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.

Molecular taxonomy (Patrick Grimont, Francine Grimont)

Quantitative DNA-DNA hybridization, rrs gene sequencing, and ribotyping allowed us to delineate two new Arthrobacter species, A. bergerei and A. arilaitensis, occurring on cheeses (collaboration with F. Irlinger, INRA).

The phylogeny of the genus Pseudomonas was examined by rpoB gene sequencing. The taxonomic resolution was three times that obtained with gene rrs. The identification of Pseudomonas species by rpoB sequencing is now possible (Linda Ait Tayeb PhD thesis). This approach was extended to the genus Proteus (collaboration with G. Giammanco and S. Pignato).

The study of complex bacterial flora is approached by cloning and sequencing the rrs gene, fluorescence in situ hybridization (FISH), and Temporal Temperature Gradient gel Electrophoresis (TTGE) under diverse contracts (Katiana Saunier, Christophe Brézillon) (figure 1).

Evolutionary genetics and identification of pathogens belonging to the genus Klebsiella (Sylvain Brisse, Cindy Fevre, Patrick Grimont)

A phylogenetic diversity study of the genus Klebsiella allowed to demonstrate ancestral presence (millions of years) and diversity of bla genes, which determine penicillin resistance. These genes are stably associated to the bacterial chromosome and evolved with it during speciation. These genes are thus good species markers. Other molecular markers allowing identification at the species level and below (phylogenetic group) have been developped.

The genetic structure of the species K. pneumoniae has been analyzed by multilocus sequence typing (MLST ; http://pubmlst.org/kpneumoniae ), which allows identification of major clones of the species and standardized strain typing. This method has been used to distinguish between the etiologic agent of ozaena (a chronic upper respiratory tract infection) from other K. pneumoniae strains.

Population genetics (Sylvain Brisse)

We have participated to the discovery and genetic characterization of Mycobacterium prototuberculosis, the ancestor of the etiologic agent of tuberculosis. Multiple gene sequencing showed that smooth strains, causing tuberculosis in Eastern Africa, and members of the " Tuberculosis complex ", causis tuberculosis on all continents, constitute a single species which may be 3 million-year old. Strains of this species seem to exchange genes by recombination, a phenomenon which was previously unknown in this species. The " Tuberculosis complex " represents an evolutionary successful clone that emerged from M. prototuberculosis more than 10,000 years ago.

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).

We have contributed to establish the international nomenclature for Salmonella species. WHOCC actively participates in training efforts of the Global Salm-Surv network.

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 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. CNR-Salm also performs pulse-field gel electrophoresis, ribotyping, and IS200 typing of diverse serotypes and phage typing for serotypes Typhi, Typhimurium, Paratyphi B, and Enteritidis. CNR-Salm participates in the design of new typing and subtyping methods (MLST, MLVA, prophage-typing). CNR-Salm participates in the Enter-Net European network.

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 studies the molecular mechanisms associated with emerging antimicrobial resistance in Salmonella. In 2005, emergence of strains producing extended-spectrum beta-lactamases CTX-M15 and CTX-M-27 and cephalosporinase CMY-2, and emergence of multi-drug-resistant Paratyphi B strains which had acquired Salmonella Genomic Island 1 on their chromosome were reported.

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

CNR-coli participates in the surveillance of shigellosis 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). The Center participates in the European network Enternet.

In 2005, 68 cases of HUS in children were serologically confirmed.

In October, an epidemic of E. coli O157:H7 occurred in South-West France. Sixty-six cases were recorded including 18 who developped HUS. All patients had eaten hamburgers of a single batch. E. coli O157:H7 carrying genes for shigatoxins 1 and 2, enterohemolysin, and intimin were isolated from patients stools and from hamburgers. This was the first O157:H7 epidemic associated with hamburgers occurring in France. Removal of the incriminated hamburger batch caused extinction of the epidemic.

Another E. coli epidemic occurred in Normandy in October. It is likely to be due to serotype O26 and associated with eating raw-milk cheese.

A group of 185 non-agglutinable, mannitol positive and indol negative Shigella strains that were received from various parts of the world (Africa, Asia, South-America) were studied. These strains had the same biotype, ribotype, restrictions patterns of the cryptic flagellar gene and the rfb region (invlolved in O specificity) and the same genes coding for invasivity and enterotoxin-2 (ShET-2). This groups corresponds to the new serotype S. boydii 20.

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 2005, a case of toxinogenous C. pseudotuberculosis has been reported to InVS. The source was an infected wound. C. pseudotuberculosis infections generally occur in horse, sheep, goat, and cattle. Contamination of man has been described in 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 2005, among 690 strains studied by sequencing, 83% were identified to 226 valid species, 8% were close to poorly delineated species, 8% represented 55 new species, and 1% represented 4 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 (infections associated with Flexispira rappini and new species of Janibacter and rod-shaped Neisseria) were published (joint publications with clinical microbiology colleagues)

During the winter 2004-2005, CMIB was contacted by Institut de Veille Sanitaire following the occurrence of invasive Enterobacter sakazakii (Es) infections in newborns. The investigation aimed at evaluating the spread of the epidemic, identify the infected product, and set up control action. CMIB definitely identified the species (Es) by rpoB sequencing, and provided rapid typing of strains by automated ribotyping. These was the first occurrence in France of Es infections associated with a feeding preparation for newborns which caused the authorities to remove the contaminated product. CMIB acted as a reference center.

Photos :

Bacterial flora comparison among mice (A1-A8 and B1, B2) by Temporal Temperature Gradient gel Electrophoresis (TTGE). Each band corresponds to a bacterial species.

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

Activity Reports 2005 - Institut Pasteur

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