EP1003765A1

EP1003765A1 - OLIGONUCLEOTIDE SPECIFIC OF THE $i(ESCHERICHIA COLI) SPECIES AND METHOD FOR DETECTING AND DISPLAYING BACTERIA OF THIS SPECIES

Info

Publication number: EP1003765A1
Application number: EP98941537A
Authority: EP
Inventors: Patrick Grimont; Béatrice REGNAULT; Monique Collin
Original assignee: Institut Pasteur de Lille
Current assignee: Institut Pasteur de Lille
Priority date: 1997-08-04
Filing date: 1998-08-04
Publication date: 2000-05-31
Also published as: AU8987998A; FR2766825B1; WO1999007722A1; US6551776B1; FR2766825A1; JP2001512665A; US20040219524A1; CA2299599A1

Abstract

The invention concerns an oligonucleotide capable of being specifically hybridised with the ribosomal RNA (RNAr) or with the corresponding gene (ADNr) of the Escherichia coli genomic species(including all the Shigella genomic species except for serotype 13 S. boydii/Escherichia fergusonii genomic species. The invention also concerns a method for detecting and displaying the bacteria of said species.

Description

SPECIFIC OLIGONUCLEOTIDE OF THE SPECIES

ESCHERICHIA COLI AND METHOD FOR DETECTION AND VISUALIZATION OF BACTERIA OF THIS SPECIES

The invention relates to oligonucleotides for the detection and visualization of bacteria belonging to the genomic species Escherichia coli in a sample. More particularly, it relates to an oligonucleotide capable of hybridizing specifically with ribosomal RNA (rRNA) or with the corresponding gene (rDNA ) of the genomic species Escherichia coli (including Shigella with the exception of S boydu serotype 13) I Escherichia fergusonii

It also relates to a method for detecting the genomic species in question using this oligonucleotide as well as the use of said oligonucleotide in a gene amplification method The term "Escherichia coli" (E. coli) denotes in this document the genomic species (genomospecies) containing the type strain Escherichia coli ATCC 11775 (= CEP 58-8) A genomic species is a set of strains whose deoxyπbonucleic acid (DNA) has a homology of more than 70% with the DNA of the type strain of the species considered with a thermal instability of the hybrid DNA of less than 5 ° C. (Grimont, 1988, Wayne et al, 1987) According to these criteria, the genomic species E. col includes, in addition to the strains usually identified as E coli, strains traditionally classified as Shigella (S. dysenteriae, S. flexneri, S boydu, S. sonne i) with the exception of serotype 13 of S. boydu (Brenner et al, 1973) By strictly applying these criteria es, it can be argued that Escherichia fergusonii belongs to the genomic species E. coli (Farmer et al, 1985) E. coli is usually a commensal bacteria from the colon of humans and warm-blooded animals. For this reason, its presence in a sample of water, food, or the environment, is interpreted as an indication of faecal contamination (indicator bacteria). Thus, a food product must not contain more than a certain number of living E. coli cells (which can form a colony on a solid culture medium) in a defined mass of product (these numbers vary according to the products). For example, drinking water should not contain a living E. coli cell in 100 ml (De Zuane, 1997). The enumeration of E. coli is essential to assess the hygienic quality of a food.

Strains of the genomic species E. coli can be pathogenic. Among these strains are all that is commonly called Shigella, agent of human bacillary dysentery. The strains commonly called E. coli, can cause different infections of humans or animals depending on the pathogenicity equipment (urinary tract infections, choleriform or hemorrhagic diarrhea, dysenteric syndrome, hemolytic uremic syndrome, sepsis, neonatal meningitis, infections various purulent).

The identification of a strain of the genomic species E. coli (taxonomic identification) is important to suspect or demonstrate fecal contamination of water or food. It is also important in the case where the bacteria is isolated from a normally sterile or almost sterile biological medium (urine, blood, cerebrospinal fluid, fluid collection in a tissue or in a closed space of the body) In the open spaces of the body (digestive tract) or faeces, the presence of E. coli is commonplace and the identification of the pathogenicity factors of E. coli takes precedence over taxonomic identification. ^' The taxonomic identification of E. coli is conventionally based on the isolation and culture of the bacteria on a solid agar medium and the application of some biochemical tests. The appearance of colonies on an agar medium requires at least 18 hours. In the case of environmental samples, a culture of a few days is often necessary so that all the colonies which have to develop appear. The application of biochemical tests from an isolated colony requires another 18 to 48 hours. For example, the enumeration of E. coli in water requires the filtration of a volume of water through a sterile membrane, the deposition of the membrane on a semi-selective and or indicator medium, the incubation (48 hours) allowing colonies to develop a characteristic color (but not absolutely specific) which are then counted. Each isolated colony being supposed to be derived from a bacterial cell, the enumeration of E. coli per unit of volume can be carried out. It is prudent to check that the isolated colonies correspond well to the species E. coli and this requires at least 18 hours more.

Recently, techniques based on the detection of nucleotide sequences specific for the genomic species E. coli have been described. Thus, detection by gene amplification (PCR type) of the gene encoding beta-glucuronidase makes it possible to identify the presence of E. coli in a sample. This method is mainly used in a qualitative way and the interpretation of gene amplification is frequently hampered by the possibility of contamination due to the dispersion on experimental devices and tools of a few nucleic acid fragments. In situ hybridization is an interesting alternative to gene amplification. A labeled oligonucleotide probe (generally with a fluorescent substance) enters the bacterial cells previously treated to facilitate this step. According to ribosomal nucleic acids may or may not have a sequence complementary (target) to the probe, the probe will bind to its target and will not be removed by washing. The bacteria which have thus retained the probe become marked (for example, fluorescent) and visible by microscopic examination. Ribosomal ribosomal acids (rRNA) constitute the preferred target in in situ hybridization because of the number of copies per cell (10,000 to 30,000), higher than the number of copies of messenger RNA after induction (100 to 200) or of a given gene (one to a few). These ribosomal RNAs (rRNA) are identified according to their sedimentation constant (for bacteria: 5S, 16S and 23 S), present in the small subunit (16S rRNA) or the large subunit (23 S and RNA 5 S) of the ribosome.

The largest rRNAs are 16S (approximately 1500 nucleotides) and 23S (approximately 3000 nucleotides). A nucleic acid probe complementary to part of an rRNA will be able to hybridize with this rRNA but also with the complementary strand of the gene (rDNA) which coded this rRNA. Various applications of this methodology have been published (Amann et al., 1990; DeLong et al., 1989; Giovannoni et al., 1988; Trebesius et al., 1994).

These rRNAs have in fact appeared as the most suitable molecules to serve as a molecular chronometer for the evolution of bacteria (Brenner et al., 1969; Doi & Iragashi, 1965; Moore and McCarthy, 1967; Pace & Campbell, 1971; Takahashi et al., 1967). The primary structure (sequence) of rRNAs contains highly conserved regions and others which are hypervariable (Sogin et al., 1972; Woese et al., 1975). The development of a DNA-rRNA hybridization method (Gillespie & Spiegelman, 1965) has been followed by a very large number of publications applying this approach to economics and phylogeny bacteria and the identification of misclassified bacteria (Johnson et al., 1970; Palleroni et al., 1973; De Smedt & De Ley, 1977).

In general, in a hybridization experiment involving given sequences, the result greatly depends on the temperature and the molarity of sodium ions in the reaction medium. For a reaction medium of given composition, an optimal hybridization temperature is defined. If the temperature is raised, the reassociated strands will eventually separate. The temperature necessary for this separation depends on the length of the perfectly hybridized sequence part (perfect pairing) and on its nucleotide composition. A temperature allowing only the hybridization of the longest sequences is said to be restrictive (as opposed to optimal). Mismatches during hybridization cause the thermal stability of the hybridized molecules to drop.

The specificity of in situ hybridization will therefore depend on the quality of the probe capable of recognizing and hybridizing with a complementary sequence present in an rRNA.

Kohne et al. (1968) described a method for preparing probes reacting with rRNA without however indicating how to detect E. coli specifically. Gόbel and Stanbridge (1984) use a cloned rDNA gene to detect mycoplasmas contaminating tissue cultures.

Galpin et al. (1981) used hybridization of genes encoding rRNA to detect Mycoplasma pidmonis infections in mice. US 4,851,330 describes a strategy for obtaining nucleic acid fragments which can be used as a probe reacting with rRNAs

WO-A-84/02721 describes methods for detecting microorganisms infecting a human or animal body, using probes which hybridize with rRNAs. There is no guidance on how to detect or identify E. coli.

Berent et al. (1985) show the advantage of oligonucleotide probes compared to cloned probes.

French patent 2,596,774 proposes the use of an oligonucleotide complementary to bacterial rRNA as a probe and describes two universal oligonucleotide probes.

Goel et al. (1987) use a synthetic oligonucleotide reacting with rRNA or its gene, in order to identify mycoplasmas. US 5,084,565 describes an oligonucleotide probe called specific for E. coli. This probe targets the nucleotide zone 465 to 477 (numbering of the nucleotides according to Brosius et al. [1978]). The probe would react with Escherichia fergusonii and Shigella boydii serotype 13 (in addition to E. coli and Shigella) and would not react with Citrobacter koseri. Nothing is said about the reaction of this probe with species of the genus Cedecea which are phylogenetically close to E. coli.

US 5,593,841 mentions a probe reacting with the 995-1030 region of the E. coli 16S rRNA. This probe reacts with E. fergusonii but does not react with all the E. coli strains tested and does not react with Shigella dysenteriae. Nothing is said about the reaction of this probe with Citrobacter koseri (= C. diversus) and the species of the genus Cedecea which are phylogenetically close to E. coli.

Kwok et al. (1990) showed that a mismatch at the 3 'end of a primer used in gene amplification (PCR) affected the efficiency of the amplification.

Cha et al (1992) described a test called "Mismatch Amplification Mutation Assay" in which a primer has a mismatch with the target sequence of a mutation to be detected, and two 5 mismatches with the corresponding sequence of the wild allele. These mismatches relate to the 3 'terminal part of the primer. Under these conditions, their PCR system detects only the mutant allele. This method was applied to the specific detection of Salmonella enterica serotype Enterinais (Lampel et al., 1996) by creating a mismatch with

10 the penultimate position of the 3 'end of a primer.

The invention provides an oligonucleotide for the specific and rapid detection and visualization of bacteria belonging to the genomic species Escherichia coli in a sample. It therefore relates to an oligonucleotide capable of carrying out specific hybridization with

15 the genomic species of Escherichia coli (i.e. specific to all strains of Escherichia coli, Shigella (except S. boydii serotype 13) and Escherichia fergusonii.

More particularly, this oligonucleotide is capable of hybridizing with the region 637-660 of the 16S RNA of E. coli ^ system of

20 numbering of Brosius et al., 1978). Indeed, this portion of 16S RNA is fairly well conserved in Enterobacteria but not enough to be specific for them. However, surprisingly, it has proved to be very interesting in that it allows the detection of the genomic species defined above without cross-reaction.

25 with other species. The oligonucleotide according to the invention may also specifically hybridize only to at least 10 consecutive nucleotides of the region 637-660 of the 16S RNA of E. coli. Indeed, with two oligonucleotides recognizing adjacent zones and then linked by a ligase, we obtain a longer oligonucleotide and therefore more

3.0 resistant to more stringent hybridization conditions.

We can thus use two oligonucleotides representing the left half and the right half of one of the oligonucleotides of the invention, make them hybridize with the target, bind them by the action of a ligase, and increase the washing temperature so as to remove any small unbound oligonucleotide. This method which reduces background noise has been proposed by Alves and Carr (1988).

Advantageously, the oligonucleotide according to the invention corresponds to SEQ ID No. 1. In fact, an oligonucleotide of 24 nucleotides, complementary to the above region 637-660 of the 16S RNA of E. coli has been synthesized. It was called Ec637 and identified SEQ ID N ° 1.

The labeling was obtained by grafting two chromophores (fluorescein or Texas Red) at each end of the oligonucleotide. The probe oligonucleotide used in in situ hybridization at 42 ° C in the presence of 22% formamide followed by washing at 60 ° C, fluoresces the cells of Escherichia coli, Shigella dysenteriae, Shigella βexneri, Shigella boydii (except the serotype 13), Shigella rings i and Escherichia fergusonii (genomic group E. coli). It does not react with most of the other species and genera tested. However, it has been observed that the Citrobacter koseri species and the Cedecea species remain fluorescent after washing at 60 ° C.

In fact, it is necessary to reach a temperature of the order of 61 ° C so that these species no longer react. However, at 61 ° C, the E. coli genomic group is made very weakly fluorescent.

The present invention therefore also relates to an oligonucleotide enabling even better results in terms of specificity to be obtained.

Indeed, the oligonucleotide Ec637 was modified at the level of a nucleotide located at a conserved position (invariant) of the corresponding 16S rRNA sequence to create a voluntary mismatch.

This mismatch was carried out in the central part of the oligonucleotide. The sequence obtained was called Colinsitu and identified SEQ TD N ° 2. The purpose of introducing a central mismatch is to weaken the hybrid that will be obtained. If a sequence differs by a single nucleotide from the sequence 637 to 660 of E. coli, this will cause two mismatches with the Colinsitu probe which will not hybridize under the experimental conditions chosen. This probe remains reactive towards the genomic species E. coli and becomes inactive towards all the other species and genera. This specificity is maintained over a wide range of washing temperatures from 51 ° C to 59 ° C.

The Colinsitu probe can be used in situ hybridization but also in hybridization on a filter, in a liquid medium, in reverse hybridization, or as a specific primer in a gene amplification system.

The invention also relates to oligonucleotides complementary to the oligonucleotides described below.

Other types of labeling of the probe (radioactivity, chemical or enzymatic labeling) can be used for in situ hybridization.

More particularly, the oligonucleotides according to the invention can be labeled at their 3 'or 5' end or at the 3 'and 5' ends.

The advantage of this probe, applied in in situ hybridization with microscopic examination of bacterial cells or detection by flow cytometry, is to be able to detect, identify, and count the cells of the genomic species E. coli in various samples such as clinical and veterinary samples (especially urine), water and other drinks, food, environment. The subject of the invention is also a method for detecting and visualizing bacteria of the genomic species Escherichia coli (including all Shigella with the exception of S. boydii serotype 13) / Escherichia fergusonii in a sample comprising a step of hybridization of the ribosomal RNA of bacteria of said genomic species with an oligonucleotic according to the invention, and more particularly with an oligonucleotic chosen from SEQ ID N ° 1 and SEQ ED N ° 2.

More particularly, the hybridization in question can be an in situ hybridization, a hybridization on a filter, a hybridization in a liquid medium or a reverse hybridization.

By reverse hybridization for the purposes of the present invention is meant a hybridization reaction in which the oligonucleotide probe of interest is immobilized on a support, the nucleic acid to be detected and / or the organism containing the nucleic acid to be detected being present in solution.

According to a particular embodiment of a reverse hybridization reaction according to the invention, the oligonucleotide probes can be implemented within a detection device comprising a matrix library of oligonucleotides. An exemplary embodiment of such a matrix bank may consist of a matrix of probe oligonucleotides fixed on a support, the sequence of each probe of a given length being located one or more bases offset from the probe. previous, each probe of the matrix arrangement thus being complementary to ^a distinct sequence of the target DNA or RNA to be detected and each probe of known sequence being attached at a predetermined position of the support. The target sequence to be detected can advantageously be radioactive or non-radioactive. When the labeled target sequence is brought into contact with the matrix device, this forms hybrids with the probes of complementary sequences. A nuclease treatment, followed by washing, eliminates the probe-target sequence hybrids that are not perfectly complementary.

Due to the precise knowledge of the sequence of a probe at a determined position in the matrix, it is then possible to deduce the nucleotide sequence from the target DNA or RNA sequence and to detect possible mutations accordingly. located in the ribosomal DNA of E. coli, and more particularly mutations affecting the 637-660 region of the DNA coding for the 16S rRNA of E. coli.

An alternative to the use of a labeled target sequence may consist of the use of a support allowing “bioelectronic” detection of the hybridization of the target sequence on the probes of the matrix support, when said support consists or comprises a material capable of acting, for example, as an electron donor at the positions of the matrix at which a hybrid has been formed. Such an electron donor material is for example gold. Detection of the nucleotide sequence of the target DNA or RNA is then determined by an electronic device.

An exemplary embodiment of a biosensor, as defined above, is described in European patent application No. EP-0721 016 (Affymax technologies NV) or also in American patent No. US 5,202,231 (Crkvenjakov and Drmanac).

The invention also relates to the use of an oligonucleotide corresponding to SEQ ID No. 1 or SEQ ID No. 2 or different from SEQ ID

No. 1 with a nucleotide or a complementary oligonucleotide as a primer for the implementation of a gene amplification method, such as PCR.

The oligonucleotides in accordance with the invention can also be used in a method of inhibiting hybridization. In fact, one can envisage fixing on a support (filter, cup or microchip) a Identical or homologous oligonucleotide of region 637-660 of E. coli 16S RNA and labeling in any way an oligonucleotide complementary to this region according to the present invention. In the absence of competing DNA or RNA, the two oligonucleotides must re-associate completely. The introduction into the system of a nucleic acid capable of reassociating with one or other of the nucleotides (for example a nucleic acid belonging to one of the species targeted by the present invention) or both (case of two separate strands) inhibits the binding of the free, labeled oligonucleotide according to the invention to the support. The present invention also relates to a method of detection and visualization of microorganisms by hybridization making it possible to optimize the specificity of the oligonucleotide probe used. Indeed, an oligonucleotide is all the more specific as it presents clear differences in its hybridization capacities with on the one hand the target sequences and on the other hand the other sequences. In the experimental hybridization conditions, this difference is all the more detectable as there are differences in sequences (or mismatch) between the aforementioned oligonucleotide and the sequence with which it is capable of hybridizing. Consequently, it becomes advantageous to artificially increase the number of these mismatches by modifying the oligonucleotide used for hybridization at the level of a nucleotide generally very conserved at the level of the sequence which it is sought to detect.

Consequently, the present invention relates to a method for detecting and visualizing microorganisms (or a group of microorganisms) by hybridization using an oligonucleotide complementary to the target sequence of the microorganism with the exception of a nucleotide located in the central part of said oligonucleotide. The The nucleotide in question is located at an invariant position of the target sequence of the microorganisms and is preferably in the central position.

For example, for a complementary oligonucleotide with a length of 20 base pairs, the non-complementary nucleotide is

10 located between positions 7 and 13 inclusive according to a numbering of the oligonucleotide starting at its N-terminal end, preferably, the nucleotide in question is located at position 10.

The invention therefore relates to a detection and visualization method as described above, applied to bacteria of the genomic species Escherichia coli (including all Shigella with the exception of S. boydii serotype 13) / Escherichia fergusonii.

Thus, in the context of the present invention, the complementary oligonucleotide used in the above method is an oligonucleotide in accordance with the present invention which does not differ from SEQ ID

No. 1 only with a nucleotide and preferably corresponding to SEQ ID No.

2.

Among the potential applications of the invention, the following will be mentioned more particularly:

- Search for confirmation that atypical strains of E. 25 coli do indeed belong to this species. Reference Centers often receive strains which could be atypical E. coli. They give unusual biochemical reactions for this species such as a negative reaction for the production of indole or gas, the hydrolysis of o-nitrophenyl-β-galactopyranoside, the hydrolysis of beta-glucuronides, unusual fermentation reactions , or very weak growth in usual environments. The Colinsitu probe can confirm whether these strains belong to the genomic species E. coli-E. fergusonii. If the reaction with the probe is positive, it is easy to distinguish E. fergusonii by the fermentation of adonitol and cellobiose;

- Detection, identification and enumeration of E. coli in the urine of infected patients or animals. Most urinary tract infections are due to E. coli and a urinary tract infection or colonization characterized by the presence of more than 1000 or 10 000 bacteria per ml, in situ hybridization with Colinsitu of an appropriate dilution of urine should allow note the presence and enumerate E. coli in the urine in 2 to 3 hours;

- Detection and enumeration of E. coli in water and food. Escherichia coli is the main biological indicator of faecal contamination of water and food. It suffices to filter a known and sufficient quantity of water and to carry out the in situ hybridization on the filters thus obtained. If, using a micrometer and a reticle, we know the filtered volume relative to a surface observed under the microscope, it is possible to quantify the number of E. coli cells in water. In the case of foods which are not filterable and which must not have an E. coli per 25 g, enrichment may be necessary from 25 g of food, the in situ hybridization then carried out on the medium of culture will indicate if E. coli is present. The invention is not limited to the above description but encompasses all the variants thereof. The examples below make it possible to understand it better while being mentioned for purely illustrative purposes.

EXAMPLES Bacterial strains used A total of 208 strains were used to assess the specificity of the probes.

The authenticity of the strains was verified by re-identifying them on Biotype-100 galleries (BioMérieux, La Balme-les-Grottes, France). The galleries were inoculated according to the manufacturer's instructions. Automatic identification was obtained thanks to the Recognizer ^" program (Taxolab Institut Pasteur, Paris) and a Macintosh Powerbook 5300ce computer (Apple Computers). EXAMPLE 1: In situ hybridization During the tests, a sequence of 23 nucleotides called

Ec465, 5 '-GGT AAC GTC AAT GAG CAA AGG TA-3', recognizing the region 465 to 487 of 16S rRNA, was also synthesized. This Ec465 sequence, corresponding to SEQ ID No. 3, was used for the purpose of comparison. A published method (Trebesius et al., 1994) was followed with some modifications. The cultures were diluted in sterile distilled water to obtain an absorbance at 600 nm of 0.010, and 100 μl were filtered through PC filters (Millipore, St Quentin-en-Yvelines, France) of 0.22 μm. The fixation was carried out with a 3% aqueous solution of paraformaldehyde. The formamide in the hybridization mixture represented 22% of the volume. The probe was added at the concentration of 25 pmole. Hybridization was performed at 42 ° C for 2 hours. After washing (step determining the specificity: 20 min at 51 ° C for Colinsitu, 60 ° C for Ec637, 48 ° C for Ec465), the filters were placed on glass slides, covered with 5 μl of Vectashield (Vector Laboratories, Burlingame, CA) and a coverslip.

The slide-mounted filters were examined by epifluorescence using an Olympus BX60 microscope equipped with a WTBA (for fluorescein) or NG (for Texas Red) filter and a DEI-470 color camera (Optronics).

The table shows the results obtained in in situ hybridization with the fluorescent probes Colinsitu, Ec637, and Ec465. All tested strains of Escherichia coli, Shigella (except S. boydii serotype 13), and Escherichia fergusonii, are made fluorescent by the hybridization reaction using the Colinsitu probe. No other genomic species reacts. When the probe is Ec637, a reaction is obtained with Citrobacter koseri and the species of the genus Cedecea.

The Ec465 probe, used in comparison, reacted weakly with E. coli and with strains of Buttiauxella and the experiment was not carried forward.

The result of in situ hybridization, that is to say the bacterial cells rendered fluorescent, can also be viewed by flow cytometry rather than by microscopy. BOARD

Reactions obtained with fluorescent probes in in situ hybridization.

Reaction with Colinsitu Strain Species Ec637 Ec465 Escherichia coli genomic species:

Escherichia coli CIP 54.8 ++++ f

2430 +++ f

CIP 54-120 + + + f

CIP 54-122 +++ f

CIP 54-124 +++ f

CIP 70-59 +++ f

CIP 70-68 + + + f

044 +

052 +

066 +

O90 +

O103 +

WHERE WHERE +

01 10 + or i +

01 13 + 01 19 +

0121 +

0127 +

0132 +

0135 +

0136 +

O140 +

0151 +

O157: H7 +

96-4597 +

6085 +

67 Tunis +

K-12 HB101 +

H19 +

PMK1 +

H30 +

E3251 1 +

B2 F1 H21 +

0X3 H21 +

412 + ffl 8 +

96-302 +

96-303 +

96-301a +

Shigella dysenteriae 1 NCDC 1007-71 ++

Y6R +

60R +

Shigella βexneri CIP 54-58 + + Shigella boydii 15 NCDC 965-58 + + Shigella sonnei CIP 52-55 + + Escherichia fergusonii 1016-74 + +

85-1 1615 ++

568-73 ++ 29586 ++

32-96 ++

1-85 ++

Other genomic species of the genus Escherichia:

Escherichia hermanii 1158-78 - -

1200-74 - -

3514-77 - -

980-72 - -

E. vulneris CDC 2524-69 - -

394-83 - -

875-72 - -

E. blattae 9005-74 - -

Other genres

Budvicia aquatica 2377 - -

20186HG - -

Buttiauxella agrestis CUETM 77-167 - -

B. brennerae Sl / 6-571 - -

B. cochleae S3 / 1-49 - -

B. ferragutiae CUETM 78-31 - -

B. gaviniae Sl / 14-669 - - + f

CUETM 77-159 - -

B. georgiana CDC 2891-76 - -

B. izardii S3 / 2-161 - -

B. nockiae NSW 1 1 - -

B. warmboldiae NSW 326 - -

Cedecea davisae 005 - +

C lapagei 004 - +

C. neteri 002 - +

Cedecea sp. 001 - +

Citrobacter amalonaticus 9020-77 -

C. braakii 80-58 -

C. farmeri 2991-81 -

C. freundii 621-64 _ C. koseri (= C. diversus) 3613-63 +

8132-86 +

8127-86 +

C. rodentium 1843-73

C. sedlakii 4696-86

C. werkmanii 876-58

C. youngae 460-61

Citrobacter species 10 4693-86

Citrobacter species 11 2970-59

Edwardsiella hoshinae 2-78

E. ictaluri 92-7041

E. tarda 10396

Enterobacter aerogenes Al

E. agglomerans group II 3123-70 group III (Pantoea dispersa) 1429-71 group IV 1471-71 group V 3482-71 group Vf (Pantoea pineapple) 6070-69 group VU 6003-71 group VÏÏI 5422-69 group IX 4388- 71 group X 1600-71 group XI 5378-71 group XII 219-71 group Xπi (Pantoea agglomerans) E20

E. amnigenus 77-118

E. asburiae 1497-78

E. cancerogenes 2176

E. cloacae CIP 60-85

77-21

E. gergoviae 16-74

E. hormaechei 491-62

E. intermedium 77-130 E. nimipressuralis E63

E. persicinus HK204

E. pyrinus 4205-93

E. sakazakii 4562-70

E. taylorae 2126-81

E vinia carotovora 495

E. carotovora subsp. betavasculorum E235

2122

E. chrysanthemi SR32

1451

E. cypripedii EC 155

E. mallotivora 2851

E. nigriβuens EN 104

E. rhapontici 1075

E. rubrifaciens ER 105

E. stewartii CNBP 3157

E. uredovora 158

Ewingella americana 23

Hafnia alvei group I 5632-72

H. alvei group II 4510-75

Klebsiella ornithinolytica 626

K. oxytoca 131-82

K. planticola CIP 100751

K. pneumoniae subsp. pneumoniae 464

K2

12-52

532

K. pneumoniae subsp. ozaenae 10-79

K. pneumoniae subsp. rhinoscleromatis 475

K. terrigena 1

Koserella trabulsii 3518-73

Kluyvera ascorbata 648-74

K. cryocrescens 2065-78 Leclercia adecarboxylata CUETM 77-3

8-82

Leminorella grimontii 1944-81 Leminorella sp. 3346-72 Moellerella wisconsensis 2897-78 Morganella morganii 25830 Obesumbacterium proteus NCIMB 8771

CIP 104862

Pragia fontium 2434 Proteus mirabilis PMI

PR14

P. myxofaciens

P. penneri 8.88

P. vulgaris PR1

Providencia alcalifaciens 3370-67

P. heimbachae 8025-83

P. rettgeri 1163

P. rustigiani 132-68

P. stuartii 282

Rahnella aquatilis 3307

Salmonella enterica subsp. arizonae 44

S. enterica subsp. diarizonae 41

S. enterica subsp. enterica serotype. 6323-88 serotype ... 122 serotype ... 119 serotype Typhimurium LT2 serotype Gallinarum 4-86

S. enterica subsp. houtenae 6700-88

S. enterica subsp. salamae 1492-74

Serratia entomophila Al

S. caria 4024

S. fond cola 5680

S. grimesii 503 S. liquefaciens ATCC 27592

275

S. marcescens 504 S. odorifera 1073 S. plymuthica 510 S. proteomaculans 3630 S. rubidaea 864 Trabulsiella guamensis 370-85

371-85

Yersinia enterocolitica ATCC 27729 Y. frederiksenii CIP 8029

Y. intermedia 29908

Y. kristensenii 9993

Y. pestis EV40

Y. pseudotuberculosis 29833 Y. ruckeri ATCC 29473

Yokenella regensburgei 2403

2405

Other family species:

Acinetobacter .... Al 745 Aeromonas caviae 67.24

Pseudomonas aeruginosa 63-52

Pseudomonas fluorescens DSM 50090

P. putida 2066

Vibrio alginolyticus LMG 4408 V. anguillarum CIP 63-36

V. cholerae CIP 62-13

V. harveyi ATCC 1426

V. hollisae CEP 101886

Xanthomonas maltophilia 2377 Legends

+: good fluorescence of bacterial cells

+ f: weak fluorescence

-: absence of fluorescence (nothing): experiment not carried out.

EXAMPLE 2 Gene amplification

The oligonucleotide Colinsitu (unlabeled) and the following nucleotide (complementary to the conserved region 8-32 of 16S rRNA): 5'-ATT TGA AGA GTT TGA TCA TGG CTC AG-3 '(SEQ ID N ° 4) were used as a primer for the specific amplification of the gene encoding the 16S rRNA of E. coli. The "GeneAmp" DNA Amplification Reagent Kit "(Perkin Elmer Cetus, Norwalk, CT) was used according to the manufacturer's instructions, with the DNA polymerase" AmpliTaq ³ "'and a" Thermal Thermal Cycler 480 "thermocycler ( Perkin Elmer Cetus). The reaction volume was 100 μl comprising 10 μl of buffer, 2.5 units of AmpliTaq, 200 μM of each nucleotide dATP, dGTP, dCTP, dTTP, 100 pMole of each primer and 30 to 50 ng of total DNA. The amplification conditions were as follows: initial denaturation at 94 ° C for 3 minutes, 25 cycles from 60 s to 94 ° C for denaturation, 60 s at 65.5 ° C for reassociation, 120 s at 72 ° C for elongation. The amplification product was subjected to electrophoresis in 1.3% agarose (Applégéne, Illkirch, France). The expected size of the amplified fragment was about 600 base pairs. The use of this system effectively makes it possible to amplify this fragment specifically for the genomic species Escherichia coli-Shigella-E. fergusonii. EXAMPLE 3 Hybridization on a Filter

Hybridization on a nitrocellulose, nylon or cellulose filter is a practical method allowing the same probe to be applied to a large number (hundreds) of DNA samples. Hybridization can be done on colonies. In this case, the membrane is applied to colonies, impregnated with sodium hydroxide (lyses the bacteria, destroys the RNA, and denatures the DNA), and brings the labeled probe into the presence of an adequate buffer. After a sufficient exposure time (several hours), the membrane is washed, dried in the oven (to irreversibly fix the DNA), and the labeling is revealed. This method requires having colonies on a dish, but allows to select a reactive colony among thousands. A similar protocol makes it possible to filter 96 samples on a membrane treated in the same way as the colonies.

EXAMPLE 4 Hybridization in Liquid Medium

If a sample is processed to lyse bacteria and extract DNA, it can be denatured and hybridized with a radioactive probe (1251, for example). The radioactivity associated with the hybrid DNA can be counted (γ count) after separation, by chromatography on hydroxyapatite, of the radioactivity associated with the non-hybridized probe.

EXAMPLE 5 Reverse reverse hybridization

Oligonucleotide probes can be fixed on a support (filter, microplate, microchip). Several probes can thus be available on the same support. The target gene is amplified and labeled, and the amplicon is put into hybridization conditions with the panel of probes. After washing and revealing the marking, fixing the marking on one of the probes allows identification. This approach also allows the simultaneous detection of several organelles when the amplification is carried out on DNA extracted from a plurimicrobial sample (Rijpens et al., 1995). Although the published work uses as a target the intergenic space between the genes coding for the 16S and 23S rRNA, this approach is applicable to the gene coding for the 16S rRNA.

EXAMPLE 6 Specificity of the Colinsitu probe

It is now well established that the species of the genus Shigella (except S. boydii serotype 13) belong to the genomic species Escherichia coli (Brenner et al., 1973). It is therefore not surprising that the Colinsitu probe reacts with Shigella. In the genus Escherichia, E. fergusonii has 59 to 63% homology with E. coli (by DNA hybridization) with a thermal instability of the hybridized molecules of 4.5 ° C (Farmer et al., 1985). So the E. fergusonii strains partially meet the criteria that would make them included in the genomic species Escherichia coli. Recall that these criteria are homology greater than or equal to 70% with thermal instability of the hybridized molecules less than or equal to 5 ° C (Wayne et al., 1987). These criteria should be interpreted flexibly (Wayne et al., 1987). The Colinsitu probe is therefore strictly specific for the genomic species E. coli if E. fergusonii is admitted in this genomic species.

The comparison of the specificity of the Colinsitu probe with that of other probes which have been or could be proposed, will appear clearly with the following reference strains given by way of example:

Strains to be viewed by Colinsitu under the conditions described: Escherichia coli CIP 54.8 (= ATCC 1 1775) Shigella dysenteriae serotype 1 NCDC 1007-71 Shigella flexneri serotype CIP 54-58

Shigella boydii serotype 15 NCDC 965-58

Shigella sonnei CIP 52-55

Escherichia fergusonii CIP 103357 (= ATCC 35469)

Strains not to be viewed by Colinsitu under the conditions described:

Shigella boydii serotype 13 CDC 1610-55

Escherichia vulneris CDC 875-72 (= ATCC 33821)

Escherichia hermanii CDC 980-72 (= ATCC 33650)

Citrobacter koseri {= C. diversus) CDC 3613-63 (= ATCC 27156) Citrobacter braakii 80-58 (= ATCC51113)

Cedecea davisae CIP 80.34 (= ATCC 33431)

Cedecea lapagei CIP 80.35 (= ATCC 33432)

Cedecea neteri CIP 103241 (= ATCC 33855)

Klebsiella pneumoniae subsp. pneumoniae K2 • Obesumbacterium proteus CIP 104862

Salmonella enterica serotype Typhimurium (= S. typhimurium) LT2 (=

CIP 60.62, ATCC 43971).

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Claims

1. Oligonucleotide capable of hybridizing specifically to ribosomal RNA (rRNA) or to the corresponding gene (rDNA) of the genomic species Escherichia coli (including all Shigella except S. boydii serotype 13) / Escherichia fergusonii .

2. An oligonucleotide according to claim 1 capable of hybridizing specifically to the region 637-660 of the 16S rRNA of E. Coli.

3. The oligonucleotide according to claim 2 capable of hybridizing specifically with at least 10 nucleotides of region 637-660 of the 16S rRNA of E. coli

4. Oligonucleotide according to one of claims 1 to 3, characterized in that it corresponds to SEQ ID No. 1.

5. Oligonucleotide according to one of claims 1 to 3, characterized in that it differs from a nucleotide of SEQ ID No. 1.

6. Oligonucleotide according to claim 3, characterized in that it corresponds to SEQ ID No. 2.

7. Oligonucleotide complementary to the oligonucleotide according to one of claims 1 to 6.

8. Oligonucleotide according to one of claims 1 to 7, characterized in that it is labeled at its 3 'or 5' end or at the 3 'and 5' ends.

9. Method for detecting and visualizing bacteria of the genomic species Escherichia coli (including all Shigella except S. boydii serotype 13) / Escherichia fergusonii in a sample comprising a step of hybridization of ribosomal RNA bacteria of said genomic species with an oligonucleotide according to one of claims 1 to 8.

10. Method according to claim 9, characterized in that the oligonucleotide is chosen from SEQ ID No. 1 and SEQ ID No. 2.

11. Method according to one of claims 9 or 10, characterized in that the hybridization is an in situ hybridization, a hybridization on a filter, a hybridization in a liquid medium or a reverse hybridization.

12. Use of an oligonucleotide according to one of claims 4 to 7 as a primer for the implementation of a gene amplification method.

13. Method for detecting and visualizing microorganisms by hybridization, characterized in that it implements an oligonucleotide complementary to the target sequence of said microorganisms, with the exception of a nucleotide located in the central part of said oligonucleotide.

14. Detection and display method according to claim 13, characterized in that the non-complementary nucleotide is located at an invariant position in said microorganisms.

15. A detection and display method according to claim 13 or 14, characterized in that the microorganisms are bacteria of the genomic species Escherichia coli (including Shigella with the exception of S. boydii serotype 13) / Escherichia fergusonii.

16. A detection and visualization method according to claim 15, characterized in that the complementary oligonucleotide used is as defined in claim 5 or 6.