EP1305447A2

EP1305447A2 - Nucleic acids and pathogenic e.coli proteins, and their uses

Info

Publication number: EP1305447A2
Application number: EP01960863A
Authority: EP
Inventors: Sylvie Perelle; Patrick Fach; Françoise Dilasser; Jo[L Grout
Original assignee: Agence Francaise de Securite Sanitaire des Aliments (AFSSA)
Current assignee: Agence Francaise de Securite Sanitaire des Aliments (AFSSA)
Priority date: 2000-08-04
Filing date: 2001-08-03
Publication date: 2003-05-02
Also published as: WO2002012538A2; FR2812656A1; WO2002012538A3; FR2812656B1; AU2001282259A1

Abstract

The invention concerns novel markers of Escherichia coli pathogenicity, useful in particular for detecting pathogenic Escherichia coli of serotype O157 producing Shiga-like toxins.

Description

NUCLEIC ACIDS AND E. PROTEINS. COLI PATHOGENES, AND THEIR USES. The present invention relates to new markers of pathogenicity to 'Escherichia coli, which can be used in particular for the detection of pathogenic Escherichia coli of serotype 0157 producing shiga-toxins.

Escherichia coli (E. coli) is a ubiquitous bacterium, frequently isolated in medical or food microbiology laboratories, and present in the normal state in the intestinal flora of man. Some strains are pathogenic, however, and associated with many intestinal and non-intestinal infections. Identification of the different strains of E. coli is conventionally based on the serological typing of their antigens O (somatic antigen) and H (flagellar antigen) by immunological methods.

E. serotypes most common coli pathogens have been classified into 5 main groups, according to their mechanism of infection of the digestive tract: - ΕPΕC, for: "enteropathogenic E. coli", which groups the classic enteropathogenic genes,

ΕTΕC, for: "enterotoxigenic E. coli", which includes E. coli producing a stable or heat-sensitive toxin,

ΕAGGΕC, for "enteroagregative E. coli", which groups together the E. coli which are characterized by a particular adhesion profile called "aggregative adhesion",

ΕIΕC, for: "enteroinvasive E. coli", which groups together E. coli which mimic the capacity of Shigella to invade and multiply in the cells of the intestinal epithelium, and

VTΕC, for: "verotoxin producing E. coli", or STΕC "shiga-toxin producing E. coli", which groups together E. coli producing cytotoxins, called "Shiga- like toxins", because of their analogy with the toxin secreted by Shigella dysenteriae type 1 or also verotoxins (VT), due to their toxic activity on Vero cells.

Among the strains of E. pathogenic coli, the colibacilli producing shiga-toxins called STΕC, are emerging microorganisms increasingly recognized as responsible for foodborne illness (GANNON et al, 1993, J. Clin. Microbiol. 31: 1268-1274; BΕGUM D . et al., 1993, J. Clin. Microbiol. 31: (12) 3153-3156). In humans, infection with STΕC results in more or less severe symptoms which can range from simple diarrhea, to hemorrhagic colitis, to hemolytic uremic syndrome (HUS) or thrombotic thrombocytopenic purpura ( PTT), and can sometimes be fatal (GRIFFIN et al., 1991, Epidemiol. Rev. 13: 60-98; NATARO et al., 1998, Microbiol. Immunol. 42: 371-376; PATON et al., 1998, Microbiol Clin, Rev. 11: 450-479). Hemolytic uremic syndrome occurs mainly in young children; it represents approximately 9% of STEC infections diagnosed in the United States. It is also the main cause of acute renal failure in children, and the fatality observed is around 3 to 5/1000. In France, recent studies have shown that the average annual incidence of HUS is 0.7 / 10,000 in children under the age of 15 and 1.8 / 10,000 in children under the age of 5 (DECLUDT et al , 2000, Epidemiol. Infect. 124: 215-220). STEC are also involved in animals, in more or less serious digestive infections. Symptoms observed include diarrhea and dysentery in calves or edema in pigs. In addition, healthy carriers of cattle could play a role in the transmission of STEC to humans, via water or contaminated foodstuffs of animal origin (meat, raw milk and milk-based products of raw milk), (BONNET et al., 1998, J.

Clin. Microbiol. 36: 1777-1780; PRADEL et al., 2000, J. Clin. Microbiol. 38: 1023-1031).

Initially, only one serotype of E. coli had been associated with HUS: it is the serotype O157: H7. It was subsequently observed that other serotypes, in particular O157: H ^" , O26: H11, Ol11: H ^" and O103: H2, could be implicated in STΕC infections (BRIAN et al., 1992, J. Clin. Microbiol. 30: 1801-1806). Among these serotypes, 0157 is the major serotype, involved in 75 to 80% of hemolytic and uremic syndromes (DΕCLUDT et al., Cited above). The shiga toxins produced by STΕC, in particular SLT1 (NT1), SLT2

(VT2) and their variants VT2 vha, VT2 vhb, SLTII-v (VTΕ) and SLT Il-va (NTΕ-va) (ZAWLIΝ et al., 1993, Microbiol. Immunol., 37: 543-548), were identified as the main pathogenicity factors involved in these infections (CALDΕRWOOD et al., 1987, Proc. Νat. Acad. Sci. 84: 4364-4368). However, the shiga-toxins are not responsible on their own for the pathogenicity of STΕC; other bacterial virulence factors could also play a role in the pathogenic power of these colibacilli:

- the enterocytic erasure locus (LΕΕ) which codes for proteins involved in the adhesion of bacteria and the alteration of the cytoskeleton of intestinal cells, in particular the intimin coded by the eae gene (BΕΕBAKHΕΕ et al., 1992, FΕMS Microbiol Lett. 70: 63-68; Me DANIEL et al., 1995, Proc. Nat. Acad., Sci. 92: 1664-1668; Me DANIEL et al., 1997, Mol. Microbiol. 23: 399-407) ,

the efa 1 gene (fEHEC factor for adhesion) has been described as a factor which contributes to the adhesion capacity of the bacterium (NICHOLLS et al., 2000, Mol. Microbiol., 35: 275-288), and

- a 90 kb plasmid (BURLAND et al., 1998, Nucleic Acids Res., 26: 4196-4204), which codes for potential virulence factors such as an enterohemolysin (SCHMIDT et al., 1995, Infect. Immun. , 63: 1055-1061; SCHMIDT et al., 1996, Microbiol., 142: 907-914)), a catalase-peroxidase (BRUNDER et al., 1998, Microbiol., 142: 3305-3315), a serine protease (BRUNDER et al., 1997, Mol. Mirobiol., 24, 767-778) and a type II secretion system (SCHMIDT et al., 1997, FEMS Microbiol. Letter, 148: 265-272). To prevent pathologies associated with colibacilli producing shiga-toxins (STEC) and in particular the most serious pathologies (hemorrhagic colitis, HUS, PTT) associated with the most pathogenic of them, it is necessary to have means of detection of these colibacilli, in order to be able to systematically control water and food likely to be contaminated, and in particular fresh products of animal origin (meat, raw milk and dairy products based on raw milk).

This control involves the sensitive and specific detection of STEC responsible for these pathologies and in particular STEC serotype 0157.

This detection is complex because, as mentioned above, the pathogenicity of STEC is not simply linked to a particular serotype, but that it results from the association of a number of virulence factors, the combination of which exact is not known. In addition, certain virulence factors are common to STEC and other groups of E. coli pathogenic or to other species of bacteria, which constitutes an additional problem for the specific detection of STΕCs and in particular STΕCs of serotype 0157. For example, the virulence plasmid of 90 kb is present in STΕC pathogens of other serotypes (BONNET et al., cited above), and the LEE locus is present both in pathogenic STECs of different serotypes (PRADEL et al., cited above) and in EPECs, notably in O127: H6 (PERNA et al., 1998, Infect. Immun., 66: 3810-3817; PRADEL et al, cited above).

Cytotoxicity tests and various biochemical, immunological (ELISA) or genetic methods have been proposed to detect STEC (NATARO et al., 1998, Clin. Microbiol. Rev., 11: 142-201; PATON et al, 1998, Clin Microbiol. Rev. 11: 450-479; FENG et al, 1993, Mol. Cell. Probes, 7: 151-154; HUCK et al., Int. J. Food Microbiol., 25: 277-287).

• Cytotoxicity tests on cell cultures constitute the reference method used in food hygiene to detect the presence of bacteria producing shiga-toxins or verotoxins. The principle of these tests is based on the cytopathogenicity of verotoxins on African green monkey kidney cells. Subsequently, confirmation of the antigenic types of verotoxin is required using anti-VT1 and anti-VT2 antibodies. This technique has the advantage of being very sensitive, however it is not specific to the colibacilli producing shiga-toxins of serotype 0157. In addition, a series of transfers in selective media is necessary in order to promote the development of verotoxic bacteria. prior to the determination of toxins on a cell culture; it takes 5 to 6 days before obtaining the first results, and costs are high. This test is therefore not a routine method usable in the food industry.

• Biochemical methods use the characteristics of the majority of E. coli O157: H7 and O157: H ^" , which do not ferment sorbitol in 24 hours, and which in addition do not produce β-glucuronidase. E. coli O157 : H7 and O157: H ^" appear as white colonies on sorbitol agars, while the colibacilli producing shiga-toxins of other serotypes (026, OU I and 0103) and the numerous non-pathogenic colibacilli, which are capable of fermenting sorbitol, appear as pigmented colonies. These methods are however insufficient because there are atypical mutants of E. coli O157: H7 and O157: H ^" which are capable of fermenting sorbitol (OUNZΕR et al, 1992, J. Clin. Microbiol, 30: 1807-1810; KARCH et al, 1997, J. Food. Prot, 60: 1454 -1457).

• The ΕLISA type immunological methods are based on the detection of O and H antigens or shiga toxins (VT1 and VT2) using specific antibodies. The immunological tests currently available are not satisfactory; in fact, other bacteria such as Citrobacter freundii, Salmonella O from group N and Hafnia senior have cross-reactivity with respect to the anti-0157 sera used (BΕTTΕLHΕIM et al, 1993, J. Clin. Microbiol, 31: 760 -761; BORCZYK et al, 1987, Int. J. Food Microbiol, 4: 347-349; CHART et al, 1992, Epidemiol. Infect, 108: 77-85; SHIMADA et al, 1992, Curr. Microbiol, 25: 215-217).

In addition, most of the available immunological tests are limited to the detection of the 0157 antigen and do not make it possible to differentiate STEC 0157 from non-pathogenic E. coli of serotype 0157. Finally, these methods are generally too insensitive or too not very specific to allow detection in food. • Polymerase chain reaction (PCR) methods, allowing the specific detection of genes coding for bacterial factors involved in virulence have been proposed. Amplification of the following genes has been reported:

- the shiga-toxin (stx) genes, (CHEN et al, 1998, Appl. Environ. Microbiol, 64: 147-152; Application EP 0 864 657 on behalf of CNEVA; GANNON et al, 1992, Appl. Environ. Microbiol, 58: 3809-3818; JACKSON, 1991; J. Clin. Microbiol, 29: 1910-1914; KARCH et al, 1989, J. Clin. Microbiol, 27: 2751-2757; LIN et al, 1993, Microbiol. Immunol, 37: 543-548; POLLARD et al, J. Clin. Microbiol, 1990, 28: 540-545; POLLARD, 1990, J. Infect. Dis, 162, 1195-1198; READ et al, 1992, Mol. Cell. Probes, 6: 153-161), - the intimin (eae) gene carried by the LEE locus (SANDHU et al, 1996,

Epidemiol. Infect, 116: 1-7; WILLSHAW et al, 1994, J. Clin. Microbiol, 32: 897-902),

- the hemolysin gene encoded by the 90 kb plasmid (E-hlya), (SCHMIDT et al, 1995, cited above), - genes involved in the biosynthesis of the O antigen (locus rfb), (DESMARCHELIER et al, 1998, J. Clin. Microbiol, 36: 1801-1804; MAURER et al, 1999, Appl. Environ. Microbiol, 65: 2954-2960), and

- the gene encoding the H7 flagellar antigen (fliC _M ), (FIELDS et al, J. Clin. Microbiol, 1997, 35: 1066-1070).

These methods are more sensitive and more specific than biochemical or immunological methods. However, STEC 0157 cannot be identified using a single PCR reaction targeting one of these genes. Indeed, the somatic antigens 0157 and flagellar H7, which can be carried by strains of E. non-pathogenic coli alone are not markers of pathogenicity. In addition, verotoxin genes are by definition carried by all STEC strains; the virulence factors described on the 90 kb plasmid (enterohemolysin, catalase peroxidase, serine protease) are present in STECs of different serotypes (BONNET et al, cited above) and a homolog of the LEE locus is present in both STECs of different serotypes (PRADEL et al, cited above) and in EPECs (PERNA et al, cited above). Consequently, the demonstration of only one of these virulence genes is not sufficient to identify a STEC of serotype 0157.

Tests allowing the simultaneous amplification of several genes in a single reaction (PCR-multiplex) have been proposed (CEBULA et al, 1995, J. Clin. Microbiol, 33: 1048; DENG et al, 1996, J. Food Prot, 59: 570-576; FRATAMICO et al, 1995, J. Clin. Microbiol, 33: 2188-2191; GANNON et al, 1997, J. Clin. Microbiol, 35: 656-662; NAGANO et al, 1998, Microbiol. Immunol, 42: 371-376; PATON et al, 1998, J. Clin. Microbiol, 36: 598-602).

These techniques, which are suitable for detecting an isolated bacterial strain, cannot be used for the analysis of complex biological samples, such as food or stool, which are likely to contain a mixture of bacteria. Indeed, as reported by DENG et al. (cited above), PCR-multiplex does not allow us to conclude whether the signals corresponding to the different genes detected come from a single bacterial strain, or else from the additive effect of genes present in different strains of bacteria. This major drawback, which involves the systematic isolation of each strain in order to verify that the different genes amplified by PCR are indeed present in the same strain, is not suitable for routine diagnosis in the laboratory of food hygiene or analyzes medical.

Comparison of the nucleotide sequence of certain genes common to STECs of serotype 0157 and to other pathogenic E. coli made it possible to identify point mutations. Thus, some authors have proposed specific primers targeting the genes carried by STEC 0157 which allow their detection in a single PCR reaction. Thus, the amplification of the following regions has been reported: the 3 'region of the eae gene (LOUIE et al, 1994, Epidemiol. Infect, 112: 449-461), the region immediately adjacent to the 5 'region of the eae gene (MENG et al, 1996, Int. J. Food Microbiol, 32, 103-113; MENG et al, 1997, Lett. Appl. Microbiol, 24: 172-176; ZHAO et al, 1995, FEMS Microbiol. Lett, 133: 35-39) or the H7 gene (WANG et al, 2000, J. Clin. Microbiol, 38: 1786-1790). It therefore appears that at present only very few specific markers are available which are easy to implement for detecting pathogenic E. coli, and in particular STECs of serotype 0157.

The inventors have identified strains of E. pathogenic coli, in particular in all pathogenic colibacilli of serotype 0157 producing shiga- toxins, the presence of a nucleic acid insert representing a new locus, absent from the non-pathogenic strains. This locus is inserted between positions 93848 and 93849 of the genome sequence of E. coli Kl 2 (GenBank U82664); this insertion site is precisely located between the stop codons of two open reading phases (Genbank AAB40241 and Genbank AAB40242) in opposite orientation, which in the genomic sequence of E. strains non-pathogenic coli such as strain K12 are directly adjacent.

The inventors also noted the presence of a similar locus, inserted at the same position, in pathogenic E. coli strains, belonging to serotypes other than 0157. On the other hand, they observed no insertion at this position in all of the non-pathogenic strains that have been tested. The presence of an insert at this position therefore constitutes a new genetic marker of virulence useful for the detection of strains of E. pathogenic coli.

The nucleic acid sequences originating from such an insert also constitute markers useful for the detection of strains of E. pathogenic coli, and in particular for the specific detection of strains of pathogenic bacteria carrying said insert; for example, nucleic acid sequences originating from the insert present in a strain of serotype 0157 producing shiga-toxins allow a specific detection of all the E. coli of serotype 0157 producing shiga-toxins.

Consequently, the present invention relates to a nucleic acid molecule derived from a pathogenic strain of E. coli, characterized in that it is selected from the group consisting of: a) a nucleic acid molecule representing a locus inserted into the genome of said pathogenic strain, at the location of said genome corresponding to that located in E. coli Kl 2, between positions 93848 and 93849 of the GenBank sequence U82664; b) a nucleic acid molecule comprising the molecule a) defined above, and between 1 and 2500 bp of the sequence upstream of the insertion site of said molecule, and / or between 1 and 2500 bp downstream of the site insertion of said molecule. c) a nucleic acid molecule constituting a fragment of at least 15 bp, preferably at least 25 to 30 bp, of the molecule a) above; d) a nucleic acid molecule constituting a fragment of at least

15 bp, preferably at least 25 to 30 bp, of the molecule b) above, said fragment comprising at least a portion of the molecule a) and at least a portion of the sequence upstream from the insertion site of said molecule a), or at least a portion of the sequence downstream of the insertion site of said molecule a).

Nucleic acid molecules in accordance with the invention can in particular be derived from the genome of a pathogenic bacterium of the species Escherichia coli, chosen from E. coli STΕC of serotype 0157, 077, 079, or 0145, the Escherichia coli STΕC or ΕPΕC of serotype 055, and Escherichia coli ΕPΕC of serotype 0127. For example, nucleic acid molecules according to the invention derived from the genome of E. coli O157: H7, are represented in the sequence list in the annex under respectively the numbers SΕQ ID NO: 1, SΕQ ID NO: 2, SΕQ ID NO: 11 to 17, SΕQ ID NO: 19, SΕQ ID NO: 21 and SΕQ ID NO: 23.

The present invention also relates to nucleic acid molecules capable of specifically hybridizing, under stringent conditions, with any of the molecules a) to d) above. ^•

This includes in particular oligonucleotides which can be used as amplification primers for obtaining a nucleic acid sequence in accordance with the invention, or as probes for the detection of said sequence, and in particular the oligonucleotides P388L, P388M, P388G, P388D, P388F, P388C and P388P, corresponding respectively to the sequences SΕQ ID NO: 3 to 9 in the list of sequences in the appendix. The oligonucleotides P388M, P388G, P388D, P388F and P388C are in particular usable as molecular hybridization probes, useful for the detection, of a nucleic acid molecule a) in accordance with the invention derived from the genome of a bacterium E coli STΕC of serotype 0157. The present invention encompasses in particular the pairs of primers which can be used for the amplification of a nucleic acid molecule a) b) c) or d) according to the invention.

The present invention further relates to the recombinant vectors, and in particular the recombinant plasmids containing a nucleic acid molecule according to the invention.

Nucleic acid molecules in accordance with the invention can for example be obtained by polymerase chain reaction, from the DNA of a pathogenic E. coli strain comprising the locus defined above. Especially :

a nucleic acid molecule a) in accordance with the invention can be obtained by using a pair of amplification primers, one of which hybridizes comprising a primer which hybridizes at each of the ends of the molecule a),

a nucleic acid molecule b) according to the invention can be obtained by using a pair of amplification primers comprising a primer hybridizing upstream of the insertion site of molecule a) and a primer hybridizing downstream of the insertion site of molecule a); suitable primers can be chosen by a person skilled in the art, respectively from the 2500 bp sequence upstream from position 93848 of the GenBank U82664 sequence, and from the 2500 bp sequence downstream from position 93849.

Amplification conditions will of course be used to amplify large fragments, and in particular comprising a step of extending primers of sufficient duration.

Advantageously, the pair of primers P388L / P388P can be used, which hybridizes on either side of the site of insertion of a molecule a) according to the invention.

This pair amplifies a fragment of 682 bp when the amplification is carried out from the genome of the non-pathogenic E. coli type strain Kl 2, whereas it amplifies a larger size with pathogenic E. coli; it amplifies in particular a fragment of 3316 bp (SΕQ ID NO: 11) with all the E. coli producing shiga-toxins of serotype 0157.

A nucleic acid molecule c) according to the invention can be obtained by using a pair of primers which hybridize with internal sequences of the molecule a)

A nucleic acid molecule d) according to the invention can be obtained using a pair of primers comprising a primer hybridizing with an internal sequence of the molecule a) and a primer hybridizing with a sequence located upstream of the insertion site of molecule a) or a primer hybridizing with a sequence located downstream of the insertion site of molecule a).

It is advantageous to use a pair of primers selected from the group consisting of: P388C / P388D (SΕQ ID NO: 8 / SΕQ ID NO: 6), P388F

/ P388G (SΕQ ID NO: 7 / SΕQ ID NO: 5), P388F / P388D (SΕQ ID NO: 7 / SΕQ ID NO: 6),

P388P / P388L (SΕQ ID NO: 9 / SΕQ ID NO: 3), P388L / P388M (SΕQ ID NO: 3 / SΕQ ID

NO: 4) and P388P / P388D (SΕQ ID NO: 9 / SΕQ ID NO: 6).

The present invention also relates to the use of a nucleic acid molecule in accordance with the invention for screening for E. pathogenic coli, and in particular for the specific screening for E. strains. coli serotype 0157 producing shiga toxins.

In particular, the present invention relates to a method for screening for E. pathogenic coli, characterized in that it comprises the detection of the presence in the genome of the E. coli bacteria present in the test sample, of a locus inserted at the location of said genome corresponding to that located in E. coli K12, between positions 93848 and 93849 of the GenBank sequence U82664. According to a preferred embodiment of the detection method according to the invention, it comprises at least one step of chain amplification by polymerase using a pair of primers comprising a primer hybridizing with the genome region of E. coli defined by the 2500 bp sequence upstream of position 93848 of the GenBank sequence U82664, and a primer hybridizing with the genome region of E. coli defined by the 2500 bp sequence downstream of position 93849 of the GenBank sequence U82664.

The determination of the size of the amplification product, and its comparison with that obtained using the same pair of primers, from the genome of a non-pathogenic strain, for example the strain type E. coli K12 makes it possible to detect insertion of a locus according to the invention.

In addition, the size of the amplification product for the same pair of primers can also make it possible to distinguish different groups of E. pathogenic coli.

For example if we use the pair of primers P388L / P388P, we observe in the presence of E. coli pathogens of serotype 0157 producing shiga-toxins, an amplification fragment of 3316 bp specific for these bacteria.

According to a variant, the amplification is carried out under conditions which do not make it possible to amplify large fragments. The absence of amplification product makes it possible, in this case, to detect the insertion of a locus in accordance with the invention. However, this mode of implementation does not make it possible to distinguish different groups of E. pathogenic coli.

According to an embodiment of the detection method according to the invention, it comprises at least one step of chain amplification by polymerase using a pair of primers which hybridize with internal sequences of a molecule a) as defined above.

For example, for the detection of STΕC of serotype 0157, the use of the following pairs of primers: P388C / P388D, P388F / P388D, P388F / P388G produces amplification fragments of 300 bp respectively (SΕQ ID NO: 12) , 125 bp (SΕQ ID NO: 13) and 578 bp (SΕQ ID NO: 14) with all STΕCs of serotype 0157 while no amplification product is observed with the type E strain. coli K12 used as control.

According to yet another embodiment of the detection method according to the invention, it comprises at least one step of chain amplification by polymerase using a pair of primers comprising a hybridizing primer with an internal sequence of a molecule a) as defined above, and a primer hybridizing with the region of the genome of E. coli defined by the 2500 bp sequence upstream of position 93848 of the GenBank sequence U82664, or a primer hybridizing with the region of the genome of E. coli defined by the 2500 bp sequence downstream of position 93849 of the GenBank sequence U82664.

For example, for the detection of STΕC of serotype 0157, the use of the following pairs of primers: P388P / P388D, P388L / P388M, produces fragments of 1299 bp (SΕQ ID NO: 15) and 419 bp (SΕQ ID respectively) NO: 16) with all STΕCs of serotype 0157 whereas no amplification product is observed with the strains of E. non-pathogenic coli.

The inventors studied the sensitivity and the specificity of the pairs of primers P388C / P388D, P388F / P388D and P388F / P388G for the detection of STΕC-0157.

Out of 211 bacterial strains tested, the three pairs of primers have equivalent sensitivity and detect the 55 STΕC-O157 tested as well as the E. coli of serotype 055, which is explained by the clonal origin of these two serotypes (FΕNG et al, 1998, J. Infect. Dis, 177: 1750-1753; WHITTAM et al, 1993, Infect. Immun, 61: 1619-1629). In addition, with the three pairs of primers, no signal is obtained with the bacteria Citrobacter and Hafnia alvei which exhibit cross-immunological reactions with the 0157 antigen.

For the specific detection of all E. coli producing shigotoxins of serotype 0157, the pair P388F / P388D is preferably used. In fact, this pair of primers has a particularly high specificity and does not give any positive reaction with STECs from other serotypes (30 serotypes tested) as well as with non-STEC E. coli of serotype 0157 or other serotypes ( 55 tested) and bacteria from other species.

The oligonucleotides in accordance with the invention can also be used as primers in a PCR-ELISA type technique. In this case, it is possible, for example, to use the oligonucleotide P388D previously immobilized on a solid support, as a capture probe for fixing the amplification product of 578 bp obtained with the primers P388G and P388F; this amplification product can then be revealed either directly (for example if it has been appropriately labeled by incorporation of labeled nucleotides during the amplification reaction) or via a labeled detection probe.

Different methods for fixing nucleic acids on a solid support, as well as those for labeling them using cold or radioactive labels are well known to those skilled in the art by way of example, those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wïley and son Inc, Library of Congress, USA).

The present invention also relates to a nucleic acid molecule obtained by polymerase chain reaction of a non-pathogenic E. coli with using the pair of primers P388P / P388L. This molecule can be used as a control, in the context of a detection method according to the invention.

Advantageously, said molecule is obtained from E. coli Kl 2 and is constituted by a fragment of 682 bp (SΕQ ID NO: 10). The present invention also relates to any protein encoded by an open reading frame present on a nucleic acid molecule according to the invention, and in particular on a molecule derived from a strain of E. coli serotype 0157 producing shiga toxins.

Indeed, from a genomic bank of E. coli O157: H7, the Inventors isolated a sequence of 5478 bp (SΕQ ID NO: 1) containing a locus in accordance with the invention called SIL ₀₁₅₇ , defined by the sequence presented in the sequence list in the appendix under the number SΕQ ID NO: 2. Figure 1 illustrates the genetic organization of the E. locus. coli O157: H7, called SIL ₀₁₅₇ , inserted into the genome of E. coli between positions 93849 and 93848, (with reference to the genome type of non-pathogenic E. coli Kl 2, Genbank U82664). The regions indicated in white represent the homologous regions between the sequence of E. non-pathogenic coli Kl 2 (Genbank U82664) and the sequence of STΕC O157.Η7. The regions indicated in black represent the SIL ₀₁₅₇ locus. The large black arrows including accession numbers represent the open reading phases described in non-pathogenic E. coli. The large white arrows represent the potential open reading phases included in locus SIL ₀₁₅₇ . The small arrows indicate the positions of the different primers used for the PCR amplifications.

The inventors analyzed this locus SIL ₀₁₅ 7, and thus highlighted highlighted open reading frames and, in particular 4 open reading frames encoding proteins hereinafter named IHP1, IHP2, IHP3 and IHP4, of respectively 338 , 140, 120 and 159 amino acids.

The amino acid sequences of the proteins IHP1, IHP2, IHP3 and IHP4 are presented in the list in the appendix, respectively under the numbers SΕQ ID NO: 18, 20, 22 and 24, the corresponding nucleotide sequences are presented in the list in the appendix, respectively under the numbers SΕQ ID NO: 17, 19, 21 and 23. The proteins according to the invention represent new antigens potentially usable for the detection of pathogenic E. coli.

In particular, in the case of proteins whose genes are carried by the locus SIL ₀₁₅₇ , mentioned above, the protein IHP1, which has homologies with proteins of the outer membrane and adhesins of other bacteria (Salmonella flagellin and adhesin from Neisseria, Yersinia, Haemophilus and Staphylococcus) constitutes a surface antigen which can potentially be used for the detection of E. coli producing shiga-toxins of serotype 0157. The present invention also relates to any peptide representing a fragment of at least 5 amino acids, and preferably at least 7 to 15 amino acids of a protein according to the invention.

The present invention further relates to: - expression cassettes comprising the nucleotide sequences coding for the proteins or peptides in accordance with the invention, placed under the transcriptional control of an appropriate promoter, in particular a promoter allowing the expression of said proteins or said peptides in transformed host cells. Advantageously, the expression cassettes contain the sequences coding for one of the proteins IHP1 to IHP4, as defined above (SEQ ID NO: 17, 19, 21 and 23).

- Recombinant vectors, comprising an insert consisting of the nucleotide sequences coding for the proteins or peptides in accordance with the invention. Advantageously, these vectors are expression vectors comprising at least one expression cassette as defined above. Many vectors into which a nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a eukaryotic or prokaryotic host cell are known in themselves; the choice of an appropriate vector depends on the intended use for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of the sequence in extrachromosomal form or else integration into the chromosomal material of the host ), as well as the nature of the host cell.

The subject of the invention is furthermore prokaryotic or eukaryotic cells transformed with a plasmid or a recombinant vector in accordance with the invention.

The recombinant vectors and the transformed cells according to the invention are useful in particular for the production of proteins and peptides according to the invention.

The present invention also relates to antibodies, polyclonal or monoclonal, directed against a protein or a peptide in accordance with the invention.

These antibodies can be obtained by conventional methods, known in themselves, comprising in particular the immunization of an animal with a protein or a peptide in accordance with the invention, in order to make it produce antibodies directed against said protein or said peptide. Where appropriate, these antibodies may undergo additional steps of purification and / or elimination of any cross-reactions with other antigens, in particular by immuno-adsorption techniques. The techniques for the production and purification of recombinant proteins, the methods of immunization and the production of antibodies and the immunological techniques based on the detection of antigen-antibody complexes (ELISA, RIA, Western-Blot) are known in themselves. ; by way of example, there may be mentioned those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wïley and son Inc, Library of Congress, USA) and in Current protocols in Immunology (John E. Coligan, 2000, Wïley and son Inc, Library of Congress, USA) .

The present invention also relates to the use of at least one protein, a peptide, or an antibody in accordance with the invention for obtaining a medicament, intended for the prevention or treatment of an infection induced by a pathogenic E. coli bacterium, in particular an E. coli bacterium of serotype 0157 producing shigatoxins.

The subject of the present invention is also the use of at least one protein, a peptide, or an antibody in accordance with the invention for the immunological detection of pathogenic E. coli, in particular for the screening of producing E. coli of serotype 0157. of shiga toxins.

The nucleic acid molecules, proteins and peptides, as well as the antibodies in accordance with the invention can also constitute reagents which can be used for the detection of strains of E. pathogenic coli, including all strains of E. coli producing shiga toxins of serotype 0157.

The reagents according to the invention can be used for the search for E. strains. pathogenic coli, including all strains of E. coli of serotype 0157 producing shiga toxins in food products, in particular water, fresh food products such as meats, raw milk and derived products (cheeses, dairy products).

These reagents can also be used for the diagnosis of pathogenic E. coli infections, in particular E. coli infections producing shigotoxins of serotype 0157 in humans and animals, from a biological sample, for example in faeces.

The invention further relates to an E. coli detection kit. pathogenic coli, including all strains of E. coli producing shiga toxins of serotype 0157, characterized in that it includes at least one reagent according to the invention.

The present invention will be better understood with the aid of the additional description which follows, which refers to nonlimiting examples illustrating the isolation and characterization of a nucleic acid molecule according to the invention, and its use for the identification & pathogenic Escherichia coli, and in particular of Escherichia coli of serotype 0157 producing shiga-toxins. EXAMPLE 1: IDENTIFICATION AND CHARACTERIZATION OF A LOCUS INSERTED IN THE CHROMOSOME OF E. COLI 0157: H7 BY ANALYSIS OF DNA POLYMORPHISM BY RAPD (RANDOMLY AMPLIFIED POL YMORPHIC DNA). 1. Identification of a specific DNA fragment from E. coli O157: H7 by RAPD

1.1. Bacterial DNA extraction and PCR amplification

12 strains of E. coli: 10 strains of STΕC [3 strains O157: H7 and 1 strain (0157: H ^" , O26: Hl l, 091: H21, O103: H2, Ol ll: H ⁺ , O113: H4 and O128: H2) J and 2 strains of E. coli serotype 0157, not producing shiga-toxins (O157-non STΕC). Are cultured in TSB medium (Tryptone Soy Broth), at 37 ° C, overnight and the total bacterial DNA is extracted using the INSTAGENE matrix (BIO-RAD), following the manufacturer's instructions. The E. coli strains are analyzed by RAPD, using the READY TO GO kit (PHARMACIA BIOTΕCH). PCR is carried out in a final volume of 25 μl, in the presence of 10 ng of purified total bacterial DNA and of 25 pmol of the oligonucleotide primer PI (PHARMACIA-BIOTΕCH). PCR amplification (GENE-AMP PCR SYSTEM 9600, PΕRKJN ΕLMΕR) is carried out under the following conditions: an initial denaturation step at 95 ° C for 5 min is followed by 45 cycles alternating a denaturation step at 95 ° C for 1 min, a step priming hybridization at 36 ° C for 1 min and an extension step at 72 ° C for 2 min. The amplification product is separated by electrophoresis in a 2% agarose gel containing ethidium bromide, in TBΕ buffer (Tris-Borate-ΕDTA). The amplified DNA fragments are visualized under an ultraviolet lamp.

The profile of the RAPD analysis shows that a 387 bp fragment is amplified randomly, only in the colibacilli producing shiga-toxins of serotype 0157 (3 strains of serotype O157.Η7 and 1 strain of serotype O157.Η ^" ).

1.2. Hybridization of the fragment obtained by RAPD

The 387 bp fragment is isolated from the agarose gel, purified using the QIAEXII extraction kit (QIAGΕN) and cloned into the pMOS Blue vector (AMΕRSHAM PHARMACIA BIOTΕCH), using the cloning kit blunt ends of AMERSHAM LIFE SCIENCE, to give the plasmid pSP7. A probe corresponding to the 387 bp insert (P7 probe) is synthesized by PCR and labeled with digoxigenin using the PCR reagent DIGOXIGENIN LABELING MIX (ROCHE MOLECULAR). This probe is hybridized, by Dot-blot, with the DNA of the 12 strains of E. coli analyzed by RAPD. The hybridization is carried out on nylon membranes (ROCHE MOLECULAR), overnight at 65 ° C., in a conventional hybridization buffer (ROCHE MOLECULAR) containing 20 ng / ml of denatured probe labeled with digoxigenin, following the protocols classics described in MANIATIS et al. (Molecular Cloning: a laboratory manual, 1982, Cold Spring Harbor Laboartory, Cold Spring Harbor, New York). Then, the hybridization of the digoxigenin-labeled probe is detected by luminescence (DIG LUMINESCENT DETECTION KIT, ROCHE MOLECULAR) on BIOMAX films (KODAK). The results of the hybridization show that the labeled P7 probe recognizes only STECs of serotype 0157. This result suggests that the 387 bp fragment obtained by RAPD could come from a specific locus of the colibacillus strains producing shiga-toxins from serotype 0157.

2. Identification and location of a locus inserted in the chromosome of E. coli O157: H7.

2.1 Construction of a DNA bank of E. coli O157: H7

The chromosomal DNA of the strain ΕDL 933 (ATCC 43895) from E. coli O157: H7, purified using the QIAGEN GENOMIC TIP SYSTEM kit (QIAGΕN) is fully digested with Hind III (ROCHE MOLECULAR), following the manufacturer's instructions. Then 300 ng of this digested DNA is ligated with 10 ng of the plasmid pUC 18, digested with Hind III and dephosphorylated (PHARMACIA-BIOTECH), in the presence of 5 IU of the T4 DNA ligase (ROCHE MOLECULAR), in a volume 20 μl reaction at 16 ° C overnight. Competent E. coli MOS Blue bacteria are transformed by the ligation product and spread on LB-agar dishes containing 100 μg / ml of ampicillin, 25 μg / ml of isopropyl β-D-thiogalacto-pyranoside and 25 μg / ml of X-gal (5-bromo-4-chloro-3-indolyl-D-galactoside) and incubated overnight at 37 ° C., according to the conventional protocols described in MANIATIS et al, cited above.

The presence of the fragment isolated by RAPD in the plasmid DNA of colonies resistant to ampicillin is analyzed by homologous hybridization using the P7 probe.

Analysis of 1000 colonies of transformed bacteria made it possible to isolate the plasmid pSP26 containing a 4.4 kb fragment of the genome of E. coli O157: H7. 2.2. Location of the locus inserted in the chromosome of E coli O157: H7

The plasmids pSP7 and pSP26 were sequenced by standard techniques and the sequence obtained was compared with the sequences available in the databases using the BLAST program.

The alignment of the sequences of the plasmids pSP7 and pSP26 shows that the sequence of the insert of 387 bp of the plasmid pSP7 comprises the 3 ′ end of the sequence of the insert of 4.4 kb of the plasmid pSP26, added with 127 nucleotides additional, located at its 3 'end (Figure 1).

Analysis of the sequence of the 4.4 kb fragment of pSP26 shows that its 5 'end has 98% identity with the genomic sequence of minutes 9 to 11 (positions 96304-93849, with reference to the genomic sequence of non-pathogenic E. coli type Kl 2, Genbank U82664). In contrast, the 3 'end of this fragment (nucleotides 2457 to 4418) and the entire 387 bp sequence of the plasmid pSP7 show no homology with the sequence of non-pathogenic strains of E. coli (Figure 1). These results demonstrate that the genome of E. coli O157: H7 contains a locus inserted between positions 93848 and 93849, with reference to the genomic sequence of E. non-pathogenic coli type K12 (Genbank U82664). The results obtained in Example 1 also indicate that this locus is also present in other STΕCs of serotype 0157 but that it is absent from colibacillus strains producing shiga-toxins from other serotypes as well as strains of E . coli of serotype 0157 which do not produce shigotoxins.

2.3. Complete sequencing of the locus inserted into the chromosome of E. coli O157: H7

The sequence of the locus inserted into the chromosome of E. coli O157: H7 was supplemented with the amplification product obtained with the primers sense P388D (position 4205 to 4230 of the 4.4kb fragment of pSP26, table I) and antisense P388P (positions 93436 to 93460 of E. coli non-pathogenic type K12, table I).

The PCR reaction is carried out in a final volume of 100 μl containing, 3 μl of total DNA purified as described in example 1, 10 μl of PCR buffer 10 times concentrated (100 mM Tris HC1 pH 8.5, 250 mM KC1, 50 mM (NH4) ₂ SO ₄ , 20 mM MgSO ₄ ), 200 μM of each deoxynucleotide, 600 nM of each primer and 2.5 IU of DNA polymerase PWO (ROCHE MOLECULAR). The PCR amplification is carried out under the following conditions: an initial denaturation step at 94 ° C for 2 min is followed by 30 cycles alternating a denaturation step at 94 ° C for 40 s, a priming hybridization step at 69 ° C, for 40 s and an extension step at 72 ° C, for 50 s and a final elongation step is carried out at 72 ° C for 10 min.

These primers do not amplify any sequence of E. non-pathogenic K12 type coli, on the other hand an amplification product of 1299 bp called Amp 388P / 388D, (FIG. 1) is amplified from DNA of E. coli O157: H7 (strain ΕDL 933).

The relative arrangement of the different sequenced fragments is illustrated in FIG. 1.

The sequence of the 1299 bp amplification product shows that the 413 bp of its 3 'end have 92% identity with the sequence of E. non-pathogenic coli (positions 93848 to 93436, with reference to the genome sequence of non-pathogenic E. coli type K12, Genbank U82664). The sequences of the 1299 bp amplification product and the fragment of

4.4 kb of pSP26 made it possible to identify a new sequence of 5478 bp (SΕQ ID NO: 1), in E. coli O157: H7. This sequence contains a small locus of 2634 bp (positions 2457 to 5090 of the sequence of 5478 bp) inserted into the chromosome of E. coli O157: H7, flanked on both sides by the conserved sequences of E. non-pathogenic coli. This locus called SIL0157 (Small Inserted Locus in STEC 0157) is presented in the list in the appendix under the number SΕQ ID NO: 2. The sequence of SILoisi has no homology with a known sequence and has a G + C content of 44 , 4%, which is significantly lower than that of the entire E. genome coli (50.8%, BLATTNΕR et al, 1997, Science, 277: 1453-1474). This locus is inserted between positions 93848 and 93849, with reference to the sequence of the genome of E. non-pathogenic coli type K12, Genbank U82664). Remarkably, in enterohemorrhagic colibacilli of serotype O157: H7, this locus is precisely inserted between the stop codons of 2 open reading phases (Genbank AAB40241, homologous to the open phase, reading HI0293 to 'Haemophilus influenzae and Genbank AAB40242) which are in opposite orientation. In contrast, in the sequence of E. non-pathogenic Kl 2 type coli, the stop codons of these 2 open reading phases are directly attached.

3. Analysis of the potential open reading phases of the SIL0 locus ₁₅ 7

The SIL ₀₁₅₇ locus includes 4 potential open reading phases of more than 100 amino acids, respectively called IHP1 (Inserted Hypothetical Protein 1), IHP2 (Inserted Hypothetical Protein 2), IHP3 (Inserted Hypothetical Protein, 3) and IHP4 (Inserted Hypothetical Protein 4), corresponding respectively to proteins of 338 (SΕQ ID NO: 18), 140 (SΕQ ID NO: 20), 120 (SΕQ ID NO: 22) and 159 (SΕQ ID NO: 24) amino acids. The positive strand codes IHP1 (nucleotides of positions 3364 to 4380 of the sequence of 5478 bp, SΕQ ID NO: 17) and IHP2 (nucleotides of positions 4465 to 4887 of the sequence of 5478 bp SΕQ ID NO: 19), in the same phase, and the negative strand code IHP3 (nucleotides of positions 2997 to 2635 of the sequence of 5478 bp SΕQ ID NO: 21) and IHP4 (nucleotides of positions 4002 to 3523 of the sequence of 5478 bp SΕQ ID NO: 23), in the same phase.

Analysis of the IHP1 protein, using the PSORT and BLAST programs, respectively indicated the presence of a cleavable N-terminal sequence of 23 amino acids, and homologies with membrane proteins and bacterial adhesins. In particular, we can note homologies with the following proteins; a potential adhesin / invasive from Neisseria meningitidis (Genbank AAF 42321 and Genbank AAF41395), invasin Yopl from Yersinia pseudotuberculosis (Genbank CAA32088), adhesin YadA from Yersinia enter ocolitica (Genbank CAA32086), adhesin HIA (Genbank AAC437) and the HSF protein (product of the fibrillus locus, called locus hsf accession Genbank AAC44560) ά 'Haemophilus influenzae, flagellin Hl of Salmonella rubislaw (Genbank CAA28190) and autolysin / adhesin of the surface protein Aas of Staphylococcus saprophyticus (Genbank CAA03852). Analysis of the IHP2 protein indicates the presence of a cleavable N-terminal sequence of 27 amino acids and little homology with known proteins, apart from the YCCF protein of Bacillus subtilis (Genbank CAB 12066).

IHP3 has homologies with a phosphate-dependent human renal sodium transporter (Genbank AAA36354), as well as with Bacillus thuringiensis transposase (Genbank Q99335).

IHP4 has no homology with known proteins.

All of these results show that the strain of E. enterohemorrhagic coli O157: H7 contains a small locus (SIL ₀₁₅₇ ) inserted into its chromosome. The insertion of this locus is specific for E. strains. coli producing shiga toxins of serotype 0157 (STΕC-O157) and includes 4 potential open reading phases, one of which (IHP1) probably codes for an adhesin-like surface membrane protein which could play a role in the pathogenicity of STΕC serotypes 0157.

EXAMPLE 2 DETECTION OF STEC-O157 BY PCR 1. DNA extraction and PCR reaction

211 strains of E. coli: 56 strains of STΕC of serotype 0157, 93 strains of STΕC of other serotypes, 55 strains of E. non-shiga-toxin-producing coli and 7 strains of bacteria of other species, originating from reference laboratories or isolated from food or biological samples, are cultured in TSB (Tryptone Soy Broth) medium, at 37 ° C, overnight. Total bacterial DNA is extracted using the INSTAGENE matrix (BIO-RAD), following the manufacturer's instructions.

The PCR is carried out using the GENEAMP PCR SYSTEM 9600 (PΕRKIN ΕLMΕR) and primers obtained by conventional techniques of oligonucleotide synthesis. The sequence of the different primers used is presented in Table I below, in which the positions in SIL ₀₁₅₇ relate to the sequence of the

The following pairs of primers: P388C / P388D, P 388F / P388D and P388F / P388G are used in a reaction volume of 50 μl containing 200 μM of each of the deoxynucleotides (dNTP), 600 nM of each of the primers, 5 μl of buffer 10X GENE AMP (PERKTN ELMER), 2.5 IU to 'TAQ GOLD AMP (PERKIN ELMER) and 3 μl (approximately 10 ng) of purified bacterial DNA. The PCR amplification is carried out in the following conditions: an initial denaturation step at 94 ° C for 10 min is followed by

35 cycles alternating a denaturation step at 94 ° C for 25 s, a priming hybridization step at 71 ° C for 30 s and an extension step at 72 ° C for 30 s and a final step for elongation is carried out at 72 ° C for 10 min. The amplification products are separated by electrophoresis in a 2% agarose gel containing ethidium bromide, in TBE buffer (Tris-Borate-EDTA) and visualized under an ultraviolet lamp. The scale of

1 kb (BIOLABS NEW ENGLAND) or the molecular weight marker VI (ROCHE

MOLECULAR) are used as a size marker.

The size of the fragments expected by amplification of a STEC of serotype O157: H7 (FIG. 1) or else of a strain of E. non-pathogenic coli type K12, with the different pairs of primers, is presented in Table II below.

TABLE II

2. Study of the specificity of the pairs of primers P388C / P388D, P388F / P388D and P388F / P388G a) pair of primers P388C / P388D

In a first series of experiments, the P388C / P388D pair was used under the amplification conditions described above. The results obtained with the 211 strains tested are as follows: a product of 300 bp is amplified in all the strains of STEC of serotype 0157, no product is amplified in the majority of the strains of colibacilli producing shiga-toxins of other serotypes (STEC-no 0157) no product is amplified in E. strains coli of serotype 0157 not producing shiga-toxins (O157-non STΕC), - no product is amplified in bacteria of other species, in particular Hafnia alvei and Citrobacter.

The results obtained show that the pair of primers used makes it possible to specifically detect STΕCs of serotype 0157. In addition, the use of this detection method avoids the problems of cross-reactions of anti-0157 sera with the & Hafnia alvei or of Citrobacter, observed with immunological methods

(ELISA).

It was also observed that when the hybridization temperature of the P388C / P388D primer couple was lowered to 60 ° C., an amplification product was observed in the E. coli of the following serotype (055, 077, 079, 145 and O127: H6). b) pairs of primers P388F / P388D and P388F / P388G

To increase the specificity of the detection of STECs of serotype 0157, two other primers were tested: the primer P388F whose position is internal to the 300 bp fragment amplified using the primers P388C / P388D, and the primer P388G , located 5 'of this sequence (Table I and Figure 1). These primers were selected so that the primers P388F, P388G and P388D which are used in pairs (P388F / P388D and P388F / P388G) have close melting temperatures.

The same 211 strains of bacteria were tested with the 2 pairs of primers P388F / P388D and P388F / P388G and the results obtained with these strains are illustrated in the first 2 columns of Tables III, IV and V below.

BOARD

cured strains of the 60 MDa plasmid TABLE IV:

a: 2 strains out of 3 are EPEC b: 3 strains are EPEC c: 1 strain out of 2 is a strain of E. colo enteroaggregative d: This strain is the reference strain of EPEC e: this strain is an EPEC

TABLE V:

These results show that: all the strains of colibacilli producing shiga-toxins of serotype 0157 (56 strains of STEC of serotype 0157 tested including 38 strains of serotype O157: H7 including 4 strains cured of the plasmid of 60 Mda, 1 strain of serotype O157 : H ^" and 17 other STEC strains of serotype 0157 (5 O157: NM (Non Mobile) and 12 0157: ND (Not Determined)] are amplified by the pairs of primers P388F / P388D and P388F / P388G. In addition, the observation of an amplification product in the E. coli O157: H7 strains healed from the 60 MDa plasmid also confirms the chromosomal location of the locus

- the strains of colibacilli producing shiga-toxins of other serotypes (30 different serotypes tested on a total of 93 strains) are not amplified with the pairs of primers P388F / P388D and P388F / P388G. In particular the serotypes 0145, 077 and 079 which are amplified with the primers P388C / P388D are not amplified with these two pairs of primers.

- none of the strains of serotype 0157 which do not produce shiga-toxins (44 strains tested) is amplified with the pairs of primers P388F / P388D and

P388F / P388G, 7 strains of 3 other bacterial genera including those which give cross-reactions with 0157 by immunological techniques (Citrobacter and Hafnia alvei) are not amplified with the pairs of primers P388F / P388D and P388F / P388G, the strains of E. coli of serotype 055 (STΕC and non-STΕC) are also amplified with the pairs of primers P388F / P388D and P388F / P388G, which is explained by the clonal origin of serotypes 055 and 0157 which has been clearly demonstrated

(FΕNG et al, 1998, J. Infect. Dis, 177: 1750-1753; WHITTAM et al, 1993, Infect.

I mun, 61: 1619-1629). E. strains. coli not producing shiga-toxins of other serotypes (non-STΕC and non-0157; 5 serotypes tested on a total of 8 strains) are not amplified by the pairs of primers P388F / P388D and P388F / P388G, at l exception of O127: H6 which is amplified with the P388F / P388G couple but not with the couple

P388F / P388D.

All the results demonstrate that the two pairs of primers P388F / P388G and P388F / P388D make it possible to detect all of the strains of colibacilli producing shiga-toxins ^• of serotype 0157. The pair P388F / P388D, however, has more specificity. higher than the P388F / P388G pair.

EXAMPLE 3: ANALYSIS OF THE CONSERVATION OF THE LOCUS SIL ₀₁₅₇ INSERTION SITE IN E. COLI AND EVIDENCE OF A HOMOLOGATED LOCUS IN THE COLIBACILLA GROUP

ENTEROPATHOGENES (EPEC)

1. Analysis of the conservation of the insertion site of the SILoι- locus; ₇ in E. coli

The conservation of the insertion site of the SIL ₀₁₅ 7 locus was studied by PCR in the 211 strains of bacteria analyzed in Example 2, using the following 3 pairs of primers: the pairs P388L / P388M and P388P / P388D which respectively amplify the left and right junctions of the SIL ₀₁₅₇ locus and the P388P / P388L pair which amplifies a product of 682 bp in all strains of E. non-pathogenic coli such as strain K12 (Figure 1).

The reactions are prepared according to the protocol described in Example 1 and the PCR amplification is carried out under the following conditions: an initial denaturation step at 94 ° C for 10 min is followed by 35 cycles alternating with a denaturation step at 94 ° C for 40 s, a priming hybridization step at 65 ° C, for 40 s and an extension step at 72 ° C, for 50 s and a final elongation step is carried out at 72 ° C for 10 min . The sequences of the primers used and the size of the fragments expected with STΕC-O157 or with the strain E. coli Kl 2 are presented in Tables VI and VII respectively below. In table VI the positions in SIL ₀₁₅₇ relate to the fragment of 5478 bp (SEQ ID NO: 1) and the positions in E. coli Kl 2 relate to the Genbank sequence U82664.

TABLE VI:

TABLE VII

The results are illustrated in the last 3 columns of Tables III, IV and V. The results obtained show that by using short elongation times (40 s) for the P388P / P388L pair, no amplification product is observed in all strains of hummingbirds producing shiga toxins of serotype 0157, and that the majority of the other strains are identical to the strain of E. coli Kl 2 in the region of minutes 9 to 11 and do not contain the SILoιs7 locus- However, some strains tested have a particular amplification profile:

1) The strains of serotype 055 (STΕC and non-STΕC), the strain of serotype 079 (STΕC) and a strain of serotype 077 (STΕC) have a profile similar to that of STΕC of serotype 0157 which suggests the presence, in these strains of a locus homologous to SIL ₁₅₇ , inserted between positions 93849 and 93848 of the genome of E. coli., with reference to the genome of E. non-pathogenic K12 coli (Genbank U82664)

Nevertheless, with the exception of the colibacillus strains of serotype 055, which by virtue of their relationship to the serotype 0157 are amplified by the primers P388F / P388D and P388F / P388G, the locus homologous to SIL _0157, present in the colibacilli of seroytype 079 and 077 is not amplified by the primers P388F / P388D and P388F / P388G. Consequently, the presence of a locus homologous to SIL ₀₁₅₇ in these colibacilli does not prevent the specific detection of STECs of serotype 0157 using the primers P388F / P388D and P388F / P388G.

2) 2 strains of serotype 077 (STEC), 3 strains of serotype 0145 (STEC) and one strain of EPEC (O127: H6) give negative results with the 3 couples primers which indicate that they have, inserted in this same region, a locus whose sequence differs significantly from that of SIL ₀₁₅₇ .

The EPEC strain E2348 / 69 (0127: H6) was amplified using the pair of primers P388P / P388L, under the following conditions which make it possible to amplify large products: an initial denaturation step at 94 ° C for 10 min is followed by 35 cycles alternating a denaturation step at 94 ° C for 25 s, a priming hybridization step at 62 ° C, for 25 s and an extension step at 72 ° C, for 2 min 15 s and a final elongation step is carried out at 72 ° C for 7 min. The strain of E. coli O157.Η7 (strain ΕDL 933) is used as a positive amplification control.

The products obtained were hybridized with a probe called FD probe, corresponding to the amplification product of the strain O157: H7 using the primers P388F and P388D, under the conditions described in Example 1. The results obtained are the following: - an amplification product of 682 bp which does not hybridize with the FD probe is observed with the strains which do not have a locus inserted between minutes 9 and 11 of the genome of E. non-pathogenic coli,

- an amplification product of 3316 bp, hybridizing with the FD probe is observed with the strain O157: H7 and - an amplification product of size slightly greater than 3316 bp, hybridizing with the FD probe is observed with the strain O127: H6.

These results demonstrate that the strain of ΕPΕC O127.Η6 contains a locus homologous to locus SIL ₀₁₅₇ .

They also demonstrate that the SIL0157 locus isolated in the ΕDL933 strain of STΕC-O157: H7 is present in:

- all the colibacilli producing shiga-toxins of serotype 0157,

- strains of E. pathogenic coli serotype 055 (STΕC or ΕPΕC); which is explained by the clonal origin of serotypes 055 and 0157, and

- in other STΕC serotypes, in particular in the colibacilli producing shiga-toxins of serotype 077, 079 and 0145.

The presence of locus SIL ₀₁₅₇ or a homologous locus inserted between positions 93848 and

93849 of the E. genome coli (with reference to the genome of non-pathogenic E. coli type

K12, Genbank U82664) therefore constitutes a new means of specifically detecting strains of E. pathogenic coli and in particular all STΕC-O157. EXAMPLE 4 PRODUCTION AND PURIFICATION OF IHP1 PROTEIN

The outer membrane protein IHP1 was produced in the form of a recombinant protein tagged with a C-terminal 6X-Histidine epitope, and purified according to the steps described below. The gene coding for the IHP1 protein was amplified with the following primers:

PQP1 (SEQ ID NO: 25):

5 'MTTTCACACAGAATrCATTA GAGGAGA TTMGCATGAAMCTGTAMœTAœT ^ and PQP3 (SEQ ID NO: 26): which carry in their 5 ′ regions respectively the EcoRl and BgUl restriction sites (indicated in bold characters).

The PCR reaction is carried out in a volume of 100 μl containing 3 μl of DNA from STΕC O157: H7 (ΕDL 933), 10 μl of 10X buffer (100 mM Tris-HCl, pH 8.85 (20 ° C), 250 mM KC1 , 50 mM (NH ₄ ) ₂ SO ₄ , 20 mM MgSO ₄ ), 200 μM of each dNTP, 600 nM of PQP1 and PQP3, and 2.5 U of DNA polymerase PWO.

The PCR amplification is carried out under the following conditions: an initial denaturation step at 94 ° C for 2 min is followed by 30 cycles alternating a denaturation step at 94 ° C for 40 s, a step of hybridization of the primers at 57 ° C for 40 s and an extension step at 72 ° C, for 50 s. A final extension step is carried out at

72 ° C for 10 min.

The amplification product obtained and the expression vector PQΕ70 (QIAGΕN) were digested with the restriction enzymes EcoRl and Bgil and ligated, so as to obtain the plasmid pSP46 containing the open reading phase of IHP1 under the control of the promoter. strong and inducible from PQΕ70.

After transformation, a strain of E. coli M15 [pRΕP4] (QIAGΕN) carrying the recombinant plasmid pSP46 was selected. The nucleotide sequence of the pSP46 insert was confirmed by sequencing. Expression of the IHP1 protein fused to the 6XHis epitope (IHP1-

6Xhis) is strongly induced by IPTG during the culture of the strain of E. coli M15 [pRΕP4] carrying the plasmid pSP46. The purification of IHPl-6Xhis was carried out with NI-NTA agarose under denaturing conditions following the recommended protocol by QIAGEN, with the difference that 18 mM imidazole and 0.1% Triton were added to the buffers used, to improve the quality of the purification of IHP1-6XHis.

The purified protein IHPl-6XHis can be detected by western-blot using anti-PentaHis antibodies

The IHP1-6XHis protein can be used to immunize animals, and produce antibodies specific for the IHP1 protein, for the immunological detection of STECs of serotype 0157.

Claims

1) Nucleic acid molecule from a pathogenic strain of E. coli, characterized in that it is selected from the group consisting of: a) a nucleic acid molecule representing a locus inserted into the genome of said pathogenic strain, at the location of said genome corresponding to that located in E. coli K12, between positions 93848 and 93849 of the GenBank sequence U82664; b) a nucleic acid molecule comprising the molecule a) defined above, and between 1 and 2500 bp of the sequence upstream of the insertion site of said molecule, and / or between 1 and 2500 bp downstream of the site insertion of said molecule. c) a nucleic acid molecule constituting a fragment of at least

15 bp, preferably at least 25 to 30 bp, of the molecule a) above; d) a nucleic acid molecule constituting a fragment of at least 15 bp, preferably at least 25 to 30 bp, of the molecule b) above, said fragment comprising at least a portion of the molecule a) and at least a portion of the sequence upstream of the insertion site of said molecule a), or at least a portion of the sequence downstream of the insertion site of said molecule a).

2) Nucleic acid molecule according to claim 1, characterized in that said pathogenic bacterium is chosen from Escherichia coli STΕC of serotype 0157, 077, 079, or 0145, Escherichia coli STΕC or ΕPΕC of serotype 055, and Escherichia coli ΕPΕC of serotype 0127.

3) Nucleic acid molecule according to any one of claims 1 or 2, characterized in that it is chosen from the sequences: SΕQ ID NO: 1, SΕQ ID NO: 2, SΕQ ID NO: 11 to SΕQ ID NO: 17, SΕQ ID NO: 19, SΕQ ID NO: 21 and SΕQ ID NO: 23. 4) Nucleic acid molecule capable of specifically hybridizing under stringent conditions with a nucleic acid molecule according to any one of claims 1 to 3.

5) Nucleic acid molecule according to claim 4, chosen from oligonucleotides P388L (SΕQ ID NO: 3), P388M (SΕQ ID NO: 4), P388G (SΕQ ID NO: 5), P388D (SΕQ ID NO: 6 ), P388F (SΕQ ID NO: 7), P388C (SΕQ ID NO: 8) and P388P (SΕQ ID NO: 9).

6) Use of a nucleic acid molecule according to any one of claims 1 to 5 for screening for E. pathogenic coli.

7) Screening process for E. pathogenic coli, characterized in that it comprises the detection of the presence in the genome of the E. coli bacteria present in the test sample, of a locus inserted at the location of said genome corresponding to that located in E. coli K12, between positions 93848 and 93849 of the GenBank sequence U82664. 8) A method according to claim 7, characterized in that it comprises at least one step of chain amplification by polymerase using a pair of primers comprising a primer hybridizing with the genome region of E. coli defined by the 2500 bp sequence upstream of position 93848 of the GenBank sequence U82664, and a primer hybridizing with the genome region of E. coli defined by the 2500 bp sequence downstream of position 93849 of the GenBank sequence U82664.

9) Method according to claim 8, characterized in that one uses the pair of primers P388L / P388P.

10) Method according to claim 7, characterized in that it comprises at least one step of chain amplification by polymerase using a pair of primers hybridizing with internal sequences of a molecule a) according to claim 1.

11) Method according to claim 10, characterized in that one uses a pair of primers chosen from the use of the following pairs of primers: P388C / P388D, P388F / P388D, P388F / P388G. 12) Method according to claim 7, characterized in that it comprises at least one step of chain amplification by polymerase using a pair of primers comprising a primer hybridizing with an internal sequence of a molecule a) as defined above, and a primer hybridizing with the region of the genome of E. coli defined by the 2500 bp sequence upstream of position 93848 of the GenBank sequence U82664, or a primer hybridizing with the E. genome region. coli defined by the 2500 bp sequence downstream of position 93849 of the GenBank sequence U82664.

13) Method according to claim 12, characterized in that said pair of primers is chosen from P388P / P388D and P388L / P388M.

14) Method according to any one of claims 7 to 13, characterized in that it is implemented for the detection of E. coli serotype 0157 producing shigotoxins

15) Protein encoded by an open reading frame present on a nucleic acid molecule according to any one of claims 1 to 3, and in particular on a molecule derived from a strain of E. coli serotype 0157 producing shiga toxins. 16) Protein according to claim 15, characterized in that it is chosen from the IHP1 proteins IHP2, IHP3, and IHP4 of respective sequences

SΕQ ID NO: 18, 20, 22 and 24.

17) Peptide, characterized in that it consists of a fragment of at least 5 amino acids of a protein according to any one of claims 15 or 16. 18) Nucleic acid molecule according to claim 1, encoding a protein or a peptide according to any of claims 15 to 17. 19) Nucleic acid molecule according to claim 18 characterized in that it comprises a sequence selected from the group consisting of the sequences SEQ ID NO: 17, 19, 21 and 23.

20) Expression cassette, characterized in that it comprises a nucleic acid molecule encoding a protein according to any one of claims 18 or 19 placed under transcriptional control of an appropriate promoter.

21) Nucleic acid vector, characterized in that it comprises an insert consisting of a nucleic acid molecule according to any one of claims 1 to 4, 18, or 19, or an expression cassette according to claim 20. 22) Cell transformed by at least one vector according to claim 21.

23) Antibody, directed against a protein or a peptide according to any one of claims 15 to 17.

24) Use of a protein or a peptide according to any one of claims 15 to 17 for the detection of strains of E. pathogenic coli. 25) Use of an antibody according to claim 23 for the detection of strains of E. pathogenic coli.

26) Use according to any one of claims 6, 24, or 25, characterized in that said strains of E. pathogenic coli are strains of E. coli of serotype 0157 producing shiga-toxins, 27) Use according to any one of claims 6, or 24 to 26, characterized in that the search for strains of E. pathogenic coli is carried out in a food product.

28) Use according to any one of claims 6, or 24 to 26, characterized in that the search for strains of E. pathogenic coli is performed in a feces sample.

29) Use of a protein or a peptide according to any one of claims 15 to 17 for the preparation of a vaccine.