Unit: Institut Pasteur Collection
Director: BIZET Chantal
The role of the Collection de l'Institut Pasteur (CIP) is to ensure the maintenance and the enrichment of its bacterial collection, the production of a catalogue, the diffusion of information on the distributed strains, the development of research on identification, taxonomy and preservation of bacteria and to participate to the teaching operations of the Institut Pasteur. The CIP has been certificated ISO 9001 version 2000 on June 2001. The CIP belongs to the Centre of Biological Resources of Institut Pasteur and participates to several international and national collection projects for the creation of the Biological Resources Centres.
1. Strain stock management
More than 8 800 strains are available on stock. Every year, new bacterial strains are added to the collection (about 300) coming either from researchers of the Institut Pasteur, or from foreign collections or from newly deposited strains after having connection with foreign or French researchers. The CIP contains also plasmids, transposons, host strains and probes. Titrated spore suspensions (species Bacillus and Clostridium) are available. Strains are preserved in two different places and by two different ways (freeze dried and deep-frozen at - 80°C, or deep-frozen in nitrogen liquid and at - 80°C for strains which cannot be freeze-dried).
2. Check up of the CIP strains
a) Total protein electrophoresis and chimiotaxonomy
This laboratory helps in developing bacterial identification methods by analysis of bacterial cell wall components : lipids, peptidoglycane, sugars... and total proteins. Main techniques used are chromatography and derivated techniques and also electrophoresis in discontinued gel. Results are used thanks to a computerized process with specific softwares.
We are still working on feeding several databanks with information regarding protein profiles and nature of cell wall components (about 4000 strains).
A database with 900 profiles obtained after ribotyping has been performed.
This laboratory is necessary for the Collection strain check and strain expertise for industrial laboratories
c) molecular characterization.
All the new incorporated strains are identified by sequencing the gene coding for ARNr 16S. We begin to sequence all the strains of the collection.
3. Quality System
Process regarding activities and tasks are written in an organized process system which suits to the CIP organization. Tasks are recorded. Implemented processes are always followed up and improved. Regularly, internal audits are made in order to check and measure differences between what is written and what is done. Quality indices are provided with laboratory problems and requests from customers. If necessary, corrective actions are undertaken. Certification (ISO 9001) has been granted for 3 years. But, every year, a follow up audit must be made in order to keep it.
The CRBIP (including the CIP) has renewed its certification in November 2005 according to the new referential ISO 9001 version 2000 and the used methods by the CIP for identifying are going to be accredited regarding 17025 standard.
4. Biological Resources Centre of the Institut Pasteur (CRBIP)
The Institut Pasteur is the French institution having on one site, the most important collections specialized in microorganisms (bacteria, fungi, viruses ).
In most of cases, these collections are kept inside research laboratories. Only four of these collections are "opened" i.e. they send strains to laboratories, pharmaceutical industries, research laboratories, education when receiving an order form. These four collections are recorded in the World Directory of Collections of Cultures of Microorganisms. It includes the collection of the Institut Pasteur (CIP), the cyanobacteria collection, the fungi collection (UMIP) and the collection of sporulated entomopathogenical bacteria (IBEC).
With the creation of the Center of Biological Resources of Institut Pasteur (CRBIP), it involves :
1) to increase the number of microorganisms strains available for the scientific researchers and industrials for which including, production, check (including molecular), preservation, stocking and distribution are made according to Quality Insurance and legal requests, specifically regarding safety,
2) to create and run : the software for the management of the collection, used by all the collections into the CRBIP. Such a software should be able to manage all data for different kind of microorganisms. These data concerning taxonomy, keywords references for bibliography, morphological, physiological, physiopathological and molecular characters, pratical information (such as used medium, temperature for growing ), as well as the management of the production, storage and distribution.
Presently two collections have been integrated into the CRBIP, CIP and UMIP, bacteria coming from Leptospira, Bordetella, Listeria, and some viruses (Retrovirus, Flavivirus and Lyssavirus) have been included into the CRBIP after having check their viability and identity and having a minimum stock. These strains come from different laboratories of the Institut Pasteur : Reference National Centers and Research laboratories. About 12.000 strains are in the CRBIP.
Moreover, CRBIP is involved in the preservation of collections such as Staphylococcus, anaerobia form Jean-Philippe Carlier's collection, Maurice Hofnung's collection, venom and counter-venom's collections ).
The main goal was to deal with the creation of a software for running all the collections (ARPAS). The database has been created for adding different collections. An improvement has been made to implement Quality Insurance.
CRBIP has been certified in March 2005 regarding ISO 9001 version 2000 standard for accession, production, preservation and distribution of biological materials.
Connections to ARPAS are done through our Internet, a web site has been created since July 2005 (http://www.crbip.pasteur.fr providing information to scientists, teachers, industrials..., regarding strains it is a positive action for the CRBIP.
5. Identification of strains belonging to Acinetobacter genus using a phenotypic and molecular approach
The genus Acinetobacter was created in 1954 by Brisou and Prévot. These Gram-negative short rods are oxidase-negative, non motile, and strictly aerobic.
Phylogenetic analysis based upon RNA 16S sequences were useful to classify Acinetobacter in the sub-specy gamma of Proteobacteria. 21 genomic groups were found out through DNA-DNA studies. However for a long time, Acinetobacter has only 7 species. Recently 9 new species of Acinetobacter were described. We can conclude on the fact that a lot of new species are not yet discovered.
22 strains of Acinetobacter of the CIP have been characterized through phenotypic and electrophoresis of global proteins methods and by sequencing RNA 16S. Results show that 4 strains can be included into genomospecies 9, 4 strains to Acinetobacter baumanii specy, 6 strains to Acinetobacter parvus specy, 1 strain to genomospecies 10 and 1 strain to genomospecies 13.
However, we are not sure of their identification.
6. Taxonomic approach of CDC group EF-4 strains
Group CDC EF-4 has been found by E.O. King by studying 85 strains isolated between 1949-1973. These Gram-negative rods are non motile, strictly aerobic, catalase and oxydase positive. Group EF-4 was separated into two biovars EF-4a and EF-4b. EF-4a biovars possess an arginine dihydrolase activity whereas EF-4b biovars do not.
18 strains of group CDC EF-4 isolated in human after animal bites and deposited into the CIP in the 80's are being studying in order to get characterization of a new bacteria taxon using phenotypic methods, gene sequencing and electrophoresis of whole-cell proteins.
By sequencing the coding gene of RNA 16S, 3 groups are different form the 3 which were identified through electrophoresis of global proteins. Moreover, sequencing the coding gene for RNAr 16S doesn't enable to distinguish the strains of EF-4 group for the species of Neisseria weaveri and Neisseria canis. Another sequencing of another coding gene is on going.
7. Taxonomic characterization of Microbacterium strains
Microbacterium genus has been described in 1919 by Orla Jensen. It is the type genus of the family Microbacteriaeceae. In 1998, the genus Aureobacterium was unified with the genus Microbacterium. In 2005, three new species of Microbacterium were described (M. natoriense, M. oleivorans and M. xylanilyticum) increasing to forty the number of species in this genus.
Forty three strains of Microbacterium were deposited into the CIP in the 80's and are not yet identified. Most of them were isolated from human blood whereas Microbacterium strains are usually found environmental samples. Using electrophoresis of total proteins, phenotypic methods and sequencing genes coding for 16S ribosomal RNA and gyrB, these strains are taxonomically studied in order to identify them and eventually describe a new taxon.
8. Sequencing of coding gene for RNAr 16S for the type strains of the CIP
With the Genopole of the Institut Pasteur and in order to increase the enrichment of the CIP data base, the coding gene for RNAr 16S of the type strains is systematically sequenced.
Bacterial strains from external collections are preserved in the CIP. It's submitted to a maintenance contract. CIP provides industrial laboratories with spore suspensions.
Since 2002, these activities have been certifying with the standard ISO 9001 version 2000.
10. National relationships
The Ministry of Research created Biological Resources Centers (BRC) at a national level which is managed by a Committee of Biological Resources. The CIP belongs to this Committee. Its missions are to create a national network for BRC, to implement a Quality Insurance and to check the launch of quotations for creation and finance of accredited BRC,
11. International relationships
The CIP is a member of different official organizations : l'ECCO (European Culture Collections' Organization - www.eccosite.org), EBRCN (European Biological Resource Centres Network - www.ebrcn.org), CABRI (Common Access to Biotechnological Resources and Information - www.cabri.org and WFCC (World Federation for Culture Collection).
1. In 1999, OECD (Organization for Economic Cooperation and Development) initialized an international meeting in Tokyo in order to look at the necessary conditions to set up Biological Resource Centers (BRC).
The Presidence of this project has been given to France and a study group is in charge of determining if it's possible to create and implement biological resources centers. The CIP belongs to the French delegation and writes documents regarding the creation of the Insurance Quality implementation in collaboration with the other delegates.
2. European Biological Resource Centers Network (EBRCN)
The CIP participates to EBRCN.
This European project enables Europe to :
. show its study skills in CRB in computing
. create global policy
. maintain a high level of quality for the services provided by European Collections.
Aims of this project are the following :
. to create a Biological Resource Centers network including alive organisms, components and associated data bases, and to answer OECD new decisions in an international context.
. to develop European standards for BRC, based upon management quality system
. to create a framework in order to increase complementarity and to decrease redundancy among European BRC.
. to introduce new techniques in Information Technology in order to bring added value to the catalogue and to give a better access.
. to get and provide information on BRC.
Keywords: taxonomy, sequencing, biodiversity, collection, bacterial strains, studies, preservation, CBR