EP0377711A1

EP0377711A1 - Serotyping dna probes

Info

Publication number: EP0377711A1
Application number: EP89907053A
Authority: EP
Inventors: Paul Alexander Manning; Michael William Heuzenroeder; Danny Werner Beger
Original assignee: Luminis Pty Ltd
Current assignee: Luminis Pty Ltd
Priority date: 1988-06-20
Filing date: 1989-06-20
Publication date: 1990-07-18
Also published as: DK37990A; DK37990D0; WO1989012693A1; JPH03501326A

Abstract

La sonde d'ADN décrite comprend un fragment d'ADN contenant des gènes qui codent pour la synthèse de l'antigène O d'un organisme d'Enterobacteriaceae, ladite sonde détectant la présence de la région rfb d'un organisme d'Enterobacteriaceae.The described DNA probe comprises a DNA fragment containing genes which code for the synthesis of the O antigen of an Enterobacteriaceae organism, said probe detecting the presence of the rfb region of an Enterobacteriaceae organism.

Description

SKROTY ING DNA PROBES The present invention relates to the identification and isolation of DNA fragments which may be utilised inter alia as DNA probes and in live vaccine carrier strains. The outer membrane of the cell wall of Gram negative bacteria comprises proteins, phospholipids and lipopolysaccharide (LPS) and of these the LPS is by far the most abundant molecule on the cell surface. The LPS is composed of three regions: the innermost is the lipid-A moiety which forms the outer leaflet of the lipid bilayer of the outer membrane; the central region is the core oligosaccharide, and outermost is the O-antigen side-chain consisting of a series of repeat units usually of specific sugars. The sequence and types of sugars and their linkages is what determines the O-serotype of the bacterium.

The diagnosis of disease requires the specific identification of the aetiological agent and this usually involves isolation of the organism on selective media and then subjecting it to a battery of biochemical tests which can take days before a positive identification can be made. It may then be necessary to serotype the organism especially if the epidemiology of the disease is to be examined. Serotyping can usually only proceed after the type of organism has been determined, and again is time consuming. Thus, a more rapid means of both diagnosis and serotyping would be of considerable benefit especially since many of the Enterobacteriaceae can cause life-threatening disease and are in fact the major causes of infant mortality in developing countries such as India, Bangladesh and much of South America. It is accordingly an object of the present invention _ _ _.

to overcome, or at least alleviate, one or more of the difficulties related to the prior art.

Accordingly, in a first aspect, there is provided a DNA probe including a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae. which probe detects the presence of the rfb region of an Enterobacteriaceae.

More preferably the fragment of DNA contains genes encoding for the synthesis of the O-antigen of the 0101, 0157 or 02 serotype of an Escherichia coli. Preferably the Escherichia coli is an Enterotoxigenic E.coli (ETEC) .

In a preferred aspect of the present invention there is provided a DNA probe kit including a first DNA probe including a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae, which probe detects the presence of the rfb region of an Enterobacteriaceae; and a second DNA probe including a fragment of DNA which detects the presence of the rfb region of an Enterobacteriaceae in a manner different to said first probe such that the first and second probes are serotype specific.

Most of the genes responsible for the production of the O-antigen of the lipopolysaccharide of members of the Enterobacteriaceae are encoded within the gene cluster referred to as the rfb locus. There is variation at this locus and as a result the structure of the O-antigen also varies. The direct consequence of this is that the serotype of the bacterium is different. Thus this region of the bacterial chromosome represents a set of genes which are directly related to the serotype of the organism, and thus variation in this region is absolutely related to the change in the serotype. Thus, the DNA probes according to the present invention make it possible to directly analyse the variation at rfb and correlate this with the serotype and thus provide a rapid means of serotyping an organism, as well as being able to differentiate one species from another.

In a still further aspect of the present invention there is provided a DNA probe including a plasmid cloning vector; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae inserted into a suitable site in the plasmid cloning vector. The fragment of DNA may include the entire rfb locus of the Enterobacteriaceae or a fragment thereof. The Enterobacteriaceae may be an E.coli of the 01, 0157 or 02 serotype. Where the Enterobacteriaceae is an E.coli of the 0101 serotype, we have found that a fragment having a minimum length of approximately 8.9 Kb and a maximum length of approximately 11.8 Kb is required for 0101 0-antigen biosynthesis when it is introduced into E.coli K-12.

The plasmid cloning vector may be of any suitable type. The plasmid cloning vector ρHC79 has been found to be suitable. The fragment of DNA may be inserted into the BamHI site on the plasmid pHC79. The plasmid so formed has been designated pPM 1301. By deleting rfb DNA a derivative pPM 1305 has been derived.

Accordingly, in a preferred form there is provided a DNA probe including the plasmid pPM 1305 or a digestion derivative thereof.

Mini cell analysis of proteins produced by the plasmid clone pPM 1305 have demonstrated that O-antigen synthesis is genetically complex with at least 6 proteins involved.

Fragments of the clone pPM 1305 may be produced by partial digestion as described below. At present six fragments of pPM 1305 have been envisaged. These are designated plasmids pPM 1321, pPM 1322, pPM 1323, pPM 1324, pPM 1325, pPM 1326, pPM 1330 and pPM 2218. Samples of the plasmids are maintained in the Culture Collection of the Universith of Adelaide, North Terrace, Adelaide, South Australia. These are illustrated in Figure 6 below.

Accordingly in a further aspect of the present invention there is provide a DNA probe including a plasmid from which rfb DNA has been deleted and selected from plasmids pPM 1305, pPM 1322, pPM 1323, pPM 1324, pPM 1325, pPM 1326, pPM 1330 and pPM 2218 and mixtures thereof.

As described above the rfb,_Q1 region has been subcloned into six parts and each has been used as a probe in Southern DNA hybridizations with a selection of E.coli and Sh.flexneri strains of different serotypes. This has enabled us to show that the different probes vary in their ability to react in a manner which shows serotype specificity. For example, Sh.flexneri strains all react with one probe whereas only serotypes 4a/y and 6 react with another and only 2a and 4 react with a third. The fragment sizes with which they react also differ markedly so that the pattern observed using several probes is quite distinctive for that particular serotype. Using these same set of probes it was also possible to very rapidly subgroup E.coli strains of the 02 and 018 serotypes into exactly the same groups as had previously been defined by using an extensive series of tests, involving analysis of both LPS and outer membrane proteins and a large number of isoenzymes and one strain had been mistyped probably because of a mutation affecting the ability to produce a specific isoenzyme. The 02 serotype strain is important as it is implicated in such diseases as septicaemia n chickens and in urinary tract infections in man. (It is a particular advantage of this aspect of the present invention that the probes are effective at differentiating serotypes even when the gene products are not made.)

By the same approach we have also cloned the genes for the 02 rfb region. The plasmids pPA1343, pPA1344 and pPA1345 are examples of clones containing the 02 rfb region. Accordingly in a preferred form, there is provided a DNA probe including a plasmid selected from plasmids pPA1343, 1344 or 1345 or a digestion derivative thereof. This has great potential in the development of vaccines against diseases caused by Enteobacteriaceae where the O-antigen is a protective antigen, that is, where the protection is serotype specific. These clones also provide a basis for developing further probes. Similarly this approach has also been used to clone the genes for the 0157 rfb region. Plasmid pPM1354 represents an example of such a clone.

Accordingly in a preferred form, there is provided a DNA probe including a plasmid pPM1354, or a digestion derivative thereof. This clone has potential for both vaccine development and for specific probes.

E.coli of the 0157 serotype are responsible for haemorrhatic uraemic syndrome and are referred to as

Enterohaemorratic E.coli and EHEC. This is a severe condition and can often lead to death, and vaccines against this disease are not presently available.

In summary, the advantages of such probes include: the presence of rfb and closely linked DNA can be detected even in the absence of gene expression, a problem which may arise if the O-antigen is incompatible with the core region of the recipient organism, or if the recipient is expressing its own O-antigen and effectively out-competing the O-antigen encoded by the cloned DNA; the identification of an rfb clone is greatly simplified; a restriction map of the region to be cloned can be determined beforehand by Southern DNA hybridization, and this could permit a better choice of restriction enzyme for cleaving the chromosomal DNA prior to cloning.

In addition the probes enabled us to differentiate two 0115 strains, one which was an enterotoxigenic E.coli (ETEC) and another which was associated with septicaemia. Thus, these results, with the 02, 018 and 0115 strains, illustrate that the probes may also enable us to differentiate organisms which are apparently of the same serotype but produce different diseases. This is extremely important because of course some E.coli serotypes are part of natural human bacterial flora.

The DNA probes according to the present invention may be labelled in any suitable manner. A radioactive or non-radioactive label may be used. Where a radioactive labelled probe is used, radioactive isotopes of phosphorus or sulphur e.g. 32 phosphorus and 35 sulphur may be used. Where non-radioactive labelling is preferred, a biotinylated photo-sensitive label may be selected.

In a still further aspect of the present invention there is provided a method for preparing a DNA probe which method includes providing a plasmid cloning vector; a first restriction enzyme; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae; partially digesting the plasmid cloning vector with the restriction enzyme; and inserting the fragment of DNA into a restriction site.

The restriction site chosen may correspond to the restriction enzyme used or be compatible therewith.

The plasmid cloning vector may be of any suitable type* ^Tne plasmid cloning vector pHC79 has been found to be suitable. Since this plasmid cloning vector includes a BamHI restriction site, a corresponding BamHI or Sau 3A restriction enzyme is preferred.

In a preferred form, the ligated DNA so formed may be packaged in vitro and transduced into a suitable bacterial strain.

An E.coli strain may be used. An E.coli K-12 strain may be used. The E.coli K-12 strain DH1 has been found to be suitable. In a preferred aspect where subclones are required the method of preparing a DNA probe may further include providing a plurality of restriction enzymes; and digesting the ligated DNA with the restriction enzymes to produce restriction fragments of differing size. The restriction enzymes may include Mlul, Kpn 1,

Sail, BamHI, Sad. PstI, Hpal, Clal, HindiII and the like.

Where the fragments include protruding ends, the fragments may be subjected to the further step of filling the protruding ends so formed. This may be undertaken utilizing the Klenow fragment of DNA polymerase 1.

The sub clones so formed may include the plasmids pPM 1321, pPM 1322, pPM 1323, pPM 1324, pPM 1325, pPM 1326, pPM 1330 and pPM 2218. The plasmids pPM 1321, pPM 1330 and pPM 1326 are preferred as they have been found to be highly conserved. It is hypothesized that the plasmids encode part of the region that specifies the OlOl serotype and may be associated with any common properties of the O-antigen chains that allow ETEC to be more efficient pathogens or inhabitants of the intestinal tract are likely to be found encoded in this region of the rfb cluster.

Accordingly in a further aspect of the present invention there is provided a method of diagnosing a disease which method includes providing a sample to be tested; and at least one DNA probe including a fragment of DNA which probe detects the presence of the rfb region of an Enterobacteriaceae; contacting the sample with the at least one DNA probe; and _ _

subjecting the product thereof to an assay. In a preferred form of this aspect of the present invention the method of diagnosis may in addition provide a method of serotyping the organism responsible for the disease. In this form the method of diagnosis may include providing a series of samples to be tested; and a DNA probe kit including a first DNA probe including a fragment of DNA which detects the presence of the rfb region of an Enterobacteriaceae: and a second DNA probe including a fragment of

DNA which detects the presence of the rfb region of an Enterobacteriaceae in a manner different to said first probe such that the first and second probes are serotype specific; contacting each sample with a different DNA probe from the kit; and subjecting the products thereof to a series of assays.

The one such sample to be tested may be of any suitable type. A faecal sample may be used.

The or each DNA probe is preferably a radioactively or non-radioactively labelled DNA probe, as described above. The product may be assayed in any suitable manner.

A Western and/or color blot analysis may be used.

The plasmids including restriction fragments of DNA including substantially the whole of the rfb locus of an

Enterobacteriacae, for example plasmid pPM 1305 may be inserted into a suitable avirulent bacterial carrier strain which may in turn function as a live vaccine. As stated above, where the Enterobacteriaceae is an E.coli of the 0101 serotype, we have found that a fragment having a minimum length of approximately 13 Kb and a maximum length of approximately 15.8 Kb is required for 0101 0-antigen biosynthesis when it is introduced into E.coli K-12.

Accordingly in a still further aspect of the present invention there is provided a method of preparing an avirulent strain of an enterobacterium which method includes providing a sample of a preselected bacterial strain; and a plasmid including a fragment of DNA containing genes encoding for the synthesis of the

O-antigen of an Enterobacteriaeae; and inserting the cloned genes on the plasmid into the chromosome of the preselected bacterial strain.

The plasmid may be plasmid pPM 1305.

The preselected bacterial strain may be selected from Salmonella and Shiqella strains. Salmonella tvphimurium. Salmonella typhi and Salmonella dublin have been found to be suitable.

Diseases of interest may include those generated by any enterobacterial strain.

The avirulent strain of an enterobacterium referred may provide the basis for a vaccine composition.

The vaccine composition may be used as a live oral vaccine against enteropathogenic diseases of man or domestic animals.

The genes for group- (rfb) and type-antigen biosynthesis of Shiqella flexneri may be introduced into the preselected strain. Further changes may include introduction of the Shiαella LPS core biosynthesis genes (rfa) so that the strain expresses the O-antigen of the appropriate Shiqella specificity on the cell surface. Analysis of the LPS by silver staining, ELISA and haemagglutination inhibition may be used to assay the production of the O-antigen. Immunogenicity may be assessed in mice, and rabbits and ultimately monkeys.

Accordingly in a still further aspect of the present invention there is provided a vaccine composition useful for effecting immunity against diseases of an Enterobacteriaceae, including a bacterial carrier strain; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae, inserted into the chromosome of the bacterial carrier strain.

The bacterial carrier strain may be selected from Salmonella and Shiqella strains.

The Enterobacteriaceae is preferably an E.coli of the 0101 serotype and the fragment of DNA has a munimum length of approximately 8.9 kilobase pairs, and a maximum length of approximately 11.8 kilobase pairs.

Alternatively the Enterobacteriaceae is an E.coli of the 0157 or 02 serotype. The vaccine composition may be provided in any suitable form. The vaccine composition may be provided in an oral or injectable form. Suitable carriers including aqueous and/or alcoholic carriers may be included. A buffered saline solution may be used. Other recipients and compounding ingredients known per se in the art may be included if desired.

In a further aspect of the present invention there is provided a method for the prophylactic treatment of diseases of an Enterobacteriaceae in mammals, including 5 humans, which method includes providing a mammal to be treated; and a vaccine composition useful for effecting immunity against diseases of an Enterobacteriaceae. including 0 a bacterial carrier strain; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an

Enterobacteriaceae. inserted into the chromosome of the bacterial carrier strain; and 5 administering to the mammal a prophylactically effective amount of the vaccine composition.

In a particularly preferred aspect of the present invention there is provided a method for the prophylactic treatment of diseases of E.coli of the 0157 serotype in o mammals, including humans, which method includes providing a mammal to be treated; and a vaccine composition including a Salmonalla or Shiqella carrier strain; and/or a fragment of DNA derived from plasmid pPM1354, or a digestion derivative thereof; and administering to the mammal a prophylatically effective amount of the vaccine composition. _Q E.coli of the 0157 serotype are responsible for haemorrhatic uraemic syndrome and are referred to as Enterohae orratic E.coli and EHEC.

This is a severe condition and can often lead to death, and vaccines against this disease are not presently available.

The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

In the Figures: Figure 1

Comparison by electrophoresis in SDS on 20% polyacrylamide gels of whole membrane material from H510a, B41, DH1, E963 (DHl[pPM1305]), VAC1676, KATI1706 and VC1751 by Western Blot analysis.

After electrophoretic transfer the LPS Material was visualized using 0101 typing serum raised against B41 (1:1000) followed by the addition of goat anti rabbit-IgG coupled to horseradish peroxidase and the addition of peroxidase substrate. Figure 2

Sarkosyl extracted membrane material from strains DH1, E11176, E963, H510a, B41, VAC1676, KATI1706, VC1751 and 8CE275/6 were solubilised in SDS and electrophoresed on 7-25% polyacrylamide exponential gradient gels as described in (10). The gels were fixed so that predominantly LPS or lipoproteins were stained with silver (28) . Figure 3

Restriction endonuclease cleavage pattern and deletion analysis of plasmid pPM 1305. The thicker region corresponds to residual pHC79. Plasmid DNA of single and double digests were analysed in 0.8% and 1.0% agarose gels in TBE buffer. The lines indicate the extent of cloned DNA retained in each of the derivatives of pPM1305. Their ability to mediate synthesis of 0101 O-antigen is indicated. pPM 1305 was initially digested with Mlul and insert DNA ligated into the Mlul sites of pLG339 (Stoker et al. 1982) forming pPM1330. Plasmids pPM2218 and pPM2219 represent Sail and Hpal deletions of pPM1305. Plasmids of pPM2220 and PPM2221 were generated by and filling of the protruding ends after BamHI cleavage and by removing the protruding ends after digesting with Sad respectively.

Six DNA fragments defined by the PatI restriction endonuclease sites of pPM1305 were subcloned into the PatI silo of pGB2 (Churchward et al., 1904). Analysis of minimal coding region for the 0101 E.coli rfb region indicates that subclones pPM1322, pPM1324, pPM1325 and pPM1326 all contain rfb coding DNA, and that pPM 1321 and pPM1323 may contain a combination of flanking and rfb DNA. The thicker line represents residual pHC79 DNA. Figure 4 An autoradiograph of an SDS polyacrylamide gel

35 showing [ S]-labelled proteins produced from whole minicells carrying plasmid pPM1305 and the cloning vector pHC79. The exposure was for seven days at room temperature without fluorography. - -

Figure 5

Autoradiographs showing Southern hybridisation analysis of restriction endonuclease-digested whole genomic

DNA from E.coli 0101 isolates, using (Pj-labelled pPM1322 (Panel A, Psi); Panel B, EcoRi) and pPM1324 (Panel C, EcoRi;

Panel D, PatI as probes. The stringency in the final washes of the nitrocellulose filter was 65°C in 2 x SSC. The developing time was 2d using Ilford intensifying screens at

-70°C. Figure 6

Chromosal DNA from serotypic variants of 018 and 02

E.coli was digested with restriction endonucleases PstI,

EcoRI and Hindlll in all combinations of single and double digests. The DNA samples were electrophoresed on 0.8% agarose and subsequently transferred to nitrocellulose by the method of Southern.

The two subclones pPM1321 and pPM1326 from the 0101 rfb clone pPM1305 are believed to represent a highly conserved region of DNA within the rfb gene cluster. These subclones were used as radio-labelled probes to detect restriction fragment length polymorphisms (RFlPs) within 018 and 02 E.coli.

The restriction maps obtained (D) from using these probes in Southern hybridisation analysis (A, B, C) show the high degree of relatedness between 018 and others not shown on E.coli and the probable close evolutionary relationships.

Figure 7

Chromosomal DNA from the Shigella flexneri strains

PE532, PE524, PE525, PE526 and PE527 corresponding to serotypes 2a, 4a, 6, 2a and 4a/y, respectively, as well as a selection of randomly chosen E.coli strains of the serotypes indicated, were digested with Pstl. the fragments separated by electrophoresis in 0.8% agarose. The DNA was then transferred to nitrocellulose and replicates were probed in Southern DNA hybridization with the various subclonal fragments of pPM1305. The radiolabelled probes used were A (PPM1321); B (pPM1326); C (pPM1325); D (pPM1322); E (pPM1324) and F (pPM1323).

The hybridizations were performed at high stringency; 0.2 x SS C at 65°C. The autoradiography was exposed for 2 days at - 70°C using intensifying screens.

EXAMPLE 1 Bacterial strains Media All strains were routinely grown in Nutrient broth

(Difco Research Laboratories Detroit, Mich.) made at double strength with 5g/l NaCl added. Nutrient agar was blood agar base (Difco) without the additoin of blood. Where appropriate, ampicillin, media (25ug/ml) and tetracycline (lOug/ml) were added. Cultures for routine use were maintained as 15% glycerols at -20 C. Antiserum

Typing serum for 0101 raised against B41 in rabbits was obtained from C. Murray, IMVS, Adelaide. This was extensively adsorbed with E.coli K-12 strain DHL Preparation of genomic DNA

The isolation of genomic DNA was performed essentially as described by Manning et al (14) . Cells from a 20ml shaking Overnight culture were pelleted (3000xg; 10 min.) and washed once, the pellet treated with lysozyme and then the sarkosyl lysing solution, followed by phenol and ether extraction. The DNA solution was then extensively dialysed against TE buffer (lOmM Tris-HKCl, ImM EDTA, pH 8.0). Plasmid DNA isolation As previously described in (14), this method is essentially a three step alkaline lysis, followed by CsCl centrifugation on a 2-step gradient (Garger et al 1983). Transformation of Plasmid DNA

Transformation of E.coli K-12 strains was perfomred as previously described (9). Competent cells were made by

MgC -CaC incubation on ice. Although cells were assumed to be competent after 60 min. on ince, they were routinely left overnight on ice.

Competent cells (0.2ml) were mixed with 1 ug DNA, incubated on ice for 30 min, then heat shocked (42°C) for 2 min. The cells were then allowed to express in nutrient broth for 60 min. at 37°C with shaking and the mixture plated onto the appropriate antibiotic plate. Molecular cloning

Using genomic DNA from B41, a cosmid library was constructed as previously described (14) . 1-2 ug of genomic DNA partially digested with Sau3A was cloned into the BamHI site of 6 ug alkaline phosphatase treated BamHI digested pHC79 DNA. The ligated DNA was packaged in vitro and transducted into E.coli K-12 strain DHL This procedure usually yielded 1000-2000 ampicillin resistant colonies. Minicell analysis and autoradiography

Plasmids were transformed into the minicell producing stran DS410. Minicells were purified on sucrose gradients and the plasmid encoded proteins were labelled with -x c t S3-methionine and solubilized in sample buffer as previously described (13) . After electrophoresis in SDS on 11-20% polyacrylamide gels, the dried gel was subjected to autoradiography using Kodak X-omat film at room temperature. Restriction analysis

Restriction enzyme digestion of DNA was carried out as recommended by the manufacturers. The digested DNA was analysed in horizontal 0.8% w/v agarose gel system in TBE buffer (67mMTris, 22mM Boric acid, 2mM EDTA ρH8.8). All gels for size determination of plasmid DNA fragments or restriction endonuclease digested genomic DNA for Southern transfer were electrophoresed overnight at 20v. Gels were stained in ethidium bromide (2ug/ml) and visualized under UV light. The sizes of restriction fragments were determined by their relative mobility to Bacillus subtilis bacteriophage SPP1 DNA cut with EcoRi 22) . End filling with Klenow fragment

Protuding ends created by cleavage with BamHI and SacI were filled or removed using the Klenow fragment of DNA polymerase I. Typically, 1.6ug of digested DNA, 2 ul of each dNTP (2mM) and 5 units of Klenow fragment (Pharmacia) were mixed and left at room temperature for 30 min. The reaction was stopped by the addition of lul of -.5mM EDIA and incubation at 65°C for 15 min. Unincorporated dNTPs and enzyme were removed by centrifugation through a Sepharose C1-6B column.

SDS-Polvacrylamide gel electrophoresis (SDS-PAGE) and silver staining for lipopolysaccharide

Analyses of lipopolysaccharides on SDS-PAGE and the preparation of samples is identical to that of Kusecek et al. _ _

(1984). Strains for analysis were grown in 100 ml of L-broth. The cell envelopes were extracted with sarkosyl to enrich for outer membranes (Achtman et al., 1983). After suitable dilution, these were loaded onto 7-25% exponential gradient gels and run at 10mA for 16h. Silver staining for lipopolysaccharide was carried out according to Tsai and Frasch (1982). Western and Colony Blot Analysis

Colony and Western blots were performed using a 1:1000 dilution of 0101 typing serum as primary antiserum. The secondary antiserum was goat anti-rabbit IgG coupled to horseradish peroxidase (Nordic Immunology) at 1:5000 final dilution. The methods in detail have been described previously (Manning et al. 1986). Protein A-colloidal gold

Colloidal gold particles (c. 15-20nm) were prepared using the citrate method as described (de Mey and Meormans, 1986) and conjugated with Protein A (Pharmacia) . Immunoqold labelling of whole cells

Cells grown on CFA agar for 18h at 37°C were resuspended and washed once in phosphate-buffered saline (pH 7.2) containing 1% bovine serum albumin (Fraction V, FLow Laboratories, North Ryde, Australia) (PBS + BSA) . A drop (35 ul) of cell suspension was placed on a sheet of parafilm. A poly-t-lysine treated formvar electron microscope grid was then placed plastic side down on the surface of this drop for 2 min. (Mazia et al 1978). This grid was then transferred to drops of the following reagents placed on the same parafilm sheet: PBS + BSA x 2; antibody (1:200 dilution in saline) for 15 min.; PBS + BSA x 3; Protein A-gold (1:50 dilution in PBS + BSA); distilled wter x 3. After completing the protocol, excess fluid was removed from the grid and the grid allowed to air-dry without negative staining. In some experiments, immunogoId-labelled preparations were negatively stained with a solution of 2% w/v uranyl acetate. Grids were examined in a JEOL JEM 100S transmission electron microscope operated with an accelerating voltage of 80kV. Nick Translation of PPM1305 and Southern Analysis

Plasmid DNA was Nick translated with DNA Polymerase i and Southern DNA hybridization analysis (Southern, 1975) was carried out according to a modification of Maniatis et.al. (1982). [³²p3 labelled DNA of pPM1305 and its subclones were used to probe suitably digested genomic DNA from a number of 0101 isolates. Autoradiography was carried out at -70°C on Kodak X-omat film, with intensifying screens. The exposure time was 3 days.

RESULTS Molecular cloning of (rfb) region for 101 LPS biosynthesis

Genetic evidence from E.coli. Salmonella and Vibrio cholerae demonstrates that the genes for the biosynthesis of the O-antigen is determined by a gene cluster referred to as the rfb (Nikaido et al. , 1987; Brahmbatt et al. 1986; Manning et al. 1986; Ward et al 1987). Thus, a cosmid genomic library of approximately 1000 colonies was screened using 0101 typing serum, made against type strain B41 (Orskov et al. , 1987), and which had been extensively adsorbed with E.coli K-12 strain DHL We detected 13 clones demonstrating various degrees of reactivity on colony blot. Initial Western blot analysis of whole membrane material showed that the majority of the clones owed their reactivity to the expression of cloned genes for proteins (data now shown) . However, one strain, DHl (pPM1301) produced a smear-like pattern, suggesting that it could mediate the expression of genes responsible for synthesis of LPS-like material. This clone was confirmed as 0101 serotype by the E.coli reference laboratory of the Institute of Medical and Veterinary Science in Adelaide, South Australia. Western blot analysis of whole-membrane material from DHl (pPM1305) a subclone of pPM1301 (see below) along with other known 0101 E.coli isolates using antiserum raised against B41 revealed that indeed DHl harbouring pPM1305 produces LPS O-antigen material similar in reactivity to the other 0101 isolates (data not shown) . Immunogold electron microscopy using the 0101 typing serum demonstrates that not only is the O-antigen of the appropriate specificity but also that it is surface-located in a similar fashion to B41 (Figure 1).

When sarkosyl-extracted membrane material is run in SDS on 7-25% polyacrylamide exponential gradient gels, there are variations in the O-antigen banding patterns of different 0101 isolates (Figure 2). For example, type strain H510a (Orskov et al., 1977) appears as a series of doublets of slightly different electrophoretic mobility. The E.coli K-12 strains harbouring pPM1305 and pPM1330 (a low copy number plasmid; see below) exhibit a singlet banding pattern which differs somewhat from the pattern of B41, although it still reacts with 0101 antiserum on a Western blot (data not shown) . These differences presumably reflect a difference in lipopolysaccharide core composition or incomplete expression or modification in E.coli K-12. _ _ _.

Restriction analysis of cloned DNA

The cosmid pPM1301 was reduced in size in a two stage process: a partial Hindlll digestion to produce

PPM1302 followed by a subsequent partial Mlul digestion to

5 give pPM1305. A restriction map of pPM1305 which contains

16.4kb of cloned DNA, is shown in Figure 3.

A more accurate estimate of the minimal coding region of 1010 O-antigen biosynthesis in pPM1305 was determined by subcloning a series of fragments into low copy 0 number vector pLG339 (Stoker et al., 1982) and by constructing a series of derivatives (Figure 3) . Only plasmids pPM1330 (Mlul-Mlul subclone) and pPM2218 (Sail deletion) expressed O-antigen, as judged by silver staining for LPS (Figure 2) . pPM1330 also exhibits superior plasmid 5 stability compared to pPM1305 (data not shown) . NOne of the other derivatives produced detectable levels of O-antigen (Figure 3). Therefore the coding region of the 0101 rfb gene cluster must extend from the region defined by the Sall-Hpal restriction endonuclease sites at (6.0-7.3kb) to the region o between the Sacl-Mlul sites (at 16.2-17.8 kb) ; i.e. a minimum of 8.9 kb and a maximum of 11.8 kb of DNA is required for

O-antigen biosynthesis.

35 Analysis of ϊ S3 - labelled proteins encoded bv pPM1305 in minicells 5 Examination of the proteins encoded by pPM1305 in minicells revealed six new polypeptides produced in various amounts with sizes of 80, 79, 52, 42, 38 and 24 kD (Figure 4) . The sum of the sizes of these proteins would require a coding region of about 9kb of DNA, assuming there are no ₀ overlaps and no non-coding regions between the structural genes. This correlates very closely to the amount of DNA required for antigen biosynthesis. Since [ 35S]-methionine was used, it is also possible that proteins lacking methionine or produced in very low amounts are also involved in 0101 synthesis and have not been detected. It has not been possible to determine whether all of these proteins have a role in O-antigen biosynthesis. Attempts using transposon insertion analysis have invariably resulted in the simultaneous generation of deletion plasmids, none of which expresses O-antigen (data not shown) .

DNA hybridization analysis using subclones of pPM1305 as probes . Colony hybridization analyses using [ 32P3 labelled pPM1305 as a probe were performed to determine whether this plasmid is useful as a specific probe for 0101 rfb region. These experiements (data not shown) revealed that pPM1305 could hybridize (in most cases weakly) to all

E.coli isolates tested, regardless of the O-serotype. There was no hybridization with eithr V.cholerae, Salmonella or the E.coli K-12 rfb delete strain, QE35 (Sunshine and Kelly, 1971) . Interestingly, this probe reacts strongly with several apparently immunologically unrelated serotypes and the basis for this is being examined.

In order to determine whether the differences in O-antigen banding pattern on SDS-PAGE gels are reflected at the DNA level in the rfb locus, the six PatI fragments of PPM1305 were subcloned into vector pGB2 (Churchward et al., 1984) (Figure 3). Southern hybridization analyses using selected subclones as probes are shown in Figure 5. The two largest PstI fragments, pPM1322 and pPM1324, known to be required for O-antigen biosynthesis were used to determine the level of homology in the rfb locus among other 0101 isolates. When the largest probe pPM1322 is employed, htere appears to be extensive conservation of restriction sites in the rfb region of B41 and the other ETEC isolates as well as isolate 8CE275/B (non-ETEC) . Strain H510a (non-ETEC) does not appear to show the same level of homology with B41 as the other isolates. Strain B41 itself, whilst sharing common restriction fragments with the other isolates, also shows evidence of DNA sequence duplication. When pPM1324 is used, the close relationship with the other 0101 isolates with the exception of strain H510a is evident, as is sequence duplication in B41. The control strains QE35 (rfb delet) and DHl have no sequences in common with these probes.

The results of the Southern hybridization analysis using the two largest internal probes pPM1322 and pPM1324 with the 0101 isolates and E.coli K-12 strains Q35 and DHl are shown in Figures 6 and 7.

Figure 6 shows that when pPM1322 is used as a probe under conditions of high stringency, this fragment appears to be highly conserved among all isolates. The strain H510a, which has a unique LPS banding pattern, while hybridizing with sequences on pPM1322 exhibits a markedly different pattern of fragments, not surpisingly there is no detectable homology with the rfb delete strain Q35 or the E.coli K-12 host strain DHL

Figure 7 shows that when pPM1324 is used as a probe under the same conditions as pPM1322, H510a exhibits a unique pattern, whereas 8CE275/6, VAC1676 and VC17651 exhibit identical patterns. Strains B41 and KAT11706 have a fragment in common with the other isolates (except H510a) . No hybridization is seen with strains Q35 or DHl. Interestingly, this probe fails to detect an identical fragment in B41, suggesting that all of the cloned DNA in ρPM1323 and pPM1324 is not contiguous, probably due to the deletion process by which they were generated. Discussion

We have described the cloning and expression in E.coli K-12 of the rfb region derived from an Enterotoxigenic E.coli of the 0101 serotype. The LPS expressed by the two clones, DHl (pPM1305) and DHl(ρPM1330) is significantly different electrophoretically from that of the parent strain B41. This may be because of a different host genetic background, possibly reflecting a difference in the LPS core structure. It is already known that several different core structures exist in E.coli (for a review, see Luderitz et al, 1982) . It may also reflect modification of the O-chain by strain DHl.

At least six proteins are encoded within the minimal coding region, implicating them in the synthesis and assembly of the O-subunit. Production of the 0101 O-antigen as LPS in an rfb delete strain QE35 implies that only the rfb genes carried by the clones are necessary for its expression and assembly on the E.coli K-12 core (data not shown). Since both structure of 0101 LPS is unknown, and the precise minimal coding region is not defined, it is not possible to say whether all of the proteins expressed in minicells or other additional undetected proteins are required for the synthesis of 0101 O-antigen. Subcloning and deletion analysis suggests that the predicted 9kb required for the _ _

synthesis of these proteins is a good estimate of the minimal amount of coding DNA.

The 0101 isolates that we have examined appear to exhibit some variability in the LPS, as judged by SDS-PAGE. This difference is not detected serologically with polyvalent antiserum. There are no reports in the literature of 0101 serotype variants, despite the fact that it is a common animal ETEC serotype. It is not yet possible to speculate on the chemical relationships between the different 0101 patterns. The genetic relatedness, as judged by DNA hybridization, one may predict that these differences may simply reflect minor modifications in the O-chain. Current data suggest that multiple 0101 LPS types exist: one type defined by H510a and other types observed in B41, KATH706, 8CE275/6, VAC1676 and VC1751. The DNA hybridization data suggest that the enzymes involved in H510a LPS biosynthesis are less closely related to the other types which share common restriction fragments. The distinct LPS pattern on SDS-PAGE of B41 may reflect the sequence of gene duplication that we have observed in this strain, since the clones of the rfb region of B41, i.e. DHl(pPM1305) and DHl(pPM1330) more closely resemble the LPS patterns of the other 0101 isolates.

The possibility tha 0101 E.coli belongs to the "clonal" groupings described by Achtman and Plsuchke (1986) has yet to be explored. It is possible that the non-H510a LPS types form a closely related "clonal" grouping that is often found in association with a specific disease in animals, and so the disease may be considered as part of the "clonal" character of the strain. Subclones of pPM1305 show homology to strains not of _ _

the 0101 serotype and may be useful in determining the relationships between strains of serotypes other than 0101. Since these probes can hybridize to serologically unrelated serotypes (data not shown; Beger et al. , 1988), they may also form the basis for developing rapi diagnostic probes capable of differentiating disease-causing organisms from the commensal flora of the same serotype. Conventional serotyping has not provided a means of identifying disease-causing organisms. The detection of O-serotype variants by DNA hybridization analysis may provide a more specific method for identifying pathogenic organisms.

The availability of cloned rfb 0101 genes also provides a basis for the construction of a hybrid vaccine strain in avirulent Salmonella (Stevenson and Manning, 1985), since it has been documented that antibodies to 0101 are more hghly protective against 010LK99/F41 challenge than antibodies to the colonization factors or adhesins K99 or F41 (Duchet-Suchaux, 1986).

TABLE 1

Bacterial Strains

Strain Genotype/Phenotype Source

DHl F^" ^"gyrA96.recAl,endAl.thi-1.hsdR17.SUPE B.Bachmann

QE35 delratt2PH-mgl387.relAl,SPOTLthi-L B.Bachmann

DS410 F~.minA.minB,rpsL D. Sherratt

B41 0101:K99/F41 F. and I. Orskov

H510a 0101:H33 F. and I. Orskov

8CE275/6 010LK103 F. and I. Orskov

VAC1676 0101:K30:H~(3P^") H. Moon

KATI1706 0101:K30:H~(3P~) H. Moon

VC1751 0101:K27:H~(3P~) H. Moon

E963 DHl(pPM1305);aml^r;0101-Oag⁺ This study

E1176 DHl(pPM1330);tet^r;0101-Oag⁺ This study

E1159 DHl(pPM1321) ;Spc^r;str^r This study

E1160 DHl(pPM1322);Spc^r;str^r This study

E1161 DHl(pPM1323);Spc^r;str^r This study

E1197 DHl(pPM1324);Spc^r;str^r This study

E1198 DHl(pPM1325);Spc^r;str^r This study

E1199 DHl(pPM1326);Spc^r;str^r This study

E1102 DHl(pPM1329);tet^r This study

E1203 DHl(pPM1331) ;tet^r This study

E1204 DHl(pPM1332) ;tet^r This study

E1205 DHl(pPM1333) ;tet^r This study

E1206 DHl(pPMΪ334);tet^r This study _ _

LITERATURE CITED

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(6) Churchwald, G., D. Belin and Y. Nagamine. 1984. A pSClOl derived plasmid which sows no sequence homology to other commonly used cloning vectors. Gene. 31 : 165-171.

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(15) Morris, J.A., and W.J. Sojka, 1985. Escherichia coli as a pathogen in animals. pp 47077 in The Virulence of Escherichia coli : Reviews and Methods. M. Sussman, Ed., Academic Press, (London).

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(17) Orskov, I., F. Orskov, B. Jann, and K. Jann, 1977. Serology, chemistry and genetics of 0 and K antigens of Escherichia coli. Bacteriol. Rev. 1 : 667-710.

(18) Osborn, M.J., 1979. Biosynthesis and assembly of the lipopolysaccharide of the outer membrane, PP15-34. In M. Inouye (Ed)., Bacterial outer membranes, John Wiley and Sons, Inc., New York.

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Rev. Microbio. 34 : 369-422.

(20) Pluschke, G., A. Moll, B. Kusecek and M. Achtman, 1986. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and monoclonal antibodies as tools for the sub-grouping of Escherichia coli lipopoly¬ saccharide 018 and 023 antigens. Infect. Immun. 51 : 286-293.

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SPP1: the arrangement of restriction endonuclease generated fragments. Mol. Gen. Genet. 168 : 165-172.

(22) Smith, H.R., S.M. Scotland and B. Rowe, 1985. Genetics of Escherichia coli virulence. pρ227-269 in The Virulence of Escherichia coli : Reviews and Methods. M. Sussman, Ed., Academic Press (London). (23) Stoker, N.G., N.F. Fairweather and B.G. Spratt, 1982. Versatile low copy number plasmid vectors for cloning in Escherichia coli: Gene 18: 335-341. (24) Stocker, B.A.D., and P.H. Makela, 1971. Genetic aspects of biosynthesis and structure of Salmonella lipopolysaccaride pp.369-438. In G. Weinbaum, S. Kadis and S.J. Ajl (Ed.), Microbial Toxins, Vol.IV, Academic Press Inc., London. (25) Sunshine, M.G., and B. Kelly, 1971. Extent of host deletions associated with bacteriophase P2-mediated education. J. Bacteriol. 108 : 695-704.

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Ed., Academic Press (London).

(27) Tsai, C-M. , AND E. Frasch, 1982. A sensitive silver stain for detecting lipopolysaccharide in polyacrylamide gels. Anal. Biochem. 119 : 115-119.

(28) Ward, H.M., G. Morelli, M. Kamke, R. Morona, J. Hackett and P.A. Manning. 1987. A physical map of the choromosdomal region determining O-antigen biosynthesis in Vibrio cholerae 01. Gene 55 : 197-204. Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims

CLAIMS :

1. A DNA probe including a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae. which probe detects the presence of the rfb region of an Enterobacteriaceae.

2. A DNA probe according to claim 1 wherein the fragment of DNA contains genes encoding for the synthesis of the O-antigen of the 0101, 0156 or 02 serotype of an Escherichia coli.

3. A DNA probe according to claim 2 wherein the 0 fragment of DNA contains genes encoding for the synthesis of the O-antigen of the 0101 serotype of an Enterotoxigenic Escherichia coli.

4. A DNA probe according to claim 3 wherein the DNA fragment has a minimum length of approximately 8.9 kilobase 5 pairs and a maximum length of approximately 11.8 kilobase pairs.

5. A DNA probe kit including a first DNA probe including a fragment of DNA containing genes encoding for the synthesis of the O-antigen o of an Enterobacteriaceae, which probe detects the presence of the rfb region of an Enterobacteriaceae: and a second DNA probe including a fragment of DNA which detects the presence of the rfb region of an Enterobacteriaceae in a manner different to said first probe, ₅ such that the first and second probes are serotype specific.

6. A DNA probe including a plasmid cloning vector; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae inserted 0 into a suitable site in the plasmid cloning vector.

7. A DNA probe according to claim 6 wherein the DNA fragment contains genes encoding for the synthesis of the O-antigen of the 0101 serotype of an Enterotoxigenic Escherichia coli and has a minimum length of approximately 8.9 kilobase pairs and a maximum length of approximately 11.8 kilobase pairs; and wherein the plasmid cloning vector is the plasmid cloning vector pHC79.

8. A DNA probe according to claim 6, further including a radioactive of non-radioactive label.

9. A DNA probe including a plasmid from which rfb DNA has been deleted, selected from plasmids pPM 1305, pPM 1322, pPM 1323, pPM 1324, pPM 1325, pPM 1326, pPM 1330 and pPM 2218 as hereinbefore described, and mixtures thereof.

10. A DNA probe containing genes encoding for the synthesis of the O-antigen of an Escherichia coli of the 0157 serotype and including a plasmid pPM 1354, or a digestion derivative thereof.

11. A DNA probe containing genes encoding for the synthesis of the O-antigen of an Escherichia coli of the 02 serotype and including a plasmid selected from plasmids pPA 1343, pPA 1344 or pPA 1345, or a digestion derivative thereof.

12. A method for preparing a DNA probe which detects the presence of the rfb region of an Enterobacteriaceae. which method includes providing a plasmid cloning vector; a first restriction enzyme; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae; partially digesting the plasmid cloning vector with the restriction enzyme; and ligating the fragment of DNA into a restriction site on the plasmid cloning vector.

13. A method according to claim 12, further including providing a plurality of restriction enzymes; and digesting the ligated DNA with the restriction enzymes to produce restriction fragments of differing size.

14. A method according to claim 13, wherein the restriction enzymes are selected from Mlul. Kpn 1, Sail. BamHI. Sad. PstI, Hpal, Clal and Hindlll.

15. A method of diagnosing a disease, which method includes providing a sample to be tested; and at least one DNA probe including a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae. which probe detects the presence of the rfb region of an Enterobacteriaceae; contacting the sample with the at least one DNA probe; and subjecting the product thereof to an assay.

16. A method according to claim 15, wherein the sample is a faecal sample; the at least one DNA probe is a radioactively or non-radioactively labelled DNA probe; and the assay is selected from Western and/or color blot analysis thereof.

17. A method of serotyping an organism responsible for a disease, which method includes providing a series of samples to be tested; and a DNA probe kit including a first DNA probe including a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae. which probe detects the presence of the rfb region of 0 an Enterobacteriaceae: and a second DNA probe including a fragment of

DNA which detects the presence of the rfb region of an Enterobacteriaceae in a manner different to said first probe, such that the first and ₅ second probes are serotype specific; contacting each sample with a different DNA probe from the kit; and subjecting the product thereof to a series of assays.

18. A method of preparing an avirulent strain of an o enterobacterium which method includes providing a sample of a preselected bacterial strain; and a plasmid including a fragment of DNA containing genes encoding for the synthesis of the ₅ O-antigen of an Enterobacteriaceae: and inserting the cloned genes on the plasmid into the chromosome of the preselected bacterial strain.

19. A method according to claim 18, wherein the preselected bacterial strain is selected from Salmonella and 0 Shigella strains.

20. A method according to claim 19 wherein the Enterobacteriaceae is an E.coli of the 0101 serotype and the fragment of DNA has a minimum length of approximately 8.9 kilobase pairs, and a maximum length of approximately 11.8 kilobase pairs.

21. A method according to claim 19 wherein the Enterobacteriaceae is an E.coli of the 0157 or 02 serotype.

22. A vaccine composition useful for effecting immunity against diseases of an Enterobacteriaceae. including a bacterial carrier strain; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an Enterobacteriaceae. inserted into the chromosome of the bacterial carrier strain.

23. A vaccine composition according to claim 22 wherein the bacterial carrier strain is selected from Salmonella and

Shigella strains.

24. A vaccine composition according to claim 23 wherein the Enterobacteriaceae is an E.coli of the 0101 serotype and the fragment of DNA has a minimum length of approximately 8.9 kilobase pairs, and a maximum length of approximately 11.8 kilobase pairs.

25. A vaccine composition according to claim 23 wherein the Enterobacteriaceae is an E.coli of the 0157 or 02 serotype.

26. A method for the prophylactic treatment of diseases of an Enterobacteriaceae in mammals, including humans, which method includes providing a mammal to be treated; and a vaccine composition useful for effecting immunity against diseases of an Enterobacteriaceae. including a bacterial carrier strain; and a fragment of DNA containing genes encoding for the synthesis of the O-antigen of an

Enterobacteriaceae, inserted into the chromosome of the bacterial carrier strain; and administering to the mammal a prophylactically effective amount of the vaccine composition.

27. A method according to claim 26 wherein the bacterial carrier strain of the vaccine composition is selected from Salmonella and Shigella strains.

28. A method according to claim 27 wherein the Enterobacteriaceae is an E.coli of the 0101 serotype and the fragment of DNA has a minimum length of approximately 8.9 kilobase pairs, and a maximum length of approximately 11.8 kilobase pairs.

29. A method according to claim 27 wherein the Enterobacteriaceae is an E.coli of the 0157 or 02 serotype.

30. A method for the prophylactic treatment of diseases of E.coli of the 0157 serotype in mammals, including humans, which method includes providing a mammal to be treated; and a vaccine composition including a Salmonella or Shigella carrier strain; and a fragment of DNA derived from plasmid PPM1354, or a digestion derivative thereof; and administering to the mammal a prophylactically effective amount of the vaccine composition.

31. A method according to claim 30 wherein the disease is haemorrhatic uraemic syndrome.