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 (gyrB, 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. New species have been described and several potentially new species are being studied.
Infraspecific diversity (Francine Grimont)
Traditional typing tools are generally limited to a single bacterial species whereas molecular methods are more widely applicable. Molecular typing system, based on the best methods such as tibotyping (initiated in our Unit in 1986) have been developped. Computer identification of patterns has been set up thanks to a home-made software (Taxotron). Databases for automatic ribotype identification have been built for Escherichia coli (correlated with serotype), Shigella, Salmonella enterica serotype Typhi (may replace phage typing), Legionella (identification of all species), Pseudomonas, vibrios and coryneforms. PCR systems for the identification of E. coli pathotypes have also been designed.
A step forward: molecular serotyping (Francine Grimont, Patrick Grimont, PhD work of Roney S. Coimbra and Jorge Machado).
Amplification and restriction of the gene coding for flagelline in Escherichia coli allowed to recognize almost all flagellar types. A cryptic flagellar gene has been studied in Shigella. It reflects the clonal history of this bacterial group. 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. It is the first time that a molecular typing system can replace a complex serotyping system with almost the same results. This approach allowed us to discover a new Shigella serotype.
Bacterial immuno-taxonomy (Philippe Bouvet)
Bacteriological immuno-diagnosis (preparation of sera for serotyping or antigens for serological tests) often uses whole bacterial cells. As a consequence, many cross reactions occur. We work with purified antigens and most recent immunological techniques . These are applied to identification and serological tests. A serological test for the detection of antibodies against the 25 E. coli O serogroups most often associated with shiga-toxin production, has been designed. Furthermore, a serum specifically agglutinating nonidentified strains of Shigella was prepared.
Metabolic biodiversity of bacteria (Odile Bouvet)
Metabolic abilities of bacteria associated with poly-beta-hydroxyalcanoate synthesis are studied (Stephane Diard's thesis). Physiological behavior (energetic and oxidative state, nutriment transport, intracellular pools, and activity of major metabolic pathways) of species subjected to different stress conditions is studied with the aim to better define the frontier between bacterial life and death (Andrea Villarino's thesis). For such project, we develop new methodologies which provide a global approach (e.g. application of nuclear magnetic resonnance).
Aquabiolab (Béatrice Regnault, Patrick Grimont)
This laboratory, supported by Vivendi Water, designs molecular methods for the bacteriological control of water. Fluorescent oligonucleotide probes are designed from 16S rRNA sequences to visualize - by Fluorescence In Situ Hybridization (FISH) - bacterial cells carrying target sequences in their ribosomes. Revivification of bacterial cells in the presence of ciprofloxacine allows to distinguish living bacteria (which elongate) from dead bacteria (unreactive). A project exploring the frontier between bacterial life and death has been initiated (Andrea Villarino's thesis). Aquabiolab research and developpment dealing with the detection and enumeration of Escherichia coli cells in water, foods, or infected urine, has been published. Some of the fluorescent probes designed by Aquabiolab are used by the Center for Molecular Identification of Bacteria.
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). Soon as a significant increase has been detected, an alert message is sent by fax. In 2000, 7 local or regional peaks have been detected. Information has been sent to health authorities about 499 case clusters mentioned by collaborating laboratories. Among the 47 Salmonella serotypes responsible for 460 case clusters, serotypes Enteritidis and Typhimurium accounted for 79%. 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. E. coli pathogenicity genes are detected. 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 has studied 1550 strains by ribotyping and/or phage typing, 238 strains or specimens by PCR (detection of pathogenicity genes), and studied new Shigella atypical strains. The NRCEMT participates in the European network Enter-Net to the surveillance of infections caused by Salmonella enterica serotypes Typhi, Typhimurium, Enteritidis, and Paratyphi B, 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 Center for Molecular Identification of Bacteria, the NRCCD identifies all coryneform species.
In association with the NRCEMT (F. Grimont) 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. P.A.D. Grimont is the coordinator of the international C. diphtheriae ribotype database and an international nomenclature is being validated for these tibotypes.
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). Within a few months, the CMIB has characterized several emerging pathogens. Strains are received from clinical, veterinary, environmental, or industrial laboratories. 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)
This laboratory characterizes industrial strains by molecular methods (double strand sequencing of 16S rRNA, quantitative DNA-DNA hybridization, full ribotyping with 10 restriction endonucleases, detection of virulence genes, determination of plasmid copy number, detection and characterization of bacteriophages), writes bibliographical reviews, offers technical counceiling, and accepts all contract work when fitting with our domain of expertise. A new program, supported by Procter & Gamble, studies the spread of marker bacilli through an experimental kitchen with the purpose of evaluating disinfection strategies. Lessons that will be learnt from this work should help prevent foodborne infections.
Taxolab software (Patrick Grimont)
Version 2000 of the Taxotron software package (for Macintosh) includes programs Recognizer (bacterial identification using Biotype-100 from BioMerieux), RestrictoScan (captures restriction fragment migration values from a gel image), RestrictoTyper (fragment size interpolation, fragment size database building, automated identification of restriction patterns), Adanson (numerical taxonomy), Dendrograf (trees and dendrogramms), Antibiotyper (entry of quantitative values, Euclidian distance), and FactorAna (factorial analyses). Improvements involved mathematical filtering of images, speedy calculations by optimisation for PowerMacintosh, and automated restriction pattern identification taking error into account.
Detection by fluorescence in situ hybridization of Escherichia coli in infected urine.