro-organisms kept in the BCCM collections is at the disposal of Biotechnology and life sciences. At the end of 2012, the total amount of genera was approximately 2,300 and 9,900 for the species. This encomprises about 4,700 bacterial species (including the Mycobacteria and Cyanobacteria), about 5,000 fungal species, 25 species of Diatoms and 2,150 plasmids.

Each year a considerable number of new species are described in the BCCM collections (see list at the end).Potential for new applications...

In the following chapter some of the important sub-collections present at BCCM are described.


Fostering bacterial biodiversity

The BCCM/LMG bacteria collection has over 25,000 bacterial strains in its stores, of which 5,000 are type strains (i.e. about 5% of all recognised type strains) and hence the bulk of the collection consists of non-type strains. Type strains are reference strains for bacterial systematics and are the name bearers of a species. A bacterial species consists of strains (isolates) that show some degree of similarity to each other and versus the type strain. The molecular definition of the bacterial species is based on a 97-98% sequence similarity of 16S rRNA gene and on 70% DNA relatedness as measured by DNA:DNA hybridisations, or on an ANI (Average Nucleotide Identity) value of 96% as based on comparative analysis of whole genome sequences.

The 25,000 bacterial strains in the BCCM/LMG bacteria collection consist of a number of diverse sub-collections that originate from collaboration between the BCCM/LMG bacteria collection and the various research groups of the Laboratory of Microbiology (hosting the BCCM/LMG bacteria collection) or from services provided by the BCCM/LMG bacteria collection. The bacterial taxa present in these sub-collections are: Pseudomonas and relatives, Bacillus and relatives, Burkholderia and relatives, Acetobacteriaceae, lactic acid bacteria (genera Bifidobacterium, Enterococcus, Lactococcus, Leuconostoc, Lactobacillus etc), plant growth promoting bacteria (Rhizobiaceae) and clinically associated bacteria (Acinetobacter, Arcobacter, Campylobacter, Helicobacter). There is also an extensive set of strains belonging to the Enterobacteriaceae, with emphasis on plant pathogenic members belonging to the genera Pantoea, Pectobacterium, Erwinia, Dickeya etc. Finally, there are plant pathogens with quarantine status belonging to Clavibacter, Xanthomonas and Ralstonia.

Having immediate access to such extensive sub-collections is of utmost importance in supporting a robust and reliable taxonomic framework, as the intra-taxon variability can be verified and taken into account for species delineation. This then leads to a better identification scheme, and it is precisely in these groups of organisms that the BCCM/LMG bacteria collection occupies a strong position for identification and typing.

An example of how the BCCM/LMG bacteria collection and the LM-UGent have contributed substantially to improving the existing taxonomy concerning plant pathogenic bacteria. Two groups were envisaged. The first group concerns the so-called quarantine bacterial species belonging to the genera Clavibacter, Xanthomonas, Ralstonia and Xylella

(A2 list of EPPO see http://www.eppo.int/QUARANTINE/listA2.htm). This study was part of an EU project (QBOL) that aimed to develop bar coding sequences for identification. The data obtained resulted in an identification scheme that is meant in the first place for NPPOs (National Plant Protection Organisations), which are the centres for identification of pest organisms in the EU. Access to the ID schemes and bar coding data is via the Q-bank bioportal (http://www.q-bank.eu/) that also refers, in the case of pest bacteria, to the reference biological material (quality checked reference strains) that is managed by three different collections (BCCM/LMG bacterial collection, CFBP –Collection Française de Bactéries Phyto pathogène and the NCPPB – The National Collection of Plant Pathogenic Bacteria of the UK) in a consortium called Q-Bacco Net.

The second group of plant pathogenic bacteria that has been intensively studied contains the various plant pathogens that belong to the Enterobacteriaceae. This study extended over a long period of time (Zaluga et al. 2011, 2013, Brady et al. 2010a, 2010b, 2012). An MLSA scheme based on four different genes (atpD, GyrB, rpoB, infB) enables differentiating at the generic level, resulting in the splitting of genera, e.g. Erwinia and Enterobacter and the creation of new genera (Brady et al. 2008, 2013). Species are delineated by DNA based methods as well as phenotypically. There is no doubt that this contribution has had a major impact on creating a more stable identification that needs further development at the strain level.

Plant pathogenic bacteria:Plant pathogenic bacteria are responsible for major economic losses in various agricultural crops. EU quarantine regulations for pests demand accurate identification of the causal organisms. The BCCM/LMG bacteria collection contributed in a major way to this process of identification by supporting the new bar-coding based identification schemes developed in an EU context by offering continual access to authentic reference cultures.The taxonomy of the plant pathogens that belong to the Enterobacteriaceae is complex and confusing because it is based on unstable phenotypic characteristics. The BCCM/LMG bacteria collection has developed an MLSA based taxonomic and identification scheme that now offers a more robust taxonomic context for reliable identifications.


Dermatophytes

The kingdom of Fungi includes unicellular yeasts, filamentous fungi and mushroom-producing species. Currently, about 100,000 species are known, but the latest estimates suggest a diversity of 5.1 million fungal species. Its diversity is therefore still largely unknown, though hundreds of new species are being discovered every year. Many species play an important ecological role, such as saprotrophs, which break down plant debris or other organic matter, thus recycling nutrients back into the ecosystem. Other species are vital symbionts for plants or animals. A minority live as parasites on other organisms. One such group of medical interest are dermatophytes, which can cause superficial mycoses (e.g. skin diseases) in humans and animals. They consist of a group of closely related species belonging to the Onygenalean order. Such superficial mycoses are mostly localised, e.g. on the scalp (called tinea capitis), on the feet (called tinea pedis) or cause nail problems (called onychomycosis). The differing clinical aspects are typically related to different species of dermatophytes. Regional shifts in the prevalence of individual species could be linked to trends in human migrations.

The BCCM/IHEM collection focuses on preserving fungi of biomedical significance: pathogens, allergenic species or toxin producers. The collection also spans a wide range of fungal biodiversity because many mycoses are in fact opportunistic infections. This is not the case for the dermatophytes, which are adapted to break down keratin. BCCM/IHEM holds over 2,500 strains of dermatophytes, covering the majority of the approximately 30 clinically relevant species. Many are of tropical origin and appear thanks to the recent integration of the Raymond Vanbreuseghem (RV) collection, which used to be housed at the Institute of Tropical Medicine in Antwerp.

The general principle of the research at BCCM/IHEM is based on identifying and characterising fungal pathogens. This can be within the framework of a clinical case study, often in collaboration with clinicians, for instance a case of canine infection by Trichophyton rubrum (Van Rooij et al 2012) or an onychomycosis by T. simii (Beguin et al

2013). On a second level, studies are performed that reassess species delimitation by phylogenetic analysis of genetic markers, or by mass spectrometry of protein extracts. This has already been done for the Trichophyton mentagrophytes complex (Beguin et al. 2012), but also for non-dermatophytes like the Candida parapsilosis group (Hendrickx et al 2011) or the Aspergillus niger group (Hendrickx et al 2012). A third level of research aims to develop or optimise new techniques for fungal species identification (Maldi-tof identification of dermatophytes, but currently also new techniques investigated for Mucorales and Fusarium).

The Trichophyton mentagrophytes complexA phylogenetic and taxonomic study of the T. mentagrophytes species complex was performed using multiple specimens of various origins of all taxa belonging to this complex. Our results show that the controversial T. quinckeanum is a species in its own right despite morphological similarity to T. mentagrophytes. Trichophyton quinckeanum is not only genetically very distinct, but also differs in being rare and zoophilic rather than common and anthropophilic. The confusion between the two species was also due to a poorly chosen neo-type for T. mentagrophytes, which now turns out to belong to T. quinckeanum.


Polypores

Fungi represent an essential component of biological diversity and are involved in many ecosystem services. The vast majority are assumed to play a key ecological role as they participate in the turnover of all organic matter, nitrogen and other nutrients. Wood-decaying fungi are particularly significant in forest ecosystem functioning; they are one of the main actors in the turnover of woody biomass. They are also taxonomically and phylogenetically very diverse and have developed many physiological adaptations to colonise and use almost all woody niches in the forest, from below-ground root systems to the small branches at canopy level. They have specific enzymatic systems able to deal with this woody biomass, especially the biodegradation-recalcitrant polymers such as lignin. Such enzymatic systems also have high potential for biotechnological applications.

The BCCM/MUCL collection is keen to promote knowledge and use of a broad fungal diversity, including wood-decaying fungi. While the collection is known for its long-standing gathering of food-related fungi and yeast, in the last 20 years wood-decaying Basidiomycetes have become a “hot” topic at BCCM/MUCL. The goal of our studies is to fill the gap in knowledge of the wood-decaying Basidiomycetes, describing their taxonomic, phylogenetic and functional diversity. Links to applied research fields and industry were also considered highly important and valuable.

BCCM/MUCL has focused on several representative, complementary functional groups of wood-decaying fungi with different life and nutritional strategies within the Polyporaceae and Hymenochaetaceae: saprophytic species growing on dead wood and parasitic species growing on living trees, causing both white and brown rot. In parallel, we investigated the relationships with phylogenetically closely related symbiotic, ectomycorrhizal species.

BCCM/MUCL also explores areas known as biodiversity “hotspots” that remain “terrae incognitae” as far as Fungi are concerned, such as the western edge of the Guineo-Congolian rainforest and the eastern edge of the Amazonian areas. Species concept is a challenging issue within Fungi. We therefore use an integrative approach, combing complementary, phenotypical data, particularly morphology and species autecology, and phylogenetic species concepts (multiple gene genealogy concordance).

A great deal of fieldwork has yielded an extensive and unique collection of wood-decaying polypores, both herbaria and living cultures, the study of which is on-going and

has already resulted in numerous publications. Many morphologically defined fungal species, often based on concepts used across the temperate Northern hemisphere, were then found to consist of “cryptic” taxa forming independent lineages with restricted, discrete distribution, revealing geographic patterns and diverse, complex evolutionary histories. (Robledo et al. 2009, Decock et al. 2010, 2011b, 2011c, 2012).

For instance, within the species rich genera Fomitiporia, Perenniporia, or Ganoderma, taxonomic studies and phylogenetic reconstruction showed a much larger than expected species diversity, and the African or South American species were seen to cluster in different, independent endemic lineages as a consequence of different, local evolutionary histories.

Studying biodiversity hotspots in South America: the Yasuni National Park, Ecuador BCCM/MUCL is involved in several fungal diversity studies in South America, in Amazonian forest areas known as biodiversity hotspots.For 4 years, BCCM/MUCL, together with the University of Liège, has been studying the diversity and ecology of wood-decaying in French Guyana, combining both field studies and modern, state of the art molecular characterisation to reach an integrated species concept. Species diversity was found to be significantly higher than reported, and for instance within Fomitiporia or Phylloporia, species, which are mostly tree parasites, the number of species for the areas rises about 10- fold with numerous undescribed taxa with rather specific ecologies. In Ecuador, together with a partner University in Belgium, the National Botanical Garden of Belgium, and two Ecuadorian universities (Pontificia Universidad católica del Ecuador and Universidad Técnica particular de Loja), BCCM/MUCL coordinates a new project dealing with fungal diversity in the Yasuni National Park and surrounding areas, one of the major world biological diversity hotspots, which is nevertheless threatened by oil exploitation. The project, funded by the CUD (Commission Universitaire pour le Développement), will study the fungal diversity in the undisturbed native forest ecosystem, and also the impact of crude oil pollution on the soil fungal communities, over a period of 5 years.


Fusarium

More than 100 Fusarium species exist, some of which can cause devastating diseases in numerous wild and cultivated plants, of which many are important crops, thus causing considerable economic losses. They also produce a remarkably wide range of secondary metabolites or mycotoxins that contaminate food/feed worldwide and can subsequently cause a variety of acute or chronic intoxications in humans and animals.

BCCM/MUCL collections preserve 890 Fusarium isolates identified to 90 different Fusarium taxa (species, variety, subspecies, …) of agronomic, food/feed and environmental interests. For a number of years, BCCM/MUCL has conducted or contributed to numerous research projects to study the diversity and to characterise Fusarium isolates contaminating important crops like maize, wheat, banana and sweet pepper using multi-disciplinary approaches based on morphological (microscopic observations), physiological (growth properties), biological (mating experiments), phytopathological (virulence and aggressiveness tests), epidemiological, biochemical (metabolite detection), and molecular (AFLP and multilocus phylogenetic studies) methods.

In this context, BCCM/MUCL characterised populations of F. xylarioides, F. verticillioides and F. lactis species complex isolates (Cumagun et al. 2009, Lepoint et al. 2005, Van Poucke et al. 2012) contaminating coffee trees, maize and sweet peppers, respectively. BCCM/MUCL also coordinated research on Fusarium isolates contaminating bananas that resulted in the description of a new Fusarium species, F. musae (Van Hove et al. 2011), as well as participating in revealing the genetic cause F. musae's inability to produce fumonisin mycotoxins, i.e. the excision of the biosynthetic gene cluster (Van Hove et al. 2008). BCCM/MUCL subsequently collaborated in a study on the evolution of the fumonisin biosynthetic gene cluster within the Fusarium species genome aimed at understanding the origin of their fumonisin toxigenic capacity (Proctor et al. 2013).

Between 2005 and 2013, BCCM/MUCL coordinated four regionally funded projects and one federally funded research project to study the biodiversity and molecular relationship of the Fusarium species on maize, leading to the discovery and complete characterisation of a new mycotoxigenic species, F. temperatum (Scauflaire et al. 2011a, 2012b), as well as the first report in Belgium of another species of agro-industrial interest, F. miscanthi (Scauflaire et al. 2013). Pursuant to modification of the Nomenclatural code, F. temperatum was taken as one of several examples put forward in support of the proposal to use the sole Fusarium name for all species of the genus, and to give up the double nomenclature based on both the anamorph (Fusarium) and the teleomorph (Gibberella) stages (Geiser et al. 2013).

Mycotoxigenic species on maize and bananaSince 2008 BCCM/MUCL has been a partner, in collaboration with other Belgian and Chinese laboratories, on two consecutive BELSPO funded research projects to study the diversity of mycotoxigenic Fusarium species contaminating cultivated maize and wild banana in China. Among the 340 Fusarium isolates collected, more than 15 different species have been identified, of which at least three are new species (Munaut et al. 2013; Van Hove et al. 2013).


Exploring yeast diversity

Yeasts are microscopic unicellular fungi that abound in many natural and man-made environments. While morphologically simple, the physiology of yeasts has adapted to persist and thrive in very different environments, ranging from terrestrial, atmospheric environments to aquatic and extreme environments, such as those with low temperatures, low oxygen and low water availability. Although the ecological functions of most yeast species are not yet fully understood, the vast majority, since they participate in the carbon, nitrogen and sulphur cycles are assumed to exert an important influence on their habitats.

Through the efforts of scientists around the world the number of known yeast species is rapidly increasing. While the most recent, 2011 monograph, ‘The Yeasts: a taxonomic study’ edited by Kurtzman, Fell & Boekhout listed 1,500 species, estimations based on yeast isolations from tree fluxes predict up to 15,000 yeast species (Lachance in Biodiversity and ecophysiology of yeasts, 2006, Ed. Rosa & Péter).

At BCCM/MUCL, more than seven hundred yeast species are represented by over 3,000 strains. While the collection is known for its long-standing gathering of brewery and other food-related yeasts, it also hosts numerous isolates from tropical to temperate environments and habitats. More recently, field trips were undertaken to biodiversity hotspots in Cuba and Australia to gather biological resources relevant to environmental yeast diversity. To name and describe the detected yeast diversity, polyphasic studies integrating genomic characters with classical analyses of physiological abilities, metabolic potential and physico-chemical growth limitations are performed. The generated data are essential to predicting where, in view of its ecology and also in view of potential industrial applications or spoilage capacity, a yeast might be able to grow. Yeast species descriptions at BCCM/MUCL pursue the goal of discovering maximum biological meaning regarding their mating behaviour, habitat and biogeography from the study of multiple genetically different individuals of the same species.

To contribute to the BCCM/MUCL collection's mission to promote knowledge and use of a broad fungal diversity, including yeasts, the information on a large number of individual strains and species needs to be effectively managed and communicated. This is reflected in the contribution to developing a stable, predictive and extendable naming system. One research focus is the reclassification of the artificial genus Candida. This gathering of currently more than 300 species unifies ascomycetous yeast species that could not be assigned to other genera due to their lack of morphological differentiation and sexual reproduction. However, this single genus combines industrially applied species and opportunistic human pathogens. Determination of natural relationships between species from gene sequences, also referred to as molecular phylogenies, and a profound change in the rules to naming fungi (McNeill et al 2012, International Code of Nomenclature for algae, fungi and plants) now increasingly allow the assignment of Candida species to the genera where they belong according to their evolutionary history. The future restriction of the genus Candida to a core of about 30 species will ensure stable names for most of the frequently encountered clinically relevant yeasts. (Rosa et al. 2009, Daniel & Prasard 2010).

Yeast diversity in floral nectar from a Cuban biodiversity hotspotYeasts were isolated from floral nectar and the insects attracted by it and several new species were discovered. 168 samples yielded 195 yeast strains representing 67 different species, some which were new (e.g. Metschnikowia cubensis sp. nov.; Starmerella neotropicalis f.a., sp. nov.) and some as yet undescribed. (Fidalgo-Jiménez et al. 2008, Daniel et al. 2013).

The future of CandidaThe genus will be reduced from currently more than 300 species to about 30 Candida sensu strico species that are most closely related to Candida albicans, also known as the ‘Lodderomyces clade’. In a transitional period the genus Candida will continue to accommodate Candida sensu lato species. These will gradually be reclassified to give around 30 existing and more than 20 new genera.


Diatoms

Diatoms are a group of unicellular algae with a unique silica cell wall and life cycle. In evolutionary and ecological terms they are an extremely successful group and have colonised a wide range of terrestrial and aquatic habitats. Although no firm figures exist, species diversity is estimated to range between 200,000 and 2,000,000 species. Diatoms account for about 20% of global primary production and play a central role in the biogeochemical cycling of carbon and silica. Genome sequencing studies have revealed that diatoms possess a highly chimeric genome, including plant- and animal-like molecular pathways as well as many bacteria-derived pathways whose functions remain largely enigmatic to date.

The BCCM/DCG collection invests in the preservation of multiple strains with different phenotypic characteristics, which we believe will greatly benefit the commercial use of microalgae in biotechnology. While most of the species in the BCCM/DCG collection are of primary importance to the scientific community, we have recently started developing targeted collections of commercially interesting species, including the astaxanthin-producing green alga Hematococcus pluvialis. We expect that a better understanding of the genetic basis of interesting phenotypic traits of strains of such species will be of great value for the optimisation of production strains.

Research at the Laboratory of Protistology and Aquatic Ecology and BCCM/DCG aims to contribute to the understanding of diatom biology and evolution. In close collaboration with the Plant Systems Department (VIB) and several international partners, we are developing Seminavis robusta as a model organism for studying the diatom life cycle.

A second principal area of research at PAE involves understanding how diatoms respond to both abiotic and biotic cues perceived in the environment. Recently we were able to establish for the first time how cell cycle progression in diatoms is strictly controlled by light. Currently, we are investigating how diatoms interact with bacteria through the production and perception of quorum-like substances excreted by bacteria and/or diatoms.

A third line of investigation aims to understand the processes underlying the geographical structuring of diatom community composition and population genetic structure. This involves targeted studies of selected lineages, including terrestrial species, freshwater species and marine species, using different approaches such as phylogeographic and phylogenetic studies, population genetics, standard experiments and quantitative genetics. The results of these studies (e.g. Casteleyn et al. 2010, Vanormelingen et al. unpubl.) have revealed a large genotypic and phenotypic diversity of natural diatom populations on both local and regional levels. This finding is not only of fundamental relevance to understanding diatom diversification and biogeography, but is also relevant in the context of culture collections and the biotechnological application of diatoms.

Seminavis robusta as a model for diatom life cycle Recently we have made significant progress in unravelling the signalling involved during the early phases of sexualisation: identification of a conditioning factor excreted by cells of both mating types (MT) which activates mating behaviour, and the first diatom pheromone, resulting in the migration of MT+ cells towards the pheromone producing MT- cells.We discovered that mating type determination in Seminavis robusta is linked to the activity of a single gene. Full genome sequencing of S. robusta is underway.


Cyanobacteria

Cyanobacteria form a well-defined phylum in the range of bacteria. Their main characteristic is that they possess chlorophyll and perform oxygenic photosynthesis. They were very important in transforming the primitive, oxygen-deprived Earth into the Green Planet that we know today. The earliest fossils attributed to cyanobacteria were found in sedimentary rocks formed 3,500 million years ago. Subsequently they have significantly enriched the terrestrial atmosphere with oxygen and enabled the evolution of algae, plants, fungi and animals (including humans). In addition they became the ancestors of plastids after joining onto a primitive eukaryote by endosymbiosis.

They have a large ecological range, as they only need light, liquid water, CO2 and some minerals to grow. Moreover, certain taxa are very resilient to harsh conditions like high or low temperatures, desiccation, UV radiation, etc. They are also characterised by the ability to form symbioses with many different plants and invertebrates.

The microscopic nature of cyanobacteria and their large biodiversity and geographical distribution mean that there is a large proportion of cyanobacterial taxa still to be discovered. Moreover, their taxonomic system is in quite a complex state as, using different approaches, both ‘botanical’ and ‘bacteriological’ classifications exist. The BCCM/ULC collection of cyanobacteria has a large core of polar strains gathered during research projects in the Antarctic continent and Arctic lakes. In addition, five sub-polar strains from Siberian lakes were collected in biotopes that endure very low temperatures in winter and very high ones in summer.

In 2012 BCCM/ULC started to diversify the origin of the strains and included isolates from Belgian surface waters, German lakes, etc.

At the taxonomic level, the three most important cyanobacterial families are represented: Chroococcales, Oscillatoriales and Nostocales.

The research at the CIP host laboratory focuses on the taxonomy, biogeography and phylogeny of cyanobacteria from polar regions. The combination of morphological and molecular characterisations is essential to build a better classification. Several new taxa were found in the collection, including Plectolyngbya hodgsonii and Wilmottia murrayi, and we are currently studying the systematics of thin filamentous taxa of the genus Leptolyngbya. Hence the collection is also contributing to safeguarding an interesting biodiversity while its biotopes are impacted by climate change.

This collection of strains is also important because they can serve as references during bioinformatic analyses of Next Generation Sequencing data now used for environmental surveys and genomic studies.

Production of secundary metabolites The richness of cyanobacteria in secondary metabolites (some with pharmaceutical potential) can also be noted. One category, the cyanotoxins, are notorious for their production by blooms of planktonic cyanobacteria in surface waters in summer and autumn.


Mycobacteria

There are about 165 species and subspecies within the genus Mycobacterium, belonging to the Actinobacteria. Some of them are found just about everywhere, such as in water (including drinking water and swimming pools) and soil. Many are harmless and useful because they degrade organic compounds in soil; few are used in industrial applications. However, various species do cause disease in both animals and humans. The most important human disease species are Mycobacterium tuberculosis (the main causative agent of tuberculosis), M. leprae (causing leprosy) and M. ulcerans (causing Buruli ulcer). In addition, there are a number of species found in water and soil that cause nontuberculous mycobacteria (NTM) disease, primarily a problem in immuno-supressed individuals such as people infected with HIV.

Untangling the structure of pathogenic bacterial populations is helpful for understanding the epidemiology and history of diseases they cause. Tuberculosis exerts a tremendous burden on global health, with approximately 9 million new infections and 1.5 million deaths annually. In recent years our understanding of the global population structure of M. tuberculosis-complex increased, revealing more genetic diversity among its members than previously thought, which translates into significant differences in immunogenicity, virulence and global gene expression among clinical isolates.

M. ulcerans, however, has been determined to exhibit a highly clonal population structure to such an extent that conventional genetic fingerprinting methods have largely failed to genetically differentiate disease isolates, complicating molecular analyses on the disease.

Research at the Mycobacteriology Unit and BCCM/ITM strives to gain more insight into the population structure of M. africanum, a member of the M. tuberculosis complex showing impaired fitness and being geographically restricted to some West African countries, and M. ulcerans, with an emphasis on African isolates. BCCM/ITM currently comprises some reference strains of both species, but intends to broaden the diversity by inclusion of the most relevant strains from the above mentioned research activities.

We applied a redesigned form of an existing typing technique (ISE-SNP) to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci to: (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and, (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans.

Genetic diversity among African M. ulcerans isolates hotspotOur analyses identified 23 different, closely-related African ISE-SNP types which dominate in different Buruli ulcer endemic areas.Different phylogenetic methods also yielded two well-supported sister clades within the African ISE-SNP types.


Culture collections for science and industry


Our Belgian biotechnology sector is a world player!

We don’t often realise it, but biotechnology is an important part of our everyday lives. It plays a part in the clothes we wear and how we wash, the food we eat and where it comes from, the medicines that cure us and keep us alive, and the fuel we use on our daily travels.

In the last decades biotechnology has been through a considerable evolution. To start with there was red biotechnology, which covers medical and pharmaceutical applications. Then attention turned to green biotechnology, which focuses on the genetic modification of plants and the use of micro-organisms in food and agriculture. More recently we have seen the rise of white biotechnology, which uses biotechnological processes to produce sustainable chemical substances, materials and bio-energy from biomass. And now blue biotechnology, the use of marine, or aquatic 'resources' in general, is growing in importance.

The expectation is that the important and most urgent global challenges - the ever growing population, aging, exhaustion of natural resources such as water and energy, climate warming - will find their solutions in biotechnology, which will help develop a sustainable economy that relies on bio-based industrial processes.

The biotechnology sector provides the basis for innovation. Modern biotechnology just about always uses micro-organisms or their molecular components. Activities begin on the strength of basic research.

In this context the evolution for BCCM is that the present bio-based economy will become ever more reliant on biological resources and processes, and this will have a huge impact, providing enormous opportunities for culture collections or ‘biological resource centres (BRCs).

As a 'centre of excellence' BCCM provides a large patrimony of microbiologic sources for biotechnological research and for industry in the various biotechnology sectors and, therefore, provides a basis for many new applications and products across a broad field of application. The scientific expertise at BCCM is put to use in various forms, from specialised services to extensive scientific collaboration contracts. In recent years more than 1,500 scientific institutes and enterprises have called on one or other of the collections held by the BCCM.

The global biotech market is worth EUR 330 billion and the European biotech companies account for 33 billion of this. Belgium represents just 2% of the European population, but our biotech companies account for 30% of the European total.


The increasing demand for high quality foods with refined and varied flavours arouses the interest of industry and academia in detecting, identifying and preserving the micro-organisms that drive fermentations of traditionally produced foods. The range of micro-organisms found in fermenting carbohydrate-rich substrates can be enormous, and only advanced analytical techniques make distinguishing between advantageous and spoiling organisms possible.

Typically, fermentations such as those found in sourdough, lambic beer and cocoa beans share the presence of yeasts, lactic acid and acetic acid bacteria that form a succession of dominating species over the duration of the process. To make microbial consortia fit for use in modern food production, quality considerations require the accurate recognition of the essential microbial players.

The microbial landscape of sourdough fermentations in artisan bakeries revealed specific in-house communities influenced by environmental and technological parameters, while a different community was seen in controlled laboratory fermentations. Ongoing studies of the distinctive Belgian acidic beers known as lambic and further processed to become Geuze, Kriek, or other types of fruit beer, revealed for the first time their species diversity, in both bacteria and yeasts, covering the entire production process. The results will identify the species involved in lambic fermentation and their origin in the spontaneous production process. (Vrancken et al. 2010, Daniel et al. 2011, Huys et al. 2013a).

Microbial activities also contribute substantially to the production of cocoa and chocolates. Research on cocoa bean fermentations in Africa, Asia and South America indicate that, despite the detected high diversity of yeast, lactic acid bacteria and acetic acid bacteria, only a few species of each group are essential for the production of high-quality cocoa beans. Although the use of starter cultures may accelerate and stabilise these fermentations, non-inoculated spontaneous fermentations can lead to excellent cocoa beans at lower cost if good practices are applied during the fermentations. The motivation for investigating microbial communities in food fermentations is to understand the origins, interactions and roles of the different members, resulting in better control of food production. The accurate detection of species and strain diversity in correlation with biochemical parameters and fermentation outcome is a first step towards this goal. (Daniel et al. 2009, Papalexandratou et al. 2011 & 2013).

Microbial diversity in spontaneous food fermentations


Probiotics are defined as living micro-organisms which, when administered in adequate amounts, confer a health benefit for the host. Their worldwide introduction on the functional food and feed market not only triggered the need for well-validated tools for the microbial characterisation of probiotics, but has also urged companies to provide scientific evidence that substantiates the health claims made for their probiotic products.

Largely grounded in their participation in the EU-FP6 projects PROSAFE and ACE-ART, the BCCM/LMG Bacteria Collection and the Laboratory of Microbiology (LM-UGent) of Ghent University have jointly developed considerable research expertise in the microbial characterisation of probiotics. This expertise covers all three main steps in the microbial quality assessment process, i.e. (i) correct species identification and strain-specific typing of bacterial strains used in probiotic applications, (ii) safety assessment of probiotic strains used for human or animal consumption, and (iii) quality of the final probiotic product in terms of its microbial composition, concentration, stability, authenticity and labelling.

Inconsistencies in the microbial identification of commercial products with probiotic claims affect their potential efficacy and safety record, and are likely to have a negative impact on consumer trust. Our research activities have contributed to comprehensive taxonomic inventories of the microbial species commonly used as probiotics, and have evaluated the use of fingerprint-based (e.g. rep-PCR and AFLP analysis) or sequenced-based (e.g. pheS gene sequencing) molecular tools for reliable identification of commercially used probiotic strains and candidate strains for probiotic use. This work has generated a number of in-house databases for species and strain comparisons which offer an invaluable addition to the use of public sequence databases. Our expertise in species identification and strain typing has also extended to a human volunteer trial aimed at determining the stability of a probiotic candidate strain.

In recognition of the importance of ensuring their safety for human or animal purposes, new and existing probiotic strains need to be characterised with respect to a number of safety aspects. BCCM/LMG and LM-UGent have actively participated in international panel discussions on the issue of acquired antibiotic resistance, and have coordinated a large intra- and inter-laboratory performance study leading to recommendations for reproducible antibiotic susceptibility testing. Both research groups have also collaborated with a number of international research groups to establish the phenotypic and/or genotypic basis of acquired antibiotic resistance in (potential) probiotic strains.

Once a probiotic strain has been approved, it must still be determined whether the methods used to characterise the strain can also be used to analyse the commercial product containing the probiotic strain. BCCM/LMG and LM-UGent have performed several of these so called product surveys, which resulted in the formulation of minimum guidelines for correct labelling of products claiming to contain probiotics. (Bourdichon et al. 2012, Huys et al. 2010a & 2013b, Wind et al. 2010, Sandera et al. 2010, Zago et al. 2010, Mayrhofer et al. 2010).

Microbial characterization of probiotics


Plant associated organisms

Micro-organisms are intimately associated with plants because of the huge diversity of habitats offered by plants (i.e. phyllosphere, rhizosphere and endosphere). The interactions may be detrimental (e.g. parasites, pathogens) or beneficial (e.g. symbionts) for the plant. Plant-associated micro-organisms are key players in agriculture and contribute to crop yield and general environmental balance. It is estimated that the number of microbial species per gram of soil exceeds 4,000, of which most are non-cultivable. Therefore, cultivation-dependent (e.g. specific growth media, growth on excised transformed roots) as well as cultivation-independent (e.g. pyrosequencing) methods are necessary.

Within BCCM/MUCL and BCCM/LMG, particular attention has been paid to the root zone harbouring beneficial micro-organisms (e.g arbuscular mycorrhizal fungi or AMF) and free-living plant growth promoting bacteria (e.g. Pseudomonas, Bacillus).For the duration of an EU project (VALORAM), the two collections pooled their efforts in unravelling some of the microbial (i.e. fungi and bacteria) diversity in potato fields of the Andean Altiplano.

The story of the potato began 8,000 years ago near Lake Titicaca. The Central Andean Highlands are where the potato plant originated, with approximately 4,000 potato varieties being grown by local farmers. Due to the long history of the potato in these regions, it is very likely that bacteria as well as fungi (i.e. AMF) associated with the plant have developed properties beneficial to the potato plant. This hypothesis motivated the setting up of a microbial isolation campaign aimed at investigating the plant growth-promotion potential of bacteria and AMF isolated in potato fields from the Andes region. A total of 58 bacterial isolates and 20 AMF were obtained from the potato rhizosphere in eight different fields located in Ecuador, Peru and Bolivia. The bacterial isolates were tested for growth suppression of two important pathogenic

organisms Phytophthora infestans and Rhizoctonia solani in dual-culture assays. The former pathogen causes potato late blight disease; the latter causes stem canker and black scurf disease. A total of 58 isolates were capable of suppressing growth of at least one of these pathogens. Subsequent testing showed that a number of isolates also produced substances that can be involved in biofertilisation, phytostimulation and stress control. All 58 isolates were identified and tested in potato microplants for growth-promotion of healthy plants and Rhizoctonia diseased plants. A number of isolates were able to significantly outperform the commercially available biocontrol agent Bacillus subtilis FZB24® WG, which is a promising result. Further testing in the field and risk assessment of these strains may aid their commercialisation. Similarly, several AMF were isolated from the field and tested for their growth promotion and suppression of P. infestans symptoms with biocontrol against phytophthora infestans (Gallou et al. 2011).

The fungal and bacterial isolates are now kept in the respective collections for further characterisation and public deposit in the near future.

Attention is also focused on the organisms of the phyllosphere, and on endophytes in particular, because they represent a rich source of new and useful compounds of pharmaceutical and agricultural interest. Medicinal plants from Madagascar were screened for their fungal endophytes. Fungi as well as bacteria were isolated. Some of them were found to confer enhanced resistance against plant pathogens and dermatophytes. Bioactive compounds with high inhibitory properties against a large number of human and plant pathogenic fungi were extracted (Rakotoniriana et al. 2013b). This research revealed the strong potential of endophytes from medicinal plants to be a source of new microbial species (Rakotoniriana et al. 2013a) and useful active compounds (Rakotoniriana et al.,2012).


Antibiotic resistance

The Mycobacteriology unit of the Institute of Tropical Medicine, Antwerp, has a long history of focusing on Tuberculosis (TB), caused by Mycobacterium tuberculosis. It houses the public collection of mycobacterial strains (BCCM/ITM), comprising strains from the TDR-TB Strain Bank. The latter is mostly restricted to M. tuberculosis, representing various drug resistance profiles for first- and second-line anti-TB drugs. Drug resistance development in M. tuberculosis is driven by a selection of mutations in genes related to the drug interaction or drug activation. TDR-TB Strain Bank strains have been documented by phenotypic drug susceptibility testing (minimal inhibitory concentration) and Sanger sequencing of the currently described relevant genes.

Building on this expertise in drug-resistant TB, the host laboratory has initiated new approaches to better understanding the mechanisms underlying drug resistance to clofazimine (CFZ) and fluoroquinolones (FQs), and to optimising detection of rifampicin (RMP) resistance. In order to understand resistance to CFZ, an old mycobacterial drug that has recently been proven by one of our senior scientists to be efficacious in the treatment of multi-drug resistant TB, we prepared isogenic mutants with an increased MIC for CFZ. By whole genome sequencing (WGS) of the mother and daughter strains, as well as paired clinical isolates of patients who acquired CFZ resistance, we aim to identify candidate genes for CFZ resistance. In addition, we investigate the role of efflux pumps in CFZ resistance. For FQs, we take a similar approach to identifying the resistance mechanism in the proportion of isolates with phenotypic resistance without mutations in the gyrA/B genes.

Phenotypic susceptibility testing is widely considered to be the gold standard for the identification of rifampicin (RMP) resistance. A senior scientist in our group has identified limitations in culture-based techniques in identifying rifampicin resistance, as up to 15% of de novo mutations in the associated rpoB gene lead to low-level resistance and/or such fitness loss that the strains fail to grow fast enough to be recognised as RMP resistant. Importantly, these studies lead to higher valorisation of currently preferred molecular diagnostics, enabling simplification of early diagnosis of MDR-TB. Further studies focus on the optimal approach to diagnosing all forms of RMP resistance.

To achieve these objectives, the host laboratory enjoys close links with BCCM/ITM. Ongoing WGS of all 235 M. tuberculosis strains from the TDR TB Strain Collection will not only be of great value for the collection, but also assist in identifying new drug-resistance mechanisms. At international level, the strains from our public collection were of great value in the development and evaluation of new diagnostics for rapid detection (drug-resistant) TB, such as the Xpert MTB/RIF assay (Cepheid, USA) (Helb et al. 2010; Rigouts et al. 2013 & Van Deun et al. 2013).


Fusarium species are widely distributed fungi primarily known as plant pathogens but now also seen as important emerging human pathogens. Clinical manifestations of Fusarium are toe nail infections and eye infections, but severe and often deadly systemic infections in immune-deficient patients occur with increasing frequency. Fast and reliable diagnosis of Fusarium infection is therefore required to start patient treatment. But not only is diagnosis important, identification of the infecting Fusarium species is also required, as different species have different optimal treatments.

Currently, diagnosis of Fusarium requires its cultivation from patient blood samples, which is hindered by the many false negative results. In addition, cultivation implies delay of diagnosis as this mould needs time to grow. Time that immune-deficient patients might not have. When eventually diagnosed, identification of the Fusarium species is also problematic – and thus neglected – as the common identification tools, such as morphological characterisation, require a certain expertise of the analyst, sequencing of DNA markers is expensive and time consuming, and MALDI-TOF mass spectrometry to generate spectra of protein content needs an elaborate database of reference spectra.

At BCCM/IHEM, our objective is to develop a new, fast and reliable identification method for Fusarium species for diagnostic purposes by circumventing the cultivation step and directly analysing patient blood samples. A reference database of well-documented and correctly identified strains is necessary to develop such a method. The BCCM/IHEM collection, with its focus on clinical strains, therefore provides a resource of unique value. Over the last 30 years, BCCM/IHEM has gathered about 340

Fusarium strains, of which approximately half are of clinical origin. Recently, all these Fusarium strains were re-identified based on morphology, sequencing of the ITS, BT, EF1α and LSU gene regions, and MALDI-TOF mass spectrometry screening, so that they now conform to the current taxonomy for this genus. In total 35 different species could be identified, capturing a large part of Fusarium diversity, and containing most of the human infection-associated species (Triest et al. 2012). These clinically isolated Fusarium strains in the BCCM/IHEM collection were also subjected to a EUCAST in vitro antifungal susceptibility testing procedure. From the results we were able to conclude that the current drug of choice for fusariosis therapy, amphotericin B, is not always the best treatment for all types of Fusarium infections. Other anti-fungals, less toxic for humans, exhibit similar or even better efficacy against particular Fusarium species (Triest et al. 2013). This observation emphasises the usefulness of developing the new diagnostic identification method at species level for Fusarium.

Fusarium as a human pathogen


Pathogenic genes

In order to help maintain biodiversity and facilitate scientific research worldwide, the BCCM consortium collects and distributes both non-pathogenic and pathogenic micro-organisms. For pathogens, distributing the living strain could compromise European biosecurity legislation and controls. These micro-organisms include the organisms considered quarantine organisms or pests in the European Union, as specified in the EU biosecurity directives and the “European and Mediterranean Plant Protection Organisation” alert lists.

Restrictions on the distribution of isolated genetic material of these strains are less strict. Therefore, the BCCM/LMBP plasmid collection selected a list of genes from human pathogenic bacteria in order to make their DNA directly available to the scientific community. These genes were isolated and cloned into the mammalian expression plasmid pCAGGS. Because these plasmids are distributed either as pure DNA, or in a bacterial strain, the genes are not expressed, and there is no risk of infections. When brought into mammalian cell lines, these genes will be expressed and the function and effects of the proteins they encode can be studied.

The availability of pathogen DNA in the form of expression plasmids provides opportunities to analyse the interactions between pathogens and their hosts, and to unravel the mechanisms of the non-specific immune system. To further understand the molecular mechanisms controlling infection, inflammation and immunity, expression plasmids with mammalian genes (encoding e.g. caspases, cytokines and their receptors, specific signalling proteins ...) involved in these processes were also created and made available through BCCM/LMBP.


Bacillus species are aerobic spore-forming bacteria with aubiquitous distribution. As spore-formers they are resistant to extreme conditions such as high temperatures and low water content. The versatile nature of their metabolic capacities makes them well adapted to a variety of ecological niches, among which are various foods and feed production processes. As a consequence these aerobic spore-formers are regarded as spoilers/contaminants that may affect the shelf life and quality of foods and feed, not only as end products but also when used in secondary processing. Furthermore, Bacillus anthracis and Bacillus cereus are regarded as important infective agents of anthrax and of foodborne infections respectively, encompassing relatively mild to extremely severe intoxication. Hence it is very important within the framework of a quality management system that these aerobic spore-forming contaminants are rapidly and reliably.

These bacilli are very well known as contaminants of gelatin production chains, where they induce enzymatic degradation of gelatin, reducing the quality of the product dramatically. In close collaboration with a gelatin production company and with the financial support of IWT, the LM-UGent developed two Q-PCR procedures to diagnose these contaminating bacilli in final and semifinal gelatins. In case of Bacillus cereus group detection, a TaqMan® probe was designed.

The role of BCCM/LMG was very important because, in the Q-PCR test development and testing phase, an extended set of Bacillus strains needed to be included to validate the inclusive and exclusive characteristics of the test. 30 well characterised Bacillus members of the Bacillus cereus group were therefore included (inclusivity), as well as an equal number of Bacillus strains belonging to species outside the Bacillus cereus group, a pre-study of which demonstrated that they were regularly present at various points of the gelatin production chain.

In a similar setting, a TaqMan® probe based Q-PCR was designed, developed and validated for general confirmation of Bacillus sensu lato contamination of gelatins. With the application of both Q-PCRs a significant reduction in stock-holding time was achieved, with a consequent positive economic effect (Reekmans et al. 2009a & 2009b).

Detection of Bacillus contamination in gelatin


Over the few past decades, culture collections have been responsible for most of the research on preservation/maintenance of biological resources (e.g. fungi, bacteria). Despite the increasing number of taxa successfully preserved, there are still many fragile groups of organisms that remain recalcitrant to the standard methodologies applied in culture collections. Although it is imperative that they be preserved/maintained for future R&D. Similarly, preservation methods may, in some cases, result in genomic as well as phenotypic instability. Thus priority must be given to assessing microbial stability following long-term preservation.

Ectomycorrhizal (ECM) fungi and arbuscular mycorrhizal fungi (AMF) are important root symbionts. They are considered to be key players in plant nutrition and resistance/tolerance to a/biotic stresses. Their current mode of preservation/maintenance is mostly based on regular sub-cultivation on a suitable host (for ECM fungi and AMF) or as vegetative mycelium (for ECM fungi). This method is time and space consuming and guarantees neither the viability of these biotechnologically important organisms, nor their genetic, phenotypic and physiological stability.

Within the framework of two European projects (VALORAM and EMBARC), two cryopreservation methods have been developed at BCCM/MUCL for ECM fungi and AMF.

For ECM fungi, Crahay et al. (2013a) developed an efficient cryopreservation protocol adequate for nearly one hundred isolates belonging to 8 species. The protocol consisted in growing the mycelium of the ECM isolates directly in cryovials filled with agar and surmounted by glycerol (10% v:v) and subsequently cryopreserved at -130 °C. The preparation of the culture prior to freezing had a significant impact on the isolates' survival. These authors further demonstrated that the cryopreserved isolates were able to colonise Pinus sylvestris roots and to transport inorganic phosphate (Pi) and NH4

+ from the substrate to the plant (Crahay et al. 2013b).

For AMF, Lalaymia et al. (2012) developed a cryopreservation method based on the encapsulation-drying of alginate beads containing propagules (i.e. spores and mycorrhizal root pieces). The optimal procedure for cryopreservation comprised five steps: (1) encapsulation of propagules isolated from 5-months-old cultures, (2) incubation overnight in trehalose (0.5M), (3) drying for 48hrs at 27°C, (4) cryopreservation in the freezer at -130°C following a two-step decrease in temperature: a fast decrease (~12°Cmin-1) from room temperature (+20°C) to -110°C followed by a slow decrease in temperature (~1°Cmin-1) from -110°C to -130°C, and (5) direct thawing in a water bath (+35°C). Lalaymia et al. (2013) further demonstrated that this method did not affect the morphology, physiological activity and genetic stability of the AMF after 6 months of preservation.

In conclusion, both groups of organisms could now be preserved at ultra-low temperature for theoretically undefined periods of time. Among the parameters which are essential to the success of the protocols are: the physiological status of the culture (late growth or stationary phase), the pre-conditioning of the isolates (by slow drying and/or application of cryoprotectants), the carrier (growth in cryovial, entrapment in beads) and the rapid thawing after cryopreservation.

Challenges in mycorrhizal preservation


It is well known that only a very small proportion of bacteria can be cultivated. This was shown during the past two decades by metagenomic studies that clearly demonstrated the existence of an enormous bacterial diversity in various niches, such as the marine and fresh water environments and soil.

Many bacteria turned out to have characteristic metabolic capacities, such as denitrification, nitrification, nitrogen fixation and anamox reactions that are very important in the context of the nitrogen cycle and methane oxidising bacteria that are active in the carbon cycle. The Laboratory of Microbiology that hosts the BCCM/LMG bacteria collection is performing research, not only on cultivation and functionality aspects of the above mentioned groups of bacteria, but also on developing durable preservation protocols through close contact with the BCCM/LMG bacteria collection.

Preservation of aerobic methane-oxidising bacteria (MOB) has proven difficult. For most, MOB freeze-drying has not yet been applied with success and only some of them can be cryopreserved for short periods at -80°C or in liquid nitrogen. Because MOB have great potential as a microbial sink for the greenhouse gas, methane, as well as for biotechnological purposes, it is of utmost importance that new isolates can be safeguarded as microbial resources for further studies.

In a large-scale study of a diverse set of MOB exploring fifteen different cryopreservation or freeze-drying conditions, preservation results, i.e. viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed after three, six and twelve months. Successful preservation without significant loss in culturability was obtained for all strains with cryopreservation using 1% trehalose in combination with 10-fold diluted Tryptic-Soy-Broth (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other successful conditions of cryopreservation and lyophilisation all included the use of TT medium but showed a considerable loss in culturability. We observed that most of these so-called non-culturable cells survived preservation but turned into a viable but non-culturable (VBNC) state. This VBNC state is known to be reversible and consequently this observation shifts the emphasis of preservation protocol design towards revival instead of survival aspects. Consequently, we demonstrated that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium. (Hoefman et al. 2012, Heylen et al. 2012).

Challenges in preservation: methane-oxidizing bacteria


As a plasmid repository, BCCM/LMBP mainly focuses on collecting recombinant plasmids that are of scientific importance for fundamental and biotechnological research or educational purposes. Over the years BCCM/LMBP has acquired a wide variety of DNA constructs. These can be classified into three main groups, each covering a broad scope of valuable tools to be used by scientists worldwide:

1. Empty cloning plasmids useful for recombinant (over)expression of proteins of interest in bacteria, yeast, fungi, insect cells, plant and animal cells, such as but not limited to: broad host range plasmids belonging to different incompatibility groups, gateway cloning plasmids, dicistronic expression plasmids, adenoviral and retroviral expression plasmids, yeast two-hybrid and three-hybrid expression plasmids, reporter plasmids …The presence of different fusion or epitope tags gives researchers the highest possible flexibility regarding methods of protein detection and purification (affinity purification, immunodetection, fluorescence, luminescence, electron microscopy, …) and targeting of cell types or organelles.

2. Expression plasmids carrying genes or sequences of interest, such as, but not limited to: genes for several cytokines and growth factors, receptors, intracellular signalling proteins, enzymes (kinases, proteases, glycosylation related genes, ubiquitin ligases…), bacterial virulence genes, microRNA sequences, promoter sequences …

3. Plasmids for mouse genetic engineering, for generation of transgenic mice.

Plasmids are important in both basic and applied research and can be used for anything from in vitro investigation of a signalling pathway to high-yield production of protein drugs such as insulin. By providing access to plasmids constructed with widely different aims, BCCM/LMBP presents scientists with the opportunity to build on previous research conducted by others. In addition, to ensure preservation of the information associated with these publicly available plasmids, BCCM/LMBP describes and visualises the DNA molecules in great molecular detail. The online accessibility of this information ensures wide distribution of these biotechnological tools in academic and industrial research and development.

Recombinant plasmids as biotechnological tools


Fungi

Filamentous fungi and yeasts can cause infections in humans and animals. Some of these infections are superficial, more or less harmless, while others are invasive and can even cause death. These systemic infections occur mainly in immuno-compromised patients, some of these fungal diseases thus progressing very rapidly. For the correct and successful treatment of these fungal infections, rapid and reliable identification of the causative agent is necessary.

Conventional microbiological identification of fungi is based on either the combination of microscopical, macroscopical and physiological characteristics, or on the sequencing of several genomic regions. DNA sequencing is however rather time-consuming and expensive.

The BCCM/IHEM collection is collaborating with the Service for Mycology and Parasitology at the CHU in Marseilles on constructing and optimising such a reference database based on the BCCM/IHEM collection's reference strains.

Currently, the database consists of spectra from over 500 different fungal species and its performance is under evaluation. Identification of fungal strains using this MALDI-TOF MS database is being validated on the one hand with other strains of the BCCM/IHEM collection and, on the other hand, with clinical isolates from various hospitals in Brussels as well as from the CHU in Marseilles. These isolates were of course previously identified by molecular biology, which remains the gold standard technique.

The results are very promising. In general very few errors are encountered and, more importantly, while in clinical routine most cryptic species are not identified, or strains are not identified up to species level, our approach allows correct and precise identification. The aim is to implement this technique as an additional quality control for the fungal strains of the BCCM/IHEM public collection.

Bacteria

Also for bacterial characterisation, MALDI-TOF MS is increasingly used in a wide spectrum of applications. This tool is becoming the method of choice for clinical diagnostics in many hospital settings, but MALDI-TOF MS is also now commonly used for bacterial identification and taxonomy in academic research (Carbonnelle et al., Sandrin et al). To this end, the availability of well-documented and up-to-date reference databases is crucial.

In collaboration with its host lab, the Laboratory of Microbiology (Faculty of Sciences, Ghent University), the BCCM/LMG Bacteria Collection is currently building dedicated databases for specific bacterial groups with functional characteristics of interest to both industry and academics, i.e. acetic acid bacteria and bifidobacteria. Following the construction of a taxonomic framework based on MALDI-TOF MS data generated from type and reference strains, the databases will also be validated by including new isolates of which the species identity was previously assessed using sequence-based approaches.

Because of its cost efficiency and speed, MALDI-TOF MS may also be an attractive approach for quality control (QC) of bacterial preservation procedures in culture collections. For this type of application, it is thus important to assess the reproducibility of MALDI-TOF MS profiling of bacterial cultures before and after preservation. As part of its autonomous research, BCCM/LMG conducted a reproducibility study where the role of several experimental variables, including the person harvesting cells, the amount of cells harvested from the growth medium and the storage time of harvested cells at -20°C prior to protein extraction, was assessed. This study indicated that some impact of person-specific operations on the reproducibility of MALDI-TOF MS profiling for QC purposes cannot be ruled out, and further highlights the importance of dedicated training.

MALDI-TOF mass spectrometry: a straightforward tool for identification and preservation quality control of microbes


Taking into account the results of the reproducibility study, BCCM/LMG has meanwhile developed a series of technical instructions that are currently being implemented for the routine QC of new preservation (lyophilisation and cryopreservation) batches of its bacterial cultures. In this way the BCCM/LMG Bacteria Collection aims to significantly reduce the delay between receipt of original material and public availability of new cultures.


BCCM facts & figures


The BCCM patrimonyAt the end of 2012, the total holdings of the BCCM consortium numbered about 173,000 different organisms. According to their status in the collection, these organisms can be categorised into 4 types of deposits of biological material:

• Public deposits: (micro)biological material that has been deposited in the collection and that may be catalogued and distributed;

• Safe deposits: (micro)biological material that is deposited in the collection for back-up safety reasons; it remains the exclusive property of the depositor, it will not be catalogued or distributed;

• Patent deposits: (micro)biological material that is deposited under Belgian patent legislation or the international Budapest Treaty;

• Research deposits: (micro)biological material that is or has been the subject of scientific research projects and that is not publicly available, as well as (micro)biological material for which a provisional restriction regarding cataloguing and/or distribution is applicable.

The BCCM holdings consists of about 37% public deposits and 63% research deposits. Due to their specific nature, the safe and patent deposits represent only a small part of the holdings.The large share of research deposits illustrates the growth potential of the public collections. This is often the case regarding organisms that are kept as research objects. The BCCM curators will gradually transfer some of these organisms to the public collection.

The BCCM public collections

The BCCM public collections contain about 64,000 different organisms, distributed over fungi, bacteria, yeasts, plasmids, diatoms and DNA libraries. These organisms are fully documented and readily available for distribution.

Figure 1 : Relative importance for the consortium of the different deposit types.

n Public deposits 36,90 %

n Patent deposits 0,50 %

n Research deposits 62,40 %

n Safe deposits 0,20 %

Total number of organisms: 173,000

Figure 2 : Relative importance of the different organism types in the public BCCM collections. DNA libraries (0.03%) are not visible in the graph.

n Fungi 45,2 %

n Bacteria, including myco- and

cyanobacteria 39,5 %

n Yeasts 11,3 %

n Plasmids 3,4 %

n Diatoms 0,5 %

Total number of organisms in the public BCCM collections: 64,000


In the period 2009 - 2012 the BCCM public collections have grown with the addition of 6,800 organisms. In absolute numbers the fungi and bacteria collections have added the most organisms. In relative numbers however, the small collections of plasmids and diatoms have expanded the most compared to their situation at the end of 2008 (+49% and +16% respectively).

The BCCM consortium represents about 2,300 genera and about 9,900 species.

In 2006 Belgian Science Policy initiated the development of 3 specialist research and quality-controlled public collections to be integrated within the BCCM consortium. In 2011 the integration of the quality management system of these 3 collections in the multi-site, ISO 9001 certified BCCM quality management system, as well as the publication of their catalogues on the BCCM website, marked their official adherence to the BCCM consortium. It concerns the Ghent University diatom collections (313 strains), the Liège University cyanobacteria collection (103 strain) and the Institute of Tropical Medicine Mycobacteria collection (258 strains). Despite their obvious interest for scientific, industrial, environmental and medical R&D applications, these organism types were under-represented in public biological resource centres worldwide. Although they still have limited resources, these collections have added value to the BCCM consortium and have growth potential.

Figure 3 : Growth of the BCCM pubic collections in the period 2009 - 2012

n Diatoms

n DNA libraries

n Plasmids

n Yeasts

n Bacteria

n Fungi

Our stakeholdersEach year between 100 and 200 different organisations deposit biological material in one of the BCCM public collections. Most organisations are from the non-profit sector. About a quarter of them are from Belgium, the remainder are equally divided among other European countries and countries outside Europe. Each organisation can represent several independent depositors.

70000

60000

50000

40000

30000

20000

10000

0


Safe deposits

Safe deposits in a supplementary professional environment have proved to be a very cost-effective means of insuring proprietary cultures (or plasmids) of particular importance. Industry in particular is increasingly keen to insure against, for example, the loss or irreversible mutation of production strains. As full confidentiality is ensured, safe-deposited material is neither catalogued nor distributed to third parties without the depositor's approval. The BCCM safe deposit is a very flexible service, providing safekeeping of the material for a defined or undefined period at the request of the customer. The viability of the material is regularly monitored.

At the end of 2012, the BCCM consortium kept 381 samples of biological material as safe deposits.

Patent deposits

Patent deposits are a compulsory part of the patent process when the patent involves the use of biological material. Within the framework of internationally prevailing legislation (e.g. the Budapest Treaty and European Directives), and under the auspices of the World Intellectual Property Organisation, the BCCM is recognised as an International Depositary Authority (IDA).

The BCCM accepts deposits of:• Bacteria• Filamentous fungi, including arbuscular myorrhizal fungi• Yeasts• Human and animal cell lines including hybridomas • Other Genetic resources (e.g. plasmids, viruses, phages, …).

Full confidentiality is ensured and distribution of samples is governed by patent law. Viability is regularly monitored. At the end of 2012, the BCCM consortium kept 781 samples of biological material as patent deposits.

Our stakeholdersEach year between 10 and 20 different organisations deposit biological material in as a safe deposit in one of the BCCM collections. About 3/4 are organisations from the profit sector. Their geographic origin varies yearly but most are from European countries. Each organisation can represent several independent depositors.

Figure 4 : Relative importance of the different organism types in the safe deposit collections.

n Bacteria 54,9 %

n Yeasts 7,1 %

n Plasmids 17,6 %

n Cell lines, nematodes, phages 0,8 %

n Fungi 19,7 %

Total number of organisms in the safe deposit collections: 381

Our stakeholdersEach year about 15 different organisations deposit biological material as a patent deposit in one of the BCCM collections. About 60% are from the profit sector. About half are from Belgium and about 1/3 from other European countries. Each organisation can represent several independent depositors.

Figure 5 : Relative importance of the different organism types in the patent deposit collections.

n Bacteria 42,6%

n Yeasts 6,0 %

n Plasmids 25,1 %

n Cell lines, nematodes, phages,

viruses 20,4 %

n Fungi 5,9 %

Total number of organisms in the patent deposit collections: 781


Distribution of material Each year the BCCM consortium distributes between 5,000 and 6,000 samples of biological material to customers in the profit and non-profit sectors, both in Belgium and abroad. In the period 2009-2012, about 21,700 samples were distributed by the BCCM consortium.

Other scientific services The BCCM consortium offers a range of services based on its scientific expertise in the field of molecular and microbiology. The most important service is the identification of microorganisms, followed by testing and microbial count services.

Our stakeholdersEach year more than 200 different organisations order scientific services from one of the BCCM collections. They are equally divided among the profit and non-profit sector. About 60% are from Belgium, 25% from other European countries and 15% from a country outside Europe. Each organisation can represent several independent users.

Figure 7 : Relative importance of the different services carried out by the BCCM consortium. The category "other services" contains about 15 other types of service which each contribute less than 2% to the total revenues of the scientific services.

n Identification 53 %

n Microbial count 11 %

n Audits 8 %

n Testing 11 %

n Isolation 3 %

n Freeze-drying 3 %

n Analysis 3 %

n Other services 8 %Our stakeholdersEach year between 600 and 700 different organisations request samples of biological material from one of the BCCM public collections. A quarter of these organisations are from the profit sector. About 27% are from Belgium, 45% from other European countries and 28% from a country outside Europe. Each organisation can represent several independent users.

Figure 6 : Share of the different organism types in the total number of distributed samples

n Fungi and yeasts 24 %

n Bacteria, including myco- and

cyanobacteria 65 %

n Plasmids 10 %

n Diatoms 1 %

21.700 samples distributed (2009-2012)


The BCCM budget The BCCM consortium is funded by the Belgian Science Policy Office (Belspo) under an annual recurrent funding system. The income that the collections generate from their services and research projects constitutes an additional source of funding. In the past four years, Belspo provided 78% of the total BCCM budget, while services and research projects generated, respectively, 10% and 12% of the total BCCM budget.

Research projects In the past 4 years the BCCM collections have been involved as a partner in 26 research projects with external funding. These projects add value to the collections by either expanding the holdings with new interesting material, further describing and characterising the biological material that is already available, contributing to the taxonomic or phylogenetic studies or developing new or optimised preservation methods.

Since the BCCM consortium has no legal status yet, the contracts related to these projects are managed by the BCCM host institutes.

Figure 8 : Relative importance of sources of funding for the BCCM consortium

n Budget provided by Belspo 78 %

n Income from services 10 %

n Income from research projects 12 %


New taxa described in BCCM

Environmental fungi

Coltricia oboensis Decock (MUCL 54810)

Coltriciella sonorensis R. Valenz. & Decock

Fomitiporia castilloi Decock & Amalfi (MUCL)

Fomitiporia cupressicola Decock (MUCL 52486)

Fomitiporia gabonensis Decock (MUCL 47576)

Fomitiporia ivindoensis Decock (MUCL 51312)

Fomitiporia neotropica Decock (MUCL )

Fomitiporia nobilissima Decock (MUCL 51289)

Fomitiporia punicata Y.C. Dai, B.K. Cui & Decock

Fusarium musae Van Hove, Waalwijk, Munaut, Logrieco & Moretti (MUCL 52574)

Fusarium temperatum Scauflaire & Munaut (MUCL 52463)

Inonotus cubensis Decock (MUCL 47079)

Inonotus rwenzorianus Balezi & Decock (MUCL 51007)

Leptographium sinoprocerum Q. Lu, Decock & Maraite (MUCL 46352)

Metschnikowia cubensis Fidalgo-Jim., H.M. Daniel, Evrard, Decock & Lachance (MUCL 45753)

Microporellus adextrinoideus Decock (MUCL 49398)

Microporellus papuensis Decock (MUCL 49405)

Moniliella fonsecae Rosa, Nakase, Jindamorakot, Limtong, Lachance, Fidalgo-Jiménez, Daniel, Pagnotta, Inacio &

Morais

Perenniporia abyssinica Decock & Bitew (MUCL 44215)

Perenniporia alboferruginea Decock (MUCL 49279)

Perenniporia guyanensis Decock & Ryvarden (MUCL 41995)

Perenniporia subovoidea Decock & Ryvarden (MUCL 54487)

Perenniporiella chaquenia Robledo & Decock (MUCL 49758)

Phellinus castanopsidis B.K. Cui, Y.C. Dai & Decock

Phellinus gabonensis Decock & Yombiyeni (MUCL 52025)

Phylloporia nouraguensis Decock (MUCL 53816)

Phylloporia rzedowskii R. Valenz. & Decock (MUCL 52868)

Phylloporia ulloai R. Valenz., T. Raymundo, Cifuentes, & Decock (MUCL 52867)

Ruwenzoria pseudoannulata J. Fourn., M. Stadler, Læssøe & Decock (MUCL 51394)

Septomyrothecium maraitiense Decock (MUCL 47202)

Trinosporium guianense Crous & Decock (MUCL 53977)

Truncospora oboensis Decock (MUCL 53565)

Medical fungi

new species

Lactarius limbatus Stubbe & Verbeken

Lactarius coniculus Stubbe & Verbeken

Lactarius leonardii Stubbe & Verbeken

Lactarius hora Stubbe & Verbeken

Lactarius genevievae Stubbe & Verbeken

Lactarius conchatulus Stubbe & H.T. Le

Lactarius leae Stubbe & Verbeken

Lactifluus parvigerardii X.H. Wang & Stubbe

new combinations

Lactifluus subg. Gerardii (A.H. Sm. & Hesler) Stubbe

Lactifluus limbatus (Stubbe & Verbeken) Stubbe

Lactifluus leonardii (Stubbe & Verbeken) Stubbe

Lactifluus hora (Stubbe & Verbeken) Stubbe

Lactifluus genevievae (Stubbe & Verbeken) Stubbe

Lactifluus conchatulus Stubbe & H.T. Le

Lactifluus petersenii (Hesler & A.H. Sm.) Stubbe

Lactifluus sepiaceus (McNabb) Stubbe

Lactifluus subgerardii (Hesler & A.H. Sm.) Stubbe

Lactifluus wirrabara (Grgur.) Stubbe

Bacteria

Arcobacter mytili Collado, Cleenwerck, Van Trappen, De Vos and Figueras 2009 VP

Arenicella chitinivorans Nedashkovskaya, Cleenwerck, Zhukova, Kim, De Vos IN

Bacillus berkeley Nedashkovskaya, Van Trappen, Frolova and De Vos 2012 VL

Bacillus cytotoxicus Guinebretière, Auger, Galleron, Contzen, De Sarrau, De Buyser, Lamberet, Fagerlund, Granum,

Lereclus, De Vos, Nguyen-The and Sorokin 2013 VP

Bacillus thermolactis Coorevits, Logan, Dinsdale, Halket, Scheldeman, Heyndrickx, Schumann, Van Landschoot and

De Vos 2011 VP

Brenneria goodwinii Denman, Brady, Kirk, Cleenwerck, Venter, Coutinho and De Vos 2012 VP


Cronobacter helveticus Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Cronobacter pulveris Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Cronobacter zurichensis Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Dickeya aquatica Parkinson, De Vos, Pirhonen and Elphinstone IN

Echinimonas agarilytica Nedashkovskaya, Stenkova, Zhukova, Van Trappen, Lee and Kim 2013 VL

Enterococcus quebecensis Sistek, Maheux, Boissinot, Bernard, Cantin, Cleenwerck, De Vos and Bergeron 2012 VP

Enterococcus ureasiticus Sistek, Maheux, Boissinot, Bernard, Cantin, Cleenwerck, De Vos and Bergeron 2012 VP

Erwinia oleae Moretii, Hosni, Vandemeulebroecke, Brady, De Vos, Buonaurio and Cleenwerck 2011 VP

Hafnia paralvei Huys, Cnockaert, Abbott, Janda and Vandamme 2010 VP

Gluconacetobacter maltaceti Slapsak, Cleenwerck, De Vos and Trcek 2013 VL

Gluconacetobacter medellinensis Castro, Cleenwerck, Trcek, Zuluaga, De Vos, Caro, Aguirre, Putaux and Ganan 2013

VP

Helicobacter heilmannii Smet, Flahou, D'Herde, Vandamme, Cleenwerck, Ducatelle, Pasmans and Haesebrouck 2012

VP

Kozakonia arachidis Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Kozakonia cowanii Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Kozakonia oryzae Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Kozakonia radicincitans Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Lelliottia aminigena Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Lelliottia nimipressuralis Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Lonsdalea quercina subsp. britannica Brady, Cleenwerck, Denman, Venter, Rodríguez-Palenzuela, Coutinho and De

Vos 2012 VP

Lonsdalea quercina subsp. iberica Brady, Cleenwerck, Denman, Venter, Rodríguez-Palenzuela, Coutinho and De Vos

2012 VP

Lonsdalea quercina subsp. quercina Brady, Cleenwerck, Denman, Venter, Rodríguez-Palenzuela, Coutinho and De

Vos IN

Marinobacterium coralli Chimetto, Cleenwerck, Brocchi, Willems, De Vos and Thompson 2011 VP

Marinomonas brasilensis Chimetto, Cleenwerck, Brocchi, Willems, De Vos and Thompson 2011 VP

Mesorhizobium abyssinicae Degefu, Wolde-meskel, Liu, Cleenwerck, Willems and Frostegård IN

Mesorhizobium hawassense Degefu, Wolde-meskel, Liu, Cleenwerck, Willems and Frostegård IN

Mesorhizobium shonense Degefu, Wolde-meskel, Liu, Cleenwerck, Willems and Frostegård IN

Pantoea alllii Brady, Goszczynska, Venter, Cleenwerck, De Vos, Gitaitis and Coutinho 2011 VP

Pantoea anthophila Brady, Venter, Cleenwerck, Engelbeen, Vancanneyt, Swings and Coutinho 2009 VP

Pantoea brenneri Brady, Cleenwerck, Venter, Engelbeen, De Vos and Coutinho 2010 VP

Pantoea calida Popp, Cleenwerck, Iversen, De Vos and Stephan 2010 VP

Pantoea conspicua Brady, Cleenwerck, Venter, Engelbeen, De Vos and Coutinho 2010 VP

Pantoea deleyi Brady, Venter, Cleenwerck, Engelbeen, Vancanneyt, Swings and Coutinho 2009 VP

Pantoea eucalypti Brady, Venter, Cleenwerck, Engelbeen, Vancanneyt, Swings and Coutinho 2009 VP

Pantoea eucrina Brady, Cleenwerck, Venter, Engelbeen, De Vos and Coutinho 2010 VP

Pantoea gaviniae Popp, Cleenwerck, Iversen, De Vos and Stephan 2010 VP

Pantoea rodasii Brady, Cleenwerck, van der Westhuizen, Venter, Coutinho and De Vos 2012 VP

Pantoea rwandensis Brady, Cleenwerck, van der Westhuizen, Venter, Coutinho and De Vos 2012 VP

Pantoea septica Brady, Cleenwerck, Venter, Engelbeen, De Vos and Coutinho 2010 VP

Pantoea vagans Brady, Venter, Cleenwerck, Engelbeen, Vancanneyt, Swings and Coutinho 2009 VP

Pantoea wallisii Brady, Cleenwerck, van der Westhuizen, Venter, Coutinho and De Vos 2012 VP

Photobacterium jeanii Chimetto, Cleenwerck, Thompson, Brocchi, Willems, De Vos and Thompson 2010 VP

Photobacterium piscicola Figge, Cleenwerck, De Vos, Huys, van Uijen and Robertson IN

Pluralibacter gergoviae Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Pluralibacter pyrinus Brady, Cleenwerck, Venter, Coutinho and De Vos IN

Pseudomonas asturiensis Gonzalez, Cleenwerck, De Vos and Fernandez-Sanz 2013 IN

Sporosarcina newyorkensis Wolfgang, Coorevits, Cole, De Vos, Dickinson, Hannett, Jose, Nazarian, Schumann, Van

Landschoot, Wirth and Musser 2012 VP

Staphylococcus devriesei Supré, De Vliegher, Cleenwerck, Engelbeen, Van Trappen, Piepers, Sampimon, Zadoks, De

Vos and Haesebrouck 2010 VP

Stenotrophomonas pavanii Ramos, Van Trappen, Thompson, Rocha, Barbosa, De Vos and Moreira-Filho 2011 VP

Streptococcus rubneri Huch, De Bruyne, Cleenwerck, Bub, Cho, Watzl, Snauwaert, Franz and Vandamme 2013 VP

Tatumella citrea (Kageyama, Nakae, Yagi and Sonoyama 1992) Brady, Venter, Cleenwerck, Vandemeulebroecke, De

Vos and Countinho 2010 VP

Tatumella punctata (Kageyama, Nakae, Yagi and Sonoyama 1992) Brady, Venter, Cleenwerck, Vandemeulebroecke, De


Tatumella terrea (Kageyama, Nakae, Yagi and Sonoyama 1992) Brady, Venter, Cleenwerck, Vandemeulebroecke, De


Tetragenococcus halophilus subsp. flandriensis Justé, Van Trappen, Verreth, Cleenwerck, de Vos, Michiels, Lievens

and Willems 2012 VP

Tetragenococcus halophilus subsp. halophilus Justé, Van Trappen, Verreth, Cleenwerck, de Vos, Michiels, Lievens and

Willems 2012 VP

Tetragenococcus osmophilus Justé, Van Trappen, Verreth, Cleenwerck, de Vos, Michiels, Lievens and Willems 2012 VP

Vibrio artabrorum Diéguez, Beaz-Hidalgo, Cleenwerck, Balboa, de Vos and Romalde 2011 VP

Vibrio atlanticus Diéguez, Beaz-Hidalgo, Cleenwerck, Balboa, de Vos and Romalde 2011 VP

Vibrio breoganii Beaz-Hidalgo, Cleenwerck, Balboa, Prado, De Vos and Romalde 2009 VP

Vibrio celticus Beaz-Hidalgo, Dieguez, Cleenwerck, Balboa, Doce, De Vos and Romalde 2011 VL

Vibrio communis Chimetto, Cleenwerck, Alves Jr, Silva, Brocchi, Willems, De Vos and Thompson 2011 VP

Vibrio maritimus Chimetto, Cleenwerck, Moreira, Brocchi, Willems, De Vos and Thompson 2011 VP

Vibrio variabilis Chimetto, Cleenwerck, Moreira, Brocchi, Willems, De Vos and Thompson 2011 VP


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Justé A., Van Trappen S., Verreth C., Cleenwerck I., De Vos P., Lievens B. and Willems K.A. 2012. Characterization of Tetragenococcus strains from sugar thick juice reveals a new species, Tetragenococcus osmophilus sp. nov., and divides Tetragenococcus halophilus into two subspecies, T. halophilus subsp. halophilus subsp. nov. and T. halophilus subsp. flandriensis subsp. nov. Int J Syst Evol Microbiol. 62(Pt 1):129-37.

Korhonen J., van Hoek A., Saarela M., Huys G., Tosi L., Mayrhofer S. and von Wright A. 2010. Antimicrobial susceptibility of Lactobacillus rhamnosus. Beneficial Microbes 1:75-80.

Labriola L., Vandercam B., Swinne D. and Jadoul M. (2009). Successful treatment with voriconazole of prolonged Paeciliomyces lilacinus Fungemia in a chronic hemodialyzed patient. Clinical Nephrology, 71(3):355-358.

Lalaymia I., Cranenbrouck S., Draye X., Declerck S. 2012. Preservation at ultralow temperature of in vitro cultured arbuscular mycorrhizal fungi via entrapmentdrying. Fungal Biology 116:1032-1041.

Lalaymia I., Declerck S., Cranenbrouck S. 2013. Cryopreservation of in vitro produced arbuscular mycorrhiza fungi has minor effects on their morphology, physiology and genetic stability. Mycorrhiza (in press).

L’Ollivier C., Cassagne C., Normand A.-C., Bouchara J.-P., Contet-Audonneau M., Hendrickx M., Fourquet P., Coulibaly O., Piarroux R. and Ranque S. (2012). A MALDI-TOF MS procedure for clinical dermatophyte species identification in the routine laboratory. Medical Mycology (accepted)

Lu Q., Decock C., Zhang X. Y., and Maraite H. 2009. Ophiostomatoid fungi (Ascomycota) associated with Pinus tabuliformis and Dendroctonus valens (Coleoptera) in northern China and assessment of their

pathogenicity on mature trees. Antonie van Leeuwenhoek 96: 275-293.

Madoroba, E., Steenkamp E.T., Theron J., Scheirlinck I., Cloete T.E. and Huys G. 2011. Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food. Syst. Appl. Microbiol. 34:227-234.

Mayrhofer S., van Hoek A., Mair C., Huys G., Aarts H., Kneifel W., Domig K. 2010. Antibiotic susceptibility of members of the Lactobacillus acidophilus group using broth microdilution and molecular identification of their resistance determinants. Int. J. Food Microbiol. 144:81-87.

Montoya P., Bandala V.M., Haug I. and Stubbe D. (2012). A new species of Lactarius (subgenus Gerardii) from two relict Fagus grandifolia var. mexicana populations in Mexican montane cloud forests. Mycologia 104(1):175-181.

Moretti C., Hosni T., Vandemeulebroecke K., Brady C., De Vos P., Buonaurio R. and Cleenwerck I. Erwinia oleae sp. nov., isolated from olive knots caused by Pseudomonas savastanoi pv. savastanoi. Int J Syst Evol Microbiol. 61(Pt 11):2745-52.

Munaut F., Moretti A. and Van Hove F. 2011. Identification of mycotoxigenic fungi in food and feed. In Determining mycotoxins and mycotoxigenic fungi in food and feed. Edited by De Saeger S., Woodhead Publishing , Cambridge, UK, pp. 427.

Munaut F., Van Hove F. and Moretti A. 2011. Molecular identification of mycotoxigenic fungi in food and feedstuffs. In S. De Saeger (ed.), Determining mycotoxins and mycotoxigenic fungi in food and feed. Woodhead Publishing Limited, Cambridge, UK, p. 298-331.

Munaut F., Scauflaire J., Gourgue M., Bivort C., Qi Y., Wu A., De Saeger S., Zhang D. and Van Hove F. 2013. Genetic and mycotoxigenic diversity of isolates belonging to the Fusarium incarnatum-equiseti species complex, and recovered from maize and banana in China. "12th European Fusarium Seminar", 12-16/05/2013, Bordeaux, France.

Nedashkovskaya O.I., Van Trappen S., Frolova G.M.and De Vos P. 2012. Bacillus berkeleyi sp. nov., isolated from the sea urchin Strongylocentrotus intermedius. Arch Microbiol. 194(3):215-221.

Ngamskulrungroj P., Himmelreich U., Breger J.A., Wilson C., Chayakulkeeree M., Krockenberger M.B., Malik R., Daniel H.M., Toffaletti D., Djordjevic J.T., Mylonakis E., Meyer W. and Perfect J.R. 2009. The trehalose synthesis pathway is an integral part of the virulence composite for Cryptococcus gattii. Infection and Immunity 77: 4584-4596.

Ouadghiri, M., Vancanneyt, M., Vandamme, P., Naser, S., Gevers, G., Lefebvre, K., Swings, J. and Amar, M. (2009). Identification of lactic acid bacteria in Moroccan raw milk and traditionally fermented skimmed milk ‘lben’. Journal of Applied Microbiology. 106: 486–495

Pablos, M., Huys G., Cnockaert M., Rodríguez-Calleja J.M., Otero A., Santos J.A. and García-López M.L.. 2011. Identification and epidemiological relationships of Aeromonas isolates from patients with diarrhoea. Int. J. Food Microbiol. 147:203-210.

Packeu A., Lebecque P., Rodriguez-Villalobos H., Boeras, A., Hendrickx M., Bouchara, J.P. and Symoens, F. (2012). Molecular typing and antifungal susceptibility of Exophiala species isolates from patients with cystic fibrosis. Journal of Medical Microbiology 61 (9):1226-33.

Packeu A., Hendrickx M., Beguin H., Martiny D., Vandenberg O. and Detandt M. (2012). Identification of the Trichophyton mentagrophytes complex species using MALDI-TOF mass spectrometry. Medical Mycology (accepted)

Papalexandratou Z., Cleenwerck I., De Vos P. and De Vuyst L. 2009. (GTG)5-PCR reference framework for acetic acid bacteria. FEMS Microbiol Lett. 301(1): 44-9.

Papalexandratou Z., Falony G., Romanens E., Jimenez J.C., Amores F., Daniel H.M., De Vuyst L. 2011. Species diversity, community dynamics, and metabolite kinetics of the microbiota associated with traditional

Ecuadorian spontaneous cocoa bean fermentations. Applied and Environmental Microbiology, 77: 7698-7714.

Papalexandratou Z., Lefeber T., Bahrim B., Lee O.S., Daniel H.M. and De Vuyst L. 2013. Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations, underlining the importance of these microbial species for a successful cocoa bean fermentation process. Food Microbiology 35: 73-85.

Pinhassi, J., Nedashkovskaya, O.I., Hagström, A. and Vancanneyt, M. (2009). Winogradskyella rapida, sp. nov., isolated from protein-enriched seawater. Int J Syst Evol Microbiol. 59: 2180-2184.

Popp A., Cleenwerck I., Iversen C., De Vos P. and Stephan R. 2010. Pantoea gaviniae sp. nov. and Pantoea calida sp. nov. isolated from infant formula and infant formula production environment. Int J Syst Evol Microbiol. 60(Pt 12):2786-92.

Proctor R.H., Van Hove F., Susca A., Stea G., Busman M., van der Lee T., Waalwijk C., Ward T.J., and Moretti A. 2013. Evidence for birth-and-death evolution of a secondary metabolite biosynthetic gene cluster and its relocation within and between genomes of the filamentous fungus Fusarium. Submitted in Molecular Microbiology, vol, blz

Rakotoniriana E.F., Chataigné G., Raoelison G., Rabemanantsoa C., Munaut F., El Jaziri M., Urveg- Ratsimamanga S., Marchand-Brynaert J., Corbisier A.M., Declerck S., Quetin-Leclercq J. 2012. Characterization of an endophytic whorl-forming Streptomyces from Catharanthus roseus stems producing polyene macrolide antibiotic. Canadian Journal of Microbiology 58: 617-627.

Rakotoniriana E.F., Scauflaire J., Rabemanantsoa C., Urveg-Ratsimamanga S., Corbisier A.M., Quetin-Leclercq J., Declerck S. and Munaut F. 2013a. Colletotrichum gigasporum sp. nov., a new species of Colletotrichum with long straight conidia. Mycological Progress 12: 403-412.

Rakotoniriana E.F., Rafamantanana M., Randriamampionona D., Rabemanantsoa


C., Urveg-Ratsimamanga S., El Jaziri M., Munaut F., Corbisier A.M., Quetin-Leclercq J. and Declerck S. 2013b. Study in vitro of the impact of endophytic bacteria isolated from Centella asiatica on the disease incidence caused by the hemibiotrophic fungus Colletotrichum higginsianum. Antonie van Leeuwenhoeck 103:121-133.

Ramos P.L., Moreira-Filho C.A., Van Trappen S., Swings J., De Vos P., Barbosa H.R. and Thompson C.C., Vasconcelos A.T. and Thompson F.L. 2011. An MLSA-based online scheme for the rapid identification of Stenotrophomonas isolates. Mem Inst Oswaldo Cruz. 106(4):394-399.

Ramos P.L., Van Trappen S., Thompson F.L., Rocha R.C., Barbosa H.R., De Vos P. and Moreira-Filho C.A. 2010. Screening for endophytic nitrogen-fixing bacteria in Brazilian sugarcane varieties used in organic farming and description of Stenotrophomonas pavanii sp. nov. Int J Syst Evol Microbiol.

Raymundo T, Decock C., Valenzuela R., Amalfi M., Cifuentes J., Pacheco-Mota L. 2012. New records of the genus Fomitiporia (Hymenochaetales, Basidiomycota) in Mexico. Revista Mexicana de Biodiversidad 83: 313–32.

Reekmans, R., Van den Plas, C., Stevens, P., Vervust, T. and De Vos, P. 2009. An alternative real-time PCR method for the detection of thermotolerant Bacillus sensu lato contaminants in naturally contaminated gelatine. Letters in Appl. Microbiol. 48. 763-769.

Reekmans R., Stevens P., Vervust T. and De Vos P. 2009. An alternative real-time PCR method to detect Bacillus cereus group in naturally contaminated food gelatine: a comparison study. Lett. Appl. Microbiol. 48: 97-104.

Robledo G., Amalfi M., Castillo G., Rajchenberg M., and Decock C. 2009. Perenniporiella chaquenia sp. nov. from Argentina, and further notes on Perenniporiella and its relationships with Perenniporia (Poriales, Basidiomycota). Mycologia 101: 657-673.

Rosa C.A., Jindamorakot S., Limtong S., Nakase T., Lachance M.A., Fidalgo-Jiménez A., Daniel H.M., Pagnocca F.C., Inácio J. and Morais P.B. 2009. Synonymy of the yeast genera Moniliella and Trichosporonoides and proposal of Moniliella fonsecae sp. nov. and five new species combinations. Int J Syst Evol Microbiol 59: 425-429.

Rouah-Martin E., Mehta J., Van Dorst B., De Saeger S., Dubruel P., Maes B.U.W., Lemiere F., Goormaghtigh E., Daems D., Herrebout W., Van Hove F., Blust R., Robbens J. 2012. Aptamer-based molecular recognition of lysergamine, metergoline and small ergot alkaloids. Int. J. Mol. Sci. 13(12), 17138-17159.

Sanders M.E., Akkermans L.M.A., Haller D., Hammerman C., Heimbach J., Hörmannsperger G., Huys G., Levy D.D., Lutgendorff F., Mack D., Phothirath P., Solano-Aguilar G. and Vaughan E. 2010. Safety assessment of probiotics for human use. Gut Microbes 1:1-22.

Savini V., Hendrickx M., Sisti M., Masciarelli G., Favaro M., fontana C., Pitzurra L., Arzeni D., Astolfi D., Catavitello C., Polilli E., Farina C., Fazii P., D'antonio D. and Stubbe D. (2012). An atypical, pigment-producing Metschnikowia strain from a leukaemia patient. Medical Mycology (early online)

Scauflaire J. and Munaut F. 2009. Etat de la fusariose sur maïs en Wallonie. Les nouvelles de l’été, 3e trimestre 2009, Publication trimestrielle de la direction générale de l’agriculture des ressources naturelles et de l’environnement, Namur, Belgique. pp34-35.

Scauflaire J., Mahieu O., Louvieaux J., Foucart G., Renard F. and Munaut F. 2011. Biodiversity of Fusarium species in ears and stalks of maize plants in Belgium. European Journal of Plant Pathology, 131:59–66.

Scauflaire J., Gourgue M. and Munaut F. 2011. Fusarium temperatum sp. nov. from maize, an emergent species closely related to Fusarium subglutinans. Mycologia 103 : 586-597.

Scauflaire, J., Gourgue, M., Callebaut, A., Pussemier, L, Munaut, F. 2011. Pathogenicity and mycotoxin profile of

Fusarium temperatum, an emergent pathogen of maize in Belgium. Journal of Plant Pathology 93 (1, supplement), 25.

Scauflaire J., Gourgue M., Callebaut A., Munaut F. 2012. Fusarium temperatum, a mycotoxin-producing pathogen of maize. European Journal of Plant Pathology 133 (4): 911-922.

Scauflaire J., Godet M., Gourgue M., Liénard Ch. and Munaut F. 2012b. A multiplex real-time PCR method using hybridization probe for the detection and quantification of Fusarium proliferatum, F. subglutinans, F. temperatum and F. verticillioides. Fungal Biology, 116, 1073-1080.

Selouane A., Bouya D., Lebrihia A., Decock C., and Bouseta A. 2009. Impact of some environmental factors on growth and production of ochratoxin A by Aspergillus tubingensis, A. niger, and A. carbonarius isolated from Moroccan grapes. Journal of Microbiology 47: 411-419.

Selouane A., Zouhair S., Bouya D., Lebrihi A., Decock C. & Bouseta A. 2009. Production de l’ochratoxine A par des espèces d’Aspergillus isolées à partir du raisin: identification moléculaire de douze isolats et impact du milieux de culture sur la croissance et la production d’OTA. Proceeding of 3d international congress of Biochemistry, Marrakesh, 20-25 avril 2009.

Sistek V., Maheux A.F., Boissinot M., Bernard K.A., Cantin P., Cleenwerck I., De Vos P. and Bergeron M.G. 2012. Enterococcus ureasiticus sp. nov. and Enterococcus quebecensis sp. nov., isolated from water. Int J Syst Evol Microbiol. 62(Pt 6):1314-20.

Smet A., Flahou B., D'Herde K., Vandamme P., Cleenwerck I., Ducatelle P., Pasmans F., Haesebrouck F. Helicobacter heilmannii sp. nov., isolated from feline gastric mucosa. Int J Syst Evol Microbiol. 62(Pt 2):299-306. Erratum in: Int J Syst Evol Microbiol. 2012, 62(Pt 4):1016.

Stadler M., Fournier J., Læssøe T., Decock C., Peršoh D., Rambold G. 2010. Ruwenzoria, a new genus of the Xylariaceae from Central Africa. Mycological Progress 9: 169–179.

Stubbe D. and Verbeken A. (2012). Lactarius subg. Plinthogalus: The European taxa and American varieties of L. lignyotus re-evaluated. Mycologia 104(6):1490-1501.

Stubbe D., Wang X.H. and Verbeken A. (2012). New combinations in Lactifluus. 2. L. subg. Gerardii. Mycotaxon 119:483-485.

Stubbe D., Le H., Wang X.H., Nuytinck J., Van de Putte K. and Verbeken A. (2012). The Australasian species of Lactarius subgenus Gerardii (Russulales). Fungal Diversity 52(1):141-167.

Supré K., De Vliegher S., Cleenwerck I., Engelbeen K., Van Trappen S., Piepers S., Sampimon O.C., Zadoks R.N., De Vos P. and Haesebrouck F. 2010. Staphylococcus devriesei sp. nov., isolated from teat apices and milk of dairy cows. Int J Syst Evol Microbiol. 60(Pt 12): 2739-44.

Swinne D., Nolard N., Van Rooij P. and Detandt M. (2009). Bloodstream yeast infections in Belgium: a 15-months survey (06.2005-09.2006). Epidemiology and Infection, 137(7):1037-40.

Symoens F., Jousson O., Planard C., Fratti M., Staib P., Bernard M. and Monod M. (2010). Molecular analysis and mating behaviour of the Trichophyton mentagrophytes species complex. International Journal of Medical Microbiology 301(3): 260-6.

Triest D., Hendrickx M., De Cremer K., Piérard D. and Detandt M. 2012. Pathogenic moulds of the BCCM/IHEM collection: screening of Fusarium. Poster at the annual meeting of the Belgian Society of Human and Animal Mycology.

Triest D., Hendrickx M., De Cremer K., Stubbe D., Piérard D. and Detandt M. 2013. In vitro antifungal susceptibility testing of the clinical Fusarium strains in the Belgian fungal BCCM/IHEM-collection suggests alternative treatments for different types of Fusarium infection. Article in preparation.

Vanden Bempt I., Van Trappen S., Cleenwerck I., De Vos P., Camps K., Celens A. and Van De Vyvere M.2011. Actinobaculum schaalii causing Fournier's gangrene. J Clin Microbiol. 49(6):2369-71.

Vanhee L., Symoens F., Jacobsen M.D., Nelis H.J. and Coenye T. (2009). Comparison of multiple typing methods for Aspergillus fumigatus. Clinical Microbiology and Infection, 15:643-650.

Vanhulle S., Trosvalet M., Enaud E., Lucas M., Mertens V., Sonveaux M., Decock C., Schneider J. and Corbisier A.M. 2008. Cytotoxicity and genotoxicity evolution during decolourization of dyes by White Rot Fungi. World Journal of Microbiology and Biotechnology 24: 337–344.

Van den Bossche D., De Bel A., Hendrickx M., De Becker A., Jacobs R., Naessens A. and Piérard D. (2012). Galactomannan enzymatic immunoassay cross-reactivity caused by Prototheca species. Journal of clinical microbiology 50 (10): 3371-3373.

Van Hoorde K., Heyndrickx M., Vandamme P. and Huys G. 2010. Influence of pasteurization, brining conditions and production environment on the microbiota of artisan Gouda-type cheeses. Food Microbiol. 27:425-433.

Van Hoorde K., Van Leuven I., Dirinck P., Heyndrickx M., Coudijzer K., Vandamme P. and Huys G. Selection, application and monitoring of Lactobacillus paracasei strains as adjunct cultures in the production of Gouda-type cheeses. Int. J. Food Microbiol. 144:226-235.

Valenzuela R., Raymundo T., Amalfi M., Castillo G. and Decock C. 2010. Two undescribed species of Phylloporia are evidenced in Mexico based on morphological, phylogenetic and ecological data. Mycological Progress 10: 341-349.

Valenzuela R., Raymundo T., Amalfi M., Decock C. 2012. Coltriciella sonorensis sp. nov, an undescribed species from Mexico: evidence from morphology and DNA sequence data. Mycological Progress 11:181–189.

Valenzuela R., Raymundo T., Decock C., Esqueda M. 2012. Aphyllophoroid fungi from Sonora, México 2. New records from Sierra of Álamos-Río Cuchujaqui Biosphere Reserve. Mycotaxon 122: 51-59.

Van Hove F. 2010. Des mycotoxines dans les parties apparemment non moisies des ensilages. Point Vet. 41(310):66-68.


Van Hove F., Waalwijk C., Logrieco A., Munaut F. and Moretti A. 2011. Gibberella musae (Fusarium musae) sp. nov.: a new species from banana closely related to F. verticillioides. Mycologia 103: 570-585.

Van Hove F., De Saeger S., Lazzaro I., Qi Y., Zhang D., Wu A., Munaut F. 2013. Phylogenetic diversity of and fumonisin gene cluster distribution within Fusarium isolates from wild banana in China. "12th European Fusarium Seminar", 12-16/05/2013, Bordeaux, France.

Van Poucke K., Monbaliu S., Munaut F., Heungens K., De Saeger S., Van Hove F. 2012. Genetic diversity and mycotoxin production of Fusarium lactis species complex isolates from sweet pepper. Int. J. Food Microbiol. 153(1-2):28-37.

Van Rooij P., Declercq J. and Beguin H. (2011). Canine dermatophytosis caused by Trichophyton rubrum: an example of man-to-dog transmission. Mycoses 55 (2): 15-17.

Van Rooij P., Declercq J., Beguin H. 2012. Canine dermatophytosis caused by Trichophyton rubrum: an example of man-to-dog transmission. Mycoses 55: e15-e17.

Vanheule A., De Boevre M., Audenaert Bekaert, B., K., Eeckhout M., Munaut F., De Saeger S. and Haesaert, G. 2013. Natural occurrence of Fusarium mycotoxins in small unprocessed cereals, food and feed products using a newly developed and validated UPLC-MS/MS. To be submitted in Journal of Agricultural and Food Chemistry.

Voets L. , de la Providencia I., Fernandez K., IJdo M, Cranenbrouck S. and Declerck S. 2009. Extraradical mycelium network of arbuscular mycorrhizal fungi allows fast colonization of seedlings under in vitro conditions. Mycorrhiza 19: 347-356.

Voyron S., Roussel S., Munaut F., Varese G.C., Declerck S. and Filipello Marchisio V. 2009. Vitality and genetic fidelity of White Rot Fungi mycelia following different methods of long-term preservation. Mycological research 113, 1027-103.

Vrancken G., De Vuyst L., Van der Meulen R., Huys G., Vandamme P. and Daniel H.M. 2010. Yeast species composition differs

between artisan bakery and spontaneous laboratory sourdoughs. FEMS Yeast Res. 10:471-481.

Vrancken G., De Vuyst L., Van der Meulen R., Huys G., Vandamme P., Daniel H.M. 2010. Yeast species composition differs between artisan bakery and spontaneous laboratory sourdoughs. FEMS Yeast Research 10: 471-481.

Wang X.H., Stubbe D. and Verbeken A. (2012). Lactifluus parvigerardii sp. nov., a new link towards the pleurotoid habit in Lactifluus subgen. Gerardii (Russulaceae, Russulales). Cryptogamie-Mycologie 33.

Weckx S., Van der Meulen R., Allemeersch J., Huys G., Vandamme P., Van Hummelen P. and De Vuyst L. 2010. Community dynamics of bacteria in sourdough fermentations as revealed by their metatranscriptome. Appl. Environ. Microbiol. 76:5402-5408.

Weckx S., Allemeersch J., Van der Meulen R., Vrancken G., Huys G., Vandamme P., Van Hummelen P. and De Vuyst L. 2011. Metatranscriptome analysis for insight into whole-ecosystem gene expression during spontaneous wheat and spelt sourdough fermentations. Appl. Environ. Microbiol. 77:618-626.Wieme A., Cleenwerck I., Van Landschoot A. and Vandamme P. 2012. Pediococcus lolii DSM 19927T and JCM 15055T are strains of Pediococcus acidilactici. Int J Syst Evol Microbiol. 62(Pt 12):3105-8.

Wind, R.D., Tolboom H., Klare I., Huys G. and Knol J. 2010. Tolerance and safety of the potentially probiotic strain Lactobacillus rhamnosus PRSF-L477: a randomized, double-blind placebo-controlled trial in healthy volunteers. British Journal of Nutrition 104:1806-1816.

Winkelhoff A.J., Cleenwerck I., De Vos P. 2010. Phylogenetic variation of Aggregatibacter actinomycetemcomitans serotype e reveals an aberrant distinct evolutionary stable lineage. Infect Genet Evol. 10(7): 1124-31.

Yombiyeni P., Douanla-Meli C., Amalfi M. and Decock C. 2010. Poroid Hymenochaetaceae from Guineo-Congolian

rainforest: Phellinus gabonensis sp. nov. from Gabon - taxonomy and phylogenetic relationships. Mycological Progress 10: 351-362.

Zago, M., Huys G. and Giraffa G. 2010. Molecular basis and transferability of tetracycline resistance in Enterococcus italicus LMG 22195 from fermented milk. International Journal of Food Microbiology 142:234-236.

Zhao J.P., Lu Q., Liang J., Decock C. and Zhang X. Y. 2010. Lasiodiplodia pseudotheobromae, a new record of pathogenic fungi from some subtropical and tropical trees in southern China. Cryptogamie Mycologie 31 (4): 431-439.


ContactsThe BCCM consortium consists of seven complementary research-based service culture collections and a Coordination Cell, financed and coordinated by the Belgian Science Policy Office.

Coordination CellBegian Science Policy officePhone: +32 (0)2 238 34 62 Email: [email protected]

DiatomsGhent UniversityPhone: +32 (0)9 264 85 10 Email: [email protected]

Biomedical Fungi & YeastsScientific Institute of Public Health, BrusselsPhone: +32 (0)2 642 55 18 E-mail: [email protected]

MycobacteriaInstitute of Tropical Medicine, AntwerpPhone: +32 (0)3 247 65 51E-mail: [email protected]

PlasmidsGhent UniversityPhone: +32 (0)9 331 38 43E-mail: [email protected]

BacteriaGhent UniversityPhone: +32 (0)9 264 51 08 E-mail: [email protected]

Environmental and applied MycologyUniversité Catholique de LouvainPhone: +32 (10) 47 37 42 E mail: [email protected]

Cyanobacteria University of LiègePhone: +32 (0)4 366 33 87 E-mail: [email protected]

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