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  Enbac


  Director : Patrick A. D. Grimont (pgrimont@pasteur.fr)


  abstract

 

The Unit includes research laboratories, three National Reference Centers, and three industry-oriented laboratories. All modern aspects of bacterial taxonomy are considered with the purposes of providing a better molecular and physiological definition of bacterial species and designing new tools for identification and epidemiological typing. Our methods have been applied to all sorts of bacteria including emerging pathogens.



  report

cale

Molecular taxonomy of bacteria (Patrick Grimont, Elisabeth Ageron, with many collaborations)

Most molecular methods are universally applicable. Comparison of rrs gene (encoding 16S rRNA) sequences allows to position a new isolate in the phylogenetic tree. However, DNA-DNA hybridization remains the essential tool for bacterial species delineation. Other gene sequences (r.g. rpoB) are studied with the aim to reach a taxonomic resolution similar to that of DNA-DNA hybridization. Once new species have been carefully delineated, discriminant phenotypic traits (Biotype-100 strips) or specific signatures in rrs or other genes are searched for. A new species of the genus Vibrio has been described in collaboration with Spanish and German teams and two new species of Legionella have been described in collaboration with our colleagues from Lyon..

Infraspecific diversity (Francine Grimont, Roney S. Coimbra)

Traditional typing tools are generally limited to a single bacterial species whereas molecular methods are more widely applicable. Performing molecular typing systems have been developped. Computer identification of patterns is currently done with softwareTaxotron. A ribotyping system for the genus Shigella has been set up (in collaboration with Giuseppe Nicastro). With endonuclease MluI, 51 discrete ribotypes were observed. A ribotype database allowed to identify 111 clinical isolates of Shigella. Somatic O antigens of Enterobacteriaceae result from the action of several enzymes. The genes encoding most of these enzymes are clustered in chromosomal region rfb. This region has been amplified and restricted for many E. coli serotypes and all Shigella serotypes. A database of restriction fragment sizes allows computer identification of E. coli and Shigella O serogroups. This approach allowed us to discover a new Shigella dysenteriae serotype represented by 22 nonagglutinable isolates. A cryptic flagellar gene (fliC) has been evidenced in 120 strains of Shigella (nonmotile bacteria). After restriction of the amplified gene, 17 patterns were observed. This approach may help uncover the evolutionary history of Shigella.

Metabolic diversity of bacteria (Odile Bouvet)

Synthesis of poly-beta-hydroxyalcanoate by Pseudomonas strains representing various known ribogroups has been studied (Stephane Diard's thesis). Methods which provide a global approach of the metabolism (e.g. application of nuclear magnetic resonnance) were set up as well as techniques allowing to evaluate the physiologic status of bacteria (energetic and oxidative state of bacteria, substrate transport, intracellular pools, activity of major metabolic pathways). These methods contributed to the study of bacterial viability developped at Aquabiolab.

Aquabiolab (Béatrice Regnault, Patrick Grimont)

This R&D laboratory is supported by industrial contracts. Within six years, it has acquired a wide experience in fluorescence in situ hybridization and has designed rapid molecular methods for detecting and enumerating specific bacterial species in water, foods, or clinical samples. A project exploring the frontier between bacterial life and death has been developped (Andrea Villarino's thesis). It has been demonstrated that revivification of bacteria in the presence of ciprofloxacine allowed to distinguish living bacteria (which elongate) from dead bacteria. This year, in collaboration with Maria C. Urdaci (Bordeaux), a fluorescent probe has been designed to visualize nonculturable segmented filamentous organisms associated with trout diarrhoea.

National Reference Center for Salmonella and Shigella (Philippe Bouvet and Patrick Grimont)

The NRCSS contributes to the surveillance of salmonellosis in France by performing complete serotyping (with 164 sera) on more than 10,000 clinical isolates of Salmonella and about 1000 isolates of Shigella each year. Strains are addressed to the NRCSS (with essential epidemiological information) on a voluntary basis by about 1600 public or private clinical laboratories. The NRCSS collects also information on strains which were serotyped locally (5000 per year). A house-made computer system for surveillance and alert of clinical salmonellosis and shigellosis, allows to document spatial and temporal tendencies of 80 major Salmonella or Shigella serotypes and to quickly detect any significant increase in number of cases at national, regional, or department level. Data reports are sent on a weekly basis to Institut de Veille Sanitaire (InVS). As soon as a significant increase has been detected, an alert message is sent by fax. The NRCSS has set up surveillance of antimicrobial resistance among Salmonella strains. A total of 1000 strains, randomly selected among 15 major serotypes, are studied each year. NRCSS participates in the Enter-Net European network.

National Reference Center for Enteric Molecular Typing (Francine Grimont, Patrick Grimont)

The NRCEMT performs ribotyping on all sorts of bacteria at the request of hospitals and health authorities. A RiboPrinter was acquired which allows us to ribotype all strains of Salmonella enterica serotype Typhi, and strains involved in nosocomial infections. The detection of E. coli pathogenicity genes is done upon request. The NRCEMT also does phage typing for Salmonella serotypes Typhi, Typhimurium, Paratyphi A, Paratyphi B, Enteritidis, and Dublin, and serotyping of Serratia strains. The NRCEMT participates in the European network Enter-Net to the surveillance of infections caused by Salmonella enterica serotypes Typhi, Typhimurium, Enteritidis, and Dublin, and that due to enterohaemorrhagic Escherichia coli.

National Reference Center for Corynebacterium diphtheriae (Patrick Grimont, Yolande Arnoux)

The NRCCD confirms identification of Corynebacterium diphtheriae and C. ulcerans strains isolated in France, determines the presence of diphtheria toxin genes by PCR, and performs molecular typing on these strains. In association with the NRCEMT and in the framework of the WHO-Europe network ELWGD (European Laboratory Working Group on Diphtheria) and two European contracts, BIOMED and INCO-Copernicus, a database of C. diphtheriae ribotypes has been built after studying 576 strains isolated in many contries including the former USSR.These strains are distributed among 86 ribotypes. The Taxotron software has been chosen for the computer identification of ribotypes.

Center for Molecular Identification of Bacteria (Anne Le Flèche, Patrick Grimont)

The CMIB was created in 2000 to identify all sorts of bacteria by molecular methods (sequencing of rrs and other genes, automated ribotyping, PCR). Strains are received from clinical, veterinary, environmental, or industrial laboratories. In 2001, among strains identified by gene sequencing, 79% corresponded to 123 different known species distributed among 62 genera, 5% corresponded to unnamed taxons with known rrs sequences, 15% corresponded to unknown species within known genera, and 1% corresponded to a new phylogenetic branch. Strains (other than industrial strains) which do not fit any described species are included in our current taxonomic research.

Taxolab (Patrick Grimont, Martine Lefèvre, Corinne Ruckly)

A program, supported by Procter & Gamble, studies the spread of marker bacilli through an experimental kitchen with the purpose of evaluating disinfection strategies. Routes of contamination have been observed and the effect of disinfection with different products has been measured. Lessons learnt from this work should help reduce the incidence of foodborne infections by disinfecting contamination hot-spots.

Photo :

Nonculturable segmented filamentous bacteria visualized in the intestine of diarrhoeic trouts by fluorescence in situ hybridization (Urdaci et al., 2001)



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

Sylvain, Chantal (for the Research Unit, csylvain@pasteur.fr)

Lebri Zoulika (for Reference Centers, zlebri@pasteur.fr)

Bouvet, Odile, INSERM (up to Dec. 31, 2001)

Bouvet, Philippe, IP (pbouvet@pasteur.fr)

Grimont, Francine, INSERM (fgrimont@pasteur.fr)

Grimont, Patrick, IP

Aslani, Mehdi, post-doc (Pasteur Institute, Tehran)

Coniglio, Maria Anna (University of Catania)

Diard, Stephane, PhD student

Fourniol, Aurelie

Gelegen, Cigdem (Istanbul)

Le Thi Anh Hong (Hanoi)

Lemaire, Céline

Loubinoux, Julien

Martinez, Olivier

Mayali, Cindy

Nassif, Nadine

Schlegel, Laurent, PhD student

Toribio, Ana Luisa (University of Montevideo)

Villarino, Andrea, PhD student

Janvier, Monique (Engineer, Teaching Department)

Le Fleche, Anne (Engineer, CMIB, lefleche@pasteur.fr)

Ageron-Ardila, Elisabeth (INSERM)

Abihssira, Marie-Valerie (NRC Salmonella - Shigella)

Arnoux, Yolande (NRC Corynebacterium diphtheriae)

Carle, Isabelle (NRC Enteric Molecular Typing)

Felix, Thomas (NRC Salmonella - Shigella)

Guesnier, Francoise (NRC Salmonella - Shigella)

Hugain, Dimitri (NRC Salmonella - Shigella)

Issenhuth-Jeanjean, Sylvie (R&D, NRC Salmonella - Shigella)

K’ouas, Guylène (NRC Salmonella - Shigella)

Lefevre, Martine (Taxolab)

Lejay-Collin, Monique (NRC Enteric Molecular Typing)

Lomprez, Fabienne (CMIB)

Martin-Delautre, Sylvie (NRC Enteric Molecular Typing)

Regnault, Béatrice (Aquabiolab, regnault@pasteur.fr)

Ruckly, Corinne (Taxolab and NRC Salmonella - Shigella)

Sanquer, Laurence (NRC Salmonella - Shigella)

Troubadour-Courcoux, Pascale (NRC Salmonella - Shigella)


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