Unit: Biodiversity of Emerging Pathogenic Bacteria - U389 INSERM

Director: GRIMONT Patrick

The Unit includes research laboratories, a WHO Collaborating Center, three National Reference Centers (CNR), and a Center providing bacterial identification services in clinical, veterinarian, and industrial fields. 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.

Taxonomy and population genetics (Sylvain Brisse, Francine Grimont, Monique Janvier, Patrick Grimont)

Our Unit participates in international efforts toward 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. An automated workstation has been acquired for the preparation of 96 sequence reactions. A study of the taxonomic structure of Klebsiella by use of multiple gene sequencing has been initiated.

The problem in detecting bacteria by molecular methods is to differentiate living bacteria from dead bacteria. Death is an irreversible state where culture, bacterial elongation, and protein synthesis are no more possible (Andrea Villarino's thesis).

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.

We have been asked by several colleagues to characterize new bacterial species by DNA-DNA hybridization or rrs gene (encoding 16S rRNA) sequencing. We have contributed to a taxonomic study on the Streptococcus bovis/S. equinus complex and described three subspecies of S. gallolyticus (collaboration with Anne Bouvet, Hôtel-Dieu hospital, Paris).

Reference centers in France or abroad have sought our collaboration for developing knowledge in their fields. As an example, most Streptococcus species can now be identified by restriction of amplified rrn operon (collaboration with the National Center for Streptococci).

The Unit provides a taxonomic support to colleagues in University Hospitals who study antibiotic resistance genes (collaboration with Alain Philippon, Cochin Hospital, Paris).

Fluorescence in situ hybridization (FISH) is applicable to the detection of bacteria in water. An OECD document stresses the advantages of FISH. The FISH methodology has also been used to detect and visualize Methylophaga in marine sediments.

WHO Collaborating Center for Salmonella (Patrick Grimont, François-Xavier Weill and Martine Guibourdenche)

WHOCC for Salmonella, which was recently integrated to our Unit, maintains and updates the White-Kauffman-Le Minor scheme which lists all known Salmonella antigenic formulae. New serotypes are characterized each year. A total of 190 sera are needed to serotype all strains of Salmonella. About 60 sera are commercially available. The other 130 sera are produced by the WHOCC for local use.

WHOCC maintains a collection of all known serotypes and antigenic variants. WHOCC now performs genetic characterization of serotypes (including flagellin gene sequencing).

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

CNR-Salm contributes to the surveillance of salmonellosis in France by performing complete serotyping (with 190 sera) of 7,000 to 10,000 clinical isolates of Salmonella each year. Strains are sent to CNR-Salm (with essential epidemiological information) on a voluntary basis by about 2000 public or private clinical laboratories. CNR-Salm also collects information on strains that were serotyped locally (15,000 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 " département " 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 is sent. All strains of Salmonella enterica serotype Typhi are ribotyped (by use of the RiboPrinter). CNR-Salm also performs pulse-field gel electrophoresis of diverse serotypes and phage typing for Salmonella serotypes Typhi, Typhimurium, Paratyphi B, and Enteritidis. CNR-Salm has set up surveillance of antimicrobial resistance of Salmonella strains. More than 1,000 strains, randomly selected among 15 major serotypes, are studied each year. CNR-Salm participates in the Enter-Net European network. CNR-Salm has started determining the molecular mechanisms associated with emerging antimicrobial resistance in Salmonella.

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 1,000 isolates of Shigella are identified and serotyped each year. Detection of E. coli virulence genes is performed as well as classical serotyping and ribotyping of selected strains. CNR-coli detects anti-LPS antibodies when haemolytic and uremic syndrome (HUS) is suspected. A molecular serotyping method was devised, which consists of characterizing the genes encoding enzymes involved in somatic (O) antigen synthesis (rfb region) and in flagellin (fliC) synthesis. CNR-coli is active in the national network for surveillance of HUS among children below 15, and has participated in a survey of risk factors for HUS in this population (collaboration with Robert Debré Hospital, Institut de Veille Sanitaire, and network of paediatric nephrologists).

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 countries 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 ELWGD network, we have characterized strains occurring in Belarus.

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 or rpoB genes, automated ribotyping, PCR). Strains are received from clinical, veterinary, environmental, or industrial sources. In 2003, among strains identified by gene sequencing, 79% corresponded to 195 different named species, 9% were close to 55 named species, 11% represented 60 new species, and 1% represented 8 new genera. Strains (other than industrial strains) which do not fit any described species are included in our current taxonomic research. Some original clinical observations were published (joint publications with clinical microbiology colleagues)

Photo :<

Pulse field gel electrophoresis performed to investigate an outbreak due to multidrug resistant Salmonella enterica serotype Newport (France, May-June 2003).

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

Activity Reports 2003 - Institut Pasteur

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