# Patent

## Title
Nucleic acid-based methods of detecting bacterial o-antigens

## Abstract

The present invention relates to method of testing a sample for the presence of nucleic acids encoding E. coli polysaccharide O-antigen serotype O111, S. enterica polysaccharide O-antigen serotype C2 and S. enterica polysaccharide O-antigen serotype B.

## Background
TECHNICAL FIELD
The invention relates to novel nucleotide sequences located in a gene cluster which controls the synthesis of a bacterial polysaccharide antigen, especially an O antigen, and the use of those nucleotide sequences for the detection of bacteria which express particular polysaccharide antigens (particularly O antigens) and for the identification of the polysaccharide antigens (particularly O antigens) of those bacteria.
BACKGROUND ART
Enteropathogenic E. coli strains are well known causes of diarrhoea and haemorrhagic colitis in humans and can lead to potentially life threatening sequelae including haemolytic uremic syndrome and thrombotic thrombocytopaenic purpura. Some of these strains are commonly found in livestock and infection in humans is usually a consequence of consumption of contaminated meat or dairy products which have been improperly processed. The O specific polysaccharide component (the “O antigen”) of lipopolysaccharide is known to be a major virulence factor of enteropathogenic E. coli strains.
The E. coli O antigen is highly polymorphic and 166 different forms of the antigen have been defined; Ewing, W. H. [in Edwards and Ewings “Identification of the Enterobacteriacea” Elsevier. Amsterdam (1986)] discusses 128 different O antigens while Lior H. (1994) extends the number to 166 [in “Classification of Escherichia coli In Escherichia coli in domestic animals and humans pp31–72. Edited by C. L. Gyles CAB International]. The genus Salmonella enterica has 46 known O antigen types [Popoff M. Y. et al (1992) “Antigenic formulas of the Salmonella enterica serovars” 6th revision WHO Collaborating Centre for Reference and Research on Salmonella enterica, Institut Pasteur Paris France].
An important step in determining the biosynthesis of O antigens-and therefore the mechanism of the polymorphism has been to characterise the gene clusters controlling O antigen biosynthesis. The genes specific for the synthesis of the O antigen are generally located in a gene cluster at map position 45 minutes on the chromosome of E. coli K-12 [Bachmann, B. J. 1990 “Linkage map of Escherichia coli K-12”. Milcrobiol. Rev. 54:130–197], and at the corresponding position in S. enterica LT2 [Sanderson et al (1995) “Genetic map of Salmonella enterica typhimurium”, Edition VIII Microbiol. Rev. 59: 241–303]. In both cases the O antigen gene cluster is close to the gnd gene as is the case in other strains of E. coli and S. enterica [Reeves P. R. (1994) “Biosynthesis and assemby of lipopolysaccharide, 281–314. in A. Neuberger and L. L. M. van Deenen (eds) “Bacterial cell wall, new comprehensive biochemistry” vol 27 Elsevier Science Publishers]. These genes encode enzymes for the synthesis of nucleotide diphosphate sugars and for assembly of the sugars into oligosaccharide units and in general for polymerisation to O antigen.
The E. coli O antigen gene clusters for a wide range of E. coli O antigens have been cloned but the O7, O9, O16 and O111 O antigens have been studied in more detail with only O9 and O16 having been fully characterised with regard to nucleotide sequence to date [Kido N., Torgov V. I., Sugiyama T., Uchiya K., Sugihara H., Komatsu T., Kato N. & Jann K. (1995) “Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterisation of mannosyl transferases, and evidence for an ATP-binding cassette transport system” J. of Bacteriol. 177 2178–2187; Stevenson G., Neal B., Liu D., Hobbs M., Packer N. H., Batley M., Redmond J. W., Lindguist L. & Reeves PR (1994) “Structure of the O antigen of E. coli K12 and the sequence of its rfb gene cluster” J. of Bacteriol. 176 4144–4156; Jayaratne, P. et al. (1991) “Cloning and analysis of duplicated rfbM and rfbK genes involved in the formation of GDP-mannose in Escherichia coli O9:K30 and participation of rfb genes in the synthesis of the group 1 K30 capsular polysaccharide” J. Bacteriol. 176: 3126–3139; Valvano, M. A. and Crosa, J. H.(1989)“Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1”. Inf and Immun. 57:937–943; Marolda C. L. And Valvano, M. A. (1993). “Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Eschericia coli O7:K1)”. J. Bacteriol. 175:148–158.1].
Bastin D. A., et al. 1991 [“Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain”. Mol. Microbiol. 5:9 2223–2231] and Bastin D. A. and Reeves, P. R. [(1995)“Sequence and analysis of the O antigen gene(rfb)cluster of Escherichia coli O111”. Gene 164: 17–23] isolated chromosomal DNA encoding the E. coli 0111 rfb region and characterised a 6962 bp fragment of E. coli 0111 rfb. Six open reading frames (orfs) were identified in the 6962 bp partial fragment and the alignment of the sequences of these orfs revealed homology with genes of the GDP-mannose pathway, rfbK and rfbM, and other rfb and cps genes.
The nucleotide sequences of the loci which control expression of Salmonella enterica B, A, D1, D2, D3, C1, C2 and E O antigens have been characterised [Brown, P. K., L. K. Romana and P. R. Reeves (1991) “Cloning of the rfb gene cluster of a group C2 Salmonella enterica”: comparison with the rfb regions of groups B and D Mol. Microbiol. 5:1873–1881; Jiang, X.-M., B. Neal, F. Santiago, S. J. Lee, L. K. Romana, and P. R. Reeves (1991) “Structure and sequence of the rfb (O antigen) gene cluster of Salmonella enterica serovar typhimurium (LT2)”. Mol. Microbiol. 5:692–713; Lee, S. J., L. K. Romana, and P. R. Reeves (1992) “Sequences and structural analysis of the rfb (O antigen)gene cluster from a group C1 Salmonella enterica enterica strain” J. Gen. Microbiol. 138: 1843–1855; Lui, D., N. K. Verma, L. K. Romana, and P. R. Reeves (1991) “Relationship among the rfb regions of Salmonella enterica serovars A, B and D” J. Bacteriol. 173: 4814–4819; Verma, N. K., and P. Reeves (1989) “Identification and sequence of rfbS and rfbE, which determine the antigenic specificity of group A and group D Salmonella entericae” J. Bacteriol. 171: 5694–5701; Wang, L., L. K. Romana, and P. R. Reeves (1992) “Molecular analysis of a Salmonella enterica enterica group E1 rfb gene cluster: O antigen and the genetic basis of the major polymorphism” Genetics 130: 429–443; Wyk, P., and P. Reeves (1989). “Identification and sequence of the gene for abequose synthase, which confers antigenic specificity on group B Salmonella entericae: homology with galactose epimerase” J. Bacteriol. 171: 5687–5693,; Xiang, S. H., M. Hobbs, and P. R. Reeves. 1994 Molecular analysis of the rfb gene luster of a group D2 Salmonella enterica strain: evidence for its origin from an insertion sequence-mediated recombination event between group E and D1 strains. J. Bacteriol. 176: 4357–4365; Curd, H., D. Liu and P. R. Reeves, 1998. Relationships among the O antigen Salmonella enterica groups B, D1, D2, and D3. J. Bacteriol. 180: 1002–1007.).
Of the closely related Shigella (which really can be considered to be part of E. coli) S. dysenteriae and S. flexneri O antigens have been fully sequenced and are next to gnd. [Klena JD & Schnaitman CA (1993) “Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1” Mol. Microbiol. 9 393–402; Morona R., Mavris M., Fallarino A. & Manning P. (1994) “Characterisation of the rifc region of Shigella flexneri” J.Bacteriol 176: 733–747].
Inasmuch as the O antigen of enteropathogenic E. coli strains and the O antigen of Salmonella enterica strains are major virulence factors and are highly polymorphic, there is a real need to develop highly specific, sensitive, rapid and inexpensive diagnostic assays to detect E. coli and assays to detect S. enterica. There is also a real need to develop diagnostic assays to identify the O antigens of E. coli strains and assays to identify the O antigens of S. enterica strains. With regard to the detection of E. coli these needs extend beyond EHFC (enteropathogenic haemorrhagic E. coli) strains but this is the area of greatest need. There is interest in diagnostics for ETEC (enterotoxigenic E. coli) etc in E. coli. 
The first diagnostic systems employed in this field used large panels of antisera raised against E. coli O antigen expressing strains or S. enterica O antigen expressing strains. This technology has inherent difficulties associated with the preparation, storage and usage of the reagents, as well as the time required to achieve a meaningful diagnostic result.
Nucleotide sequences derived from the O antigen gene clusters of S. enterica strains have been used to determine S. enterica O antigens in a PCR assay [Luk, J. M. C. et al. (1993) “Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of S. enterica major serogoups (A, B, C2, and D)”, J. Clin. Microbiol. 31:2118–2123 ]. The prior complete nucleotide sequence characterisation of the entire rfb locus of serovars Typhimurium, Paratyphi A, Typhi, Muenchen, and Anatum; representing groups B, A, D1, C2 and E1 respectively enabled Luk et al. to select oligonucleotide primers specific for those serogroups. Thus the approach of Luk et al. was based on aligning known nucleotide sequences corresponding to CDP-abequose and CDP-paratose synthesis genes within the O antigen regions of S. enterica serogroups E1, D1, A, B and C2 and exploiting the observed nucleotide sequence differences in order to identify serotype-specific oligonucleotides.
In an attempt to determine the O antigen serotype of a Shiga-like toxin producing E. coli strain, Paton, A. W., et al. 1996 [“Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-like toxin producing Escherichia coli”. J. Clin. Microbiol. 34: 1622–1627], used oligonucleotides derived from the wbdI (orf6) region, which were believed to be specific to the E. coli O111 antigen and which were derived from E. coli O111 sequence, in a PCR diagnostic assay. Unpublished reports indicate that the approach of Paton et al. is deficient in that the nucleotide sequences derived from wbdI may not specifically identify the O111 antigen and in fact lead to detection of false positive results. Paton et al. disclose the detection of 5 O111 antigen isolates by PCR when in fact from only 3 of those isolates did they detect bacteria which reacted with O111 specific antiserum.
DESCRIPTION OF THE INVENTION
Whilst not wanting to be held to a particular hypothesis, the present inventors now believe that the reported false positives found with the Paton et al. method are due to the fact that the nucleic acid molecules employed by Paton et al. were derived from genes which have a putative function as a sugar pathway gene, [Bastin D. A. and Reeves, P. R. (1995) Sequence and analysis of the O antigen gene(rfb) cluster of Escherichia coli O111. Gene 164: 17–23] which they now believe to lack the necessary nucleotide sequence specificity to identify the E. coli O antigen. The inventors now believe that many of the nucleic acid molecules derived from sugar pathway genes expressed in S. enterica or other enterobacteria are also likely to lack the necessary nucleotide sequence specificity to identify specific O antigens or specific serotypes.
In this regard it is important to note that the genes for the synthesis of a polysaccharide antigen include those related to the synthesis of the sugars present in the antigen (sugar pathway genes) and those related to the manipulation of those sugars to form the polysaccharide. The present invention is predominantly concerned with the latter group of genes, particularly the assembly and transport genes such as transferase, polymerase and flippase genes.
The present inventors have surprisingly found that the use of nucleic acid molecules derived from particular assembly and transport genes, particularly transferase, wzx and wzy genes, within O antigen gene clusters can improve the specificity of the detection and identification of O antigens. The present inventors believe that the invention is not necessarily limited to the detection of the particular O antigens which are encoded by the nucleic acid molecules exemplified herein, but has broad application for the detection of bacteria which express an O antigen and the identification of O antigens in general. Further because of the similarities between the gene clusters involved in the synthesis of O antigens and other polymorphic polysaccharide antigens, such as bacterial capsular antigens, the inventors believe that the methods and molecules of the present invention are also applicable to these other polysaccharide antigens.
Accordingly, in one aspect the present invention relates to the identification of nucleic acid molecules which are useful for the detection and identification of specific bacterial polysaccharide antigens.
The invention provides a nucleic acid molecule derived from: a gene encoding a transferase; or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit, including a wzx gene, wzy gene, or a gene with a similar function; the gene being involved in the synthesis of a particular bacterial polysaccharide antigen, wherein the sequence of the nucleic acid molecule is specific to the particular bacterial polysaccharide antigen.
Polysaccharide antigens, such as capsular antigens of E. coli (Type I and Type II), the Virulence capsule of S. enterica sv Typhi and the capsules of species such as Streptococcus pneumoniae and Staphylococcus albus are encoded by genes which include nucleotide sugar pathway genes, sugar transferase genes and genes for the transport and processing of the polysaccharide or oligosaccharide unit. In some cases these are wzx or wzy but in other cases they are quite different because a different processing pathway is used. Examples of other gene clusters include the gene clusters for an extracellular polysaccharide of Streptococcus thermothilus, an exopolysaccharide of Rhizobium melilotti and the K2 capsule of Klebsiella pneumoniae. These all have genes which by experimental analysis, comparison of nucleotide sequence or predicted protein structure, can be seen to include nucleotide sugar pathway genes, sugar transferase genes and genes for oligosaccharide or polysaccharide processing.
In the case of the E. coli K-12 colanic acid capsule gene cluster [Stevenson et al (1996) “Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid”. J. Bacteriol 178: 4885–4893] genes from the three classes were identified either provisionally or definitively. Colanic acid capsule is classified with the Type I capsule of E. coli. 
The present inventors believe that, in general, transferase genes and genes for oligosaccharide processing will be more specific for a given capsule than the genes coding for the nucleotide sugar synthetic pathways as most sugars present in such capsules occur in the capsules of different serotypes. Thus the nucleotide sugar synthesis pathway genes could now be predicted to be common to more than one capsule type.
As elaborated below the present inventors recognise that there may be polysaccharide antigen gene clusters which share transferase genes and/or genes for oligosaccharide or polysaccharide processing so that completely random selection of nucleotide sequences from within these genes may still lead to cross-reaction; an example with respect to capsular antigens is provided by the E. coli type II capsules for which only transferase genes are sufficiently specific. However, the present inventors in light of their current results nonetheless consider the transferase genes or genes controlling oligosaccharide or polysaccharide processing to be superior targets for nucleotide sequence selection for the specific detection and characterisation of polysaccharide antigen types. Thus where there is similarity between particular genes, selection of nucleotide sequences from within other transferase genes or genes for oligosaccharide or polysaccharide processing from within the relevant gene cluster will still provide specificity, or alternatively the use of combinations of nucleotide sequences will provied the desired specificity. The combinations of nucleotide sequences may include nucleotide sequences derived from pathway genes together with nucleotide sequences derived from transferase, wzx or wzy genes.
Thus the invention also provides a panel of nucleic acid molecules wherein the nucleic acid molecules are derived from a combination of genes encoding transferases and/or enzymes for the transport or processing of a polysaccharide or oligosaccharide unit including wzx or wzy genes; wherein the combination of genes is specific to the synthesis of a particular bacterial polysaccharide antigen and wherein the panel of nucleic acid molecules is specific to a bacterial polysaccharied antigen. In another preferred form, the nucleic acid molecules are derived from a combination of genes encoding transferases and/or enzymes for the transport or processing of a polysaccharide or oligosaccharide unit including wzx or wzy genes, together with nucleic acid molecules derived from pathway genes.
In a second aspect the present invention relates to the identification of nucleic acid molecules which are useful for the detection of bacteria which express O antigens and for the identification of the O antigens of those bacteria in diagnostic assays.
The invention provides a nucleic acid molecule derived from: a gene encoding a transferase; or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit such as a wzx or wzy gene, the gene being involved in the synthesis of a particular bacterial O antigen, wherein the sequence of the nucleic acid molecule is specific to the particular bacterial O antigen.
The nucleic acids of the invention may be variable in length. In one embodiment they are from about 10 to about 20 nucleotides in length.
In one preferred embodiment, the invention provides a nucleic acid molecule derived from: a gene encoding a transferase; or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit including a wzx or wzy gene the gene being involved in the synthesis of an O antigen expressed by E. coli, wherein the sequence of the nucleic acid molecule is specific to the O antigen.
In one more preferred embodiment, the sequence of the nucleic acid molecule is specific to the nucleotide sequence encoding the O111 antigen (SEQ ID NO:1). More preferably, the sequence is derived from a gene selected from the group consisting of wbdh (nucleotide position 739 to 1932 of SEQ ID NO:1), wzx (nucleotide position 8646 to 9911 of SEQ ID NO:1), wzy (nucleotide position 9901 to 10953 of SEQ ID NO:1), wbdM (nucleotide position 11821 to 12945 of SEQ ID NO:1) and fragments of those molecules of at least 10–12 nucleotides in length. Particularly preferred nucleic acid molecules are those set out in Table 5 and 5A, with respect to the above mentioned genes.
In another more preferred embodiment, the sequence of the nucleic acid molecule is specific to the nucleotide sequence encoding the 0157 antigen (SEQ ID NO:2). More preferably the sequence is derived from a gene selected from the group consisting of wbdN (nucleotide position 79 to 861 of SEQ ID NO:2), wbdO, (nucleotide position 2011 to 2757 of SEQ ID NO:2), wbdP (nucleotide position 5257 to 6471 of SEQ ID NO:2)), wbdR (13156 to 13821 of SEQ ID NO:2), wzx (nucleotide position 2744 to 4135 of SEQ ID NO:2) and wzy (nucleotide position 858 to 2042 of SEQ ID NO:2). Particularly preferred nucleic acid molecules are those set out in Table 6 and 6A.
The invention also provides in a further preferred embodiment a nucleic acid molecule derived from: a gene encoding a transferase; or a gene encoding an enzyme for the transport or processing of a polysaccharide or oligosaccharide unit including a wzx or wzy gene; the gene being involved in the synthesis of an O antigen expressed by Salmonella enterica, wherein the sequence of the nucleic acid molecule is specific to the O antigen.
In one more preferred form of this embodiment, the sequence of the nucleic acid molecule is specific to the nucleotide sequence encoding the S. enterica C2 antigen (SEQ ID NO:3). More preferably the sequence of the nucleic acid molecule is derived from a gene selected from the group consisting of wbaR (nucleotide position 2352 to 3314 of SEQ ID NO:3), wbaL (nucleotide position 3361 to 3875 of SEQ ID NO:3), wbaQ (nucleotide position 3977 to 5020 of SEQ ID NO:3), wbaW (nucleotide position 6313 to 7323 of SEQ ID NO:3), wbaZ (nucleotide position 7310 to 8467 of SEQ ID NO:3), wzx (nucleotide position 1019 to 2359 of SEQ ID NO:3)and wzy (nucleotide position 5114 to 6313 of SEQ ID NO:3). Particularly preferred nucleic acid molecules are those set out in Table 7.
In another more preferred form of this embodiment, the sequence of the nucleic acid molecule is specific to the nucleotide sequence encoding the S. enterica B antigen (SEQ ID NO:4). More preferably the sequence is derived from wzx (nucleotide position 12762 to 14054 of SEQ ID NO:4) or wbav (nucleotide position 14059 to 15060 of SEQ ID NO:4). Particularly preferred nucleic acid molecules are those set out in Table 8 which are derived from wzx and wbaV genes.
In a further more preferred form of this embodiment, the sequence of the nucleic acid molecule is specific to the S. enterica D3 O antigen and is derived from the wzy gene.
In yet a further preferred form of this embodiment, the sequence of the nucleic acid molecule is specific to the S. enterica E1 O antigen and is derived from the wzx gene.
While transferase genes, or genes coding for the transport or processing of a polysaccharide or oligosaccharide unit, such as a wzx or wzy gene, are superior targets for specific detection of individual O antigen types there may well be individual genes or parts of them within this group that can be demonstrated to be the same or closely related,between different O antigen types such that cross-reactions can occur. Cross reactions should be avoided by the selection of a different target within the group or the use of multiple targets within the group.
Further, it is recognised that there are cases where O antigen gene clusters have arisen from recombination of at least two strains such that the unique O antigen type is provided by a combination of gene products shared with at least two other O antigen types. The recognised example of this phenomenon is the S. enterica O antigen serotype D2 which has genes from D1 and E1 but none unique to D2. In these circumstances the detection of the O antigen type can still be achieved in accordance with the invention, but requires the use of a combination of nucleic acid molecules to detect a specific combination of genes that exists only in that particular O antigen gene cluster.
Thus, the invention also provides a panel of nucleic acid molecules wherein the nucleic acid molecules are derived from genes encoding transferases and/or enzymes for the transport or processing of a polysaccharide or oligosaccharide unit including wzx or wzy genes, wherein the panel of nucleic acid molecules is specific to a bacterial O antigen. Preferably the particular bacterial O antigen is expressed by S enterica. More preferably, the panel of nucleic acid molecules is specific to the D2 O antigen and is derived from the E1 wzy gene and the D1 wzx gene.
The combinations of nucleotide sequences may include nucleotide sequences derived from pathway genes, together with nucleotide sequences derived from transferase, wzx or wzy genes.
Thus, the invention also provides a panel of nucleic acid molecules, wherein the nucleic acid molecules are derived from genes encoding transferases and/or enzymes for the transport or processing of a polysaccharide or oligosaccharide unit including wzx or wzy genes, and sugar pathway genes, wherein the panel of nucleic acid molecules is specific to a particular bacterial O antigen. Preferably the O antigen is expressed S. enterica. 
Further it is recognised that there may be instances where spurious hybridisation will arise through initial selection of a sequence found in many different genes but this is typically recognisable by, for instance, comparison of band sizes against controls in PCR gels, and an alternative sequence can be selected.
The present inventors believe that based on the teachings of the present invention and available information concerning polysaccharide antigen gene clusters (including O antigen gene clusters), and through use of experimental analysis, comparison of nucleic acid sequences or predicted protein structures, nucleic acid molecules in accordance with the invention can be readily derived for any particular polysaccharide antigen of interest. Suitable bacterial strains can typically be acquired commercially from depositary institutions.
As mentioned above there are currently 166 defined E. coli O antigens while the S. enterica has 46 known O antigen types [Popoff M. Y. et al (1992) “Antigenic formulas of the Salmonella serovars” 6th revision WHO Collaborating centre for Reference and Research on Salmonella, Institut Pasteur Paris France]. Many other genera of bacteria are known to have O antigens and these include Citrobacter, Shigella, Yersinia, Plesiomonas, Vibrio and Proteus. 
Samples of the 166 different E. coli O antigen serotypes are available from Statens Serum Institut, Copenhagen, Denmark.
The 46 S. enterica serotypes are available from Institute of Medical and Veterinary Science, Adelaide, Australia.
In another aspect, the invention relates to a method of testing a sample for the presence of one or more bacterial polysaccharide antigens comprising contacting the sample with at least one oligonucleotide molecule capable of specifically hybridising to: (i) a gene encoding a transferase, or (ii) a gene encoding an enzyme for transport or processing of oligosaccharide or polysaccharide units, including a wzx or wzy gene; wherein said gene is involved in the synthesis of the bacterial polysaccharide antigen; under conditions suitable to permit the at least one oligonucleotide molecule to specifically hybridise to at least one such gene of any bacteria expressing the particular bacterial polysaccharide antigen present in the sample and detecting any specifically hybridised oligonucleotide molecules.
Where a single specific oligonucleotide molecule is unavailable a combination of molecules hybridising specifically to the target region may be used. Thus the invention provides a panel of nucleic acid molecules for use in the method of testing of the invention, wherein the nucleic acid molecules are derived from genes encoding transferases and/or enzymes for the transport or processing of a polysaccharide or oligosaccharide unit including wzx or wzy genes, wherein the panel of nucleic acid molecules is specific to a particular bacterial polysaccharide. The panel of nucleic acid molecules can include nucleic acid molecules derived from sugar pathway genes where necessary.
In another aspect, the invention relates to a method of testing a sample for the presence of one or more bacterial polysaccharide antigens comprising contacting the sample with at least one pair of oligonucleotide molecules, with at least one oligonucleotide molecule of the pair capable of specifically hybridising to: (i) a gene encoding a transferase, or (ii) a gene encoding an enzyme for transport or processing oligosaccharide or polysaccharide units, including a wzx or wzy gene; wherein said gene is involved in the synthesis of the bacterial polysaccharide antigen; under conditions suitable to permit the at least one oligonucleotide molecule of the pair of molecules to specifically hybridise to at least one such gene of any bacteria expressing the particular bacterial polysaccharide antigen present in the sample and detecting any specifically hybridised oligonucleotide molecules.
The pair of oligonucleotide molecules may both hybridise to the same gene or to different genes. Only one oligonucleotide molecule of the pair need hybridise specifically to sequence specific for the particular antigen type. The other molecule can hybridise to a non-specific region.
Where the particular polysaccharide antigen gene cluster has arisen through recombination, the at least one pair of oligonucleotide molecules may be selected to be capable of hybridising to a specific combination of genes in the cluster specific to that polysaccharide antigen, or multiple pairs may be selected to provide hybridisation to the specific combination of genes. Even where all the genes in a particular cluster are unique, the method may be carried out using nucleotide molecules which recognise a combination of genes within the cluster.
Thus the invention provides a panel containing pairs of nucleic acid molecules for use in the method of testing of the invention, wherein the pairs of nucleic acid molecules are derived from genes encoding transferases and/or enzymes for the transport or processing of a polysaccharide or oligosaccharide unit including wzx or wzy genes, wherein the panel of nucleic acid molecules is specific to a particular bacterial polysaccharide antigen. The panel of nucleic acid molecules can include pairs of nucleic acid molecules derived from sugar pathway genes where necessary.
In another aspect, the invention relates to a method of testing a sample for the presence of one or more particular bacterial O antigens comprising contacting the sample with at least one oligonucleotide molecule capable of specifically hybridising to: (i) a gene encoding an O antigen transferase, or (ii) a gene encoding an enzyme for transport or processing of the oligosaccharide or polysaccharide unit, including a wzx or wzy gene; wherein said gene is involved in the synthesis of the particular O antigen; under conditions suitable to permit the at least one oligonucleotide molecule to specifically hybridise to at least one such gene of any bacteria expressing the particular bacterial O antigen present in the sample and detecting any specifically hybridised oligonucleotide molecules. Preferably the bacteria are E. coli or S. enterica. More preferably, the E. coli express the 0157 serotype or the 0111 serotype. More preferably the S. enterica express the C2 or B serotype. Preferably, the method is a Southern blot method. More preferably, the nucleic acid molecule is labelled and hybridisation of the nucleic acid molecule is detected by autoradiography or detection of fluorescence.
The inventors envisage circumstances where a single specific oligonucleotide molecule is unavailable. In these circumstances a combination of molecules hybridising specifically to the target region may be used. Thus the invention provides a panel of nucleic acid molecules for use in the method of testing of the invention, wherein the nucleic acid molecules are derived from genes encoding transferases and/or enzymes for the transport or processing of a polysaccharide or oligosaccharide unit including wzx or wzy genes, wherein the panel of nucleic acid molecules is specific to a particular bacterial O antigen. Preferably the particular bacterial O antigen is expressed by S. enterica. The panel of nucleic acid molecules can include nucleic acid molecules derived from sugar pathway genes where necessary.
In another aspect, the invention relates to a method of testing a sample for the presence of one or more particular bacterial O antigens comprising contacting the sample with at least one pair of oligonucleotide molecules with at least one oligonucleotide molecule of the pair being capable of specifically hybridising to: (i) a gene encoding an O antigen transferase, or (ii) a gene encoding an enzyme for transport or processing of the oligosaccharide or polysaccharide unit, including a wzx or wzy gene; wherein said gene is involved in the synthesis of the particular O antigen; under conditions suitable to permit the at least one oligonucleotide molecule to specifically hybridise to at least one such gene of any bacteria expressing the particular bacterial O antigen present in the sample and detecting any specifically hybridised oligonucleotide molecules.
Preferably the bacteria are E coli or S. enterica. More preferably, the E. coli are of the 0111 or the 0157 serotype. More preferably the S. enterica express the C2 or B serotype. Preferably, the method is a polymerase chain reaction method. More preferably the oligonucleotide molecules for use in the method of the invention are labelled. Even more preferably the hybridised oligonucleotide molecules are detected by electrophoresis. Preferred oligonucleotides for use with 0111 which provide for specific detection of 0111 are illustrated in Table 5 and 5A with respect to the genes wbdH, wzx, wzy and wbdM. Preferred oligonucleotide molecules for use with O157 which provide for specific detection of O157 are illustrated in Table 6 and 6A.
With respect to serotypes C2 and B, suitable oligonucleotide molecules can be selected from appropriate regions described in column 3 of Tables 7 and 8.
The inventors envisage rare circumstances whereby two genetically similar gene clusters encoding serologically different O antigens have arisen through recombination of genes or mutation so as to generate polymorphic variants. In these circumstances multiple pairs of oligonucleotides may be selected to provide hybridisation to the specific combination of genes. The invention thus provides a panel containing pairs of nucleic acid molecules for use in the method of testing of the invention, wherein the pairs of nucleic acid molecules are derived from genes encoding transferases and/or enzymes for the transport or processing of a polysaccharide or oligosaccharide unit including wzx or wzy genes, wherein the panel of nucleic acid molecules is specific to a particular bacterial O antigen. Preferably the particular bacterial O antigen is expressed by S. enterica. The panel of nucleic acid molecules can include pairs of nucleic acid molecules derived from sugar pathway genes where necessary.
In another aspect, the invention relates to a method for testing a food derived sample for the presence of one or more particular bacterial O antigens comprising contacting the sample with at least one pair of oligonucleotide molecules with at least one oligonucleotide molecule of the pair being capable of specifically hybridising to: (i) a gene encoding an O antigen transferase, or (ii) a gene encoding an enzyme for transport or processing of the oligosaccharide or polysaccharide unit, including a wzx or wzy gene; wherein the gene is involved in the synthesis of the particular O antigen; under conditions suitable to permit the at least one oligonucleotide molecule to specifically hybridise to at least one such gene of any bacteria expressing the particular bacterial polysaccharide antigen present in the sample and detecting any specifically hybridised oligonucleotide molecules. Preferably the bacteria are E. coli or S. enterica. More preferably, the E. coli are of the 0111 or 0157 serotype. More preferably the S. enterica are of the C2 or B serotype. Preferably, the method is a polymerase chain reaction method. More preferably the oligonucleotide molecules for use in the method of the invention are labelled. Even more preferably the hybridised oligonucleotide molecules are detected by electrophoresis.
In another aspect the present invention relates to a method for testing a faecal derived sample for the presence of one or more particular bacterial O antigens comprising contacting the sample with at least one pair of oligonucleotide molecules with at least one oligonucleotide molecule of the pair being capable of specifically hybridising to: (i) a gene encoding an O antigen transferase, or (ii) a gene encoding an enzyme for transport or processing of the oligosaccharide or polysaccharide unit, including a wzx or wzy gene; wherein said gene is involved with synthesis of the particular O antigen; under conditions suitable to permit the at least one oligonucleotide molecule to specifically hybridise to at least one of said gene of any bacteria expressing the particular bacterial O antigen present in the sample and detecting any specifically hybridised oligonucleotide molecules. Preferably the bacteria are E. coli or S. enterica. More preferably, the E. coli are of the 0111 or 0157 serotype. More preferably, the S. enterica are of the C2 or B serotype. Preferably, the method is a polymerase chain reaction method. More preferably the oligonucleotide molecules for use in the method of the invention are labelled. Even more preferably the hybridised oligonucleotide molecules are detected by electrophoresis.
In another aspect, the present invention relates to a method for testing a sample derived from a patient for the presence of one or more particular bacterial O antigens comprising contacting the sample with at least one pair of oligonucleotide molecules with at least one oligonucleotide molecule of the pair being capable of specifically hybridising to: (i) a gene encoding an O antigen transferase, or (ii) a gene encoding an enzyme for transport or processing of the oligosaccharide or polysaccharide unit, including a wzx or wzy gene; wherein said gene is involved in the synthesis of the particular O antigen; under conditions suitable to permit the at least one oligonucleotide molecule to specifically hybridise to at least one such gene of any bacteria expressing the particular bacterial O antigen present in the sample and detecting any specifically hybridised oligonucleotide molecules. Preferably the bacteria are E. coli or S. enterica. More preferably, the E. coli are of the 0111 or 0157 serotype. More preferably, the S. enterica are of the C2 or B serotype. Preferably, the method is a polymerase chain reaction method. More preferably the oligonucleotide molecules for use in the method of the invention are labelled. Even more preferably the hybridised oligonucleotide molecules are detected by electrophoresis.
In the above described methods it will be understood that where pairs of oligonucleotides are used one of the oligonucleotide sequences may hybridise to a sequence that is not from a transferase, wzx or wzy gene. Further where both hybridise to one of these gene products they may hybridise to the same or a different one of these genes.
In addition it will be understood that where cross reactivity is an issue a combination of oligonucleotides may be chosen to detect a combination of genes to provide specificity.
The invention further relates to a diagnostic kit which can be used for the detection of bacteria which express bacterial polysaccharide antigens and the identification of the bacterial polysaccharide type of those bacteria.
Thus in a further aspect, the invention relates to a kit comprising a first vial containing a first nucleic acid molecule capable of specifically hybridising to: (i) a gene encoding a transferase, or (ii) a gene encoding an enzyme for transport or processing oligosaccharide or polysaccharide, including a wzx or wzy gene, wherein the said gene is involved in the synthesis of a bacterial polysaccharide. The kit may also provide in the same or a separate vial a second specific nucleic acid capable of specifically hybridising to: (i) a gene encoding a transferase, or (ii) a gene encoding an enzyme for transport or processing oligosaccharide or polysaccharide, including a wzx or wzy gene, wherein the said gene is involved in the synthesis of a bacterial polysaccharide, wherein the sequence of the second nucleic acid molecule is different from the sequence of the first nucleic acid molecule.
In a further aspect the invention relates to a kit comprising a first vial containing a first nucleic acid molecule capable of specifically hybridising to: (i) a gene encoding a transferase, or (ii) a gene encoding an enzyme for transport or processing oligosaccharide or polysaccharide including wzx or wzy, wherein the said gene is involved in the synthesis of a bacterial O antigen. The kit may also provide in the same or a separate vial a second specific nucleic acid capable of specifically hybridising to: (i) a gene encoding a transferase, or (ii) a gene encoding an enzyme for transport or processing oligosaccharide or polysaccharide including wzx or wzy, wherein the said gene is involved in the synthesis of O antigen, wherein the sequence of the second nucleic acid molecule is different from the sequence of the first nucleic acid molecule. Preferably the first and second nucleic acid sequences are derived from E. coli or the first and second nucleic acid sequences are derived from S. enterica. 
The present inventors provide full length sequence of the O157 gene cluster for the first time and recognise that from this sequence of this previously uncloned full gene cluster appropriate recombinant molecules can be generated and inserted for expression to provide expressed O157 antigens useful in applications such as vaccines.
DEFINITIONS
The phrase, “a nucleic acid molecule derived from a gene” means that the nucleic acid molecule has a nucleotide sequence which is either identical or substantially similar to all or part of the identified gene. Thus a nucleic acid molecule derived from a gene can be a molecule which is isolated from the identified gene by physical separation from that gene, or a molecule which is artificially, svnthesised and has a nucleotide sequence which is either identical to or substantially similar to all or part of the identified gene. While some workers consider only the DNA strand with the same sequence as the mRNA transcribed from the gene, here either strand is intended.
Transferase genes are regions of nucleic acid which have a nucleotide sequence which encodes gene products that transfer monomeric sugar units.
Flippase or wzx genes are regions of nucleic acid which have a nucleotide sequence which encodes a gene product that flips oligosaccharide repeat units generally composed of three to six monomeric sugar units to the external surface of the membrane.
Polymerase or wzy genes are regions of nucleic acid which have a nucleotide sequence which encodes gene products that polymerise repeating oligosaccharide units generally composed of 3–6monomeric sugar units.
The nucleotide sequence provided in this specification are described in the sequence listing as anti-sense sequences. This term is used in the same manner as it is used in Glossary of Biochemistry and Molecular Biology Revised Edition, David M. Glick, 1997 Portland Press Ltd., London on page 11 where the term is described as referring to one of the two strands of double-stranded DNA usually that which has the same sequence as the mRNA. We use it to describe this strand which has the same sequence as the mRNA.










NOMENCLATURE


Synonyms for E. coli O111 rfb











Current names
Our names
Bastin et al. 1991







wbdH
orf1




gmd
orf2



wbdI
orf3
orf3.4*



manC
orf4
rfbM*



manB
orf5
rfbK*



wbdJ
orf6
orf6.7*



wbdK
orf7
orf7.7*



wzx
orf8
orf8.9 and rfbX*



wzy
orf9



wbdL
orf10



wbdM
orf11







*Nomenclature according to Bastin D.A., et al. 1991 “Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain”. Mol. Microbiol. 5:9 2223–2231.


















Other Synonyms










wzy
rfc



wzx
rfbX



rmlA
rfbA



rmlB
rfbB



rmlC
rfbC



rmlD
rfbD



glf
orf6*



wbbI
orf3#, orf8* of E. coli K-12



wbbJ
orf2#, orf9* of E. coli K-12



wbbK
orf1#, orfl0* of E. coli K-12



wbbL
orf5#, orf 11* of E. coli K-12







#Nomenclature according to Yao, Z. And M. A. Valvano 1994. “Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Eschericia coli K-12 W3110: identification of genes the confer groups-specificty to Shigella flexineri serotypes Y and 4a”. J. Bacteriol. 176: 4133–4143.



*Nomenclature according to Stevenson et al. 1994. “Structure of the O-antigen of E. coli K-12 and the sequence of its rfb gene cluster”. J. Bacteriol 176: 4144–4156.



• S. enterica is a name introduced in 1987 to replace the many other names such as Salmonella typhi and Salmonella typhimurium, the old species names becoming serovar names as in S. enterica sv Typhi. However, the traditional names are still widely used.



• The O antigen genes of many species were given rfb names (rfbA etc) and the O antigen gene cluster was often referred to as the rfb cluster. There are now new names for the rfb genes as shown in the table. Both terminologies have been used herein, depending on the source of the information.









BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows Eco R1 restriction maps of cosmid clones pPR1054, pPR1055, pPR1056, pPR1058, pPR1287 which are subclones of E. coli O111 O antigen gene cluster. The thickened line is the region common to all clones Broken lines show segments that are non-contiguous on the chromosome. The deduced restriction map for E. coli strain M92 is shown above.
FIG. 2 shows a restriction mapping analysis of E. coli O111 O antigen gene cluster within the cosmid clone pPR1058. Restriction enzymes are: (B: BamH1; Bg: Bg1II, E: EcoR1; H: HindIII; K: KpnI; P: PstI; S: SalI and X: Xho1. Plasmids pPR1230, pPR1231, and pPR1288 are deletion derivatives of pPR1058. Plasmids pPR 1237, pPR1238, pPR1239 and pPR1240 are in pUC19. Plasmids pPR1243, pPR1244, pPR1245, pPR1246 and pPR1248 are in pUC18, and pPR1292 is in pUC19. Plasmid pPR1270 is in pT7T319U. Probes 1, 2 and 3 were isolated as internal fragments of pPR1246, pPR1243 and pPR1237 respectively. Dotted lines indicate that subclone DNA extends to the left of the map into attached vector.
FIG. 3 shows the structure of E. coli O111 O antigen gene cluster.
FIG. 4 shows the structure of E. coli O157 O antigen gene cluster.
FIG. 5 shows the structure S. enterica locus encoding the serogroup C2O antigen gene cluster.
FIG. 6 shows the structure S. enterica locus encoding the serogroup B O antigen gene cluster.
FIG. 7 shows the nucleotide sequence of the E. coli O111 O antigen gene cluster. Note: (1) The first and last three bases of a gene are underlined and of italic respectively.; (2) The region which was previously sequenced by Bastin and Reeves 1995 “Sequence and anlysis of the O antigen gene (rfb) cluster of Eschericia coli o111” Gene 164: 17–23 is marked.
FIG. 8 shows the nucleotide sequence of the E. coli O157 O antigen gene cluster. Note: (1) The first and last three bases of a gene (region) are underlined and of italic respectively (2) The region previously sequenced by Bilge et al. 1996 “Role of the Eschericia coli O157-H7O side chain in adherence and analysis of an rfb locus”. Inf. and Immun 64:4795–4801 is marked.
FIG. 9 shows the nucleotide sequence of S. enterica serogroup C2O antigen gene cluster. Note:
(1) The numbering is as in Brown et al. 1992. “Molecular analysis of the rfb gene cluster of Salmonella serovar muenchen (strain M67): the genetic basis of the polymorphism between groups C2 and B”. Mol. Microbiol. 6: 1385–1394(2) The first and last three bases of a gene are underlined and in italics respectively.(3) Only that part of the group C2 gene cluster, which differs from that of group B, was sequenced and is presented here.
FIG. 10 shows the nucleotide sequence of S. enterica serogroup B O antigen gene cluster Note: (1) The numbering is as in Jiang et al. 1991. “Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2)”. Mol. Microbiol. 5: 695–713. The first gene in the O antigen gene cluster is rmlB which starts at base 4099. (2) The first and last three bases of a gene are underlined and in italics respectively.



BEST METHOD FOR CARRYING OUT THE INVENTION
Materials and Methods-part 1
The experimental procedures for the isolation and characterisation of the E. coli O111 O antigen gene cluster (position 3,021–9,981) are according to Bastin D. A., et al. 1991 “Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain”. Mol. Microbiol. 5:9 2223–2231 and Bastin D. A. and Reeves, P. R. 1995 “Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111”. Gene 164: 17–23.
A. Bacterial strains and growth media
Bacteria were grown in Luria broth supplemented as required.
B. Cosmids and phage
Cosmids in the host-strain x2819 were repackaged in vivo. Cells were grown in 250mL flasks containing 30 mL of culture, with moderate shaking at 30° C. to an optical density of 0.3 at 580 nm. The defective lambda prophage was induced by heating in a water bath at 45° C. for 15min followed by an incubation at 37° C. with vigorous shaking for 2hr. Cells were then lysed by the addition of 0.3 mL chloroform and shaking for a further 10 min. Cell debris were removed from 1 mL of lysate by a 5 min spin in a microcentrifuge, and the supernatant removed to a fresh microfuge tube. One drop of chloroform was added then shaken vigorously through the tube contents.
C. DNA preparation
Chromosomal DNA was prepared from bacteria grown overnight at 37° C. in a volume of 30 mL of Luria broth. After harvesting by centrifugation, cells were washed and resuspended in 10 mL of 50 mM Tris-HCl pH 8.0. EDTA was added and the mixture incubated for 20 min. Then lysozyme was added and incubation continued for a further 10 min. Proteinase K, SDS, and ribonuclease were then added and the mixture incubated for up to 2 hr for lysis to occur. All incubations were at 37° C. The mixture was then heated to 65° C. and extracted once with 8 mL of phenol at the same temperature. The mixture was extracted once with 50 mL of phenol/chloroform/iso-amyl alcohol at 4° C. Residual phenol was removed by two ether extractions. DNA was precipitated with 2 vols. of ethanol at 4° C., spooled and washed in 70% ethanol, resuspended in 1–2mL of TE and dialysed. Plasmid and cosmid DNA was prepared by a modification of the Birnboim and Doly method (Birnhoim, H. C. And Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA Nuci. Acid Res. 7:1513–1523. The volume of culture was 10 nmL and the lysate was extracted with phenol/chloroform/iso-amyl alcohol before precipitation with isopropanol. Plasmid DNA to be used as vector was isolated on a continuous caesium chloride gradient following alkaline lysis of cells grown in 1 L of culture.
D. Enzymes and buffers
Restriction endonucleases and DNA T4 ligase were purchased from Boehringer Mannheim (Castle Hill, NSW, Australia)or Pharmacia LKB (Melbourne, VIC Australia). Restriction enzymes were used in the recommended commercial buffer.
E. Construction of a gene bank.
Individual aliquots of M92 chromosomal DNA (strain Stoke W, from Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen S, Denmark) were partially digested with 0.2U Sau3A1 for 1–15 mins. Aliquots giving the greatest proportion of fragments in the size range of approximately 40–50 kb were selected and ligated to vector pPR691 previously digested with BamH1and PvuII. Ligation mixtures were packaged in vitro with packaging extract. The host strain for transduction was x2819 and recombinants were selected with kanamycin.
F. Serological procedures.
Colonies were screened for the presence of the O111 antigen by immunblotting. Colonies were grown overnight, up to 100 per plate then transferred to nitrocellulose discs and lysed with 0.5N HCl. Polyoxyethylene (20)-sorbitan monolaurate (TWEEN 20) was added to TBS at 0.05% final concentration for blocking, incubating and washing steps. Primary antibody was E. coli O group 111antiserm, diluted 1:800. The secundary antibody was goat anti-rabbit IgG labelled with horseradish peroxidase diluted 1:5000. The staining substrate was 4-chloro-1-naptol. Slide agglutination was performed according to the standard procedure.
G. Recombinant DNA methods.
Restriction mapping was based on a combination of standard methods including single and double digests and sub-cloning. Deletion derivatives of entire cosmids were produced as follows: aliquots of 1.8 μg of cosmid DNA were digested in a volume of 20 μl with 0.25U of restriction enzyme for 5–80 min. One half of each aliquot was used to check the degree of digestion on an agarose gel. The sample which appeared to give a representative range of fragments was ligated at 4° C. overnight and transformed by the CaCl2 method into JM109. Selected plasmids were transformed into sφ174 by the same method. P4657 was transformed with pPR1244 by electroporation.
H. DNA hybridisation
Probe DNA was extracted from agarose gels by electroelution and was nick-translated using [α-32P]-dCTP. Chromosomal or plasmid DNA was electrophoresed in 0.8% agarose and transferred to a nitrocellulose membrane. The hybridisation and pre-hybridisation buffers contained either 30% or 50% formamide for low and high stringency probing respectively. Incubation temperatures were 42° C. and 37° C. for pre-hybridisation and hybridisation respectively. Low stringency washing of filters consisted of 3×20 min washes in 2 ×SSC and 0.1% SDS. High-stringency washing consisted of 3×5 min washes in 2 ×SSC and 0.1% SDS at room temperature, a 1hr wash in 1 ×SSC and 0.1% SDS at 58° C. and 15 min wash in 0.1 ×SSC and 0.1% SDS at 58° C.
I. Nucleotide Sequencing of E. coli 0111 O Antigen Gene Cluster (position 3,021–9,981)
Nucleotide sequencing was performed using an ABI 373 automated sequencer (CA, USA). The region between map positions 3.30 and 7.90 was sequenced using uni-directional exonuclease III digestion of deletion families made in PT7T3190 from clones pPR1270 and pPR1272. Gaps were filled largely by cloning of selected fragments into M13mp18 or M13mp19. The region from map positions 7.90–10.2 was sequenced from restriction fragments in M13mp18 or M13mp19. Remaining gaps in both the regions were filled by priming from synthetic oligonucleotides complementary to determined positions along the sequence, using a single stranded DNA template in M13 or phagemid The oligonucleotides were designed after analysing the adjacent sequence. All sequencing was performed by the chain termination method. Sequences were aligned using SAP [Staden, R., 1982 “Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing”. Nuc. Acid Res. 10: 4731–4751; Staden, R., 1986 “The current status and portability of our sequence handling software”. Nuc. Acid Res. 14: 217–231]. The program NIP [Staden, R. 1982 “An interactive graphics program for comparing and aligning nucleic acid and amino acid sequence”. Nuc. Acid Res. 10: 2951–2961] was used to find open reading frames and translate them into proteins.
J. Isolation of Clones Carrying E. Coli O111 O Antigen Gene Cluster.
The E. coli O antigen gene cluster was isolated according to the method of Bastin D. A., et al. [1991 “Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain”. Mol. Microbiol. 5(9), 2223–2231]. Cosmid gene banks of M92 chromosomal DNA were established in the in vivo packaging strain ×2819. From the genomic bank, 3.3×103 colonies were screened with E. coli 0111 antiserum using an immuno-blotting procedure: 5 colonies (pPR1054, pPR1055, pPR1056, pPR1058 and pPR1287) were positive. The cosmids from these strains were packaged in vivo into lambda particles and transduced into the E. coli deletion mutant Sφ174 which lacks all O antigen genes. In this host strain, all plasmids gave positive agglutination with 0111 antiserum. An Eco R1 restriction map of the 5 independent cosmids showed that they have a region of approximately 11.5 kb in common (FIG. 1). Cosmid pPR1058 included sufficiant flanking DNA to identify several chromosomal markers linked to O antigen gene cluster and was selected for analysis of the O antigen gene cluster region.
K. Restriction Mapping of Cosmid pPR1058
Cosmid pPR1058 was mapped in two stages. A preliminary map was constructed first, and then the region between map positions 0.00 and 23.10 was mapped in detail, since it was shown to be sufficient for O111 antigen expression. Restriction sites for both stages are shown in FIG. 2. The region common to the five cosmid clones was between map positions 1.35 and 12.95 of pPR1058.
To locate the O antigen gene cluster within pPR1058, pPR1058 cosmid was probed with DNA probes covering O antigen gene cluster flanking regions from S. enterica LT2 and E. coli K-12. Capsular polysaccharide (cps) genes lie upstream of O antigen gene cluster while the gluconate dehydrogenase (gnd) gene and the histidine (his) operon are downstream, the latter being further from the O antigen gene cluster. The probes used were pPR472 (3.35kb), carrying the gnd gene of LT2, pPR685(5.3kb) carrying two genes of the cps cluster, cpsB and cpsG of LT2, and K350 (16.5kb) carrying all of the his operon of K-12. Probes hybridised as follows: pPR472 hybridised to 1.55kb and 3.5 kb (including 2.7 kb of vector) fragments of Pst1and HindIII double digests of pPR1246 (a HindIII/EcoR1subclone derived from pPR1058, FIG. 2), which could be located at map positions 12.95–15.1; pPR685 hybridized to a 4.4 kb EcoR1fragment of pPR1058 (including 1.3 kb of vector) located at map position 0.00–3.05; and K350 hybridised with a 32 kb EcoR1 fragment of pPR1058 (including 4.0 kb of vector), located at map position 17.30–45.90. Subclones containing the presumed gnd region complemented a gnd edd strain GB23152. On gluconate bromothymol blue plates, pPR1244 and pPR1292 in this host strain gave the green colonies expected of a gnd edd genotype. The his phenotype was restored by plasmid pPR1058 in the his deletion strain Sφ174 on minimal medium plates, showing that the plasmid carries the entire his operon.
It is likely that the O antigen gene cluster region lies between gnd and cps, as in other E. coli and S. enterica strains, and hence between the approximate map positions 3.05 and 12.95. To confirm this, deletion derivatives of pPR1058 were made as follows: first, pPR1058 was partially digested with HindIII and self ligated. Transformants were selected for kanamycin resistance and screened for expression of O111 antigen. Two colonies gave a positive reaction. EcoR1digestion showed that the two colonies hosted identical plasmids, one of which was designated pPR1230, with an insert which extended from map positions 0.00 to 23.10. Second pPR1058 was digested with Sal1 and partially digested with Xho1 and the compatible ends were re-ligated. Transformants were selected with kanamycin and screened for 0111 antigen expression. Plasmid DNA of 8 positively reacting clones was checked using EcoR1 and Xho1 digestion and appeared to be identical. The cosmid of one was designated pPR1231. The insert of pPR1231 contained the DNA region between map positions 0.00 and 15.10. Third, pPR1231 was partially digested with Xho1, self-ligated, and transformants selected on spectinomycin/streptomycin plates. Clones were screened for kanamycin sensitivity and of 10 selected, all had the DNA region from the Xho1 site in the vector to the Xho1 site at position 4.00 deleted. These clones did not express the O111 antigen, showing that the Xho1 site at position 4.00 is within the O antigen gene cluster. One clone was selected and named pPR1288. Plasmids pPR1230, pPR1231, and pPR1288 are shown in FIG. 2.
L. Analysis of the E. coli O111 O antigen gene cluster (position 3,021–9,981) nucleotide sequence data
Bastin and Reeves [1995 “Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111”. Gene 164: 17–23] partially characterised the E. coli O111 O antigen gene cluster by sequencing a fragment from map position 3,021–9,981. FIG. 3 shows the gene organisation of position 3,021–9,981 of E. coli O111 O antigen gene cluster. orf3 and orf6 have high level amino acid identity with wcaH and wcaG (46.3% and 37.2% respectively), and are likely to be similar in function to sugar biosynthetic pathway genes in the E. coli K-12 colanic gene cluster. orf4 and orf5 show high levels of amino acid homology to manC and manB genes respectively. orf7 shows high level homology with rfbH which is an abequose pathway gene. orf8 encodes a protein with 12 transmembrane segments and has similarity in secondary structure to other wzx ,ens and is likely therefore to be the O antigen flippase gene.
Materials and Methods-part 2
A. Nucleotide Sequencing of 1 to 3,020 and 9,982 to 14,516 of the E. Coli O111 O Antigen Gene Cluster
The sub clones which contained novel nucleotide sequences, pPR1231(map position 0 and 1,510), pPR1237 (map position −300 to 2,744), pPR1239 (map position 2,744 to 4,168), pPR1245 (map, position 9,736 to 12,007) and pPR1246 (map position 12,007 to 15,300) (FIG. 2), were characterised as follows: the distal ends of the inserts of pPR1237, pPR1239 an pPR1245 were sequenced using the M13 forward and reverse primers located in the vector. PCR walking was carried out to sequence further into each insert using primers based on the sequence data and the primers were tagged with M13 forward or reverse primer sequences for sequencing. This PCR walking procedure was repeated until the entire insert was sequenced. pPR1246 was characterised from position 12,007 to 14,516. The DNA of these sub clones was sequenced in both directions. The sequencing reactions were performed using the dideoxy termination method and thermocycling and reaction products were analysed using flouorescent dye and an ABI automated sequencer (CA, USA).
B. Analysis of the E. Coli O111 O Antigen Gene Cluster (Positions 1 to 3,020 and 9,982 to 14,516 of SEQ ID NO:1) Nucleotide Sequence Data.
The gene organisation of regions of E. coli O111 O antigen gene cluster which were not characterised by Bastin and Reeves [1995 “Squence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111.”Gene 164: 17–23],(positions 1 to 3,020 and 9,982 to 14,516) is shown in FIG. 3. Thereare two open reading frames in region 1. Four open reading frames are predicted in region 2. The position of each gene is listed in Table 5.
The deduced amino acid sequence of orf1 (wbdH) shares about 64% similarity with that of the rfp gene of Shigella dysenteriae. Rfp and WbdH have very similar hydrophobicity plots and both have a very convincing predicted transmembrane segment in a corresponding position. rfp is a galactosyl transferase involved in the synthesis of LPS core, thus wbdH is likely to be a galactosyl transferase gene. orf2 has 85.7% identity at amino acid level to the gmd gene identified in the E. coli K-12 colanic acid gene cluster and is likely to be a gmd gene. orf9 encodes a protein with 10 predicted transmembrane segments and a large cytoplasmic loop. This inner membrane topology is a characteristic feature of all known O antigen polymerases thus it is likely that orf9 encodes an O antigen polymerase gene, wzy. orf10 (wbdL) has a deduced amino acid sequence with low homology with Lsi2 of Neisseria gonorrhoeae. Lsi2 is responsible for adding GlcNAc to galactose in the synthesis of lipooligosaccharide. Thus it is likely that wbdL is either a colitose or glucose transferase gene. orf11 (wbdM) shares high level nucleotide and amino acid similarity with TrsE of Yersinia enterocholitica. TrsE is a putative sugar transferase thus it is likely that wbdM encodes the colitose or glucose transferase.
In summary three putative transferase genes and an O antigen polymerase gene were identified at map position 1 to 3,020 and 9,982 to 14,516 of E. coli O111 O antigen gene cluster. A search of GenBank has shown that there are no genes with significant similarity at the nucleotide sequence level for two of the three putative transferase genes or the polymerase gene. SEQ ID NO:1 and FIG. 7 provide the nucleotide sequence of the O111 antigen gene cluster.
Materials and Methods-part 3
A. PCR Amplification of O157 Antigen gene Cluster from an E. Coli O157:H7 Strain (Strain C664–1992, from Statens Serum Institut, 5 Artillerivej, 2300, Copenhagen S, Denmark).
E. coli O157 O antigen gene cluster was amplified by using long PCR (Cheng et al. 1994, “Effective amplification of long targets from cloned inserts and human and genomic DNA” P.N.A.S. USA 91: 5695–569) with one primer (primer #412: att ggt agc tgt aag cca agg gcg gta gcg t (SEQ ID NO:5)) based on the JumpStart sequence usually found in the promoter region of O antigen gene clusters (Hobbs, et al. 1994 “The JumpStart sequence: a 39 bp element common to several polysaccharide gene dusted” Mol. Microbiol. 12: 855–856), and another primer #482 (cac tgc cat acc gac gac gcc gat ctg ttg ctt gg (SEQ ID NO:6)) based on the gnd gene usually found downstream of the O antigen gene cluster. Long PCR was carried out using the Expand Long Template PCR System from Boehringer Mannheim (Castle Hill NSW Australia), and products, 14 kb in length, from several reactions were combined and purified using the Promega Wizard PCR preps DNA purification System (Madison WI USA). The PCR product was then extracted with phenol and twice with ether, precipitated with 70% ethanol, and resuspended in 40 μL of water.
B. Construction of a Random DNase I Bank:
Two aliquots containing about 150 ng of DNA each were subjected to DNase I digestion using the Novagen DNase I Shotgun Cleavage (Madison Wis. USA) with a modified protocol as described. Each aliquot was diluted into 45 μl of 0.05 M Tris -HCl (pH7.5), 0.05 mg/mL BSA and 10 mM MnCl25 μL of 1:3000 or 1:4500 dilution of DNaseI (Novagen) (Madison Wis. USA) in the same buffer was added into each tube respectively and 10μl of stop buffer (100mM EDTA), 30% glycerol, 0.5% Orange G, 0.075% xylene and cyanol (Novagen) (Madison Wis. USA) was added after incubation at 15° C. for 5 min. The DNA from the two DNaseI reaction tubes were then combined and fractionated on a 0.8% LMT agarose gel, and the gelsegment with DNA of about 1 kb in size (about 1.5 mL agarose) was excised. DNA was extracted from agarose using Promega Wizard PCR Preps DNA Purification (Madison Wis. USA) and resuspended in 200 μL water, before being extracted with phenol and twice with ether, and precipitated. The DNA was then resuspended in 17.25 μL water and subjected to T4 DNA polymerase repair and single dA tailing using the Novagen Single dA Tailing Kit (Madison Wis. USA). The reaction product (85 μl containing about 8 ng DNA) was then extracted with chloroform:isoamyl alcohol (24:1) once and ligated to 3×10−3 pmol PGEM-T (Promega) (Madison Wis. USA) in a total volume of 100 μL. Ligation was carried out overnight at 4° C. and the ligated DNA was precipitated and resuspended in 20 μL water before being electroporated into E. coli strain JM109 and plated out on BCIG-IPTG plates to give a bank.
C. Sequencing
DNA templates from clones of the bank were prepared for sequencing using the 96-well format plasmid DNA miniprep kit from Advanced Genetic Technologies Corp (Gaithersburg Md. USA) The inserts of these clones were sequenced from one or both ends using the standard M13 sequencing primer sites located in the pGEM-T vector. Sequencing was carried out on an ABI377 automated sequencer (CA USA) as described above, after carrying out the sequencing reaction on an ABI Catalyst (CA USA). Sequence gaps and areas of inadequate coverage were PCR amplified directly from O157 chromosomal DNA using primers based on the already obtained sequencing data and sequenced using the standard M13 sequencing primer sites attached to the PCR primers.
D. Analysis of the E. Coli O157 O Antigen Gene Cluster Nucleotide Sequence Data
Sequence data were processed and analysed using the Staden programs [Staden, R., 1982 “Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing.” Nuc. Acid Res. 10: 4731–4751; Staden, R., 1986 “The current status and portability of our sequence handling software”. Nuc. Acid Res. 14: 217–231; Staden, R. 1982 “An interactive graphics program for comparing and aligning nucleic acid and amino acid sequence”. Nuc. Acid Res. 10: 2951–2961]. FIG. 4 shows the structure of E. coli O157 O antigen gene cluster. Twelve open reading frames were predicted from the sequence data, and the nucleotide and amino acid sequences of all these genes were then used to search the GenBank database for indication of possible function and specificity of these genes. The position of each gene is listed in Table 6. The nucleotide sequence is presented in SEQ ID NO:2 and FIG. 8.
orfs 10 and 11 showed high level identity to manC and manB and were named manC and manb respectively. orf7 showed 89% identity (at amino acid level) to the gmd gene of the E. coli colanic acid capsule gene cluster (Stevenson G., K. et al. 1996 “Organisation of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid”.J. Bacteriol. 178:4885–4893) and was named gmd. orf8 showed 79% and 69% identity (at amino acid level) respectively to wcaG of the E. coli colanic acid capsule gene cluster and to wbcJ (orf14.8) gene of the Yersinia enterocolitica O8 O antigen gene cluster (Zhang, L. et al. 1997 “Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Y. enterocolitica serotype O8”Mol. Microbiol. 23:63–76). Colanic acid and the Yersinia O8 O antigen both contain fucose as does the O157 O antigen. There are two enzymatic steps required for GDP-L-fucose synthesis from GDP-4-keto-6-deoxy-D-mannose, the product of the gmd gene product. However, it has been shown recently (Tonetti, M et al. 1996 Synthesis of GDP-L-fucose by the human FX protein J. Biol. Chem. 271:27274–27279) that the human FX protein has “significant homology” with the wcaG gene (referred to as Yefb in that paper), and that the FX protein carries out both reactions to convert GDP-4-keto-6-deoxy-D-mannose to GDP-L-fucose. We believe that this makes a very strong case for orf8 carrying out these two steps and propose to name the gene fcl. In support of the one enzyme carrying out both functions is the observation that there are no genes other than manB, manC, gmd and fcl with similar levels of similarity between the three bacterial gene clusters for fucose containing structures.
orf5 is very similar to wbeE (rfbE) of Vibrio cholerae 01, which is thought to be the perosamine synthetase, which converts GDP-4-keto-6-deoxy-D-mannose to GDP-perosamine (Stroeher, U.H et al. 1995 “A putative pathway for perosamine biosynthesis is the first function encoded within the rfb region of Vibrio choleraef”O1. Gene 166: 33–42). V. cholerae O1 and E. coli O157 O antigens contain perosamine and N-acetyl-perosamine respectively. The V. cholerae O1 manA, manB, gmd and wbeE genes are the only genes of the V. cholerae O1 gene cluster with significant similarity to genes of the E. coli O157 gene cluster and we believe that our observations both confirm the prediction made for the function of wbe of V. cholerae, and show that orf5 of the O157 gene cluster encodes GDP-perosamine synthetase. orf5 is therefore named per. orf5 plus about 100 bp of the upstream region (postion 4022–5308)was previously sequenced by Bilge, S.S. et al. [1996 “Role of the Escherichia coli O157-H7 O side chain in adherence and analysis of an rfb locus”.Infect. Immun. 64:4795–4801].
orf12 shows high level similarity to the conserved region of about 50 amino acids of various members of an acetyltransferase family (Lin, W., et al. 1994 “Sequence analysis and molecular characterisation of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus”. J. Bateriol. 176: 7005–7016) and we believe it is the N-acetyltransferase to convert GDP-perosamine to GDP-perNAc. orf12 has been named wbdR.
The genes manB, manC, gmd, fcl, per and wbdR account for all of the expected biosynthetic pathway genes of the O157 gene cluster.
The remaining biosynthetic step(s) required are for synthesis of UDP-GalNAc from UDP-Glc. It has been proposed (Zhang, L., et al. 1997 “Molecular and chemical characterisation of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O8”.Mol. Microbiol. 23:63–76) that in Yersinia enterocolitica UDP-GalNAc is synthesised from UDP-GlcNAc by a homologue of galactose epimerase (GalE), for which there is a galE like gene in the Yersinia enterocolitica O8 gene cluster. In the case of O157 there is no galE homologue in the gene cluster and it is not clear how UDP-GalNAc is synthesised. It is possible that the galactose epimerase encoded by the galE gene in the gal operon, can carry out conversion of UDP-GlcNAc to UDP-GalNAc in addition to conversion of UDP-Glc to UDP-Gal. There do not appear to be any gene(s) responsible for UDP-GalNAc synthesis in the O157 gene cluster.
orf4 shows similarity to many wzx genes and is named wzx and orf2 which shows similarity of secondary structure in the predicted protein to other wzy genes and is for that reason named wzy.
The orf1, orf3 and orf6 gene products all have characteristics of transferases, and have been named wbdN, wbdO and wbdp respectively. The O157 O antigen has 4 sugars and 4 transferases are expected. The first transferase to act would put a sugar phosphate onto undecaprenol phosphate. The two transferases known to perform this function, WbaP (RfbP) and WecA (Rfe) transfer galactose phosphate and N-acetyl-glucosamine phosphate respectively to undecaprenol phosphate. Neither of these sugars is present in the O157 structure.
Further, none of the presumptive transferases in the O157 gene cluster has the transmembrane segments found in WecA and WbaP which transfer a sugar phosphate to undecaprenol phosphate and expected for any protein which transferred a sugar to undecaprenol phosphate which is embedded within the membrane.
The WecA gene which transfers GlcNAc-P to undecaprenol phosphate is located in the Enterobactereal Common Antigen (ECA) gene cluster and it functions in ECA synthesis in most and perhaps all E. coli strains, and also in O antigen synthesis for those strains which have GlcNAc as the first sugar in the O unit.
It appears that WecA acts as the transferase for addition of GalNAc-1-P to undecaprenol phosphate for the Yersinia enterocolitica O8 O antigen [Zhang et al. 1997 “Molecular and chemical characterisation of the lipopolysaccharide O antigen and its role in the virulence of Yersinia enterocolitica serotype O8Mol. Microbiol. 23: 63–76.] and perhaps does so here as the O157 structure includes GalNAc. WecA has also been reported to add Glucose-1-P phosphate to undecaprenol phosphate in E. coli O8 and O9 strains, and an alternative possibility for transfer of the first sugar to undecaprenol phosphate is WecA mediated transfer of glucose, as there is a glucose residue in the O157 O antigen. In either case the requisite number of transferase genes are present if GalNAc or Glc is transferred by WecA and the side chain Glc is transferrd by a transferase outside of the O antigen gene cluster.
orf9 shows high level similarity (44% identity at amino acid level, sa engt) with wcah gene of the E. coli colanic acid capsule gene cluster. The function of this gene is unknown, and we give off orf9 the name wbdQ.
The DNA between manB and wdbR has strong sequence similarity to one of the H-repeat units of E. coli K12. Both of the inverted repeat sequences flanking this region are still recognisable, each with two of the 11 bases being changed. The H-repeat associated protein encoding gene located within this region has a 267 base deletion and mutations in various positions. It seems that the H-repeat unit has been associated with this gene cluster for a long period of time since it translocated to the gene cluster, perhaps playing a role in assembly of the gene cluster ashas been proposed in other cases.
Materials and Methods - part 4
To test our hypothesis that O antigen genes for transferases and the wzx, wzy genes were more specific than pathway genes for diagnostic PCR, we first carried out PCR using primers for all the E. coli 016 O antigen genes (Table 4). The PCR was then carried out using PCR primers for E. coli O111 transferase, wzx and wzy genes (Table 5, 5A). PCR was also carried out using PCR primers for the E. coli 0157 transferase, wzx and wzy genes (Table 6, 6A).
Chromosomal DNA from the 166 serotypes of E. coli available from Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen Denmark was isolated using the Promega Genomic (Madison Wis. USA) isolation kit. Note that 164 of the serogroups are described by Ewing W. H.: Edwards and Ewings “Identification of the Enterobacteriacea” Elsevier, Amsterdam 1986 and that they are numbered 1–171 with numbers 31, 47, 67, 72, 93, 94 and 122 no longer valid. Of the two serogroup 19 strains we used 19ab strain F8188–41. Lior H. 1994 [“Classification of Eschericia coli In Eschericia coli in domestic animals and humans pp 31–72. Edited by C. L. Gyles CAB international] adds two more numbered 172 and 173 to give the 166 serogroups used. Pools containing 5 to 8 samples of DNA per pool were made. Pool numbers 1 to 19 (Table 1) were used in the E. coli 0111 and 0157 assay. Pool numbers 20 to 28 were also used in the 0111 assay, and pool numbers 22 to 24 contained E. coli 0111 DNA and were used as positive controls (Table 2). Pool numbers 29 to 42 were also used in the 0157 assay, and pool numbers 31 to 36 contained E. coli 0157 DNA, and were used as positive controls (Table 3). Pool numbers 2 to 20, 30, 43 and 44 were used in the E. coli 016 assay (Tables 1 to 3). Pool number 44 contained DNA of E. coli K-12 strains C600 and WGI and was used as a positive control as between them they have all of the E. coli K-12 O16 O antigen genes.
PCR reactions were carried out under the following conditions: denaturing 94° C./30”; annealing, temperature varies (refer to Tables 4 to 8)/30”; extension, 72° C./1′; 30 cycles. PCR reaction was carried out in an volume of 25μL for each pool. After the PCR reaction, 10μL PCR product from each pool was run on an agarose gel to check for amplified DNA.
Each E. coli and S. enterica chromosomal DNA sample was checked by gel electrophoresis for the presence of chromosomal DNA and by PCR amplification of the E. coli or S. enterica mdh gene using oligonucleotides based on E. coli K-12 or Salmonella enterica LT2 [Boyd et al. (1994) “Molecular genetic basis of allelic polymorphism in malate degydrogenase (mdh) in natural populations of Escherichia coli and Salmonella enterica” Proc. Nat. Acad. Sci. USA. 91:1280–1284.] Chromosomal DNA samples from other bacteria were only checked by gel electrophoresis of chromosomal DNA.
A. Primers based on E. Coli O16 O Antigen Gene Cluster Sequence.
The O antigen gene cluster of E. coli O16 was the only typical E. coli O antigen gene cluster that had been fully sequenced prior to that of O111, and we chose it for testing our hypothesis. One pair of primers for each gene was tested against pools 2 to 20, 30 and 43 of E. coli chromosomal DNA. The primers, annealing temperatures and functional information for each gene are listed in Table 4.
For the five pathway genes, there were 17/21, 13/21, 0/21, 0/21, 0/21 positive pools for rmlB, rmlD, rmlA, rmlC and glf respectively (Table 4). For the wzx, wzy and three transferase genes there were no positives amongst the 21 pools of E. coli chromosomal DNA tested (Table 4). In each case the #44 pool gave a positive result.
B Primers Based on the E. Coli 0111 O Antigen Gene Clsuter Sequence.
One to four pairs of primers for each of the transferase, wzx and wzy genes of O111 were tested against the pools 1 to 21 of E. coli chromosomal DNA (Table 5). For wbdh, four pairs of primers, which bind to various regions of this gene, were tested and found to be specific for O111 as there was no amplified DNA of the correct size in any of those 21 pools of E. coli chromosomal DNA tested. Three pairs of primers for wbdM were tested, and they are all specific although primers #985/#986 produced a band of the wrong size from one pool. Three pairs of primers for wzx were tested and they all were specific. Two pairs of primers were betlted for wzy, both are specific although #980/#983 gave a band of the wrong size in all pools. One pair of primers for wbdL was tested and found unspecific and therefore further test was carried out. Thus, wzx, wzy and two of the three transferase genes are highly specific to O111. Bands of the wrong size found in amplified DNA are assumed to be due to chance hybridisation of genes widely present in E. coli. The primers, annealing temperatures and positions for each gene are in (Table 5).
The 0111 assay was also performed using pools including DNA from O antigen expressing Yersinia pseudotuberculosis, Shiaella boydii and Salmonella enterica strains (Table 5A). None of the oligonucleotides derived from wbdH, wzx, wzy or wbdM gave amplified DNA of the correct size with these pools. Notably, pool number 25 ncludes S. enterica Adelaide which has the same O antigen as E. coli 0111: this pool did not give a positive PCR result for any primers tested indicating that these genes are highly specifiic for E. coli 0111.
Each of the 12 pairs binding to wbdH, wzx, wzy and wbdM produces a band a predicted size with the pools containing 0111 DNA (pool number 22 to 24). As pools 22 to 24 included DNA from all strains present in pool 21 plus 0111 strain DNA (Table 2), we conclude that the 12 pairs of primers all give a positive PCR test with each of three unrelated 0111 strains but not with any other strains tested. Thus these genes are highly specific for E. coli O111.
C. Primers Based on the E. Coli 0157 O Antigen Gene Cluster Sequence.
Two or three primer pairs for each of the transferase, wzx and wzy genes of O157 were tested against E. coli chromosomal DNA of pools 1 to 19, 29 and 30 (Table 6). For wbdN, three pairs of primers, which bind to various regions of this gene, were tested and found to be specific for O157 as there was no amplified DNA in any of those 21 pools of E. coli chromosomal DNA tested. Three pairs of primers for wbdO were tested, and they are all specific although primers #1211/#1212 produced two or three bands of the wrong size from all pools. Three pairs of primers were tested for wbdP and they all were specific. Two pairs of primers were tested for wbdR and they were all specific. For wzy, three pairs of primers were tested and all were specific although primer pair #1203/#1204 produced one or three bands of the wrong size in each pool. For wzx, two pairs of primers were tested and both were specific although primer pair #1217/#1218 produced 2 bands of wrong size in 2 pools, and 1 band of wrong size in 7 pools. Bands of the wrong size found in amplified DNA are assumed to be due to chance hybridisation of genes widely present in E. coli. The primers, annealing temperatures and function information for each gene are in Table 6.
The 0157 assay was also performed using pools 37 to 42, including DNA from O antigen expressing Yersinia pseudotuberculosis, Shiaella boydii, Yersinia enterocolitica 09, Brucella abortus and Salmonella enterica strains (Table 6A). None of the oligonucleotides derived from wbdN, wzy, wbdO, wzx, wbdP or wbdr reacted specifically with these pools, except that primer pair #1203/#1204 produced two bands with Y. enterocolitica O9 and one of the bands is of the same size with that from the positive control. Primer pair #1203/#1204 binds to wzy. The predicted secondary structures of Wzy proteins are generally similar, although there is very low similarity at amino acid or DNA level among the sequenced wzy genes. Thus, it is possible that Y. enterolcolitica O9 has a wzy gene closely related to that of E. coli O157. It is also possible that this band is due to chance hybridization of another gene, as the other two wzy primer pairs (#1205/#1206 and #1207/#1208) did not produce any band with Y. enterocolitica O9. Notably, pool number 37 includes S. enterica Landau which has the same O antigen as E. coli O157, and pool 38 and 39 contain DNA of B. abortus and Y. enterocolitica O9 which cross react serologically with E. coli O157. This result indicates that these genes are highly O157 specific, although one primer pair may have cross reacted with Y. enterocolitica O9.
Each of the 16 pairs binding to wbdN, wzx, wzy, wbdO, wbdP and wbdR produces a band of predicted size with the pools containing 0157 DNA (pools number 31 to 36). As pool 29 included DNA from all strains present in pools 31 to 36 other than 0157 strain DNA (Table 3), we conclude that the 16 pairs of primers all give a positive PCR test with each of the five unrelated 0157 strains.
Thus PCR using primers based on genes wbdN, wzy, wbdO, wzx, wbdP and wbdR is highly specific for E. coli O157, giving positive results with each of six unrelated O157 strains while only one primer pair gave a band of the expected size with one of three strains with O antigens known to cross-react serologically with E. coli O157.
D. Primers Based on the Salmonella Enterica Serotype C2 and B O Antigen Gene Cluster Sequences.
We also performed a PCR using primers for the S. enterica C2 and B serogroup transferases, wzx, wzy and genes (Tables 7 to 9). The nucleotide sequences of C2 and B O antigen gene clusters are listed as SEQ ID NO: 3 (FIG. 9) and SEQ ID NO:4 (FIG. 10) respectively. Chromosomal DNA from all the 46 serotypes of Salmonella enterica (Table 9) was isolated using the Promega Genomic isolation kit, 7 pools of 4 to 8 samples per pool were made. Salmonella enterica serotype B or C2 DNA was omitted from the pool for testing primers of 46 respective serotypes but added to a pool containing 6 other samples to give pool number 8 for use as a positive control.
PCR reactions were carried out under the following conditions: denaturing, 94° C./30′′; annealing, temperature varies (see below)/30′′; extension, 72° C./1′; 30 cycles. PCR reaction was carried out in a volume of 25μL for each pool. After the PCR reaction, 10μL PCR product from each pool was run on an agarose gel to check for amplified DNA. For pools which gave a band of correct size, PCR was repeated using individual chromosomal samples of that pool, and agarose gel was run to check for amplified DNA from each sample.
The Salmonella enterica serotype B O antigen gene cluster (of strain LT2) was the first O antigen gene cluster to be fully sequenced, and the function of each gene has been identified experimentally [Jiang, X. M., Neal, B., Santiago, F., Lee, S. J., Romana, L. K., and Reeves, P. R. (1991) “Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2).” Mol. Microbiol. 5(3), 695–713; Liu, D., Cole, R., and Reeves, P. R. (1996). “An O antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase” J. Bacteriol., 178(7),2102–2107; Liu, D., Haase, A. M., Lindqvist, L., Lindberg, A. A., and Reeves, P. R. (1993). “Glycosyl transferases of O-antigen biosynthesis in S. enterica: identification and characterisation of transferase genes of groups B, C2 and E1.” J. Bacteriol., 175, 3408–3413; Liu, D., Lindquist, L., and Reeves P. R. (1995). “Transferases of O-antigen biosynthesis in Salmonella enterica:dideoxhexosyl transferases of groups B and C2 and acetyltransferase of group C2. ” J. Bacteriol. 177, 4084–4088; Romana, L. K., Santiago, F S., and Reeves, P. R. (1991). “High level expression and purification dThymidine-diphospho-D-glucose 4,6 dehydratase (rfbB) from Salmonella serovar typhimurium LT2.” BBRC, 174, 846–852]. One pair of primers for each of the pathway genes and wbaP was tested against the pools of Salmonella enterica DNA, two to three pairs of primers for each of the other transferases and wzx genes were also tested. See Table 8 for a list of primers and functional information of each gene, as well as the annealing temperature of the PCR reaction for each pair of primers.
For pathway genes of group B strain LT2, there are 19/45, 14/45, 15/45, 12/45, 6/45, 6/45, 6/45, 6/45, 1/45, 9/45, 8/45 positives for rmlB, zmlD, rmlA, rmlC, ddhD, ddhA, ddhb, ddhC, abe, manC, and manB repsectively (Table 9).
For the LT2 wzx gene we used three primer pairs each of which gave 1/45 positive. For the 4 transferase genes we used a total of 9 primer pairs. 2 primer pairs for wbaV gave 2/90 positives. For 3 primer pairs of wbaN, 11/135 gave a positive result. For the wbaP primer pair 10/45 gave a positive result (Table 9).
The experimental data show that oligonucleotides derived from the wzx and wbaV group B O antigen genes are specific for group B O antigen amongst all 45 Salmonella enterica O antigen groups except O group 67. The oligonucleotides derived from Salmonella enterica B group wbaN and wbaU genes detected B group O antigen and also produced positive results with groups A, D1 and D3. WbaU encodes a transferase for a Mannose α(1–4) Mannose linkage and is expressed in groups A, B and D1 while wbaN, which encodes a transferase for Rhamnose α(1–3) Galactose linkage is present in groups A, B, D1, D2, D3 and E1. This accounts for the positive results with the group B wbau and wbaN genes. The wbaN gene of groups E and D2 has considerable sequence differences from that of groups A, B, D1 and D3 and this accounts for the positive results only with groups B, D1 and D3.
The Salmonella enterica B primers derived from wzx and transferase genes produced a positive result with Salmonella enterica 067. We find that Salmonella enterica O67 has all the genes of the group B O antigen cluster. There are several possible explanations for this finding including the possibility that the gene cluster is not functional due to mutation and the group 067 antigenicity is due to another antigen, or the O antigen is modified after synthesis such that its antigenicity is changed. Salmonella enterica 067 would therefore be scored as Salmonella enterica group B in the PCR diagnostic assay. However, this is of little importance because Salmonella enterica 067 is a rare O antigen and only one (serovar Crossness) of the 2324 known serovars has the 067 serotype [Popoff M. Y. et al (1992) “Antigenic formulas of the Salmonella enterica serovars” 6th revision WHO Collaborating Centre for Reference and Research on Salmonella enterica, Institut Pasteur Paris France], and serovar Crossness had only been isolated once [M. Popoff, personal communication].
The Salmonella enterica B primers derived from wbaP reacted with group A, C2, D1, D2, D3, E1, 54, 55, 67 and E4 O antigen groups. WbaP encodes the galactosyl transferase which initiates O unit synthesis by transfer of Galactose phosphate to the lipid carrier Undecaprenol phosphate. This reaction is common to the synthesis of several O antigens. As such wbaP is distinguished from other transferases of the invention as it does not make a linkage within an O antigen.
We also tested 20 primer pairs for the wzx, wzy and 5 transferase genes of serotype C2 and found no positives in all the 7 pools (Table 7).
Groups A, B, D1, D2, D3, C2 and E1 share many genes in common. Some of these genes occur with more than one sequence in which case each specific sequence can be named after one of the serogroups in which it occurs. The distribution of these sequence specificities is shown in Table 10. The inventors have aligned the nucleotide sequences of Salmonella enterica wzy, wzx genes and transferase genes so as to determine specific combinations of nucleic acid molecules which can be employed to specifically detect and identify the Salmonella enterica groups A, B, D1, D2, D3, C2 and E1 (Table 10). The results show that many of the O antigen groups can be detected and identified using a single specific nucleic acid molecule although other groups in particular D2 and E1, and A and D1 require a panel of nucleic acid molecules derived from a combination of genes.
It will be understood that in carrying out the methods of the invention with respect to the testing of particular sample types including samples from food, patients and faeces the samples are prepared by routine techniques routinely used in the preparation of such samples for DNA based testing.









TABLE 1





Pool




No.
Strains of which chromosonal DNA included in the pool
Source*

















1
E. coli type strains for O serotypes 1, 2, 3, 4, 10, 16, 18 and 39
IMVSa


2
E. coli type strains for O serotypes 40, 41, 48, 49, 71, 73, 88 and 100
IMVS


3
E. coli type strains for O serotypes 102, 109, 119, 120, 121, 125, 126 and
IMVS



137


4
E. coli type strains for O serotypes 138, 139, 149, 7, 5, 6, 11 and 12
IMVS


5
E. coli type strains for O serotypes 13, 14, 15, 17, l9ab, 20, 21 and 22
IMVS


6
E. coli type strains for O serotypes 23, 24, 25, 26, 27, 28, 29 and 30
IMVS


7
E. coli type strains for O serotypes 32, 33, 34, 35, 36, 37, 38 and 42
IMVS


8
E. coli type strains for O serotypes 43, 44, 45, 46, 50, 51, 52 and 53
IMVS


9
E. coli type strains for O serotypes 54, 55, 56, 57, 58, 59, 60 and 61
IMVS


10
E. coli type strains for O serotypes 62, 63, 64, 65, 66, 68, 69 and 70
IMVS


11
E. coli type strains for O serotypes 74, 75, 76, 77, 78, 79, 80 and 81
IMVS


12
E. coli type strains for O serotypes 82, 83, 84, 85, 86, 87, 89 and 90
IMVS


13
E. coli type strains for O serotypes 91, 92, 95, 96, 97, 98, 99 and 101
IMVS


14
E. coli type strains for O serotypes 103, 104, 105, 106, 107, 108 and 110
IMVS


15
E. coli type strains for O serotypes 112, 162, 113, 114, 115, 116, 117 and
IMVS



118


16
E. coli type strains for O serotypes 123, 165, 166, 167, 168, 169, 170 and
See b



171


17
E. coli type strains for O serotypes 172, 173, 127, 128, 129, 130, 131 and
See c



132


18
E. coli type strains for O serotypes 133, 134, 135, 136, 140, 141, 142 and
IMVS



143


19
E. coli type strains for O serotypes 144, 145, 146, 147, 148, 150, 151 and
IMVS



152





*


aInstitute of Medical and Veterinary Science, Adelaide, Australia


b 123 from IMVS; the rest from Statens Serum Institut, Copenhagen, Denmark


c 172 and 173 from Statens Serum Institut, Copenhagen, Denmark, the rest from IMVS















TABLE 2





Pool




No.
Strains of which chromosonal DNA included in the pool
Source*

















20
E. coli type strains for O serotypes 153, 154, 155, 156, 157, 158, 159 and
IMVS



160


21
E. coli type strains for O serotypes 161, 163, 164, 8, 9 and 124
IMVS


22
As pool #21, plus E. coli 0111 type strain Stoke W.
IMVS


23
As pool #21, plus E. coil 0111:H2 strain C1250–1991
See d


24
As pool #21, plus E. coli 0111:H12 strain C156–1989
See e


25
As pool #21, plus S. enterica serovar Adelaide
See f


26
Y. pseudotuberculosis strains of O groups IA, IIA, IIB, IIC, III, IVA, IVB,
See g



VA, VB, VI and VII


27
S. boydii strains of serogroups 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 14 and 15
See h


28
S. enterica strains of serovars (each representing a different O group) Typhi,
IMVS



Montevideo, Ferruch, Jangwani, Raus, Hvittingfoss, Waycross, Dan,



Dugbe, Basel, 65,:i:e,n,z,15 and 52:d:e,n,x,z15





*


d C1250–1991 from Statens Serum Institut, Copenhagen, Denmark


e C156–1989 from Statens Serum Institut, Copenhagen, Denmark


f S. enterica serovar Adelaide from IMVS


g Dr. S. Aleksic of Institute of Hygiene, Germany


h Dr. J. Lefebvre of Bacterial Identification Section, Laboratoroie de Santè Publique du Quèbec, Canada















TABLE 3





Pool




No.
Strains of which chromosonal DNA included in the pool
Source*

















29
E. coli type strains for O serotypes 153, 154, 155, 156, 158, 159 and 160
IMVS


30
E. coli type strains for O serotypes 161, 163, 164, 8, 9, 111 and 124
IMVS


31
As pool #29, plus E. coli O157 type strain A2 (O157:H19)
IMVS


32
As pool #29, plus E. coli O157:H16 strain C475–89
See d


33
As pool #29, plus E. coli O157:H45 strain C727–89
See d


34
As pool #29, plus E. coli O157:H2 strain C252–94
See d


35
As pool #29, plus E. coli O157:H39 strain C258–94
See d


36
As pool #29, plus E. coli O157:H26
See e


37
As pool #29, plus S. enterica serovar Landau
See f


38
As pool #29, plus Brucella abortus
See g


39
As pool #29, plus Y. enterocolitica O9
See h


40
Y. pseudotuberculosis strains of O groups IA, IIA, IIB, IIC, III, IVA, IVB, VA,
See i



VB, VI and VII


41
S. boydii strains of serogroups 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 14 and 15
See j


42
S. enterica strains of serovars (each representing a different O group) Typhi,
IMVS



Montevideo, Ferruch, Jangwani, Raus, Hvittingfoss, Waycross, Dan, Dugbe,



Basel, 65:i:e,n,z15 and 52:d:e,n,x,z15


43
E. coli type strains for O serotypes 1, 2, 3, 4, 10, 18 and 29
IMVS


44
As pool #43, plus E. coli K-12 strains C600 and WG1
IVMS




See k





*


d O157 strains from Statens Serum Institut, Copenhagen, Denmark


e O157:H26 from Dr. R. Brown of Royal Children's Hospital, Melbourne, Victoria


f S. enterica serovar Landau from Dr. M. Poppoff of Institut Pasteur, Paris, France


g B. Abortus from the culture collection of The University of Sydney, Sydney, Australia


h Y. enterocolitica O9 from Dr. K. Bettelheim of Victorian Infectious Diseases Reference Laboratory Victoria, Australia.


i Dr. S. Aleksic of Institute of Hygiene, Germany


j Dr. J. Lefebvre of Bacterial Identification Section, Laboratoroie de Santè Publique du Quèbec, Canada


k Strains C600 and WG1 from Dr. B.J. Backmann of Department of Biology, Yale University, USA.













TABLE 4







PCR assay result using primers based on the E. coli serotype O16 (strain K-12) O antigen gene cluster sequence




















Number of pools





Base


Length of
(out of 21) giving
Annealing




positions of
Forward primer
Reverse primer
the PCR
band of correct
temperature


Gene
Function
the gene
(base positions)
(base positions)
fragment
size
of the PCR





rmlB*
TDP-rhamnose pathway
 90–1175
#1064(91–109)
#1065(1175–1157)
1085 bp
17
60° C.


rmlD*
TDP-rhamnose pathway
1175–2074
#1066(1175–1193)
#1067(2075–2058)
 901 bp
13
60° C.


rmlA*
TDP-rhamnose pathway
2132–3013
#1068(2131–2148)
#1069(3013–2995)
 883 bp
 0
60° C.


rmlC*
TDP-rhamnose pathway
3013–3570
#1070(3012–3029)
#1071(3570–3551)
 559 bp
 0
60° C.


gtf*
Galactofuranose pathway
4822–5925
#1074(4822–4840)
#1075(5925–5908)
1104 bp
 0
55° C.


wzx*
Flippase
3567–4814
#1072(3567–3586)
#1073(4814–4797)
1248 bp
 0
55° C.


wzy*
O polymerase
5925–7091
#1076(5925–5944)
#1077(7091–7074)
1167 bp
 0
60° C.


wbbl*
Galactofuranosyl
7094–8086
#1078(7094–7111)
#1079(8086–8069)
 993 bp
 0
50° C.



transferase


wbbJ*
Acetyltransferase
8067–8654
#1080(8067–8084)
#1081(8654–8632)
 588 bp
 0
60° C.


wbbK**
Glucosyl transferase
5770–6888
#1082(5770–5787)
#1083(6888–6871)
1119 bp
 0
55° C.


wbbL***
Rhamanosyltransferase
 679–1437
#1084(679–697)
#1085(1473–1456)
 795 bp
   0****
55° C.





*,**,***Base positions based on GenBank entry U09876, U03041 and L19537 respectively


****19 pools giving a band of wrong size













TABLE 5







PCR assay data using 0111 primers














Base positions of



Number of pools
Annealing



the gene according
Forward primer
Reverse primer
Length of the
(out of 21) giving
temperature


Gene
to SEQ ID NO: 1
(base positions)
(base positions)
PCR fragment
band of correct size
of the PCR





wbdH
 739–1932
 #866(739–757)
 #867(1941–1924)
1203 bp
0
60° C.




 #976(925–942)
 #978(1731–1714)
 807 bp
0
60° C.




 #976(925–942)
 #979(1347–1330)
 423 bp
0
60° C.




 #977(1165–1182)
 #978(1731–1714)
 567 bp
0
60° C.


wzx
 8646–9911
 #969(8646–8663)
 #970(9908–9891)
1263 bp
0
50° C.




#1060(8906–8923)
#1062(9468–9451)
 563 bp
0
60° C.




#1061(9150–9167)
#1063(9754–9737)
 605 bp
0
50° C.


wzy
 9901–10953
 #900(9976–9996)
 #901(10827–10807)
 852 bp
0
60° C.




 #980(10113–10130)
 #983(10484–10467)
 372 bp
 0*
61° C.


wbdL
10931–11824
 #870(10931–10949)
 #871(11824–11796)
 894 bp
7
60° C.


wbdM
11821–12945
 #868(11821–11844)
 #869(12945–12924)
1125 bp
0
60° C.




 #984(12042–12059)
 #987(12447–12430)
 406 bp
0
60° C.




 #985(12258–12275)
 #986(12698–12681)
 441 bp
 0**
65° C.





*Giving a band of wrong size in all pools


**One pool giving a band of wrong size













TABLE 5A







PCR specificity test data using 0111 primers














Base positions



Number of pools




of the gene



(pools no. 25–28)
Annealing



according to
Forward primer
Reverse primer
Length of the
giving band
Temperature


Gene
SEQ ID NO: 1
(base positions)
(base positions)
PCR Fragment
of correct size
of the PCR





wbdH
 739–1932
 #866(739–757)
 #867(1941–1924)
1203 bp
 0*
60° C.




 #976(925–942)
 #978(1731–1714)
 807 bp
0
60° C.




 #976(925–942)
 #979(1347–1330)
 423 bp
0
60° C.




 #977(1165–1182)
 #978(1731–1714)
 567 bp
0
60° C.


wzx
 8646–9911
 #969(8646–8663)
 #970(9908–9891)
1263 bp
0
55° C.




#1060(8906–8923)
#1062(9468–9451)
 563 bp
0
60° C.




#1061(9150–9167)
#1063(9754–9737)
 605 bp
 0*
50° C.


wzy
 9901–10953
 #900(9976–9996)
 #901(10827–10807)
 852 bp
0
60° C.




 #980(10113–10130)
 #983(10484–10467)
 372 bp
 0**
60° C.


wbdL
10931–11824
 #870(10931–10949)
 #871(11824–11796)
 894 bp
0
60° C.


wbdM
11821–12945
 #868(11821–11844)
 #869(12945–12924)
1125 bp
0
60° C.




 #984(12042–12059)
 #987(12447–12430)
 406 bp
0
60° C.




 #985(12258–12275)
 #986(12698–12681)
 441 bp
 0*
65° C.





*1 pool giving a band of wrong size


**2 pools giving 3 bands of wrong sizes, 1 pool giving 2 bands of wrong sizes













TABLE 6







PCR results using primers based on the E. coli O157 sequence
















Base position



Number of pools





of the gene


Length of
(out of 21)
Annealing




according to
Forward primer
Reverse primer
the PCR
giving band of
Temperature


Gene
Function
SEQ ID NO: 2
(base position)
(base position)
fragment
correct size
of the PCR

















wbdN
Sugar transferase
  79–861
#1197(79–96)
#1198(861–844)
783
0
55° C.





#1199(184–201)
#1200(531–514)
348
0
55° C.





#1201(310–327)
#1202(768–751)
459
0
55° C.


wzy
O antigen
 858–2042
#1203(858–875)
#1204(2042–2025)
1185
 0*
50° C.





#1205(1053–1070)
#1206(1619–1602)
567
0
63° C.





#1207(1278–1295)
#1208(1913–1896)
636
0
60° C.


wbdO
Sugar transferase
 2011–2757
#1209(2011–2028)
#1210(2757–2740)
747
0
50° C.





#1211(2110–2127)
#1212(2493–2476)
384
 0**
62° C.





#1213(2305–2322)
#1214(2682–2665)
378
0
60° C.


wzx
O antigen flippase
 2744–4135
#1215(2744–2761)
#1216(4135–4118)
1392
0
50° C.





#1217(2942–2959)
#1218(3628–3611)
687
  0***
63° C.


wbdP
Sugar transferase
 5257–6471
#1221(5257–5274)
#1222(6471–6454)
1215
0
55° C.





#1223(5440–5457)
#1224(5973–5956)
534
0
55° C.





#1225(5707–5724)
#1226(6231–6214)
525
0
55° C.


wbdR
N-acetyl transferase
13156–13821
#1229(13261–13278)
#1230(13629–13612)
369
0
55° C.





#1231(13384–13401)
#1232(13731–13714)
348
0
60° C.





*3 bands of wrong size in one pool, 1 band of wrong size in all other pools


**3 bands of wrong sizes in 9 pools, 2 bands of wrong size in all other pools


***2 bands of wrong sizes in 2 pools, 1 band of wrong size in 7 pools













TABLE 6A







PCR results using primers based on the E. coli O157 sequence
















Base position



Number or pools





of the gene


Length of
(pools no. 37–42)
Annealing




according to
Forward primer
Reverse primer
the PCR
giving band of
temperature


Gene
Function
SEQ ID NO: 2
(base positions)
(base positions)
fragment
correct size
of the PCR

















wbdN
Sugar transferase
  79–861
#1197(79–96)
#1198(861–844)
783
 0*
55° C.





#1199(184–201)
#1200(531–514)
348
 0*
55° C.





#1201(310–327)
#1202(768–751)
459
0
61° C.


wzy
O antigen polymerase
 858–2042
#1203(858–875)
#1204(2042–2025)
1185
 1**
50° C.





#1205(1053–1070)
#1206(1619–1602)
567
  0***
60° C.





#1207(1278–1295)
#1208(1913–1896)
636
0
60° C.


wbdO
Sugar transferase
 2011–2757
#1209(2011–2028)
#1210(2757–2740)
747
0
50° C.





#1211(2110–2127)
#1212(2493–2476)
384
  0****
61° C.





#1213(2305–2322)
#1214(2682–2665)
378
0
60° C.


wzx
O antigen flippase
 2744–4135
#1215(2744–2761)
#1216(4135–4118)
1392
0
50° C.





#1217(2942–2959)
#1218(3628–3611)
687
0
63° C.


wbdP
Sugar transferase
 5257–6471
#1221(5257–5274)
#1222(6471–6454)
1215
0
55° C.





#1223(5440–5457)
#1224(5973–5956)
534
 0*
60° C.





#1225(5707–5724)
#1226(6231–6214)
525
0
55° C.


wbdR
N-acetyl transferase
13156–13821
#1229(13261–13278)
#1230(13629–
369
0
50° C.





#1231(13384–13401)
#1232(13731–
348
0
60° C.





*1 band of wrong size in one pool


**pool #39 giving two bands, one band of correct size, the other band of wrong size in another pool


***2 bands of wrong sizes in one pool


****3 bands of wrong sizes in 2 pools, 2 bands of wrong sizes in 2 other pools













TABLE 7







PCR assay data using primers based on the Salmonella enterica serotype C2 (strain M67)


O antigen gene cluster sequence
















Base positions



Number of





of the gene


Length of
pools (out of 7)
Annealing




according to
Forward primer
Reverse primer
the PCR
giving band of
temperature


Gene
Function
SEQ ID NO: 3
(base position)
(base position)
fragment
correct size
of the PCR





wzx
Flippase
1019–2359
#1144(1019–1036)
#1145(1414–1397)
396 bp
0
55° C.





#1146(1708–1725)
#1147(2170–2153)
463 bp
0
55° C.





#1148(1938–1955)
#1149(2356–2339)
419 bp
0
55° C.


wbaR
Abequosyl transferase
2352–3314
#1150(2352–2369)
#1151(2759–2742)
408 bp
0
55° C.





#1152(2601–2618)
#1153(3047–3030)
447 bp
0
55° C.





#1154(2910–2927)
#1155(3311–3294)
402 bp
0
55° C.


wbaL
Acetyl transferase
3361–3875
#1156(3361–3378)
#1157(3759–3742)
399 bp
0
55° C.





#1158(3578–3595)
#1159(3972–3955)
395 bp
0
50° C.


wbaQ
Rhamnosyl
3977–5020
#1160(3977–3994)
#1161(4378–4361)
402 bp
0
55° C.





#1162(4167–4184)
#1163(4774–4757)
608 bp
0
55° C.





#1164(4603–4620)
#1165(5017–5000)
415 bp
 0*
60° C.


wzy
O polymerase
5114–6313
#1166(5114–5131)
#1167(5515–5498)
402 bp
 0**
55° C.





#1168(5664–5681)
#1169(6112–6095)
449 bp
0
55° C.





#1170(5907–5924)
#1171(6310–6293)
404 bp
0
55° C.


wbaW
Mannosyl transferase
6313–7323
#1172(6313–6330)
#1173(6805–6788)
493 bp
0
50° C.





#1174(6697–6714)
#1175(7068–7051)
372 bp
0
55° C.





#1176(6905–6922)
#1177(7320–7303)
416 bp
0
55° C.


wbaZ
Mannosyl transferase
7310–8467
#1178(7310–7327)
#1179(7775–7758)
466 bp
0
50° C.





#1180(7530–7547)
#1181(7907–7890)
378 bp
0
55° C.





#1182(8007–8024)
#1183(8464–8447)
458 bp
0
55° C.





*Positive pool gives another band, which is also present in another pool. All other pools gave bands of wrong size.


**Band of wrong size in 6 other pools.













TABLE 8







PCR primers based on the Salmonella enterica serotype B (strain LT2) O antigen gene cluster sequence















Base position of








the gene


Length of
Annealing




according to
Forward primer
Reverse primer
the PCR
temperature of


Gene
Function
SEQ ID NO: 4
(base position)
(base position)
fragment
the PCR





rmlB
TDP-rhamnose pathway
 4099–5184
#1094(4100–4117)
#1095(4499–4482)
400 bp
55° C.


rmlD
TDP-rhamnose pathway
 5184–6083
#1092(5186–5203)
#1093(5543–5526)
358 bp
50° C.


rmlA
TDP-rhamnose pathway
 6131–7009
#1090(6531–6548)
#1091(6837–6820)
308 bp
55° C.


rmlC
TDP-rhamnose pathway
 7010–7561
#1088(7013–7030)
#1089(7372–7355)
360 bp
55° C.


ddhD
CDP-abequose pathway
 7567–8559
#1112(7567–7584)
#1113(7970–7953)
404 bp
55° C.


ddhA
CDP-adequose pathway
 8556–9329
#1114(8556–8573)
#1115(8975–8958)
420 bp
60° C.


ddhB
CDP-adequose pathway
 9334–10413
#1116(9334–9351)
#1117(9816–9799)
483 bp
45° C.


ddhC
CDP-adequose pathway
10440–11753
#1118(10440–10457)
#1119(10871–10854)
432 bp
60° C.


abe
CDP-adequose pathway
11781–12680
#1100(12008–12025)
#1101(12388–12371)
381 bp
55° C.


wzx
Flippase
12762–14054
#1120(12762–12779)
#1121(13150–13133)
389 bp
55° C.





#1122(12993–13010)
#1123(13417–13400)
425 bp
55° C.





#1124(13635–13652)
#1125(14051–14034)
417 bp
55° C.


wbaV
Abequosyl transferase
14059–15060
#1126(14059–14076)
#1127(14421–14404)
363 bp
45° C.





#1128(14688–14705)
#1129(15057–15040)
370 bp
45° C.


wbaU
Mannosyl transferase
15379–16440
#1130(15379–15396)
#1131(15768–15751)
390 bp
60° C.





#1132(15850–15867)
#1133(16262–16245)
413 bp
50° C.





#1134(16027–16044)
#1135(16437–16420)
411 bp
60° C.


wbaN
Rhamnosyl transferase
16441–17385
#1136(16441–16458)
#1137(16851–16834)
411 bp
45° C.





#1138(16630–16647)
#1139(17087–17070)
458 bp
55° C.





#1140(16978–16995)
#1141(17382–17365)
405 bp
50° C.


manC
GDP-mannose pathway
17386–18825
#1098(17457–17474)
#1099(18143–18126)
687 bp
60° C.


mabB
GDP-mannose pathway
18812–20245
#1096(18991–19008)
#1097(19345–19328)
355 bp
55° C.


wbaP
Galactosyl transferase
20317–21747
#1142(20389–20406)
#1143(20709–20692)
321 bp
55° C.
















TABLE 9





PCR results using LT2 primers*































1094
1092
1090
1088
1112
1114
1116
1118
1100
1120
1122
1124
1126




–
–
–
–
–
–
–
–
–
–
–
–
–


Strain
O
1095
1093
1091
1089
1113
1115
1117
1119
1101
1121
1123
1125
1127


name
group
rmlB
rmlD
rmlA
rmlC
ddhD
ddhA
ddhB
ddhC
abe
wzx
wzx
wzx
wbaV





M8
A
y
y
y
y
y
y
y
y


P9003
B
y
y
y
y
y
y
y
y
y
y
y
y
y


M40
C1


M67
C2
y
y
y
y
y
y
y
y


M18
D1
y
y
y
y
y
y
y
y


M388
D2
y

y
y
y
y
y
y


M344
D3
y
y
y
y
y
y
y
y


M32
E1
y
y
y
y


M324
F
y
y
y
y


M258
G


M252
H


M264
I


M254
J


M255
K


M7
L


M269
M
y
y
y


M270
N


M95
O


M260
P


M273
Q
y


M261
R


M282
S


M287
T
y
y
y
y


M295
U


M289
V


M296
W


M278
X


M298
Y


M93
Z


M291
51


M309
52


M303
53
y
y
y


M292
54
y
y
y
y


M304
56
y


M293
57
y
y
y


M305
58


M285
59
y
y
y
y


M306
60
y


M328
61


M330
65


M322
66


M1408
33


M1409
62


M1410
63
y


M1413
67
y
y
y
y
y
y
y
y
y
y
y
y
y


M74
E4
y
y
y
y

























1128
1130
1132
1134
1136
1138
1140
1098
1096
1142





–
–
–
–
–
–
–
–
–
–



Strain
O
1129
1131
1133
1135
1137
1139
1141
1099
1097
1143



name
group
wbaV
wbaU
wbaU
wbaU
wbaN
wbaN
wbaN
mmC
mmB
wbap







M8
A

y
y
y
y
y
y
y
y
y



P9003
B
y
y
y
y
y
y
y
y
y
y



M40
C1



M67
C2







y
y
y



M18
D1

y
y
y
y
y
y
y
y
y



M388
D2







y
y
y



M344
D3

y
y


y
y
y

y



M32
E1







y
y
y



M324
F



M258
G



M252
H



M264
I



M254
J



M255
K



M7
L



M269
M



M270
N



M95
O



M260
P



M273
Q



M261
R



M282
S



M287
T



M295
U



M289
V



M296
W



M278
X



M298
Y



M93
Z



M291
51



M309
52



M303
53



M292
54







y
y
y



M304
56



M293
57



M305
58



M285
59



M306
60



M328
61



M330
65



M322
66



M1408
33









y



M1409
62



M1410
63



M1413
67
y
y
y
y
y
y
y
y
y
y



M74
E4







y
y
y







*y indicates a positive PCR result. Blank indicates a negative result.













TABLE 10







Gene specificities in Salmonella enterica serogroups









Genes


















Serogroup
wzy
wzx
wbaP
wbaU
wbaN
wbaV
wbaO
wbaW
wbaZ
wbaQ
wbaR





A
B
D
B
B
B
D
—
—
—
—
—


B
B
B
B
B
B
B
—
—
—
—
—


D1
B
D
B
B
B
D
—
—
—
—
—


D2
E1
D
B
—
E1
D
E1
—
—
—
—


D3
D3
D
B
B
B
D
—
—
—
—
—


C2
C2
C2
B
—
—
—
—
C2
C2
C2
C2


E1
E1
E1
B
—
E1
—
E1





— means ‘not present’

## Claims

1. A method of testing a sample for the presence of E. coli encoding bacterial polysaccharide O-antigen serotype O111, the method comprising the steps of:
(a) providing a sample to be tested;
(b) providing at least one oligonucleotide molecule which is about 17 to 28 nucleotides in length, and hybridizes using highly stringent wash conditions, to a nucleic acid sequence selected from the group consisting of:
wbdH (nucleotide positions 739 to 1932 of SEQ ID NO:1);
wzx (nucleotide positions 8646 to 9911 of SEQ ID NO:1) wzy (nucleotide positions 9901 to 10953 of SEQ ID NO:1); and
wbdM (nucleotide positions 11821 to 12945 of SEQ ID NO:1)

or to at least one nucleic acid sequence complementary to the group consisting of:
wbdH (nucleotide positions 739 to 1932 of SEQ ID NO:1);
wzx (nucleotide positions 8646 to 9911 of SEQ ID NO:1)
wzy (nucleotide positions 9901 to 10953 of SEQ ID NO:1); and
wbdM (nucleotide positions 11821 to 12945 of SEQ ID NO:1),

(c) contacting said sample with said at least one oligonucleotide molecule to permit said oligonucleotide molecule to hybridize under said highly stringent wash conditions to said nucleic acid sequence when present in said sample; and
(d) detecting any hybridized oligonucleotide molecules, wherein detection of said hybridized oligonucleotide molecules indicates the presence of said E. coli in said sample.

2. The method as claimed in claim 1, wherein step (b) involves providing one pair of oligonucleotide molecules, and wherein at least one oligonucleotide molecule of said pair hybridizes to one of said nucleic acid sequence.

3. The method as claimed in claim 2, wherein said pair of oligonucleotide molecules is a pair of polymerase chain reaction primers.

4. The method as claimed in claim 1, wherein said at least one oligonucleotide molecule is selected from the group consisting of positions 739–757 of SEQ ID NO:1, positions 925–942 of SEQ ID NO:1, positions 1165–1182 of SEQ ID NO:1, positions 8646–8663 of SEQ ID NO:1, positions 8906–8923 of SEQ ID NO:1, positions 9150–9167 of SEQ ID NO:1, positions 9976–9996 of SEQ ID NO:1, positions 10113–10130 of SEQ ID NO:1, positions 11821–11844 of SEQ ID NO:1, positions 12042–10259 of SEQ ID NO:1, positions 12258–12275 of SEQ ID NO:1, positions 1941–1924 of SEQ ID NO:1, positions 1731–1714 of SEQ ID NO:1, positions 1347–1330 of SEQ ID NO:1, positions 9908–9891 of SEQ ID NO:1, positions 9468–9451 of SEQ ID NO:1, positions 9754–9737 of SEQ ID NO:1, positions 10827–10807 of SEQ ID NO:1, positions 10484–10467 of SEQ ID NO:1, positions 12945–12924 of SEQ ID NO:1, positions 12447–12430 of SEQ ID NO:1and positions 12698–12681 of SEQ ID NO:1.

5. A method of testing a sample for the presence of E. coli encoding bacterial polysaccharide O-antigen serotype 0157, the method comprising the steps of:
(a) providing a sample to be tested;
(b) providing at least one oligonucleotide molecule which is about 17 to 28 nucleotides in length and hybridizes using highly stringent wash conditions, to a nucleic acid sequence selected from the group consisting of:
wbdN (nucleotide position 79 to 861 of SEQ ID NO:2);
wbdO (nucleotide positions 2011 to 2757 of SEQ ID NO:2);
wbdP (nucleotide positions 5365 to 6471 of SEQ ID NO:2);
wbdR (nucleotide positions 13156 to 13821 of SEQ ID NO:2);
wzx (nucleotide positions 2744 to 3109 of SEQ ID NO:2); and
wzy (nucleotide positions 858 to 2042 of SEQ ID NO:2),

or to at least one nucleic acid sequence complementary to the group consisting of:
wbdN (nucleotide position 79 to 861 of SEQ ID NO:2);
wbdO (nucleotide positions 2011 to 2757 of SEQ ID NO:2);
wbdP (nucleotide positions 5365 to 6471 of SEQ ID NO:2);
wbdR (nucleotide positions 13156 to 13821 of SEQ ID NO:2);
wzx (nucleotide positions 2744 to 3109 of SEQ ID NO:2); and
wzy (nucleotide positions 858 to 2042 of SEQ ID NO:2),

(c) contacting said sample with said at least one oligonucleotide molecule to permit said oligonucleotide molecule to hybridize under said highly stringent wash conditions to said nucleic acid sequence when present in said sample; and
(d) detecting any hybridized oligonucleotide molecules, wherein detection of said hybridized oligonucleotide molecules indicates the presence of said E. coli in said sample.

6. The method as claimed in claim 5, wherein step (b) involves providing one pair of oligonucleotide molecules, and wherein at least one oligonucleotide molecule of said pair hybridizes to one of said nucleic acid sequence.

7. The method as claimed in claim 6, wherein said pair of oligonucleotide molecules is a pair of polymerase chain reaction primers.

8. The method as claimed in claim 5, wherein said at least one oligonucleotide molecule is selected from the group consisting of positions 79–96 of SEQ ID NO:2, positions 184–201 of SEQ ID NO:2, positions 310–327 of SEQ ID NO:2, positions 858–875 of SEQ ID NO:2, positions 1053–1070 of SEQ ID NO:2, positions 1278–1295 of SEQ ID NO:2, positions 2011–2028 of SEQ ID NO:2, positions 2110–2127 of SEQ ID NO:2, positions 2305–2322 of SEQ ID NO:2, positions 2744–2761 of SEQ ID NO:2, positions 2942–2959 of SEQ ID NO:2, positions 5440–5457 of SEQ ID NO:2, positions 5707–5724 of SEQ ID NO:2, positions 13261–13278 of SEQ ID NO:2, positions 13384–13401 of SEQ ID NO:2, positions 861–844 of SEQ ID NO:2, positions 531–514 of SEQ ID NO:2, positions 768–751 of SEQ ID NO:2, positions 2042–2025 of SEQ ID NO:2, positions 1619–1602 of SEQ ID NO:2, positions 1913–1896 of SEQ ID NO:2, positions 2757–2740 of SEQ ID NO:2, positions 2493–2476 of SEQ ID NO:2, positions 2682–2665 of SEQ ID NO:2, positions 6471–6454 of SEQ ID NO:2, positions 5973–5956 of SEQ ID NO:2, positions 6231–6214 of SEQ ID NO:2, positions 13629–13612 of SEQ ID NO:2 and positions 13731–13714 of SEQ ID NO:2.

9. A method of testing a sample for the presence of S. enterica encoding bacterial polysaccharide O-antigen serotype C2, the method comprising the steps of:
(a) providing a sample to be tested;
(b) providing at least one oligonucleotide molecule which is about 17 to 28 nucleotides in length, and hybridizes using highly stringent wash conditions, to a nucleic acid sequence selected from the group consisting of:
wbaR (nucleotide positions at 2352 to 3314 of SEQ ID NO:3);
wbaL (nucleotide positions 3361 to 3875 of SEQ ID NO:3);
wbaQ (nucleotide positions 3977 to 5020 of SEQ ID NO:3);
wbaW (nucleotide positions 6313 to 7323 of SEQ ID NO:3);
wbaZ (nucleotide positions 7310 to 8467 of SEQ ID NO:3);
wzx (nucleotide positions 1019 to 2359 of SEQ ID NO:3); and
wzy (nucleotide positions 5114 to 6313 of SEQ ID NO:3),

or to at least one nucleic acid sequence complementary to the group consisting of:
wbaR (nucleotide positions at 2352 to 3314 of SEQ ID NO:3);
wbaL (nucleotide positions 3361 to 3875 of SEQ ID NO:3);
wbaQ (nucleotide positions 3977 to 5020 of SEQ ID NO:3);
wbaW (nucleotide positions 6313 to 7323 of SEQ ID NO:3);
wbaZ (riucleotide positions 7310 to 8467 of SEQ ID NO:3);
wzx (nucleotide positions 1019 to 2359 of SEQ ID NO:3); and
wzy (nucleotide positions 5114 to 6313 of SEQ ID NO:3),

(c) contacting said sample with said at least one oligonucleotide molecule to permit said oligonucleotide molecule to hybridize under said highly stringent wash conditions to said nucleic acid sequence when present in said sample; and
(d) detecting any hybridized oligonucleotide molecules, wherein detection of said hybridized oligonucleotide molecules indicates the presence of said S. enteria in said sample.

10. The method as claimed in claim 9, wherein step (b) involves providing one pair of oligonucleotide molecules, and wherein at least one oligonucleotide molecule of the pair hybridizes to one of said nucleic acid sequence.

11. The method as claimed in claim 10, wherein said pair of oligonucleotide molecules is a pair of polymerase chain reaction primers.

12. The method as claimed in claim 9, wherein said at least one oligonucleotide molecule is selected from the group consisting of positions 1019–1036 of SEQ ID NO:3, positions 1708–1725 of SEQ ID NO:3, positions 1938–1955 of SEQ ID NO:3, positions 2352–2369 of SEQ ID NO:3, positions 2601–2618 of SEQ ID NO:3, positions 2910–2927 of SEQ ID NO:3, positions 3361–3378 of SEQ ID NO:3, positions 3578–3595 of SEQ ID NO:3, positions 3977–3994 of SEQ ID NO:3, positions 4167–4184 of SEQ ID NO:3, positions 4603–4620 of SEQ ID NO:3, positions 5114–5131 of SEQ ID NO:3, positions 5664–5681 of SEQ ID NO:3, positions 5907–5924 of SEQ ID NO:3, positions 6313–6330 of SEQ ID NO:3, positions 6697–6714 of SEQ ID NO:3, positions 6905–6922 of SEQ ID NO:3, positions 7310–7327 of SEQ ID NO:3, positions 7530–7547 of SEQ ID NO:3, positions 8007–8024 of SEQ ID NO:3, positions 1414–1397 of SEQ ID NO:3, positions 2170–2153 of SEQ ID NO:3, positions 2356–2339 of SEQ ID NO:3, positions 2759–2742 of SEQ ID NO:3, positions 3047–3030 of SEQ ID NO:3, positions 3311–3294 of SEQ ID NO:3, positions 3759–3742 of SEQ ID NO:3, positions 4378–4361 of SEQ ID NO:3, positions 4774–4757 of SEQ ID NO:3, positions 5017–5000 of SEQ ID NO:3, positions 5515–5498 of SEQ ID NO:3, positions 6112–6095 of SEQ ID NO:3, positions 6310–6293 of SEQ ID NO:3, positions 6805–6788 of SEQ ID NO:3, positions 7068–7051 of SEQ ID NO:3, positions 7320–7303 of SEQ ID NO:3, positions 7775–7758 of SEQ ID NO:3, positions 7907–7890 of SEQ ID NO:3 and positions 8464–8447 of SEQ ID NO:3.

13. A method of testing a sample for the presence of S. enteria encoding bacterial polysaccharide O-antigen serotype B, the method comprising the steps:
(a) providing a sample to be tested;
(b) providing at least one oligonucleotide molecule which is about 17 to 28 nucleotides in length and hybridizes using highly stringent wash conditions to a nucleic acid sequence selected from the group consisting of:
wzx (nucleotide positions 12762 to 14054 of SEQ ID NO: 4); and
wbaV (nucleotide positions 14059 to 15060 of SEQ ID NO: 4), or to at least one nucleic acid sequence complementary to the group consisting of:
wzx (nucleotide positions 12762 to 14054 of SEQ ID NO: 4); and
wbaV (nucleotide positions 14059 to 15060 of SEQ ID NO: 4),

(c) contacting said sample with said at least one oligonucleotide molecule to permit said oligonucleotide molecule to hybridize under said highly stringent wash conditions to said nucleic acid sequence when present in said genomic DNA; and
(d) detecting any hybridized oligonucleotide molecules, wherein detection of said hybridized oligonucleotide molecules indicates the presence of said S. enteria in said samples.

14. The method as claimed in claim 13, wherein step (b) involves providing one pair of oligonucleotide molecules, and wherein at least one oligonucleotide molecule of said pair hybridizes to one of said nucleic acid sequence.

15. The method as claimed in claim 14, wherein said pair of oligonucleotide molecules is a pair of polymerase chain reaction primers.

16. The method as claimed in claim 13, wherein said at least one oligonucleotide molecule is selected from the group consisting of positions 12762–12779 of SEQ ID NO:4, positions 12993–13010 of SEQ ID NO:4, positions 13635–13652 of SEQ ID NO:4, positions 14059–14076 of SEQ ID NO:4, positions 14688–14705 of SEQ ID NO:4, positions 13150–13133 of SEQ ID 140:4, positions 13417–13400 of SEQ ID NO:4, positions 14051–14034 of SEQ ID NO:4, positions 14421–14404 of SEQ ID NO:4, and positions 15057–15040 of SEQ ID NO:4.

17. The method as claimed in any one of claims 1, 5, 9 or 13, wherein the method further comprises providing at least one further oligonucleotide molecule, said further oligonucleotide molecule hybridizes using highly stringent wash conditions to a sugar-pathway gene specific to the bacterial strain to be detected, wherein said sugar-pathway gene is selected from the group consisting of rmlB, rmlD, rmlA, rmlC, gtf, manC, manB, ddhD, ddhA, ddhB, ddhC and abe, and contacting said further oligonucleotide molecule with said sample to permit said further oligonucleotide molecule to hybridize under said highly stringent wash conditions to said sugar-pathway gene, and detecting any specifically hybridized oligonucleotide molecules.

18. The method according to any one of claims 1, 5, 9 or 13, wherein the hybridized oligonucleotide molecules are detected by Southern blot analysis.

19. The method as claimed in any one of claims 3, 7, 11 or 15, wherein the method is performed using a polymerase chain reaction.

20. The method as claimed in any one of claims 1, 5, 9 or 13, wherein said sample is a food derived sample.

21. The method as claimed in any one of claims 1, 5, 9 or 13, wherein said sample is a faecal derived sample.

22. The method as claimed in any one of claims 1, 5, 9 or 13, wherein said sample is derived from a patient.