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. Multiple gene sequencing is an approach which needs to be validated. Algorithms for computer interpretation will be essential. An automated workstation for the preparation of 96 sequence reactions is used in our laboratory. A study of the taxonomic structure of Klebsiella by use of multiple gene sequencing has been initiated. A MLST scheme, which consists of typing strains by sequencing internal portions of seven housekeeping genes, was developed for Klebsiella pneumoniae. This method will allow to define the genetic structure of K. pneumoniae and to better characterize strains that cause nosocomial infections.
We have been asked by several colleagues (in France or out of France, in universities or hospitals or reference centers) to characterize new bacterial species by DNA-DNA hybridization or rrs gene (encoding 16S rRNA) sequencing. We have contributed to the description of Aeromonas simiae (collaboration with Henri Monteil, Institut de Microbiologie, Strasbourg), Vibrio ponticus (collaboration with Ezperanza Garay, Universitat de Valencia), and group D Streptococcus spp (collaboration with Anne Bouvet, Hôtel-Dieu hospital, Paris). Strains degrading hydrocarbons (collaboration with J. Oudot, Museum d'Histoire Naturelle, Paris) or involved in metal corrosion (collaboration with Corrodis) were characterized. Identification tools were developped for the identification of Klebsiella phylogenetic groups and molecular methods were used to study populations of Burkholderia cepacia, Klebsiella pneumoniae, Acinetobacter baumannii, and Agrobacterium spp.
Repeatability and reproducibility of ribotyping and its computer interpretation were studied using several software packages. Taxotron (a software developed in the Unit) was found to perform well.
Molecular serotyping of Klebsiella (S. Brisse, P.A.D. Grimont)
Capsular serotyping is the reference method for strain typing in Klebsiella pneumoniae. A major drawback of capsular serotyping is the large number of cross-reactions that occur among the 77 capsule types (K-types). Serotyping is tedious and its interpretation is subjective. The method is limited to a few centers which can produce antisera. To overcome these limitations, a molecular method has been developped which consists in PCR amplification of the capsular antigen gene cluster (cps) followed by digestion with the restriction enzyme HincII (cps PCR-RFLP). A total of 97 distinct profiles (C-patterns) were obtained for 224 strains representing the 77 known K-types. The C-patterns of any K-type was distinct from the C-patterns of all other K-types (except for K22 and K37 which are known to cross react). The new cps PCR-RFLP method allows determination of the K serotype and is easier to perform and more discriminatory than classical capsular serotyping.
WHO Collaborating Center for Salmonella (Patrick Grimont, François-Xavier Weill and Martine Guibourdenche)
WHOCC for Salmonella 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 1500 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 local 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 studies the molecular mechanisms associated with emerging antimicrobial resistance in Salmonella. In 2004, the emergence of genes encoding extended spectrum beta-lactamases TEM-52, SHV-12-like, CTX-M-9, and CTX-M-15, and aminoside resistance gene aac(3)-Id, were published. Epidemiology of antibiotic multiresistance in serotype Typhi in Vietnam was studied (collaboration with M. Scavizzi).
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, which consists of characterizing the genes encoding enzymes involved in somatic (O) antigen synthesis (rfb region) and in flagellin (fliC) synthesis, is used. CNR-coli is active in the national network for surveillance of HUS among children below 15 (collaboration with Robert Debré Hospital, Institut de Veille Sanitaire, and network of paediatric nephrologists). In 2004, 58 cases of HUS in children were confirmed by demonstrating the production of anti-O157 (54 cases), O145 (2 cases), O91 (one case), and O55 (one case) antibodies. Ten strains were isolated which possessed genes stx2 and eae. A group of cases infected with E. coli O157 carrying genes stx2 and eae, was studied in the East of France. Fresh goat cheese was the source. Results from our National Reference Center allowed Health Authorities to prevent an epidemic.
A total of 180 isolates of nonagglutinable Shigella, received in 5 years form diverse countries (mostly Africa, some from Asia or South America) are being studied. These isolates have the same biotype, ribotype, and cryptic flagellar gene and rfb cluster (involved in O antigen specificity) restriction patterns.
National Reference Center for Corynebacterium diphtheriae (Patrick Grimont and Anne Le Flèche)
CNR-Cd confirms the identification of Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis strains isolated in France, determines the presence of diphtheria toxin genes by PCR, and performs molecular typing on these strains.
In 2004, 4 cases of infection with toxinogenic C. ulcerans, one with toxinogenic C. diphtheriae biotype mitis, and one with toxinogenic C. pseudotuberculosis, fit with the new European and French definition of diphtheria. These cases were then declared to InVS. In 3/4 cases of tox+ C. ulcerans infection, patients were in contact with dogs. In one case, the same C. ulcerans strain was isolated from the dog's nostril (collaboration with M-N Bachelier, CHG Bourges). The C. diphtheriae strain reported was found in the throat of a vaccinated child returning from Madagascar. The C. pseudotuberculosis strain was from caseous adenitis. Contact with goats was suspected. Cases reported in the world literature were mostly from Australia.
In the framework of WHO-Europe network ELWGD (European Laboratory Working Group on Diphtheria) and DG-SANCO network DIPNET, a C. diphtheriae ribotype database was built after the study of 576 strains, isolated in various countries including the former USSR. These strains were distributed into 86 ribotypes. Software Taxotron was chosen for the automatic identification of ribotypes. An international nomenclature for C. diphtheriae ribotypes has been published.
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 2004, among 832 strains identified by gene sequencing, 75% corresponded to 228 different named species, 10% were close to poorly delineated named species, 14% represented 85 new species, and 1% represented 6 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)
Antibiotic susceptibility of a strain of Salmonella enterica serotype Livingstone producing an extended spectrum beta-lactamase type CTX-M-27. IMP, imipenem; AMC, amoxicillin-clavulanic acid; CRO, ceftriaxone; CAZ, ceftazidime; AMX, amoxicillin; TIC, ticarcillin; PIP, piperacillin. A synergy (Champagne cork looking) between AMC and CRO, and between AMC and CAZ, can be observed.
Keywords: Bacteriology, Taxonomy, phylogenomics, Enterobacteriaceae, molecular typing, identification, population genetics