EP1328641A2 - Polynukleotidem aus b2/d+ a- e. coli und deren verwndungen - Google Patents

Polynukleotidem aus b2/d+ a- e. coli und deren verwndungen

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Publication number
EP1328641A2
EP1328641A2 EP01917119A EP01917119A EP1328641A2 EP 1328641 A2 EP1328641 A2 EP 1328641A2 EP 01917119 A EP01917119 A EP 01917119A EP 01917119 A EP01917119 A EP 01917119A EP 1328641 A2 EP1328641 A2 EP 1328641A2
Authority
EP
European Patent Office
Prior art keywords
polynucleotides
coli
group
sequence
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP01917119A
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English (en)
French (fr)
Inventor
Edouard Bingen
Stéphane BONACORSI
Olivier Clermont
Xavier Nassif
Colin Tinsley
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Original Assignee
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
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Filing date
Publication date
Priority claimed from FR0003145A external-priority patent/FR2806096A1/fr
Application filed by Assistance Publique Hopitaux de Paris APHP, Institut National de la Sante et de la Recherche Medicale INSERM filed Critical Assistance Publique Hopitaux de Paris APHP
Publication of EP1328641A2 publication Critical patent/EP1328641A2/de
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates, in general, to polynucleotides which are of nature B2/D+ A- and which are isolated from E. coli, and to the biological uses, in particular medical and biotechnological uses, of these polynucleotides and of the polypeptides which they encode.
  • the expression "the nature B2/D+ A-" is intended to mean that the polynucleotide is present with greater frequency such as observed in ECOR E. coli of group B2 (ECOR B2 frequency) and/or in ECOR E. coli of group D (ECOR D frequency) , with respect to the frequency observed in ECOR E. coli of group A (ECOR A frequency) .
  • the ECOR B2 frequency and/or the ECOR D frequency is greater than the ECOR A frequency by. a factor of 2, preferably of 3, more preferably of 3.5, very preferably by a factor of 4.
  • the set of polynucleotides which are of nature B2/D+ A-, provided by the present invention, comprises in particular products the ECOR B2 frequency and/or the ECOR D frequency of which is greater than 10%, and the ECOR A frequency of which is less than 25%, preferably less than 20%, more preferably less than 10%, and very preferably less than 5%, while always remaining less than the ECOR B2 frequency and/or the ECOR D frequency.
  • the ECOR collection is a collection which represents the genetic diversity of the E. coli species; it is available from banks such as the ATCC.
  • the E. coli species is currently divided into four main phylogenic groups termed groups A, Bl, B2 and D.
  • groups A, Bl, B2 and D The E. coli species are currently divided into four main phylogenic groups termed groups A, Bl, B2 and D.
  • MLEE multilocus enzymatic electrophoresis
  • Descriptions of these techniques are in particular described in Herzer et al . 1990 J. Bacteriol. 172:6175-6181 and in Selander et al . 1986 Appl. Environ. Microbiol. 51:873-884 for MLEE, and in Bingen et al . 1994 Clin. Microbiol. Rev. 7:311-317, Bingen et al . Clin. Infect. Dis.
  • MLEE is based on the analysis of the migration polymorphism of bacterial enzymes. For each strain, a large number of enzymes characteristic of the species (often greater than or equal to 20) are characterized by their electrophoretic mobility. The existence of migration variants for these enzymes makes it possible to characterize each strain by an electrophoretic type.
  • ribotyping it. ⁇ s based on the analysis of the restriction polymorphism of the chromosomal regions which include the genes encoding the 16S and 23S RNAs. This polymorphism is revealed using a labelled cDNA probe, prepared from the 16S and 23S RNA of -257. coli, which can be hybridized to the DNA of the strain studied, after digestion of this DNA with various restriction enzymes and Southern transfer.
  • a collection termed ECOR has, moreover, been constituted in such a way as to represent the genetic diversity of the E. coli species. This collection is available in particular from the ATCC (ATCC No. 35320 to No. 35391) . It comprises:
  • E-7- coli strains not assigned to one of the four groups A, Bl, B2 and D.
  • the present invention makes the original and pertinent choice of isolating the entire set of its polynucleotides which are of nature B2/D+ A-, as defined above. Such a solution has never, to the Applicant's knowledge, been proposed. Now, the present invention demonstrates that this particular choice not only makes it possible to solve the problem of the phylogenic determination of a given E. coli strain, but also makes it possible to detect and to treat an undesired development of i57. coli, and more particularly a development of E. coli in a human or animal compartment which is extra-intestinal
  • the present invention in fact provides, in addition to the diagnostic and therapeutic means directed against E. coli in general, specific diagnostic and therapeutic means which have the advantage of distinguishing between E. coli capable of infecting the extra-intestinal compartment (J57. coli of group B2 and D) and -57. coli which are part of the normal physiological flora of humans and animals. Besides the less deleterious effects that such treatments offer patients suffering from an extra-intestinal E. coli infection, these specific diagnostic methods and treatments according to the invention make it possible to limit the development of bacterial resistances which are more and more frequently observed as broad-spectrum antibiotic treatments are used.
  • the inventors have therefore developed means which provide access to the entire set of polynucleotides which are of nature B2/D+ A-, as defined above. To the Applicant's knowledge, this is the first description of such means and the first description of such a set of products. These means also demonstrate that there are genomic regions which are of nature B2/D+ A-.
  • a concept which is common to the various aspects of the invention corresponds to producing phylogenic markers of i57. coli by choosing to isolate the set of the chromosomal and plasmid DNA fragments of E. coli which are of nature B2/D+ A- as defined above, and developing, for this purpose, a method which is capable of allowing the entire set of these fragments to be isolated.
  • the method used consists in subtracting the polynucleotide population of one or more E. coli strain (s) of group A, randomly sheared, from the polynucleotide population of one or more E. coli strain (s) of group B2 or D, cleaved with a restriction endonuclease allowing fragments comprising from 100 to 1 500 bp approximately, with a mean of 300 to 500 bp approximately, to be produced, such as 5au3AI, Tsp509I, Mspl or Maell (any restriction enzyme with short restriction sites, for example for bp) .
  • This method makes it possible to obtain a library of products which are of nature B2/D+ A- as defined above.
  • Example 1 below gives an illustration thereof, for which the i57. coli strain subjected to subtraction is the i57. coli strain C5.
  • This particular strain is classified, according to reference techniques, in the phylogenic group B2, yet it comprises, in particular at the plasmid level, DNAs of D origin.
  • the specific choice of E. coli C5 therefore has the advantage of isolating polynucleotides which are of nature B2+ A- and/or D+ A-, using a single strain.
  • Such a subtraction can be repeated over and over until the desired level of exhaustiveness is obtained (the level of exhaustiveness of the library can be estimated by searching for the presence or absence of products known to have the nature B2/D+ A-, such as sfa, ibelO ) . If the intention is to increase the level of exhaustiveness, it is desirable to use different restriction enzymes between the various subtraction repetitions. Examples of implementation of such a method are in particular described in Example 1 which follows.
  • the present invention proposes, for the first time, to apply a subtractive method to a specific choice of E. coli strains: firstly, an isolate of E. coli of group B2, associated with neonatal meningitis (E. coli C5 assigned to the phylogenic group B2 and comprising, in particular at the plasmid level, DNAs of D origin) , and secondly, E. coli strains of group A from the ECOR collection.
  • the present invention therefore offers the means for obtaining all of the products which are of nature B2/D+ A-.
  • the polynucleotides targeted by the present application correspond to the set of polynucleotides which are of novel nature B2/D+ A- .
  • Some of these polynucleotides correspond to products which are novel in themselves (SEQ ID NO: 1 to NO: 153, sequences corresponding to regions 1, 3, 4 and 5 identified in the following examples, orf sequences containing these sequences) .
  • Others correspond to products which have homologies with known products, but which are of novel nature B2/D+ A- (in particular SEQ ID NO: 170, 171, 174,
  • a subject of the present application is therefore any isolated polynucleotide which is of novel nature B2/D+ A- and which corresponds to a novel product in itself. It is in particular aimed towards any isolated polynucleotide the sequence of which corresponds to a sequence chosen from the group consisting of :
  • polynucleotide sequences which can be obtained by digesting the total DNA of an E. coli of group B2 or group D (such as, for example, an E. coli strain isolated from the blood of a patient suffering from a septicaemic or meningeal infection) with a restriction enzyme chosen from the group consisting of otl and Blnl , selecting those of the fragments obtained which hybridize with at least one sequence chosen from the group consisting of SEQ ID NO: 134, 144, 109, 115, 140, 135, 33, 56, 122, 130, 141, 25, 48, 51, 57, 121, 44, 45, 113, 119, 120, 23, 52, and
  • this polynucleotide group corresponds to the polynucleotides which are of novel nature B2/D+ A- and which are also novel as products.
  • SEQ ID NO:l to NO: 153 it comprises the novel regions 1, 3, 4 and 5 identified according to the invention, and the orfs which can be identified on these polynucleotides (cf. examples) .
  • nature-conserving sequence of a sequence is herein intended to mean any sequence which has conserved the nature B2/D+ A- (as defined above) of the polynucleotide corresponding to this parent sequence.
  • variant is herein intended to mean any sequence having nucleotide insertions and/or deletions and/or substitutions with respect to the parent sequence, this includes any sequence which is complementary to the parent sequence.
  • fractional is herein intended to mean any fragment of the parent sequence.
  • any strain is considered to be E. coli if it can be considered to belong to the E. coli species according to the criteria given by Bergey' s Manual of systemic bacteriology ⁇ cf. in particular Volume 1) .
  • any E. coli strain is considered to belong to the group B2 or to the group A if it is considered as such after carrying out a reference phylogenic test suitable for discriminating between B2/D and A, such as for example multilocus enzymatic electrophoresis (MLEE) and/or ribotyping.
  • MLEE multilocus enzymatic electrophoresis
  • Such phylogenic techniques are well known to those skilled in the art. Examples of suitable protocols are in particular described in Herzer et al . 1990 J. Bacteriol. 172:6175-6181 and in Selander et al . 1986 Appl. Environ. Microbiol. 51:873-884 for MLEE, and in Bingen et al . 1994 Clin. Microbiol. Rev.
  • the fact of being or of not being present in a given bacterial strain or sample corresponds to a notion which is common to those skilled in the art in the field.
  • the determination can in particular be carried out by Southern transfer of the nucleotide population of said E. coli strain, and bringing this transfer into contact with the polynucleotide tested, or with a probe derived from this polynucleotide, under conditions suitable for polynucleotide hybridization reactions. Examples of such probes and such conditions are given in Example 1. A positive hybridization will then be interpreted as the presence of said polynucleotide in said E.
  • the determination can also be carried out by PCR detection of a positive or negative amplification using amplification primers constructed on the basis of said polynucleotide and placed in contact with the nucleotide population of said i57. coli strain, under conditions which are favourable for the amplification, using these primers, of a target out of this population. Illustrations of such PCR procedures are given in the examples which follow.
  • the majority of the polynucleotides which are of nature B2/D+ A- according to the invention are advantageously present in ECOR -57.
  • coli of group B2 with a frequency greater than 10%, particularly greater than 40%, preferably greater than 50%, more preferably greater than 60%, and even more preferably greater than 70% ⁇ cf. Example 2 and Table 3) . Some of them are present in ECOR -57.
  • the polynucleotides which are of nature B2/D+ A- according to the invention can be used for simply detecting the presence of E. coli bacteria of the group B2 or D with a probability of at least 90% (presence of said polynucleotides) . Taken in combination together, or with other products, they make it possible to completely distinguish between the groups A, Bl, B2 and D of i57. coli ⁇ cf. Example 5) . They also have therapeutic, palliative and/or preventive applications of interest. These polynucleotides will be referred to hereinafter as: novel polynucleotides which are of novel nature B2/D+ A-.
  • the present application is also aimed towards any pair of primers which allows the PCR amplification of at least one novel polynucleotide which is of novel nature B2/D+ A- as defined above.
  • the setting up of suitable experimental PCR conditions is accessible to those skilled in the art ⁇ cf. in particular Molecular cloning - a laboratory manual, Sambrook, Fritsch, Maniatis, and in particular Vol. 2, Chap. 14 2nd edition; cf. Ausubel et al . 1989 Current protocols in molecular biology; John Wiley and Sons Ed., and in particular Vol. 2 Chap. 15). Examples thereof are given in the examples which follow.
  • the present application is also aimed towards any nucleotide probe comprising at least one novel polynucleotide which is of novel nature B2/D+ A- as defined above. It is in particular aimed towards any probe as obtained using at least one pair of primers according to the invention, in particular by PCR amplification ⁇ cf. examples), such as (SEQ ID NO: 160; 161), (SEQ ID NO: 162; 163) or (SEQ ID NO: 164; 165) .
  • any pair of primers which allows the PCR amplification, under conditions as mentioned above, of at least one polynucleotide which is of novel nature B2/D+ A-, and also any probe directed against such a polynucleotide, and any antibody directed against a polypeptide encoded by such a polynucleotide can, in accordance with the present invention, be used for the phylogenic determination of a bacterium of the E. coli species.
  • the present invention also demonstrates that the y ' aA gene (SEQ ID NO: 254; protein of SEQ ID NO: 255) is a phylogenic tool of interest for E. coli .
  • coli of any product which makes it possible to detect y aA ⁇ cf. Example 5 therefore enters into the domain of the present application. It is in particular aimed towards any pharmaceutical composition comprising such products, such as a pair of primers (e.g. SEQ ID NO: 162; NO: 163), a probe or a specific antibody.
  • a pair of primers e.g. SEQ ID NO: 162; NO: 163
  • a probe or a specific antibody e.g. SEQ ID NO: 162; NO: 163
  • the present application is also aimed towards any antisense polynucleotide, characterized in that its sequence corresponds to the antisense sequence of at least one novel polynucleotide which is of novel nature B2/D+ A- and any isolated polypeptide, characterized in that its amino acid sequence corresponds to a sequence encoded, according to the universal genetic code and taking into account the degeneracy of this code, by at least one novel polynucleotide which is of novel nature B2/D+ A-.
  • the present application is also aimed towards any combination of the polypeptides encoded by the polynucleotides of the invention with a binding product, capable of binding to at least one novel polypeptide which is of novel nature B2/D+ A-, and which will inhibit an important biological function, such as the extra- intestinal growth by growth in serum, or the multiplication in animal, such as disclosed in example 6.
  • a binding product capable of binding to at least one novel polypeptide which is of novel nature B2/D+ A-, and which will inhibit an important biological function, such as the extra- intestinal growth by growth in serum, or the multiplication in animal, such as disclosed in example 6.
  • Such products correspond in particular to antibodies or monoclonal antibodies and compounds inhibiting the biological function of the polypeptides.
  • Methods for manufacturing such binding products using the polypeptides according to the invention are available to those skilled in the art. They are conventional methods which comprise, in particular, the immunization of animals such as rabbits and the harvesting of the serum produced, followed optionally by the purification of the serum obtained
  • the expression "capable of binding” is intended to mean physiological-type conditions
  • binding product when said binding product is intended to be administered to a human or animal organism, and ELISA-type conditions when said binding product is intended to be used in assays and methods in vi tro, for example for determining the phylogenic group of an E. coli bacterium.
  • the present application is also aimed towards any vector comprising at least one novel polynucleotide which is of novel nature B2/D+ A-, and also any cell transformed by genetic engineering, characterized in that it comprises, by transfection, at least one novel polynucleotide which is of novel nature B2/D+ A- and/or at least one vector according to the invention, and/or in that said transformation induces the production by this cell of at least one polypeptide corresponding to a novel polynucleotide which is of novel nature B2/D+ A-.
  • a subject of the present application is also any composition, in particular any pharmaceutical composition, comprising at least one compound chosen from the group consisting of the novel polynucleotides which are of novel nature B2/D+ A-, the polypeptides corresponding to at least one novel polynucleotide which is of novel nature B2/D+ A-, and the vectors and cells according to the invention, mentioned above.
  • Such compositions are in particular useful for treating and/or for alleviating and/or for preventing E. coli infections, and in particular of infection by extra-intestinal E. coli . (systemic and non-diarrhoeal infections) .
  • said compound is immunogenic or is made immunogenic, these compositions can correspond to vaccines.
  • a subject of the present application is also any composition, in particular any pharmaceutical composition, comprising at least one compound chosen from the group consisting of the novel polynucleotides which are of novel nature B2/D+ A-, the pairs of primers and probes according to the invention, mentioned above, and the binding products according to the invention, mentioned above.
  • Such a composition is in particular useful for the phylogenic determination of an E. coli bacterium. It can therefore correspond to a diagnostic kit or composition.
  • a subject of the present application is also any composition, in particular any pharmaceutical composition, comprising, in association with an inert carrier, at least one compound chosen from the group consisting of the antisense polynucleotides according to the invention, mentioned above, and the binding products according to the invention, mentioned above.
  • Such compositions can also be used for the phylogenic determination of an E. coli bacterium, and can take the form of a diagnostic kit or composition. They can also be used in a therapeutic capacity, in order to treat and/or to alleviate and/or to prevent any undesirable growth of £7. coli, such as a sanitary contamination, an E. coli infection, and in particular the presence of extra-intestinal E. coli ⁇ cf. examples) .
  • a subject of the present application is also any use of these products according to the invention for manufacturing such compositions.
  • the present invention also provides products for which the sequence exhibits homologies with known products, but for which the nature B2/D+ A- is novel.
  • the chuA gene is a known product (Genbank Accession No. U67920) : it has been described in a few E. coli responsible for intestinal and extra-intestinal infections, in Shigella as a gene involved in iron metabolism, and has been described as being homologous to a Yersinia gene. However, it has never been described as a phylogenic marker.
  • the present invention reports, for the first time, its presence in the urophathogenic E. coli strain J96 and reports, for the first time, that it is present in 100% of the B2s and of the Ds and in 0% of the
  • the majority of the polynucleotides according to the invention which exhibit homology but which are of novel nature B2/D+ A- are advantageously present in ECOR E. coli of group B2 with a frequency greater than 40%, preferably greater than 50%, more preferably greater than 60% and even more preferably greater than 70%. Some of them are present in ECOR E. coli of group B2 with a frequency greater than 80%, or even at a frequency equal to 100%, while at the same time remaining present in ECOR E. coli of group A at a frequency of less than 5%, or even less than 3%, or even equal to 0% ( cf. Example 2) .
  • the present application is thus aimed towards any use of a compound which is of novel nature B2/D+ A- for the phylogenic determination of an E. coli bacterium, for the diagnosis of the presence or absence of undesirable J57. coli bacteria, such as contaminant E. coli or extra- intestinal .57. coli, and/or for the diagnosis of an E. coli infection, and/or for the manufacture of a composition intended for such a phylogenic determination and/or for such a diagnosis.
  • polynucleotides the sequence of which corresponds to SEQ ID NO: 170, 171, 174, 175, 178, 179, 183, 185, 186, 190, 191, 193, 194, 195, 196, 199, 200, 202, 205, 206, 208, 209, 211, 214, 218, 220, 221, 233, 234, 235, 241, 244, 246, 247, 248 and 250,
  • polynucleotides which can be obtained by digesting the total DNA of an E. coli of group B2 or D with a restriction enzyme chosen from the group consisting of WotI and Blnl , and selecting those of the fragments obtained which hybridize to at least one sequence chosen from the group consisting of SEQ ID NO: 125, 123, 116, 43, 40, 127, 133, 27, 34, 36, 42, 46, 54, 55, 38, 128 and 151, with the exception of the polynucleotides sfa , hly r cnfl , pap/prs r hra and ibe 10,
  • polynucleotides the sequence of which corresponds to a nature-conserving variant or fractional sequence of the sequence of at least one of these polynucleotides (and in particular SEQ ID NO: 241, 195, 185 and 248),
  • the present invention is also aimed towards the use of at least one of these compounds for the diagnosis of the presence or absence of undesirable E. coli bacteria, such as contaminant J57. coli or extra-intestinal E. coli, and/or for the diagnosis of an E. coli infection. It is also aimed towards any use of at least one of these compounds for the manufacture of a composition intended for such a phylogenic determination and/or for such a diagnosis.
  • the detection of the presence or absence of such compounds can in particular be carried out by nucleotide hybridization, by PCR amplification or by detection of their polypeptide products. Detection of the presence of such compounds makes it possible to conclude that the B2 or D i57. coli bacterium is present.
  • the combined use of the detection of the presence or absence of chuA and of yjaA makes it possible in particular to conclude as to whether £7. coli of group B2 and/or £7. coli of group D are present or absent ( cf. Example 5) .
  • the present application is also aimed towards any use of a compound which is of novel nature B2/D+ A- for the manufacture of a composition, in particular of a pharmaceutical composition, intended to alleviate and/or to prevent and/or to treat an undesirable growth of E. coli, such as an E. coli infection, the presence of extra-intestinal E. coli or a sanitary contamination. It is aimed in particular towards any use of at least one compound chosen from the group consisting of: - the polynucleotides corresponding to the ⁇ uA gene and to its operon,
  • polynucleotides the sequence of which corresponds to SEQ ID NO: 170, 171, 174, 175, 178, 179, 183, 185, 186,
  • polynucleotides which can be obtained by digesting the total DNA of an E. coli of group B2 or D with a restriction enzyme chosen from the group consisting of WotJ and Blnl, and selecting those of the fragments obtained which hybridize to at least one sequence chosen from the group consisting of SEQ ID NO: 125, 123, 116, 43, 40, 127, 133, 27, 34, 36, 42, 46, 54, 55, 38, 128 and 151, with the exception of the polynucleotides sfa , hly r cnfl , pap/prs, hra and ibe 10,
  • polynucleotides the sequence of which corresponds to a nature-conserving variant or fractional sequence of the sequence of at least one of these polynucleotides (and in particular SEQ ID NO: 241, 195, 185 and 248),
  • polypeptides the sequence of which corresponds, according to the universal genetic code and taking into account the degeneracy of this code, to these polynucleotides,
  • binding products capable of binding to a polypeptide encoded by a polynucleotide which is of novel nature B2/D+ A-,
  • the cells transformed by genetic engineering which comprise at least one of these polynucleotides and/or at least one of these vectors, and/or the transformation of which induces the production of at least one of these polypeptides, for the manufacture of a composition, in particular of a pharmaceutical composition, intended to alleviate and/or to prevent and/or to treat an undesirable growth of E. coli, such as an J57. coli infection, the presence of extra-intestinal E. coli or a sanitary contamination.
  • E. coli such as an J57. coli infection, the presence of extra-intestinal E. coli or a sanitary contamination.
  • the present application is aimed towards any method which makes it possible to identify a compound capable of inhibiting the growth of E. coli , and in particular of inhibiting its extra-intestinal development, which comprises the detection of at least one compound:
  • polynucleotide sequences which can be obtained by digesting the total DNA of an E. coli of group B2 or group D (such as, for example, an E. coli strain isolated from the blood of a patient suffering from a septicaemic or meningeal infection) with a restriction enzyme chosen from the group consisting of Wotl and Blnl, selecting those of the fragments obtained which hybridize with at least one sequence chosen from the group consisting of SEQ ID NO: 134, 144, 109, 115, 140, 135, 33, 56, 122, 130, 141, 25, 48, 51, 57, 121, 44, 45, 113, 119, 120, 23, 52 and sequencing selected fragments, and
  • polynucleotides the sequence of which corresponds to SEQ ID NO: 170, 171, 174, 175, 178, 179, 183, 185, 186,
  • polynucleotides which are as obtained by digesting the total DNA of an E. coli of group B2 or D with a restriction enzyme chosen from the group consisting of Wotl and Blnl , and selecting those of the fragments obtained which hybridize to at least one sequence chosen from the group consisting of SEQ ID NO: 125, 123, 116, 43, 40, 127, 133, 27, 34, 36, 42, 46, 54, 55, 38, 128 and 151, with the exception of the polynucleotides sfa , hly r cnfl f pap/prS f hra and ibe 10,
  • polynucleotides the sequence of which corresponds to an orf comprising the sequence of one of the polynucleotides cited in the preceding four paragraphs, - the polynucleotides the sequence of which corresponds to a nature-conserving variant or ' fractional sequence of the sequence of at least one of these polynucleotides and in particular (SEQ ID NO: 241, 195, 185 and 248),
  • the present invention is also aimed towards any method which makes it possible to identify a compound capable of inhibiting the growth of an £7. coli bacterium, and in particular its extra-intestinal development, characterized in that it comprises the detection of at least one compound capable of inhibiting the activity of a protein encoded by a polynucleotide the orf of which comprises a polynucleotide chosen from the group consisting of :
  • polynucleotides the sequence of which corresponds to SEQ ID NO: 170, 171, 174, 175, 178, 179, 183, 185, 186, 190, 191, 193, 194, 195, 196, 199, 200, 202, 205, 206, 208, 209, 211, 214, 218, 220, 221, 233, 234, 235, 241, 244, 246, 247, 248 and 250,
  • polynucleotides which are as obtained by digesting the total DNA of an -57. coli of group B2 or D with a restriction enzyme chosen from the group consisting of Wotl and Blnl , and selecting those of the fragments obtained which hybridize to at least one sequence chosen from the group consisting of SEQ ID NO: 125, 123, 116, 43, 40, 127, 133, 27, 34, 36, 42, 46, 54, 55, 38, 128 and 151, with the exception of the polynucleotides sfa r hly r cnfl , pap/prs, hra and ibe 10,
  • polynucleotides the sequence of which corresponds to an orf (open reading frame) comprising the sequence of one of these polynucleotides
  • polynucleotides the sequence of which corresponds to a nature-conserving variant or fractional sequence of the sequence of at least one of these polynucleotides.
  • Such compounds can in particular be obtained by screening chemical and/or biological libraries.
  • the present application is also directed towards any compound as identified by one or other of these methods, and any composition, in particular any pharmaceutical composition, ' comprising at least one such compound.
  • Such compositions are in particular useful for treating and/or for alleviating and/or for preventing an undesirable growth of E. coli, such as E. coli infections, or an E. coli contamination, and especially useful for treating and/or for alleviating and/or for preventing the presence of extra-intestinal E. coli bacteria.
  • a subject of the present invention is also, according to a notable aspect of the invention, phylogenic identification methods which implement the detection of the presence or absence of at least one of the polynucleotides which are of novel nature B2/D+ A- (whether these polynucleotides are novel as products, or whether they exhibit homologies with known products) : - the novel polynucleotides according to the invention, i.e.
  • polynucleotide sequences which can be obtained by digesting the total DNA of an E. coli of group B2 or group D (such as, for example, an E. coli strain isolated from the blood of a patient suffering from a septicaemic or meningeal infection) with a restriction enzyme chosen from the group consisting of Wotl and Blnl, selecting those of the fragments obtained which hybridize with at least one sequence chosen from the group consisting of SEQ ID NO: 134, 144, 109, 115, 140, 135, 33, 56, 122, 130, 141, 25, 48, 51, 57, 121, 44, 45, 113, 119, 120, 23, 52 and sequencing selected fragments, and
  • polynucleotides the sequence of which corresponds to SEQ ID NO: 170, 171, 174, 175, 178, 179, 183, 185, 186, 190, 191, 193, 194, 195, 196, 199, 200, 202, 205, 206, 208, 209, 211, 214, 218, 220, 221, 233, 234, 235, 241, 244, 246, 247, 248 and 250, - the polynucleotides which can be obtained by digesting the total DNA of an E.
  • coli of group B2 or D with a restriction enzyme chosen from the group consisting of Wotl and Blnl, and selecting those of the fragments obtained which hybridize to at least one sequence chosen from the group consisting of SEQ ID NO: 125, 123, 116, 43, 40, 127, 133, 27, 34, 36, 42, 46, 54, 55, 38, 128 and 151, with the exception of the polynucleotides sfa r hly r cnfl , pap/prs, hra and ibe 10,
  • polynucleotides the sequence of which corresponds to an orf (open reading frame) comprising the sequence of one of these polynucleotides
  • polynucleotides the sequence of which corresponds to a nature-conserving variant or fractional sequence of the sequence of at least one of these polynucleotides.
  • This detection can be carried out by direct detection of said polynucleotide, or of its fragments, or by detection of one or more polypeptide (s) corresponding to it
  • polypeptides encoded by a polynucleotide which is of novel nature B2/D+ A- polypeptides encoded by a polynucleotide which is of novel nature B2/D+ A-.
  • the present application is aimed towards any phylogenic identification method characterized in that it comprises the use of at least one compound chosen from the group consisting of :
  • sequences SEQ ID NO: 1 to NO: 153 • the polynucleotide sequences which can be obtained by digesting the total DNA of an E. coli of group B2 or group D (such as, for example, an E. coli strain isolated from the blood of a patient suffering from a septicaemic or meningeal infection) with a restriction enzyme chosen from the group consisting of Wotl and Blnl, selecting those of the fragments obtained which hybridize with at least one sequence chosen from the group consisting of SEQ ID NO: 134, 144, 109, 115, 140, 135, 33, 56, 122, 130, 141, 25, 48, 51, 57, 121, 44, 45, 113, 119, 120, 23, 52 and sequencing selected fragments, and
  • polynucleotides the sequence of which corresponds to SEQ ID NO: 170, 171, 174, 175, 178, 179, 183, 185, 186, 190, 191, 193, 194, 195, 196, 199, 200, 202, 205, 206, 208, 209, 211, 214, 218, 220, 221, 233, 234, 235, 241, 244, 246, 247, 248 and 250,
  • polynucleotides which are as obtained by digesting the total DNA of an E. coli of group B2 or D with a restriction enzyme chosen from the group consisting of Wotl and Blnl, and selecting those of the fragments obtained which hybridize to at least one sequence chosen from the group consisting of SEQ ID NO: 125, 123, 116, 43, 40, 127, 133, 27, 34, 36, 42, 46, 54, 55, 38, 128, 151 and 211, with the exception of the polynucleotides sfa, hly, cnfl , pap/prs , hra and ibe 10,
  • polynucleotides the sequence of which corresponds to a nature-conserving variant or fractional sequence of the sequence of a polynucleotide of this group (and in particular SEQ ID NO: 241, 195, 185 and 248),
  • polynucleotides the sequence of which corresponds to an orf comprising the sequence of a polynucleotide of this group
  • the probes comprising a polynucleotide which is of novel nature B2/D+ A-, as defined above,
  • binding products which are capable of binding to a polynucleotide which is of novel nature B2/D+ A-, as defined above, - the binding products (for example, antibodies) capable of binding to a polypeptide encoded by a polynucleotide which is of novel nature B2/D+ A- as defined above.
  • This implementation can be carried out according to any technique known to those skilled in the art so as to allow said "at least one compound" to detect, in a biological sample, the presence or absence of the polynucleotide (s) or, where appropriate, the polypeptide (s) which constitute (s) the target thereof: ELISA for reactions of antibody-antigen type, Southern-type hybridization or PCR for reactions of polynucleotide hybridization and/or amplification type.
  • Suitable biological samples comprise, in particular, all samples of human origin originating from sites which are normally sterile (blood, cerebrospinal fluid -CSF-, liquid from an effusion, etc.) or non-sterile (stooTs, oropharynx, skin, etc.), and also samples of environmental origin (soils, water, etc.), of food origin and of animal origin, and microbiological cultures.
  • This method makes it possible to conclude, when said detection is positive, that £7. coli bacteria of group B2 and/or D are present. With only the detection of the presence or absence of chuA, this method makes it possible to conclude, when this detection is positive, that £7. coli bacteria of group B2 or D are present. When this detection is negative, and insofar as the sample effectively contains £7. coli, it makes it possible to conclude that bacteria of group Bl or A are present.
  • This method can also comprise the use of several of said compounds, for example in such a way as to detect the presence or absence of the ch ⁇ K gene, and also that of the fragment SEQ ID NO: 119. It makes it possible then to conclude :
  • the phylogenic identification method according to the invention can also implement the detection of the presence or absence of the yjaA gene.
  • the yjaA gene ⁇ cf. Figure 5 is, in the prior art, known to be present in the £7. coli strain K12 (group A) . There is nothing in the prior art which would suggest that it may constitute a phylogenic marker capable of distinguishing between group B2 and group D.
  • Figure 5 shows the yjaA sequence (SEQ ID NO: 254) and that of the corresponding ORF (SEQ ID NO: 255) .
  • the detection of the presence or absence of the yjaA gene can take place by any technique accessible to those skilled in the art. It can in particular take place by detection of the polynucleotide or of its fragments (SEQ ID NO: 254), or by detection of the polypeptides which correspond to it (SEQ ID NO: 255) .
  • the development of reagents which allow such a detection is accessible to those skilled in the art: amplification probes or primers for the detection of polynucleotides, compound of serum or antibody type for the detection of polypeptides.
  • the polynucleotide hybridization reactions can, for example, be carried out by Southern and/or by PCR, and those of antigen-antibody type can be carried out by ELISA. Examples of implementation of such a detection step are given in the examples which follow.
  • a pair of primers SEQ ID NO: 162 and 163 which makes it possible to amplify yjaA are described in particular.
  • the present invention is also aimed towards any phylogenic identification method which implements at least the detection of the presence or absence of the yjaA gene. This detection makes it possible to distinguish between £7. coli of group B2 and £7. coli of group D ⁇ cf. examples) .
  • the present invention provides a phylogenic detection method which makes it possible to completely distinguish the £7. coli groups A, Bl, B2 and D.
  • This method implements the detection of the presence or absence of the ⁇ uA gene, of the yjaA gene and of SEQ ID NO: 119.
  • This method is described in detail in the examples.
  • Advantageous PCR conditions are in particular are mentioned therein (“simplified PCR”) .
  • this detection can be carried out by PCR amplification.
  • suitable PCR conditions are given in a purely illustrative capacity in the examples.
  • the present application is in particular aimed towards an identification method as defined above which implements the simultaneous detection of one or more of said polynucleotides which is/are of nature B2/D+ A-, and of yjaA, preferably by triplex PCR.
  • this method can be applied directly to the bacteria or to the sample analysed. It does not require the availability of a reference strain collection.
  • the present application is also aimed towards any method for detecting an undesirable growth of £7. coli, any method for diagnosing an £7. coli infection, any method for sanitary control or detection (foodstuffs, liquids intended for consumption, soils) and any method for selecting £7. coli strains suitable for biotechnological manipulations, which implement the phylogenic identification method according to the invention.
  • kits can in particular comprise at least one of the compounds used in said method. They can also comprise instructions for use featuring one or more of the profiles given in Figure 4 ⁇ cf. Example 5), or describing one or more of these profiles.
  • An advantageous kit comprises at least one of the pairs of primers (SEQ ID NO: 160, 161), (SEQ ID NO: 162, 163) and (SEQ ID NO: 164, 165) , preferably two of these pairs, more preferably the three pairs of primers.
  • FIG. 1 represents the distribution of B2/D+ A- clones along the chromosome of the £7. coli strain RS218, - Figure 2 gives the sequence listing for isolated B2/D+ A- fragments, and indicates their respective SEQ ID NOS,
  • FIG. 3 illustrates a decision diagram for the phylogenic analysis of an £7. coli strain
  • FIG. 4 represents the various phylogenic profiles obtained using a triplex PCR method according to the invention (lanes 1 and 2: group A; lane 3: group Bl; lanes 4 and 5: group D; lanes 6 and 7: group B2) ,
  • Figure 5 represents the polynucleotide sequence of the yjaA gene (SEQ ID NO: 254) and the corresponding ORF sequence,
  • Figure 6 represents the sequences of the CTF073+ K12- regions and RS218+ K12- regions obtained using the clones of Figure 2,
  • Figure 7 represents the position of the genomic fragment specific to £7. coli CFT073 and RS218 strains on the chromosome of £7. coll K12 strain.
  • EXAMPLE 1 Production of a C5+ A- library and of a RS218+ K12- library
  • the strains used for this subtractive hybridization are an £7. coli isolated from the cerebrospinal fluid (CSF) of a newborn (£7. coli C5 of serotype 018:K1:H7; group B2) and two £7. coli strains of group A, ECOR4 and ECOR15, which belong to the ECOR collection (ATCC) .
  • £7. coli C5 harbours several virulent factors, such as the Kl capsular antigen, an sfa operon, the ibe 10 gene, Pap pili and the haemolysin ⁇ hly) gene.
  • This strain belongs to the phylogenic group B2.
  • the ECOR4 and ECOR15 strains, which belong to the phylogenic group A express no identified virulence factor.
  • Other £7. coli strains were used: strain RS218
  • Southern transfer The Southern transfers were carried out by capillary transfer onto positively charged nylon membranes. The hybridizations were carried out at 65°C in 1% sodium dodecyl sulphate (SDS) - IM NaCI - 50 mM Tris- HCI (pH 7.5) - 1% blocking reagent (Boehringer Mannheim, Mannheim, Germany) . The membranes were washed, first in
  • 2x SSC (lx SSC corresponds to 0.15M NaCI + 0.015M sodium citrate) for 15 min at room temperature, and then in 2x SSC - 0.1% SDS for 30 min at 65°C and finally, in O.lx SSC for 5 min at room temperature.
  • the ' ' detection by chemi- luminescence was carried out with the DIG luminescence detection kit for nucleic acid (Boehringer Mannheim) according to the manufacturer's instructions.
  • the sfa and ije 10 probes were produced by PCR using primers and an amplification method described previously (Bingen et al . 1997, J. Clin. Microbiol. 35: 2981-2982).
  • the chromosomal DNA of the ECOR strains was randomly sheared by repeatedly pushing it through a hypodermic needle so as to obtain fragments with a length of between approximately 3 and 10 kb. This digested DNA was purified by phenol extraction. The chromosomal DNA of £7. coli C5 was cleaved with the Sau3AI or Tsp5091 restriction endonuclease.
  • This DNA (20 ⁇ g) was ligated with 10 nmol of hybridized oligonucleotides RBaml2 (5' -GATCCTCGGTGA-3' ; SEQ ID NO: 154) and RBam24 (5' -AGGACTCTCCAGCCTCTCACCGAG-3' ; SEQ ID NO: 155) or REcol2 (5' -AATTCTCGGTGA-3' ; SEQ ID NO: 156) and REco24 (5' -AGCACTCTCCAGCCTCTCACCGAG-3' ; SEQ ID NO: 157) when the restriction was carried out with SauAI or Tsp5091, respectively.
  • the DNA was separated from the excess primers by electrophoresis in a 2% low melting point agarose gel. The portion of the gel containing fragments longer than 200 bp was excized and digested with ⁇ -agarase. This DNA was purified by phenol extraction.
  • the solution was covered with mineral oil, and the DNA was denatured by heating to 100°C for 2 min; 2 ⁇ l of 5M NaCI were added and the mixture was left to hybridize at 55°C for 48 h.
  • the reaction mixture was diluted 10 times in preheated 3x EE buffer - IM NaCI and immediately placed in ice. A portion of the dilution (10 ⁇ l) was added to 400 ⁇ l of PCR reaction mixture (10 mM Tris HCl [pH 9.0], 50 mM KCI, 1.5 mM MgCl 2 , 0.1% Triton X-100, a 0.25 mM concentration of each deoxynucleotide triphosphate and 50 U of Taq polymerase per ml) and the whole mixture was incubated for 3 min at 70 °C in order to fill the ends of the re-hybridized £7. coli C5 fragments. After denaturation at 94°C for 5 min an addition of RBam24 or REco24 oligonucleotides (0.1 mmol per 100 ⁇ l) , the hybridizations were amplified by PCR
  • the PCR products were purified on a gel in order to separate the £7. coli C5 fragments from the primer and from the high molecular weight ECOR subtraction DNA.
  • the second round of subtractive hybridization was carried out using 40 ⁇ g of fragmented ECOR4 or ECOR15 £7. coli DNA and 25 ng of DNA ligated to RBam24 or REco24 obtained from the first round.
  • the gels were 1% agarose in 0.5x Tris-borate-EDTA buffer, and they were subjected to electrophoresis at 6 V/cm for 27 h, with pulsed durations varying in a linear manner between 2 and 49 s.
  • the positions on the RS218 chromosome of sequences which are reactive with each of the clones were determined by comparing the Blnl and Wotl recognized restriction fragments with the published macrorestriction map (Rode et al . 1999) .
  • Some of the clones correspond to genes which are already known to be present in the strain C5, such as pap, hly and kps .
  • the 494 clones none proved to be homologous to sfa or ibel O . Additional rounds of subtraction and/or the sequencing of additional clones make it possible to obtain greater exhaustiveness, until this is complete.
  • sequences are found which correspond to virulence factors described in strains which, to date, have never been associated with neonatal meningitis: (i) prs, cnf and hra, all of which form part of a PAI (pathogenicity island) in the uropathogenic £7.
  • each of these clones was used to probe the ral-digested DNA of strains EC0R4, EC0R15 and MG1655: they proved to be nonreactive with respect to these strains.
  • TspE4.A7 was positioned by SauE15.F12 overlap; 2, SauE15.F12 also exhibits reactivity on the plasmid; and 3, TspE4.A8 also exhibits weak reactivity on Notl fragment P.
  • Region 1 is contained in Blnl fragment m (85 kb) .
  • the clones of region 2 were mapped on Notl fragments p and n ( ⁇ 240 kb) .
  • Region 3 is contained in Notl fragment a ( ⁇ 310 kb) and region 4 is contained in Blnl fragment h (210 kb) .
  • Region 5 is located on Blnl fragment j (135 kb) .
  • Region 6 which overlaps Notl fragment b and Blnl fragment b ( ⁇ 550 kb) , was divided into two subregions, regions 6a and 6b, with the Xbal enzyme. This latter subregion contains clones which exhibit homologies with Cnfl, hly, prs and hra . The sfa and ibel O probes hybridize in regions 2 and 6a, respectively. The genes encoding the capsule were not linked to any of these regions. Six clones exhibiting no homology with known sequences and the senB gene were all located on a large plasmid present in the strain RS218.
  • a The prevalence of PAI V was evaluated using clones TspE4.Dll, TspE4.D10 and TspE15.Cl, which are homologous to the hly, cnfl and hra genes, respectively.
  • b p ⁇ 0.05, with respect to the strains of the ECOR collection (the existence of a difference in the distribution of the clones studied was tested using the ⁇ 2 test) .
  • regions 1, 3 and 4 appeared with a frequency which was notably higher (p ⁇ 0.05) among the NMEC than among the other B2 strains, thus suggesting that these regions contain DNAs encoding NMEC-specific factors. These regions, their portions and the clones which they contain therefore constitute a source of polynucleotides involved in neonatal meningitis.
  • any restriction enzyme having short restriction sites for example 4 bp, such as Tsp509I or 5au3AI, and also Mspl or aell
  • two to three rounds of subtraction followed by the elimination of those of the clones which exhibit homology with a strain of group A such as £7. coli K12 gives a very good level of specificity.
  • Varying the nature of the restriction enzymes used, in particular between each subtraction cycle, further increases the level of exhaustiveness and/or of specificity of the set obtained. In order to evaluate the level of exhaustive- ness effectively obtained, this evaluation can be carried out by investigating the presence or absence of known markers such as sfa or ibel O .
  • Region 5 has a high prevalence, but it is similar in both collections and may thus correspond to segments which are highly characteristic of the phylogenic group B2 with respect to the group A.
  • Region 5 of £7. coli thus appears to be an advantageous source of DNA fragments present in a great majority (more than 80%) of £7. coli of group B2 of the ECOR collection, and absent from the majority of £7. coli of group A of the ECOR collection. The same distribution was observed for ⁇ uA, but interestingly, these genes were present, without exception, in all the strains of group B2 tested.
  • regions 1, 3 and 4 they appear to be clearly more common in NMEC than in the other B2 £7. coli . Given that regions 1, 3 and 4 do not, moreover, contain any known virulence factors, these regions appear to correspond to DNA islands associated with invasion of the meninges by £7. coli in infants and newborn.
  • Regions 1, 3, 4 and 5 were not available, that they had never been described or isolated and that no function was known for them. Regions 1, 3, 4 and 5 therefore appear to be novel. As regards regions 2, 6a and 6b, they comprise DNAs which were known as products, but this is the first description of the existence of such regions and the first description of their nature C5+ A-. EXAMPLE 2: Distribution of the C5+ A- fragments among the ECOR strains
  • Example 1 The frequency of presence of the fragments obtained in Example 1 (C5+ A- fragments) among ECOR £7. coli of groups Bl and D was then measured by Southern hybridization as described in Example 1.
  • the results obtained for 14 of them (SEQ ID NO: 56, 116, 43, 51, 141, 130, 45, 50, 52, 119, 127, 125, 55 and 37) are given in Table 3 below.
  • 15 £7. coli strains of group B2 which the ECOR collection contains. More than 75% of the fragments are present in the 15 ECOR £7. coli strains of group B2 at a frequency greater than 40%, 50% of them are present therein at a frequency greater than 70% and approximately 35% of them are present therein at a frequency greater than or equal to 80%.
  • the various results obtained indicate that the set of DNAs isolated according to the invention constitute, taken alone or in combination, particularly suitable tools for the phylogenic identification of £7. coli strains.
  • TspE15.G6 is however present at a frequency of 12% in the £7. coli of group A, at a frequency of 53.3% in the ECOR £7. coli of group B2, and at a frequency of 83.3% in the ECOR £7. coli of group D.
  • the choice of the £7. coli strain C5 as the strain of group B2 for the subtractive hybridization which enabled the isolation of these fragments ⁇ cf. Example 1 above) is probably not unrelated to these results.
  • the fragments isolated in accordance with the invention using a B2/D £7. coli strain such as £7. coli C5 have, in addition to phylogenic diagnostic applications ( cf. Example 5 below) , applications of particular value for preventing, alleviating and combating any extra-intestinal development of £7. coli (systemic and nondiarrhoeal development) .
  • This B2/D+ A- set according to the invention in particular comprises mostly fragments which are present at a frequency greater than 10% in the ECOR £7. coll of group B2 and/or D, and at a frequency of less than 25%, preferably 20%, more preferably 10%, and even more preferably 5%, in the ECOR £7. coli of group A (with the proviso that this A frequency is always lower than that in the B2s and/or Ds) .
  • the present invention also provides means which give access to the entire set of these polynucleotides which are of nature B2/D+ A-.
  • EXAMPLE 3 Example of medical application of the B2/D+ A- fragments isolated (systemic and non- diarrhoeal development of E. col ⁇ in an extra- intestinal compartment)
  • B2/D+A- E. coli are those which are particularly responsible for extra-intestinal infections
  • DTA differential transcriptional analysis
  • the bacteraemia phase is compared to the growth of the bacterium in the presence of human serum
  • the £7. coli strain C5 (serotype 018:K1:H7) was isolated from the CSF of a newborn. This virulent strain belongs to the phylogenic group B2 and exhibits the following virulent factors: The Kl capsular polysaccharide, S adhesin, IbelO invasin, the type P pilus and haemolysin, but does not produce aerobactin.
  • The- subtractive fragment library was obtained using the C5 strain.
  • the nonpathogenic strains used were the £7. coli K12 strain MG1655 and the strain ECOR15 belonging to the so-called nonpathogenic phylogenic group A, originating from the ECOR reference collection.
  • the bacterial inocula were prepared from 18 h cultures on tryptocasein-soybean agar (Sanofi Pasteur) , with the colonies being resuspended in sterile water. After measuring the OD, this bacterial suspension, pure or diluted, was used to prepare the inoculum in the various culture media, adding a volume which was always less than 1/1Oth of the final volume.
  • the bacterial cultures were prepared at 37°C with shaking, using either pure nutrient broth (Sanofi Pasteur) or nutrient broth supplemented with an iron chelator: 2, 2'-dipyridyl (Sigma) at a concentration of 200 ⁇ M.
  • the bacterial strains were also cultured in the presence of human serum. Two types of serum were obtained: one consisting of a pool of 4 sera originating from donor blood, the other corresponding to a single donor. The serum was collected after harvesting the blood in a dry tube, spontaneous coagulation for approximately 3 hours at room temperature, and then decanting. The serum was then stored in the form of 2.5 ml aliquots at -80°C. Decomplemented serum was obtained, from the pool, after incubation at 56°C for 30 minutes.
  • the subcultures of the clones of the B2/D+ A- library were prepared in nutrient broth enriched with ampicillin (50 ⁇ g/ml) at 37°C.
  • the subtractive DNA fragments cloned into the plasmid pUC19 were amplified by PCR reaction, without DNA extraction, directly from a 1/lOth dilution of an 18 h culture broth of each clone. 5 ⁇ l of this bacterial solution were added to the reaction mixture, with a final volume of 50 ⁇ l, comprising: IX PCR buffer (10 mM Tris-HCI pH 9; 1.5 mM MgC12; 50 mM KCI; 1% Triton X100; 0.1% gelatin); 2U SuperTaq polymerase (ATCG Biotechnologie) ; 200 ⁇ M deoxynucleotide triphosphates; 6 ⁇ M specific primers.
  • the primers used were as follows : PI (5'-CATGCCTGCAGGTCGACTCT-3'; SEQ ID NO: 728), P2 (5'-CGTTGTAAAACGACGGCCAG-3' ; SEQ ID NO: 729).
  • the PCR reaction consisted of 30 cycles: 30 s at 95°C (denaturation) , 30 s at 55°C (hybridization) and 30 s at 72°C (elongation) .
  • Membranes comprising the set of specific amplified fragments of the £7. coli strain C5 were manufactured by the company Eurogentec. 180 nl of each of the PCR products were deposited in duplicate, in the form of microspots, onto 6 cm by 11 cm nylon membranes. In order to be able to normalize the signals recorded for each reverse transcript, spots consisting of 4-fold serial dilutions of the chromosomal DNA of the £7. coli strain C5, on the one hand, and of dilutions of the product of a PCR of the 16S rRNA, on the other hand, were deposited. In addition, a negative control corresponding to the PCR product of a subtractive fragment of 17 bp was also deposited. These membranes are conserved sealed, at +4°C.
  • RNA is extracted from a bacterial pellet obtained after centrifugation (4 700 rpm at 4°C for 3 min) of 5 ml of a 4-hour culture (approximately 10 8 CFU/ml) .
  • this reagent consists of a monophasic solution of phenol and of guanidine isothiocyanate, which allows extraction of the total RNA in a single step according to the method developed by Chomczynski and Sacchi.
  • the bacterial cells are lysed by adding the TRIZOL reagent, vortexed for 30 s and subjected to a heat shock (incubation at 65°C followed by freezing at -80°C) .
  • the following steps are those of a conventional phenol-chloroform extraction.
  • the recovered aqueous phase contains the RNA.
  • this kit contains no phenol-based solution. It consists in using a solution which allows the cells to be lysed during an incubation at 65°C for 5 minutes. Next, a solution for precipitating the DNA and the proteins makes it possible to then recover the RNA which is in the aqueous phase.
  • RNA is precipitated using isopropanol: the isopropanol is added to the aqueous solution containing the RNA, volume for volume. This mixture is incubated at -20°C for approximately 15 h, and after centrifugation (13 000 rpm at 4°C for 5 min), the RNA is obtained in the form of a pellet. The RNA is then washed with 70% ethanol and precipitated by centrifugation (13 000 rpm at 4°C for 2 min) . Finally, the RNA is solubilized in water in the first case
  • RNA samples are stored at -80°C.
  • RNA assay and puri ty The RNA is assayed using a spectophotometer, by measuring the absorbance at 260 nm, it being known that an optical density value at 260 nm corresponds to an RNA concentration of 40 ⁇ g/ml.
  • the purity of the sample is estimated by calculating the OD 26 o nm/OD 2 8o nm ratio: a ratio greater than 1.6 reflects an acceptable purity.
  • RNA preparation obtained is of good quality.
  • the cDNA probe is synthesized using random priming. 10 ⁇ g of RNA are mixed together, in a final volume of 50 ⁇ l, with 10 ⁇ g of random hexamers; dATP, dTTP and dGTP, final concentration of 10 ⁇ M (Boehringer Mannheim) ; 40 U of Rnasin ribonuclease inhibitor (Promega) ; IX RT buffer; 10 ⁇ g of bovine serum albumin
  • the membrane is prehybridized in 5 ml of Church and Gilbert hybridization buffer (0.5 M NaPi, pH 7.2; 1 mM EDTA; 0.7% SDS) for a minimum of 30 minutes at 65°C.
  • Church and Gilbert hybridization buffer 0.5 M NaPi, pH 7.2; 1 mM EDTA; 0.7% SDS
  • the 33 P- labelled cDNA probe is directly added to 5 ml of hybridization buffer.
  • the hybridization reaction is carried out at 65°C for 15 to 18 h.
  • the membrane is first rinsed twice with washing buffer (40 mM NaPi, pH 7.2; 1 mM EDTA; 1% SDS). Then, four washing steps are carried out (30 min at 65°C) .
  • washing buffer 40 mM NaPi, pH 7.2; 1 mM EDTA; 1% SDS. Then, four washing steps are carried out (30 min at 65°C) .
  • the membrane is exposed to a 33 P-sensitive screen (Molecular Dynamics) for 48 h to 90 h.
  • the membrane is incubated twice for 15 min at 37 °C in the presence of a solution of 0.2N NaOH and 0.1% SDS, and then it is rinsed with distilled water for 5 min.
  • the exposed 33 P-sensitive screen is scanned using a Phosphorlmager (Storm 840, Molecular Dynamics) with a pixel size of 50 urn, and then the image obtained is analysed using the XdotsReader software (Cose) .
  • This software allows automatic recognition of the spots and is capable of calculating the pixel-density for each of the spots. It also makes it possible to subtract the background noise and to normalize the signal intensity. For each spot, the local background noise was subtracted and the signal intensity was normalized using the signal of the chromosome diluted 400-fold.
  • the disparity between two spots from the same clone was calculated in order to eliminate the spots with a significant disparity, reflecting aberrant signals. Then, the mean of the intensity of the signals- from a same pair was defined, and it is this mean intensity, net (after subtraction of the local background noise) and normalized, which will be considered for the data analysis.
  • the intensity obtained for the 17 bp clone made it possible to define a positivity threshold, the signals lower than this intensity being considered as negative or "undetected".
  • ⁇ £7. coli strain K12 is killed in less than 2 hours and the strain ECOR15 experiences a decrease in growth of more than 2 logs in 2 hours, persisting at a level of between 10 4 and 10 5 CFU/ml.
  • the strain C5 persists without growing for the first two hours of culturing, and then experiences a growth of 1 log in the 4th hour.
  • the inoculum of 10 7 CFU/ml was therefore selected for the transcriptome * study.
  • the growth of the strains K12 and ECOR15 is similar to that of the strain C5, which suggests that the bactericidal effect observed was due to the lytic activity of the complement.
  • RNA extraction step is a fundamental step of the DTA, it appeared to us to be necessary to compare two extraction methods in order to determine whether or not the mode of extraction could have an influence on the transcriptome results.
  • Preparations of good quality are characterized by the presence of the 23S, 16S and 5S rRNAs, detected in the form of clear bands by agarose gel electrophoresis.
  • the bands obtained for the 23S and 16S rRNAs, and a band corresponding to the 5S rRNA and to the tRNAs reflect the good quality of the RNA preparations and of the equivalence of the extraction methods .
  • the detection of RNA of low- molecular weight shows that the extraction method makes it possible to isolate the mRNAs which are small in size, unlike other systems and in particular those using columns which retain only the mRNAs longer than 200 bp.
  • a high molecular weight band corresponding to the chromosomal DNA appears clearly on the lane of the RNA obtained using the Biorad kit.
  • the two transcriptomes obtained from these two types of RNA were compared visually (without integrating the signals), and appear to be identical.
  • the transcriptomes obtained by hybridization of the reverse transcripts originating from £7. coli C5, under two different culture conditions, on the high density membranes comprising the set of clones of the B2/D+ A- library were analysed with the aid of the Cose software, using the 400-fold dilution of the chromosome as the normalization spot, the signal of which is close to the median of all of the signals. It appears clearly that a certain number of spots are lacking in signals.
  • the normalized intensity recorded on the 17 bp clone (0.05) was used. In order to be safe, this threshold was doubled and, therefore, any spot with arr intensity of less than 0.1 was considered to be "undetected".
  • the normalized intensities of the signals obtained for all the clones for which the transcript was detected under at least one of the culturing conditions make it possible to visualize the level of transcription of the set of subtractive clones in the course of the three respective experimental conditions: growth in nutrient broth, in nutrient broth supplemented with 2,2' -dipyridyl (iron chelator) and in serum. It is noted that most of the signals detected have similar intensities whatever the culturing conditions, whereas certain fragments exhibit levels of transcription which vary by a factor of ten according to the culturing conditions. These results therefore suggest good reproducibility of the technique, with a capacity to detect different transcriptional levels.
  • the transcriptome obtained in nutrient broth was considered to reflect the basal level of transcription of the bacterium in favourable medium.
  • the expression profile obtained under conditions of stress consisting of culturing in serum was analysed with respect to this control transcriptome.
  • the transcriptomes obtained when culturing in nutrient broth and iron chelator were also prepared, in order to determine the respective roles of iron deficiency and of complement in the serum.
  • Table 5 gives the ratios of the intensities of the signals obtained for some of the clones of the B2/D+ A- library under the various experimental conditions, with respect to the control condition represented by the growth in nutrient broth (NB) .
  • the transcripts induced in the- serum (ser) are most commonly also induced in the presence of dipyridyl (dip) and of decomplemented serum (DC serum), with the exception of the SauE15.A12, SauE4.E6, SauE4.Cll and TspE15.G6 clones.
  • a new probe was prepared from a culture of £7. coli C5 in a serum originating from a single donor.
  • the transcriptome obtained with this new probe was compared with the transcriptome produced with the pool consisting of several sera.
  • the straight line of regression obtained, and also the regression coefficient with a value of 0.85, indicate the excellent reproducibility of the technique.
  • the intensities obtained for the chromosomal range points consisting of l/100th, l/400th and 1/1 600th dilutions were recorded on a graph.
  • the graph obtained shows the existence of a linear relationship between these values, making it possible to deduce an induction factor directly from the normalized intensities recorded.
  • the DTA technique described herein is a reliable method for selecting, from the B2/D+ A- library obtained from £7. coli C5, DNA fragments the transcription of which is increased in the presence of serum. These fragments make up genes which participate specifically in the systemic and non- diarrhoeal extra-intestinal development of £7. coli in humans and animals. These genes can be isolated using said DNA fragments the transcription of which is increased in the presence of serum, according to conventional techniques of those skilled in the art ⁇ cf. Example 6) .
  • fragments and the genes which bear them can be used as active principles (in the form of naked DNA placed under the control of a eukaryotic promoter or in the form of DNA transfected into a cell) in a vaccine composition intended to prevent, alleviate or combat the systemic and non-diarrhoeal development of £7. coli in a human or animal extra-intestinal compartment.
  • these fragments and genes can, if desired, be modified, for example so as to produce inactivated isogenic mutants.
  • polypeptides which they encode can also be used in such vaccine compositions, in an inactivated and immunogenic form.
  • DNA fragments and these genes can also be used as anti-pathogenicity targets (with the objective of preventing the development of £7. coli in an extra- intestinal area, and not in the intra-intestinal area) : they allow the identification of compounds capable of specifically inhibiting their transcription and/or translation, or of compounds capable of inhibiting the activity of the proteins encoded by these genes.
  • compounds can be used as active principles in pharmaceutical compositions (medicinal products) in order to prevent, alleviate or treat the systemic non- diarrhoeal development of £7. coli in a human or animal extra-intestinal compartment.
  • fragments which are of nature B2/D+ A-, and the transcripts of which are increased in the serum those which are not present in £7. coli which are agents of infections localized in the intestine, such as £7. coli 0157 :H7, are more particularly preferred.
  • a systemic non-diarrhoeal development of £7. coli in an extra-intestinal compartment is in particular observed in the context of diseases such as neonatal meningitis, septiceamias, sepsis or pyelonephritis.
  • Such vaccines and pharmaceutical compositions are particularly useful in the context of hospital-acquired infections.
  • Such vaccines are most particularly valuable for the vaccination of women (from adolescent to adult, and more particularly before and during a gestation period) as a prevention against pyelonephritis, in order to avoid contamination of the newborn during birth.
  • the vaccines and pharmaceutical compositions according to the invention are therefore particularly suitable for such pathologies.
  • the present invention is therefore aimed towards any polynucleotide capable of being obtained :
  • the DNAs given in Table 5 above are examples of such polynucleotides .
  • any vaccine composition comprising such a polynucleotide, or an inactivated isogenic mutant of such a polynucleotide (inactivation of the possible pathogenic potency) , in particular for preventing, alleviating or combating the development (systemic and non-diarrhoeal) of £7. coli in a human or animal extra-intestinal compartment,
  • any pharmaceutical composition comprising a compound capable of inhibiting the transcription and/or translation of such a polynucleotide or mutant, or capable of inhibiting the activity of a polypeptide encoded by such a polynucleotide or mutant.
  • the present invention is also aimed towards a method for identifying compounds which can be used as active principles in a pharmaceutical composition (medicinal product) intended to prevent, alleviate or combat the development (systemic and non-diarrhoeal) of £7. coli in a human or animal extra-intestinal compartment.
  • This method comprises detecting and selecting compounds capable of inhibiting the transcription and/or translation of said polynucleotides, or capable of inhibiting the activity of the polypeptides which they encode.
  • the present invention is also aimed towards any kit suitable for the implementation of this method, said kits comprising at least one of said polynucleotides or polypeptides.
  • Such cells and transgenic animals are particularly useful for selecting active principles of interest.
  • Example 1 the isolation and the sequence of 259 DNA fragments which are of nature B2/D+ A- (153 of which are novel as products) are described, and means for producing the entire set of these products are provided.
  • Example 1 and Figure 1 give the sequence of eight (novel) fragments belonging to region 1 (SEQ ID NO: 134, 144, 109, 115, 140, 135, 33 and 56) .
  • region 2 5 novel fragments (SEQ ID NO: 125, 123, 116, 43 and 40), and the presence of the sfa gene, are indicated.
  • region 3 7 (novel) fragments (SEQ ID NO: 122, 130, 141, 25, 48, 51 and 57) are indicated.
  • region 4 three (novel) fragments (SEQ ID NO: 121, 44 and 45) are indicated.
  • region 5 (novel) fragments SEQ ID NO: 113, 119, 120, 23 and 52) are indicated.
  • region 6a eight novel fragments (SEQ ID NO: 127, 133, 27, 34, 36, 42, 46 and 54), and the presence of the known iielO product, are indicated.
  • region 6b four (novel) fragments (SEQ ID NO: 55, 38, 128 and 151), and the presence of four known products (SEQ ID NO: 212, 226, 201 and 229), are indicated.
  • One of the conventional techniques consists in developing primers for amplifying the desired region, based on the sequences of the fragments of this region, and in carrying out a PCR amplification using various combinations of said primers placed in contact with the polynucleotide population of an £7. coli strain of group B2 or D (which may or not be ECOR) , under conventional PCR conditions.
  • the PCR products which overlap are sequenced on both strands, using the chain termination technique and automated sequencing.
  • the sequence obtained can be extended beyond the limit of the clones available by cloning, for example in lambda DASH-II, partial fragments of approximately 15 kb obtained by restriction carried out on said polynucleotide population.
  • the inserts overlapping the desired region are then identified by hybridization with clones of this region.
  • the inserted DNA is then sequenced from the end of the inserts, and these sequences are used to develop novel primers which" will be used to directly amplify the chromosomal (and non-phage) DNA. Amplification of the chromosomal DNA is then obtained using these novel primers and those of the shorter sequence already obtained.
  • These PCR strands are also sequenced on both strands, which then produces the complete sequence of the desired region.
  • sequence of such regions can be obtained by extension of the sequencing of the chromosome of an £7. coli strain of group B2 or D, from points at which a clone which is of nature B2/D+ A- is located (cf. Example 6 below) .
  • the open reading frames of the sequences obtained for the regions can then be analysed according to conventional techniques for seeking ORFs.
  • a search is carried out in particular for ORFs which begin with ATG or CTG and which have a high codon use index.
  • EXAMPLE 5 Method and kit for identifying the phylogenic group of an E. col ⁇ strain
  • the polynucleotides which are of nature B2/D+ A- can be used to determine the phylogenic group of any £7. coli strain. They can be used alone, in combination together or in combination with other products, depending on the result and the precision of phylogeny desired. In the event of positive detection, the set of DNAs which are of nature B2/D+ A- makes it possible to eliminate the hypothesis that a strain belongs to the group A.
  • probes can in particular be carried out using said polynucleotides as probes (Southern technique) , or by constructing amplification primers capable of amplifying one of said polynucleotides, so as to carry out a PCR.
  • the construction of probes and of primers can be carried out according to any technique known to those skilled in the art; examples of such constructions are given below.
  • a phylogenic identification method which allows a phylogenic precision of at least 99% for £7. coli .
  • This method is based on the PCR detection of two genes (chuA and yjaA) , the sequence of which is known, but which- are of novel nature, and on the detection of the
  • TspE4.C2 novel DNA fragment (SEQ ID NO: 119). The method was evaluated by testing 220 strains which had already been grouped together using reference methods
  • the inventors in fact demonstrated that the known cnuA gene is present in 100% of the ECOR strains of group D, in 100% of the ECOR strains of group B2, and in 0% of the ECOR strains of group A and of the ECOR strains of group Bl. They also demonstrated that the y aA gene, the sequence of which is known, is present in 100% of the ECOR B2 strains and in 0% of the
  • Bacterial strains The 72 strains of the ECOR- collection are available from the ATCC. These reference strains, isolated from various hosts and various geographical locations, are representative of the range of genotypic variation of the species. Sixty-eight of these strains belong to the four main phylogenic groups (A, Bl, B2 and D) , and 4 are unclassified.
  • the other 17 NMEC and the remaining 65 clinical isolates were classified by means of ribotyping as previously described in Binger et al . (ref. above) .
  • the bacteria were cultured at 37°C on Luria Bertani broth medium or agar. If necessary, ampicillin (100 ⁇ g per ml) was used.
  • PCR amplification In a first step, the PCR was carried out according to a standard protocol. The reaction was carried out in a volume of 20 ⁇ l containing 2 ⁇ l of 10X buffer (supplied with Taq polymerase) , 20 pmol of each primer, 2 ⁇ M of each dNTP, 2.5 U of Taq polymerase (ATGC Biotechnologie, noisysy-le-Grand, France) and 200 ng of genomic DNA.
  • the PCR was carried out using a Perkin-Elmer GeneAmp 9600 thermal cycling machine, with MicroAm tubes, under the following conditions ⁇ denaturation for 5 minutes at 94°C, 30 cycles of 30 seconds at 94 °C, 30 seconds at 55°C and 30 seconds at 72°C, and a final extension step of 7 minutes at 72°C, using the pairs of primers :
  • chuA.l (5'-GACGAACCAACGGTCAGGAT-3' ; SEQ ID NO: 160) and ChuA.2 (5'-TGCCGCCAGTACCAAAGACA-3' ; SEQ ID NO: 161), for chuA
  • yjaA.l (5'-TGAAGTGTCAGGAGACGCTG-3' ; SEQ ID NO: 162) and yjaA.2 (5'-ATGGAGAATGCGTTCCTCAAC-3' ; SEQ ID NO: 163), for yjaA and
  • TspE4C2.1 (5'-GAGTAATGTCGGGGCATTCA-3' ; SEQ ID NO: 164) and TspE4C2.2 (5' -CGCGCCAACAAAGTATTACG-3' ; SEQ ID NO: 165) for TspE4C2 (SEQ ID NO: 119) .
  • These pairs of primers generate, by amplification, fragments of 279 bp, of 211 bp and of 152 bp, respectively.
  • the PCR steps were as follows: denaturation for 4 minutes at 94°C, 30 cycles of 5 seconds at 94°C and 10 seconds at 59°C, and a final extension step of 5 minutes at 72°C.
  • the Southern transfer was carried out by transfer by capillarity onto positively charged nylon membranes.
  • the hybridization was carried out at 65°C in 1% SDS/1M NaCl/50 mM Tris HC1, pH 7.5/1% of blocking agent (Boehringer Mannheim, Mannheim, Germany) .
  • the membranes were washed in 2x SSC for 15 minutes at room temperature; then in 2x SSC/0.1% SDS for 30 minutes at 65°C and finally, in O.lx SSC for 5 minutes at room temperature.
  • the detection of chemiluminescence was carried out according to the manufacturer's instructions (DIG Luminescence Detection Kit for nucleic acids, Boehringer Mannheim) .
  • the probes were produced by PCR according to the manufacturer' s instructions (PCR DIG Probe Synthesis Kit, Boehringer Mannheim) and using the primers and the amplification procedure described above for the standard protocol.
  • R.M phylogenic groups assessed by reference methods (MLEE or ribotyping)
  • Table 6 above shows the results obtained with the method according to the invention and the reference methods for the complete set of strains, according to phylogenic group.
  • the chuA gene is present in all the strains belonging to groups B2 and D, and absent from all the strains of groups A and Bl. This makes it possible to effectively separate groups B2/D from groups A/Bl.
  • the yjaA gene allows complete distinction between group B2 (100% positive strains) and group D (100% negative strains) .
  • the novel TspE4.C2 clone is present in all the strains of group Bl except 2, and absent from all the strains of group A. All the PCR results were confirmed by Southern hybridization.
  • the inventors have developed a PCR method for rapidly determining the phylogenic group of £7. coli strains. Using two genes, chuA and yjaA, and a novel DNA fragment named TspE4.C2 (SEQ ID NO: 119, cf. Example 1) , the phylogenic groups of 220 strains which had previously been assigned to phylogenic groups determined using known methods were determined. The precision of analysis obtained according to the invention exceeds 99% with respect to the grouping established with the methods of the prior art. In addition, the same results were observed with a technically very simple triplex PCR method which is used directly on the bacterial colonies.
  • the phylogenic characterization of the £7. coli strains based on a few genotypic or phenotypic characteristics, appeared, in the prior art, to be very difficult.
  • Such genotypic characteristics Presence or absence of a gene, for example
  • the gene must have been acquired, or have been deleted, when the group that i- characterizes emerged.
  • this same gene must have been "stabilized” so as to exclude any phenomenon of subsequent deletion or horizontal transfer towards bacteria belonging to other phylogenic groups.
  • recombination phenomena must be very rare in the candidate gene. In other words, the gene product must not be a target for natural selection, which would favour novel genetic recombinations.
  • ECOR 70 can be considered to be a "hybrid" strain in which a few housekeeping genes have nucleotide sequences which are common with ECOR strains of group A, and in which few other genes haver nucleotide sequences which are common with the ECOR strains of group Bl.
  • the phylogenic membership of ECOR 70 to group Bl is not clearly determined; it is, in any event, considered to be part of group A using the method according to the invention.
  • the invention has the advantage of not requiring the use of a reference collection such as the ECOR strain or another collection, which means that the analysis can be easily carried out in the laboratory and in particular for routine analyses.
  • the allocation to the phylogenic groups is unequivocal. Specifically, the 4 strains which, until now, were unclassified in the ECOR collection (E31, E37, E42 and E43) can be classified using the method of the invention; the first three strains belong to group D, and the fourth to group A. It can also be noted that all the sequences of the housekeeping genes studied in the latter strain appear to be characteristic of those found in the strains of group A.
  • the first use is, of course, the bioclinical use, taking into account the link which can be established between the phylogenic group and the possible virulence or dangerousness.
  • the second use corresponds to a biotechnological screening tool which makes it possible to. eliminate potentially pathogenic strains, i.e. strains which are highly dangerous, when candidate strains are being sought for cloning. It was possible to develop such screening tools in the prior art, for example for identifying the £7. coli K12 strains by PCR- or for detecting £7. coli strains which exhibit none of the virulence genes which were, until then, known due to a reverse dot blot procedure.
  • the method described herein has the notable advantage of allowing the identification of nonpathogenic strains other than £7. coli K12, and of being suitable for screening strains on a large scale.
  • EXAMPLE 6 Production of seventy CFT073+ K12- zones and thirty one RS218+/K12- zones
  • These clones are present with greater frequency in the ECOR £7. coli of group B2 and/or in the ECOR £7. coli of group D with respect to the ECOR £7. coli of group A (preferably 2 times greater, more preferably 3 times greater, even more preferably 3.5 times greater, and very preferably 4 times greater) . They are, in particular, present at a frequency greater than 10% in the ECOR £7. coli of group B2 and/or in the ECOR £7. coli of group D, and at a frequency of less than 25%, preferably less than 20%, more preferably less than 10%, and even more preferably less than 5% in the ECOR £7. coli of group A, the frequency observed in the ECOR £7. coli of group A always remaining less than that observed in the ECOR £7. coli of group B2 and/or in the ECOR £7. coli of group D.
  • the chromosomal position of each of these seventy CFT073+ K12- zones was then determined ("K12 coordinates" column in Table 8 below) with respect to the chromosomal map of £7. coli K12, which constitutes a reference in the field.
  • These seventy CFT073+ K12- zones were then analysed in order to determine the possible presence of open reading frames (orf/ORF) using programs such as CodonUseTM (£7. coli codon use) and ORF Finder (www : _ncbi .nliti•.nih_gov/gqr;f/ orf._html, bacterial genetic code) .
  • ORFs The corresponding protein sequences (ORFs) were extracted compared to various databases in order to search for homology compared to known sequences.
  • the protein sequence (ORF) and polynucleotide sequence (orf) of the open reading frame identified is shown, indicating the start and end positions (Pos.) of these. frames with respect to the complete sequence of the zone concerned.
  • Some of these ORFs are encoded by orfs which are, in fact, located on the strand complementary to the indicated sequence of the region (the "frame start" position then has a number which is higher than that of the "frame end” position) .
  • Table 7 summarizes the SEQ ID NOS assigned to each of the seventy CFT073+ K12- zones and to their ORFs and orfs, and each thirty one RS218+/K12- zones.
  • zone 24 and its orfs/ORFs which, correspond to known products which are of known nature B2+ A-, none of the sequences identified herein is strictly identical to a sequence of the prior art (comparison of the nucleotide series over their entire length, independently of the function of the process) .
  • some homologies are found as it appears from table 9 above.
  • the CFT073+ K12- and RS218+/K12- zones isolated herein all comprise, in the vicinity of their chromosome, at least one DNA fragment which is of nature B2/D+ A- as defined above (ECOR B2 frequency and/or ECOR D frequency higher than ECOR A frequency SEQ ID NO 1 to NO. 550, the zones and orfs isolated herein are excellent candidates as polynucleotides which are of nature B2/D+ A-.
  • Verifying that the CFT073+ K12- or RS218+/K12- zones, ORFs and orfs isolated herein are effectively of nature B2/D+ A- can be easily carried out by those skilled in the art according to techniques which are conventional in the domain of phylogeny.
  • One way of making sure may, for example, consists in applying to at least one other E. coli strain of group B2 or D, for example E. coli C5, the procedure which has been described herein for the E. coli strain CFT073 and RS218, in such ' a way as to identify and isolate the zones, ORFs and orfs which are present in E. coli C5 and absent in E. coli K12 (or another E. coli strain of group A) .
  • sequences of these C5+ K12- zones, ORFs and orfs are then compared with those of the CFT073+ K12- and RS218+ K12- groups described herein, while searching for homologous sequences and sequence fragments.
  • This comparison can, for example, be carried out using the BLAST program (National Centre for Biotechnology Information, NCBI, Altschul et al . 1997, Nucleic Acids Res. 25: 3389- 3402), comparing each sequence of the C5+ K12- group with those of the CFT073+ K12- (or RS218+ K12-) group.
  • polynucleotide sequences, or fragments of polynucleotide sequence, with a low probability of the significant homology (for example, identity of 80% or more) being linked by chance can then be selected as being of nature B2/D+ A-, as defined above. If desired, the process can be repeated on a fourth or fifth E. coli strain of group B2 or D.
  • Such B2/D+ A- products are particularly useful for the phylogenic determination of E. coli .
  • the polynucleotides which are of nature B2/D+ A- and their inactivated isogenic mutants can be used, in the form of isolated DNAs (placed under the control of a eukaryotic promoter or in the ' form of DNA transfected into a cell) , as active principles in a vaccine composition intended to prevent, alleviate or combat an undesirable development of E. coli, and in particular a development of E. coli in an extra-intestinal compartment.
  • the ORFs themselves, administered in an optionally inactivated and immunogenic form are active principles which are very promising for the development of a vaccine composition intended to prevent, alleviate or combat an undesirable development of E. coli , and in particular a development of E. coli in an extra- intestinal compartment.
  • polynucleotides and polypeptides which are of nature B2/D+ A- can also be used as anti-pathogenicity targets (with extra-intestinal and non intra-intestinal targeting) / they make it possible to identify and isolate capable active principles which can be used in pharmaceutical compositions (medicinal products in particular) intended to inhibit the growth of an E. coli bacterium and, notably, its extra-intestinal development, by selecting, from candidate active principles, those which inhibit or block the correct transcription and/or translation of these zones or orfs, or which inhibit or block the activity of the ORFs.
  • the present application is aimed towards each of these CFT073+ K12- and RS218+/K12- zones, numbers 1 to 23, 25 to 48, and 49 to 101, also their ORFs and orfs, as products. It is also aimed towards any vaccine or pharmaceutical composition intended to prevent, alleviate or combat an undesirable development of E. coli, and in particular an extra-intestinal development
  • E. coli which comprises at least one of said zones or at least one of said orfs and ORFs.
  • zones, ORFs and orfs which are both CFT073+ K12- and RS218+ K12-.
  • the applications targeted concern a development of E. coli in an extra-intestinal compartment (systemic and non- diarrhoeal E. coli infection)
  • those of said CFT073+ K12-, RS218+ K12- zones, orfs and ORFs which are also 0157 :H7- are then preferred.
  • sequence or sequence fragment derived from the set obtained in i. and a sequence or sequence fragment obtained in ii. being due to chance are sequence or sequence fragment derived from the set obtained in i. and a sequence or sequence fragment obtained in ii. being due to chance.
  • the present application is also aimed towards the possible orfs identifiable on such a polynucleotide, and the ORFs and polypeptides encoded by these orfs .
  • the B2/D+ A- DNAs isolated according to the invention are useful as active principles in the context of a vaccine composition intended to prevent, alleviate or combat an undesirable development of E. coli , and in particular a development of E. coli in an extra- intestinal compartment (systemic and non-diarrhoeal E. coli infections) . They can then be used in an isolated ⁇ ("naked" DNA) form or in the form of DNA transfected into a cell chosen for its physiological innocuity and its capacity to secrete the polypeptide encoded by the transfected B2/D+ A- fragment.
  • polypeptides encoded by the DNAs which are of nature B2/D+ A- can also be used as active principles in the context of such vaccine compositions. They are then used in an inactivated and immunogenic form.
  • E. coli Examples of extra-intestinal development of E. coli comprise, in particular, septicaemias, pyelonephritis, and meningeal infections in newborns. Recognized or potential hospital-acquired infections are most commonly the product of such extra-intestinal developments of E. coli .
  • Region 5 appears to be an • advantageous source for manufacturing broad- spectrum anti-B2-coli products.
  • regions 1, 3 and 4 they appear to be advantageous sources for manufacturing products against septicaemia with meningitis and in particular against meningitis in newborns and in adults.
  • the invention is aimed in particular towards the compositions, in particular ' • the vaccine and pharmaceutical compositions, comprising at least one of said novel DNAs or at least one of said novel ORFs or at least one polypeptide the sequence of which can be considered to correspond, according to the universal genetic code and taking into account the degeneracy of this code, to one of these DNAs or ORFs. It is also aimed towards the use of the known DNAs which are of novel nature B2/D+ A-, such as chuA, the chuA fragments which are of nature B2/D+ A-, and of the polypeptides corresponding to these DNAs, for manufacturing such compositions .
  • DNAs or polypeptides which are of nature B2/D+ A- can be used for screening chemical and/or biological libraries in such a way as to identify compounds capable of binding to them and of blocking an undesirable development of E. coli bacteria
  • compositions can be developed and adjusted by those skilled in the art as a function of the medical indication targeted, of the method of administration desired, and of the patient under consideration (age, weight, sex, condition) .
  • compositions can also comprise- one or more physiologically inert vehicles, and in particular any excipient suitable for the pharmaceutical formulation and/or for the method of administration desired
  • B2/D+ A- compound can also comprise one or more 'active co-agent (s) in order to modify the intensity of activity of the B2/D+ A- compound according to the invention, or to modify its system of activity, or alternatively to modify the targeting of its activity.
  • s 'active co-agent
  • They can also comprise other agents useful for preventing and/or alleviating and/or treating an infection, without these agents having any interaction with the B2/D+ A- compounds according to the invention.
  • the polynucleotides and polypeptides which are of nature B2/D+ A- according to the invention are used for identifying, for example by screening chemical and/or biological libraries, compounds capable of inhibiting, in vivo or under conditions mimicking as closely as possible the in vivo state, the activity of a protein the ORF of which comprises a polynucleotide which is of nature B2/D+ A- according to the invention.
  • Such compounds can, in particular, be used in compositions, in particular pharmaceutical compositions, intended to inhibit the growth of E. coli , and in particular its extra-intestinal growth.
  • a subject of the present invention is such an identification method, the compounds as obtained by such a method, and such compositions.
  • Example 8 Animal models for studying E. col ⁇ virulence
  • Newborn rats (Sprague Dawley - January breeding) after 24 hours of acclimatisation in animal houses were infected when 5 days old.
  • the injected inoculum was prepared from dilutions in physiological serum in a nutritive culture medium of 2 h.
  • the animals were infected by intra-peritoneal route after anesthesia with ether, then put back to their mother after- randomization by brood of ten.
  • the numeration of the bacteraemia at 18h was obtained by taking of 5 ⁇ l otf blood after incision of the tail.
  • mice Mortality of adult mice (%) at D7 after intra peritoneal injection of 3 strains of E. coli .
  • the inoculum was prepared as above-mentioned and the injection made by the intra peritoneal route.
  • E. coli B2 responsible for neonatal infection ' (C5 strain) are capable inducing a bacteraemia after injection of a weak inoculum in the newborn rat and that the mortality in mice is 10 times higher.
  • CFT073 i.e. the E. coli B2 responsible for sepsis in the adult, it is necessary to use a stronger inoculum. But said coli is still pathogenic in mice.
  • An E. coli of group A is not .pathogenic even in mice.

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EP01917119A 2000-03-10 2001-03-12 Polynukleotidem aus b2/d+ a- e. coli und deren verwndungen Ceased EP1328641A2 (de)

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FR0003145 2000-03-10
FR0003145A FR2806096A1 (fr) 2000-03-10 2000-03-10 Produits a propriete b2+ a-isoles a partir de e.coli, et leurs applications biologiques
FR0101449 2001-02-02
FR0101449 2001-02-02
PCT/EP2001/003445 WO2001066572A2 (en) 2000-03-10 2001-03-12 Polynucleotides isolated from e. coli of nature b2/d+ a-, and uses thereof

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EP1342784A1 (de) * 2002-03-06 2003-09-10 Mutabilis S.A. ExPEC-spezifische Proteine, entsprechende Gene und ihre Verwendungen
EP1858919B1 (de) 2005-02-18 2012-04-04 Novartis Vaccines and Diagnostics, Inc. Immungene von uropathogenen escherichia coli
EP1858920B1 (de) * 2005-02-18 2016-02-03 GlaxoSmithKline Biologicals SA Proteine und nukleinsäuren von meningitis-/sepsis-assoziierten escherichia coli
JP2010503380A (ja) * 2006-05-10 2010-02-04 アンスティテュ ナシオナル ドゥ ラ ルシェルシュ アグロノミック 細胞の増殖を阻害するための細胞変性物質としての特異的ｄｎａ分子を含む細胞の使用法
EP2586790A3 (de) 2006-08-16 2013-08-14 Novartis AG Immunogene von uropathogenen Escherichia coli
ITMI20090946A1 (it) 2009-05-28 2010-11-29 Novartis Ag Espressione di proteine ricombinanti
JP2012532626A (ja) 2009-07-16 2012-12-20 ノバルティス アーゲー 無毒化されたＥｓｃｈｅｒｉｃｈｉａｃｏｌｉ免疫原
EP2298798B1 (de) * 2009-09-10 2012-05-09 Freie Universität Berlin Identifizierung und Charakterisierung eines neuartigen aviären pathogenen E. coli (APEC) Fimbrien-Adhäsins
WO2013014261A1 (en) 2011-07-28 2013-01-31 Universiteit Gent Prevention of salmonella recrudescense
GB2554742A (en) * 2016-10-07 2018-04-11 Bhongir Ravi Treatment
TW202304504A (zh) * 2021-04-01 2023-02-01 美商詹森藥物公司 大腸桿菌ｏ１８生物軛合物之生產

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CA2078716A1 (en) * 1992-09-21 1994-03-22 Joyce De Azavedo Attaching and effacing protein of enterohemorrhagic e. coli
ES2076895B1 (es) * 1994-02-04 1996-08-16 Ind Farma Especial Fimbrias-adhesinas de tipo 1 y p aisladas de nuevas cepas de e. coli, procedimiento para su obtencion y aplicaciones.
CA2218601A1 (en) * 1995-03-24 1996-10-03 Ophidian Pharmaceuticals, Inc. Treatment for verotoxin-producing escherichia coli
WO1998022575A2 (en) * 1996-11-22 1998-05-28 Human Genome Sciences, Inc. Nucleotide sequence of escherichia coli pathogenicity islands

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