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  Director : Grimont Patrick (pgrimont@pasteur.fr )


  abstract

 

The Unit includes research laboratories, three National Reference Centers, a Center providing bacterial identification services in clinical, veterinarian, and industrial fields, and R&D projects. 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 molecular identification and typing. Our methods have been applied to all sorts of bacteria including emerging pathogens.



  report

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Taxonomy and phylogenomics (Patrick Grimont, Francine Grimont and, since 2003, Sylvain Brisse)

Our Unit participates in international efforts targeted to the molecular and phylogenomic definition of the bacterial species. An ad-hoc committee met in 2002 to evaluate the possibility of replacing DNA-DNA hybridization by easier methodologies. Multiple gene sequencing is an approach which needs to be validated. Algorithms for computer interpretation will be essential.

The expertise built up in the domain of bacterial detection and identification is used for preparedness in responding to the bioterrorism threat. Several molecular methods can be proposed for rapid detection of pathogens. Most efforts are needed for laboratory organization (dispatch of strains and information) and plans to mobilize personnel and space.

We have been asked by several colleagues to characterize new bacterial species by DNA-DNA hybridization or 16S rRNA gene sequencing. We have participated in the description of Afipia birgiae and Afipia massiliensis and a new unnamed genospecies which had been isolated by coculture with amoebae by colleagues from Marseille. However, two serotypes of Bartonella henselae which differed in some gene sequences were found to belong to a single DNA hybridization group and thus to a single species. With colleagues from Nancy (France), we demonstrated that the genus Desulfomonas does not exist and that Desulfomonas pigra is in fact a Desulfovibrio (Desulfovibrio piger). The rare species, Corynebacterium macginleyi, causing ocular infections and which was believed to be geographically limited, was isolated in Italy and its identification was confirmed by rrs gene sequencing.

We have collaborated with National Reference Centers in France or abroad. With the Pasteur Institute in Iran, we have characterized Vibrio cholerae strains circulating in Iran. The presence of four virulence genes and a large plasmid was examined. The strains typed by ribotyping and pulse field gel electrophoresis showed limited diversity with a dominant ribotype, typical of the region. With the Legionella National Reference Center in Lyon, we have built a ribotype database allowing for the identification of all known species of Legionella. This approach is being automatized.

The Unit provides a taxonomic support to colleagues in University Hospitals who study antibiotic resistance genes. We have thus collaborated to the discovery that plasmid genes encoding CTX-M beta-lactamases originated in a Kluyvera ascorbata chromosomal gene. An AmpC gene encoding an inducible beta-lactamase was found in a new species of Enterobacteriaceae.

Taxonomic research may have biotechnological interest. The production of poly(3-hydroxybutyrate) and poly(3-hydroxyoctanoate) has been studied in the genus Pseudomonas and differ according to phylogenetic branches. In collaboration with colleagues in Clermont-Ferrand (France), a strain producing isonovalal from alpha-pinene oxide has been identified by rrs sequencing as Pseudomonas rhodesiae. This species was not known to biotransform terpenes.

National Reference Center for Salmonella (Philippe Bouvet and later François-Xavier Weill and Patrick Grimont)

CNR-Salm contributes to the surveillance of salmonellosis in France by performing complete serotyping (with 164 sera) on more than 10,000 clinical isolates of Salmonella each year. Strains are sent to CNR-Salm (with essential epidemiological information) on a voluntary basis by about 1600 public or private clinical laboratories. CNR-Salm also collects information on strains which were serotyped locally (5000 per year). A house-made computer system for surveillance and alert of clinical salmonellosis allows to document spatial and temporal tendencies of major Salmonella serotypes and to quickly detect any significant increase in the number of cases at national, regional, or department levels. Data reports are sent on a weekly basis to the Institut de Veille Sanitaire (InVS). As soon as a significant increase is detected, an alert message is sent. A RiboPrinter was acquired which allows us to ribotype all strains of Salmonella enterica serotype Typhi. CNR-Salm also performs phage typing for Salmonella serotypes Typhi, Typhimurium, Paratyphi B, and Enteritidis. CNR-Salm 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. CNR-Salm participates in the Enter-Net European network. Research work derived from these activities included the characterization, with Italian colleagues, of S. bongori (a rare species) occurring in man, animals and the environment, in Southern Italy where infections were observed. By combining phage typing, pulse-field gel electrophoresis and antibiotic resistance patterns, the spread of multiresistant S. enterica ser. Typhimurium clones was followed in collaboration with the French Agency for Food Safety (AFSSA).

National Reference Center for Escherichia coli-Shigella (Francine Grimont and Patrick Grimont)

CNR-coli participates in the surveillance of shigelloses and infections due to shigatoxin-producing E. coli (haemorrhagic colitis and haemolytic and uremic syndromes). About 1000 isolates of Shigella are serotyped each year. Detection of E. coli virulence genes is performed as well as classical serotyping and ribotyping. Serological tests for haemolytic and uremic syndromes are performed. A method was devised for molecular serotyping with the study of genes encoding enzymes involved in somatic (O) polysaccharide synthesis (rfb region) and genes encoding flagellin (fliC). Isolates of E. coli O157 :H7 from diverse regions of France and Italy were compared for the presence of virulence genes, pulse-field gel electrophoresis patterns, ERIC-PCR patterns and antibiotic susceptibility patterns. The spread of some strains could thus be followed. In addition, a case of haemolytic and uremic syndrome due to a shigatoxin-nonproducing strain was studied.

National Reference Center for Corynebacterium diphtheriae (Patrick Grimont and Anne Le Flèche)

CNR-Cd confirms the 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 Francine Grimont and in the framework of the WHO-Europe network ELWGD (European Laboratory Working Group on Diphtheria) and the DIPNET network (DG-SANCO), a database of C. diphtheriae ribotypes was 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. With colleagues from the International Network of Pasteur Institutes, we have participated in a study of strains circulating in North-West Russia, Romania, and Moldavia. Romanian strains differed from Russian and Moldavian strains in their ribotypes, pulse-field types, and RAPD patterns.

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

CMIB was created in 2000 to identify all kinds of bacteria by molecular methods (sequencing of rrs and other genes, automated ribotyping, PCR). Strains are received from clinical, veterinary, environmental, or industrial sources. 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 new phylogenetic branches. Strains (other than industrial strains) which do not fit any described species are included in our current taxonomic research.

The Experimental Kitchen Project (Patrick Grimont, Martine Le Fèvre, Corinne Ruckly)

The project, supported by Procter & Gamble, allowed to study the spread of marker bacilli through an experimental kitchen with the purpose of evaluating disinfection strategies. Routes of contamination were observed and the effect of disinfection with different products was measured. Overall, surface contamination is reduced by one- to three-log through dessication (depending on the bacterial species studied), reduced by three-log when a common cleaning detergent is used, and reduced by six-log (i.e. totally suppressed) when an antibacterial product is used. Lessons learnt from this work should help reduce the incidence of foodborne infections by disinfecting contamination hot-spots.

R&D laboratory (Béatrice Regnault and Patrick Grimont)

This R&D laboratory is supported by industrial contracts. Within seven 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. The frontier between bacterial life and death has been explored. It has been demonstrated that revivification of bacteria in the presence of ciprofloxacine allowed to distinguish living bacteria (which elongate) from dead bacteria. Activities of this laboratory will be continued by CIMB in 2003.

Photo: Ribotypes of Corynebacterium diphtheriae produced by the RiboPrinter System and treated by Taxotron (with mathematical filtering). Size marker is in lanes 1, 4, 7, 10, and 13.

Keywords: Bacteriology, Taxonomy, phylogenomics, enterobacteria, molecular typing, identification, population genetics



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Sylvain, Chantal (csylvain@pasteur.fr )

Lebri, Zoulika (leaving January 2003)

Valérie Abihssira ( abihssir@pasteur.fr )

Grimont, Patrick, Institut Pasteur (Professor, pgrimont@pasteur.fr )

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

Bouvet, Philippe, Institut Pasteur (left the Unit in September 2002)

Brisse, Sylvain, Institut Pasteur (Researcher, arrived January 2003, sbrisse@pasteur.fr )

Weill, François-Xavier, Institut Pasteur (Clinical Microbiologist, arrived January 2003, fxweill@pasteur.fr )

Ait-Tayeb, Lineda (PhD student)

Bermond-Tilly, Delphine (post-doc)

Jamin, Yann

Koneva, Natalia

Lemaire, Céline

Loubinoux, Julien

Luc, Audrey

Villarino, Andrea

Le Flèche, Anne (Engineer, CIMB, lefleche@pasteur.fr)

Regnault, Béatrice (Engineer, R&D, until January 2003)

Janvier, Monique (Engineer, Teaching Center)

Arnoux, Yolande (Tech., CIMB)

Carle, Isabelle (Tech., CNR E. coli-Shigella)

Demartin, Marie (Tech., CNR Salmonella)

Fabre, Laetitia (Tech., CNR Salmonella)

Guesnier, Françoise (Tech., CNR Salmonella)

Issenhuth-Jeanjean, Sylvie (Tech., R&D CNR Salmonella)

Lefevre, Martine (Tech., CIMB)

Lejay-Collin, Monique (Tech., CNR E. coli-Shigella)

Lomprez, Fabienne (Tech., CIMB)

Martin-Delautre, Sylvie (Tech., CNR E. coli-Shigella)

Passet, Virginie (Tech, Research)

Polomack, Bernadette (Tech., CNR Salmonella)

Ruckly, Corinne (Tech., CIMB and CNR Salmonella)

Sanquer, Laurence (Tech., CNR Salmonella)


Activity Reports 2002 - Institut Pasteur
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