AU2005203358A1 - Clonal identification of bacteria - Google Patents

Description

0 P/00/011 Regulation 3.2

SAUSTRALIA

00 Patents Act 1990 00 SCOMPLETE SPECIFICATION Niq SFOR A STANDARD PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicants: Medvet Science Pty Ltd and Rural Industries Research and Development Corporation Actual Inventors: Michael W Heuzenroeder and Ian L Ross Address for Service: A.P.T. Patent and Trade Mark Attorneys PO Box 222, Mitcham, SA 5062 Invention Title: Clonal Identification of Bacteria Details of Associated Provisional Application No 2004904184 dated 28th July 2004 The following statement is a full description of this invention, including the best method of performing it known to us:- 2 This invention relates to a method of identifying clones of bacteria that might, for example, be responsible for outbreaks of disease, which method is useful for

OO

00 epidemiological and other purposes.

0 0 5 BACKGROUND OF THE INVENTION Identifying specific clones of pathogens responsible for outbreaks of disease has important public health implications, such as assisting with identifying pivotal events or breakdowns of sanitary conditions leading to the outbreaks. There continue to be barriers ,i to effective clonal identification.

Principally the technique of choice for clonal identification early last century was serological. Thus antibodies specific for surface polymers were elicited against a range of clonal variants of a pathogen species or genera and schemes were developed for serotyping these pathogens. Such schemes have been developed for a wide range of pathogens, for example, Escherichia coli, Vibrio cholera, and Salmonellae. Thus the so called Kaufmann and White scheme was developed for Salmonella enterica species and identify such serovars as Typhimirium, Heidelberg, Bovismorbificans, Enteriditis, Chester, Saintpaul, Anatum, Infantis, Muenchen, Ball, Mgulani and Stanley based on the combination of antigenic properties of flagellar H1 and H2 antigens and polysaccharide O antigen. Over 2000 Salmonella enterica serovars are now recognised (Popoff and Minor, 1997).

These serologically based schemes were, however, relatively early recognised as not giving, in many instances, adequate discrimination for specifically identifying epidemiological clones. One reason is that certain serovars are predominantly responsible for outbreaks of relevant disease. Thus Salmonella enterica serovar Typhimurium often predominates in human cases, although serovars Bovismorbificans, Enteriditis and Heidelberg also occur with significant frequency (Australian Salmonella Reference Centre Annual Report 2003). For a number of bacteria further methods of discrimination were developed. Thus in Salmonellae phage typing schemes have been widely used. A Typhimurium phage typing scheme was established in 1943 defining 12 phage types and this approach has been expanded until the presently used Anderson scheme was introduced in 1977 defining 207 definitive types (DTs) using 34 phages 3 (Anderson et al J Hyg 78 (1977) 297-300). There are also well known phage typing schemes used for other salmonella serovars. Other pathogens are also discriminated by 00 phage typing such as Staphylococcus aureus. Less well known phage typing schemes are

(N

used in a range of other pathogens.

00 Mf: The principal difficulty with phage typing schemes is however that they still lack the discriminating power required to identify clonal variants of pathogens causing relevant Soutbreaks in a significant number of cases. It is recognised that salmonellosis outbreaks in particular geographic regions have a predominance of particular phage types, making discrimination between outbreak and non outbreak strains difficult (Threfall et al, 1990, Ridley et al, 1998).

There are additional technical difficulties associated with phage typing in terms of subjectivity of interpretation of results because the typing schemes rely not only on the presence or absence of sensitivity for typing but also qualitative interpretation of parameters such as the degree of sensitivity and turbidity of plaques. Additionally propagation of phage on bacterial propagating strains can lead to variation in phage stocks because certain of the phage are known to have undergone recombination with either cryptic or other phage loads of the propagating bacterial strain or with other elements within the propagating bacterial strain.

These latter technical difficulties associated with phage typing have been sought to be addressed by using molecular typing means in Typhimurium (Lan et al, 2003 Microbes and Infection 5, 841-850), using AFLP of DNA fragments found associated with virus phage types. Fragments useful for the purpose were identified but the authors recognised that AFLP was probably not the technique of choice for routine screening. Many of the fragments were either phage or plasmid related and generally reflected the presence or absence of the genes or other fragments represented. This still does not address the inadequate discrimination power of phage typing.

A number of molecular techniques have been proposed in recent times to address the issue of better discrimination. Typically assessment of these techniques has been on whether the techniques as such are capable of providing suitable discrimination.

4 Techniques proposed for typing include multilocus enzyme electrophoresis, plasmic profile analysis, site specific and arbitrary primed polymerase chain reaction evaluation, 00 ribotyping, insertion sequence typing, DNA fingerprinting based on standard restriction fragment length polymophisms, amplified fragment length polymorphism and pulsed 00 5 field analysis of large DNA fragments, as well as Multilocus sequence typing. Whilst c n some of these techniques represent an advance on phage typing none has yet been O considered suitable as a stand alone assessment to replace and surpass the current combination of serotyping and phage typing.

(N

Probably the currently accepted best practice in typing of the Salmonellae is a combination of serotyping, phage typing and pulsed-field gel electrophoresis (PFGE), although some prefer the use of amplified fragment length polymorphis (AFLP) to supplement traditional methods. A significant drawback of PFGE is the complexity and time frame required to conduct the procedure, taking up to 5 days to perform, additionally the fragments detected can be very large, in the order of hundreds of kilobases, so that minor changes in size remain undetected. With respect to AFLP again, small changes can be detected, but the method is not in widespread use because it is slow, labour intensive and open to subjective interpretation. Hu et al ((2003), J Clin Microbiol, 3406-3415) in their investigation of fragments suitable for discrimination conclude that AFLP is probably not suitable for routine subdivision of a S. enterica phage types.

Multilocus Sequence Typing (MLST) has also recently been used for both evolutionary and epidemiological studies of microorganism, (Maiden et al, 1998; Kotetishvili et al, 2002). MLST involves the nucleotide sequencing of approximately 400bp regions of at least seven genes. Sequencing of Salmonella genes has generally been restricted to comparing sequences of housekeeping genes between the major groups of Salmonella or serovars of S. enterica subsp. 1, including serovars Typhimurium and Enteritidis (Boyd et al, 1994; Nelson and Sealander et al, 1994; Wang et al, 1997; Kotetishvili et al 2002).

Kotetishivili et al (2002) suggest the use of MLST based on ribosomal and other biosynthetic genes as a means of discriminating between strains of Salmonella. The problem with this approach is that the genes chosen are not subject to sufficient variation to give adequate discrimination within phage types.

SUMMARY OF THE INVENTION The present invention arises from the finding that isolates of Salmonella enterica serovar oO 00 Typhimurium from separate outbreaks have a greater polymorphism of DNA sequence

(N

within several prophage markers, than for general bacterial housekeeping genes. This greater extent of polymorphism relative to bacterial sequences is such that methods of r3 screening for them provide finer discrimination of outbreak strains than has been possible Susing traditional phage typing regimes.

In a first aspect the invention might be said to reside in a method of identifying a clone of a bacterial target species or species variant in a sample, said bacterial target carrying a prophage, and said method including the step of testing for sequence differences in one or more prophage nucelotide sequences between the clone and others of the bacterial target.

In a very specific form of the first aspect, the invention relates to detecting a clone within a phage type of a target Salmonella enterica serovar using multilocus sequence typing of two or more prophage nucleotide sequences to detect sequence variation between the clone and others of the target.

In a second aspect the invention might be said to reside in a method of developing a test for discriminating clones of a bacterial target species or species variant, the method comprising the steps of ascertaining the presence and distribution of one or more prophage within the bacterial target, ascertaining the nucleotide sequence of the prophage present in one of the clones, developing either primers for amplification of or probes for hybridisation with specifically a plurality of prophage nucleotide sequences of said one or more prophage, taking a plurality of distinct clones of the bacterial target and checking for polymorphism in the plurality prophage sequences, identifying sequences that exhibit the largest polymorphisms or identifying specific allelelic variation or both, and optionally developing further primers or probes specific for differentiating between alleles by their differential binding capacity or to enable ascertainment of 6 sequence differences directly or indirectly, to thereby enabling differentiation between clones of said bacterial target.

00

(N

In a third aspect the invention might be said to reside in a plurality of probes or primers 00 5 useful for identifying clones of a bacterial target species or species variant, said bacterial c target carrying a prophage, said primers or probes specific for differentiating between O polymorphic alleles of a plurality of prophage nucleic acid sequences by their differential Sbinding capacity or to enable ascertainement of sequence differences directly or indirectly, to thereby enable differentiation between alleles of said prophage nucleic acid sequences, said plurality being such that the primers and probes are collectively able to identify a unique allele or combination of alleles specific to each clone of the bacterial target.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Dendogram of PCR profiles for separation of S. Typhimurium isolates.

Dendogram generated by Dice coefficient with clustering by UPMGA based on presence or absence of amplified product. A total of 6 clusters of isolates with identical PCR profiles was generated and a total of 21 isolates with unique profiles. See Tables 3 and 4 for separation of the clustered iolaes by MLST. Abbreviates. New South Wales; Northern Territory; Qld., Queensland; South Australia; Vie., Victoria. unk=unknown Figure 2: Separation of S. Typhimurium isolates based on PFGE. Dendogram generated by DICE coefficient with clustering by UPMGA based on presence or absence of amplified product. Five different profiles were generated. Profiles 2 and 3 had >90% similarity suggesting isolates with these profiles are genetically closely related. S. Typhimurium DT 126 isolates were either profile 4 or 5, the remaining Typhimurium isolates were found in all 5 profiles, Figure 3 Is a diagrammatic representation of nucleotide differences found in certain prophage gene variants. The upper row refers to clones. Cl cro 7 SB41 refers to prohpage genes or regions. The band further down is a representation of the position of individual alleles found and

O

0, the numbers directed to those bands such as 61, 154 and 247 refers to the position of the nucleotide variation. The number used are 00 5 somewhat arbitrary, so that position 1 in this figure is equivalent to Cc position 28047 in Genbank accession number AY055382 for ST64B.

The table below is indicative of the base changes present. The first vertical column refers to the allele number and the remainder the table represent base changes from the most common type (allele number 1), and Figure 4: Separation of S. Enteriditis isolates based on PFGE. Dendogram generated by DICE coefficient with clustering by UPMGA based on presence or absence of amplified product.

DETAILED DESCRIPTION OF THE ILLUSTRATED AND EXEMPLIED EMBODIMENTS OF THE INVENTION In a first aspect the invention might be said to reside in a method of identifying a clone of a bacterial target species or species variant in a sample, said bacterial target carrying a prophage, and said method including the step of testing for sequence differences in one or more prophage test sequences between the clone and others of the bacterial target.

The principal sequence differences that are found at the clonal level are minor changes.

The majority of those found are single nucleotide base pair substitutions although other mutations likely to occur with sufficient frequency are anticipated to include, small deletions, inversion and duplications. The nucleotide changes most likely to occur frequently are likely to be in the order of one to a few and up to perhaps tens of nucleotide bases rather than rearrangements encompassing hundreds or thousands of nucleotides. However, the vast majority of changes are likely to be only a few nucleotide changes. Several molecular methods for distinguishing nucleic acid sequence differences of the magnitude above are in use and many of these will be applicable, some of these methods are elaborated below.

The present invention may be applicable to instances where a nucleotide sequence difference characteristic of the clone sought to be identified is known. In such instance a 00 single nucleotide difference may be an adequate identifier of the clone, and binding by a

(N

single probe or primer may be adequate to distinguish the clone. Given however that 00 5 there may be other closely related clones involved it is preferable even where the Cc nucleotide sequence difference is known to determine at least two such nucleotide O sequences as a more definite test.

More commonly, in particular from an epidemiological stand point, specific allelic variation of a clone is not known when an outbreak clone is first isolated, and typically many outbreaks are relatively short lived, so that it is not necessarily worthwhile to develop specific probes or primers capable of detecting specific known allelic variants. It is more useful to check through two or more prophage nucleic acid sequences to scan for any changes that might be present. This multilocus approach therefore looks at sequences that the present invention has shown vary more than others in the bacterial nucleic acid complement, for any differences in sequence to others of the same bacterial target species or species variant. If the outbreak is of significant magnitude a specific probe or primer set might be developed as a more efficient means of tracking the outbreak than the screening for unknown allelic polymorphism.

It is anticipated that more than two sequences will need to be checked in screening for allelic polymorphisms and typically more than 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide sequence might be used. Generally practice is to use 7 sequences as a minimum with differences in 2 sequences taken to mean a possible relationship between the strains, differences in three is taken to mean they are unrelated. In one form of the invention the nucleotide sequences are distinct non-overlapping sequences, particularly where the sequences are longer than about 40 bases. Each test sequence might alternatively be longer than about 50, 100, 200, 300 or 400 bases, but will typically be less than about 600 bases. In these cases the screening may comprise sequencing the test sequence, by a technique such as PCR sequencing or multilocus sequence typing (MLST).

Alternatively the checking may be undertaken by using micro-array technology with a large number of primers or probes set out on a solid surface in an array each perhaps overlapping and groups of them collectively testing for the presence of allelic variation with a test sequence by their hybridisation capacity.

00 The prophage may be a non-defective prophage. Many such phage are known across a 00 5 large range of bacterial species. They may be relatively easily isolated from a test rn, organism by culturing the organism under inductive conditions such as, for example, by an elevated temperature such as at 42*C or in the presence of ultra violet light, or an Sinductive compound such as mitomycin C. Plaques will form in overlay culture plates, and these may be isolated using standard methods. Additionally nucleic acid sequence can be determined by standard sequencing methods such as set by manufacturers of Automated DNA sequencing equipment such as Applied Biosystems.

The prophage may alternatively be defective. In some respects it is preferable that the prophage is defective because they are generally stably inserted, and thus there are unlikely to be positional effects, or total excision from the bacterial genome potentially leading to questions of whether the assay was properly performed, because positive standards reliant thereon do not hybridise. Defective phage may be screened for by hybridisation probes or primers of nucleotide sequences of known phage, or alternatively where sequence data is available this may be compared with available sequences in databases such as the Genbank database.

The one or more prophage test nucleic acid sequence may include at least one from a coding region. The coding regions might be selected from the genes selected from the group consisting of c2, cl, cro and mnt. The one or more prophage test nucleic acid sequence may alternatively or additionally include a sequence from a one or more noncoding regions. It is thought that sequences of most prophage genes or non coding sequences should be suitable for use in the present invention.

It might be preferred additionally to test at the same time for the presence or absence of prophage genes to tie in with an existing phage typing scheme. The genes concerned could include immunity genes and other genes suspected or known to be involved in determining phage type. The method might additionally look at the presence or absence of one or more bacterial, of plasmid gene sequences that are suspected or known to be involved in determining phage type.

00

(N

The method might additionally test for polymorphism in one or more bacterial, plasmid 00 5 or transposable element sequences nucleic acid sequences unrelated to phage type.

The test on the samples could ascertain the sequence variation in either DNA or RNA t molecules the later perhaps being amplified by reverse transcriptase PCR (RT-PCR) before a hybridisation step. The test samples may comprise pure cultures of bacteria, or might be a biological or other sample which may be treated to release nucleic acid without first preparing pure cultures of the bacteria.

In a specific form the invention is applicable to certain Salmonella serotypes, in particular S enterica serotypes. ST64B is found to be a particularly useful bacteriophage for typing S enterica serovar Typhimurium and perhaps also serovar Enteritidis because it is widespread amongst strains of these serovars. It is defective and thus there is no impact on the method by variation in the position of insertion into the chromosome.

Other bacteriophage P22 and DT64T are non-defective and also have a relatively high frequency of allelic polymorphism. It is anticipated that further bacteriophage will also similarly show this relatively high frequency of allelic polymorphism, so that the method is readily applicable to other Salmonella enterica serovars.

The present work has provided for resolving Salmonella serovars, in particular Typhimurium. As indicated above it is equally likely to enable resolving other Salmonella enteric serovars, for example Enteriditis, Virchow, Heidelberg, Bovismorbificans, Chester, Saintpaul, Anatum, Infantis, Muenchen, Ball, Mgulani Stanley Ohio, Singapore, London, Kiambu, Derby, Seftenburg, Oranienburg, Dublin, Newport, Agona, Aberdeen, Havana, Adelaide, Bredeney, Kottbus, Mbandaka, Livingstone, Orion, II Sofia.

11 Additionally the method is likely to be useful for other pathogens in which phage typing is used such as for Staphylococcus aureus strain involved in nosocomial outbreaks, or in 00 typing Corynebacteria pathogenesis.

(N

00 5 It is to be noted that the present method does not rely upon the bacterial species being one Cc on which phage typing is routinely conducted. Bacteriophage are somewhat ubiquitous in their distribution in bacteria from pathogens, to soil commensal to cheese making Sbacteria. It is anticipated that this method is applicable where it is desired to distinguish between closely related strains of bacteria. The method is typically applicable to strains of the same species, serovar or other variant of the same species, and in particular to differentiate clonal variants. Thus it is envisaged as being important for pathogens, but it may additionally be applicable to following divergence or variation in strains used in industrial, or food applications. Methods for discrimination are readily developed for a wide range of bacteria.

In another form the invention might be said to reside in method of developing a test for discriminating clones of a bacterial target species or species variant, the method comprising the steps of; ascertaining the presence and distribution of one or more prophage within the bacterial target, ascertaining the nucleotide sequence of the prophage present in one of the clones, developing either primers for amplification of or probes for hybridisation with specifically a plurality of prophage nucleotide sequences of said one or more prophage, taking a plurality of distinct clones of the bacterial target and checking for polymorphism in the plurality prophage sequences, identifying sequences that exhibit the largest polymorphisms or identifying specific allelelic variation or both, and optionally developing further primers or probes specific for differentiating between alleles by their differential binding capacity or to enable ascertainment of sequence differences directly or indirectly, to thereby enabling differentiation between clones of said bacterial target.

The invention might also be said to reside in a plurality of probes or primers useful for identifying clones of a bacterial target species or species variant, said bacterial target carrying a prophage, said primers or probes specific for differentiating between polymorphic alleles of a plurality of prophage nucleic acid sequences by their differential 12 binding capacity or to enable ascertainement of sequence differences directly or indirectly, to thereby enable differentiation between alleles of said prophage nucleic acid

O

00sequences, said plurality being such that the primers and probes are collectively able to identify a unique allele or combination of alleles specific to each clone of the bacterial 00 5 target.

These primers/probes may be used to additionally ascertain the presence of absence of Scertain prophage genes, and perhaps also used in conjunction with the presence or absence of plasmid sequences that are determinative of phage type. Thus the invention might encompass a micro-array having sequences specific for the presence and absence of such sequences to correlate phage type, and additionally include sequences for discriminating within each phage type to confirm the epidemiological link of a particular isolate. It may also be useful at the same time to include primers or probes to confirm the serotype.

A microarray is generally a substrate with a plurality of molecules nucleotides) bound to its surface. Microarrays, for example, are described generally in Schena, Microarray Biochip Technology, Eaton Publishing, Natick, MA, (2000).

The term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.

Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent.

The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

0 13 Primers are generally generated using software for example Primer3 software (Rosen and Skaletsky (1998) Primer3. Code available at http://www- 00 genome.wi.mit.edu/genome software/other/primer3.html.) having regards to the sequence of the bacteriophage gene. The program parameters may be selected to be more 00 5 or less specific and may be chosen in such a way that the melting temperature of the c amplicon should be close to 80C but not more than 88°C or less than 75°C., the length of Sthe amplicon was to be generally around 450 bp, with primer annealing temperature t about 60*C., and average length of primers 23 bp. Sequences of all amplicons have been Scarefully verified using proprietary software (BLASTN, FASTA), to avoid homology with repetitive elements and other related sequences, and also to distinguish between genes from the same family.

The term "probe" refers to a molecule an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification), that is capable of hybridizing to another molecule of interest another oligonucleotide). When probes are oligonucleotides they may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular targets gene sequences). In some embodiments, it is contemplated that probes used in the present invention are labelled with any "reporter molecule," so that is detectable in any detection system, including, but not limited to enzyme ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular label.

With respect to microarrays, the term probe is used to refer to any hybrididizable material that is affixed to the microarray for the purpose of detecting "target" sequences in the analyte.

EXAMPLE 1 Previous studies by this laboratory have identified two bacteriophages (ST64T and ST64B) induced from a strain of S. Typhimurium DT 64 (Mmolawa et al. 2003a; Mmolawa et al. 2003b). These two temperate phages were separated from each other with a caesium chloride gradient. The two bacteriophages have been fully sequenced and the sequences deposited in Genebank (www.ncbi.nlm.nib.gov); accession number AY052766 (ST64T) and accession number AY055382 (ST64B). Southern hybridisation 0 14 studies have suggested that these bacteriophages are present in a number of S.

Typhimurium phage types as well as other S. enterica subsp. 1 serovars including 00 Enteritidis, Virchow, Heidelberg and Hadar (Mmolawa etal. 2002; Tucker and Heuzenroeder, 2004). Based on the widespread nature of these phages, we have used the 00 5 sequence data to design a number of primer sets to analyse sequence variation of c n prophage genes. As well as using gene sequence from bacteriophages ST64B and ST64T we have also examined a range of similar genes from bacteriophage P22. A number of bacterial housekeeping genes were also examined to compare the level of sequence homology of Salmonella housekeeping genes between closely related isolates to the prophage genes. We have used this data to discriminate closely related but epidemiologically distinct strains of S. Typhimurium.

METHODS

Strains and Culture Conditions A total of 73 S. Typhimurium isolates were used in this study. Thirty six isolates were a selection of mostly epidemiologically-unrelated definitive phage types (DT) 108 (18 isolates, including 2 isolates (03-108-022 and 03-108-023) from an outbreak in New South Wales in 2003), DT 12 (9 isolates) and DT 12a (3 isolates). Six isolates comprised Typhimurium isolates of various phage types (one each of DT 64, DT 9, DT 135 DT 185 and two DT 170 isolates). Thirty seven DT 126 were also included in the study. This group comprised thirteen isolates from a 2001 restaurant outbreak in N.S.W. Another DT 126 isolates were obtained from an outbreak in 2003. Four of these isolates were designated DT 126var due to a variation in reaction to the Anderson typing panel. A further 14 DT 126 isolates were epidemiologically-unrelated.

All S. Typhimurium isolates used in this study were provided by the Australian Salmonella Reference Centre (ASRC), Institute of Medical and Veterinary Science, Adelaide, South Australia. Serotyping had previously been undertaken using the Kaufmann-White scheme and bacteriophage typing was performed using the Anderson scheme of 31 phages (Anderson et al. 1977), both by the ASRC.

N PCR of Salmonella nPCR of Salmonella was performed directly from cell lysates. Isolates were grown

OO

00 overnight in LB broth at 37°C with gentle shaking. A 30tl PCR reaction mixture was prepared as follows: 3.0l 1Ox MgCl 2 -free buffer, 1.0mM each forward and reverse 0 0 5 primer, 3.0[l 200liM each dNTP, 1.5mM MgClI, 1.OU Taq polymerase and 2.0[1 overnight cell culture and the volume taken up to 30pl with H 2 0. Buffer, MgCl 2 and Taq polymerase were supplied by Roche. Primers were designed based on published sequences (Table All primers were supplied by Geneworks, Adelaide, South CNi Australia. The dNTPs were supplied by Pharmacia.

2005203358 28 Jul 2005 Table 1. Primers used in this study Source of Sequence Gene/Region Primer name Primer Sequence SEQ ID No Sequenced Fragment Location in published (Genebank Acc. No.) Size (base pairs) sequence (inc. primers) Bacteriophage ST64B immC ci1 gene Forward: BIMIFI ATGGTGGCCTTGTCGACGC 13 82 t end) Reverse: BIMIRI GCrAACGTGAAGGATTTGYI7CCG 2 43208t 80 inmC ci gene Forward: BIM2F1 CCATTACCGGCGCTTGCAC3 31 84 t89 end) Reverse: BIM2R1 TAACGTATAACCATGCGATTTCCG 4 40241t89 immC cro gene Forward: BIM3F71 GCGATATACGCAAAAGAAGGAGG 5 475 28819 to 29336 Reverse: BIM3R1 TGGCTACTGAATGTGCCAGG 6 inun pu c2 Forward: BIM4F1 GCTGGTAC7GCAACGTGCC7 72 98 ~mm pu c2 Reverse: BIM4R1 CGAATGACATGGACATAAAGTCC 8 56224t95 ORFSB6 Forward: SB6F1 ACGACAAGCGCGTTGAGGC9 91 34o44 ORF 5B6 Reverse: SB6R1 GCTTCCACGTTGAAGAAGGC 10 5249 o44 ORF SB26 Forward: SB26F1 GACACCATCAATGTATGGATCGCl 47116 t 98 ORF 5B26 Reverse: SB26RI AGGTTATCZTATAATTCCGACCTGG 12 47146t 98 ORF SB28 Forward: SB28F1 TGCAGTCAAGAGGACGTCC 13 59236t 19 ORF 5B28 Reverse: SB28R1 TGCCGATATGCrGATC7GGC 14 59236t 19 ORF SB37 Forward: SB37F1 TGGTAGTGAATTGGTTAGCTGCG 15 439 26841 to 27322 Reverse: SB37RI CGGAAAGCrGTTACAGCAGG 16 ORF SB46 Forward: SB46F1 CATTGATGGTATCGAAGTTCGCC 17 448 33640 to 34130 Reverse: SB46RI CCTGGAGTTTCI'GGCACGC 18 Bacteniophage ST64T gene 9 end)' Forward: G9F1 GCRATTCCTTGCATCTrGGAGC 19 562 38606 to 39208 G9R1I GCAATGCGGGAACCTTTGCC gen 9(Yen)'Forward: G9F2 TACCGTAGAAGATTGCGCrGG 21 44395t 00 gen 9(3'en)~Reverse: G9R2 GGRACAAATGGTATCTCTGCCC 22 44395t 00 gene 17 Forward: F17A GGCTGTYGTTTCTT=TICAGGC 23 gene___17 Reverse: R17A AGGAAATATGAAATTACGTGTCI'GGC 24 264 10609 to 10921 gtA Forward: GTRAF1 AGACCTTTCCGAATCCGCTG 25 2224 o28 gtrA Reverse: GTRAR1 TAATTGCCGAGAAAGTGATAAGGG 26 2224 o28 gtB Forward: GTRBF1 CTTTCTCGGCAATTAGCCTG 27 3324 o26 gtrB~ Reverse: GTRBR1 TTAGCCAGCACCATATCCGC 28 3324 o26 Forward: GTRCF1 CTACTACTCGC7ATTCI=GCGC 29 gtrC Reverse: GTRCR1 CATTAACACCTCTGACCACATCC 30 492 152 to 689 mnt Forward: TMNTF1 GAGTAAAGCCCGGTTCGCC 31 20328t mnt ~Reverse: TMNTR1I TATAACCAGTAGATCATATGATGCCG 32 20328t 85 2005203358 28 Jul 2005 c2 ~Forward: TC2F1 GGAATTGTTAGAGGCCTTGCC 33 32186t 38 c2 ~Reverse: TC2R1 GATTTCCCCTGATTAGCTGGG 34 32186t 38 Forward: TCROF1 CCATCCTGAGGAGATATACCG 35 CTO Reverse: TCRORI GGTTCAGATTGGTAAAGAGCGG 36 222 13653 to 13917 c1i Forward: PTC1F1 CTTTACCAATCTGAACCGCCG 37 _35191t 43 Reverse: PTC1IR I CTGAGTTGTTTTGGCATAATTAC7CC 38 35191t 43 Bacteriophage P22 mtForward: PM4NTF1 TTATAAGTAGTCAATATGGCCCAGG 39 25396t 86 mnt Reverse: PMNTR1 AATACACrAACrTGGAGTGATGGC 40 25396t 86 int Forward: PINTF1 CATTTCCTGCAATACCGAAATCGG 41 4135 o31 lflf Reverse: PINTRI GCTGGCTTGAGCCTCACG 42 4135 o31 Forward: PCROF1 AGTGTTCTTTAATTTCGGAGCGAG 4315136 o cro Reverse: PCROR1 CGGTTCAGATTGGTAAAGAGCG 44 15148t c3 ~Forward: PC3F1 CTGCACAAGGATGGTTCCG 4525902t113 c3 ~Reverse: PO3RI ATTGATTGGTATAGCGAGTGCC4625902t113 sieN Forward: SIEAFI GCGCTATAAGCCAAGGACGG 47 42378t 79 sieA' Reverse: SIEARI TGAGTTATGCTGTGCTFAGTTGCC 442368 o 79 sieff. Forward: SIEBF1 CGATGAACAACTCATGGTGGC 49 58157t 21 sieB Reverse: SIEBRI AGCGAGGTAAGGTATTTGTCG 50 58157t 21 Salmonella housekeeping Forward: PHUAFi AGAAGAAACCATTACCGTAACCG 51 genes ]huA Reverse: FHUAR1 TGCTAACCATCGAAATGATACCG 52 402 223851 to 224296 sucA Forward: AROCFI GCACCGAAGAGAAACGC'G 53 60827 o821 sucA Reverse: AROCRI GGTTGTTGATAACGATACGTAC 54 60827 o821 tonB Forward: SB46F1 AGAATCTGTACATTTTCCAUI7CGC 55 431339t 816 tonB Reverse: SB46R1 CAGCGCAGCCTATCACGG 56 431339t 816 Forward: MANBF1 GGCAGCTACAGACAAATCAGC 57 manB" Reverse: MANBRI GCCATAAATGGCATCCTCCG 58 508 2187413 to 2187961 g~~nA Forward: FHUAF1 GTTATCGACCCGTT1CTTCGC 59 594116t 268 ginA rReverse: FUAR1 GTTGGTGCCGTTCTTCGCC 6594266 o 268 Y=CorT R=AorG sequences from bacteriophages and Salmonella housekeeping genes derived from following Genebank (www.ncbi.nlm.nih.gov) accession numbers: ST64B (AY055382), ST64T (AY052766), P22 (NC 00237 and Salmonella housekeeping genes (NC-003 197).

the primers derived from bacteriphage ST64T are also used to detect similar regions in bacteriophage P22.

"the phosphomannomnutase gene manB is designated cpsG in the refered Genebank accession number 18 Touchdown PCR was performed in an Corbett Research PC-960G gradient thermal 00 cycler as follows: 94°C 10 minutes, then 40 cycles comprising 94°C dsDNA melting for cl 30 secs. and 72°C elongation for 1 minute. Primer annealing temperatures were as follows: 59 0 C (1 cycle), 58°C (1 cycle), 57°C (2 cycles), 56°C (3 cycles), 55 0 C (5 cycles), 00 54°C (8 cycles), 53 0 C (10 cycles) and 52°C (10 cycles). All annealing steps were for c second duration. A final elongation step at 72 0 C for 5 minutes was also performed.

0 Amplification product was detected and prepared for sequencing by running 5.0tl of CI PCR product on a 2.0% w/v agarose gel (Progen, Darra, Queensland) in 1.0x TBE (Sambrook) buffer at 5.0V.cm-1 with puC19 digested with HpaII (Biotech, Belmont, Western Australia) as a marker. Bands were visualized with UV light after staining with ethidium bromide. The remaining 25[il PCR product of positive samples was prepared for sequencing by passing through a Qiaquick PCR purification column (Qiagen, Hilden, Germany) and collecting in 30[l elution buffer as per the manufacturer's instructions.

Amplicons were stored at -20 0 C prior to sequencing.

MLST

Sequencing was performed in both directions with Big Dye Terminator v3-1 (BD3-1) (Applied Biosystems). A 20tl reaction mix comprised 4.0il BD3-1 master mix, 3.0mM either forward or reverse primer, 12.5 1 l H 2 0 and 2.0[l template. Sequencing was performed on a Corbett Research PC-960G Gradient thermal cycler and comprised cycles, 96°C 30secs, 50°C 15 sees and 60°C 4.0 mins. Sequence product was precipitated and washed with 75% isopropanol and then dried. Sequencing was performed on an Applied Biosystems 3700DNA Analyzer.

Pulsed-field Gel Electrophoresis The protocol for PFGE followed that of Maslow et al. (1993). Briefly, cells grown overnight in brain heart infusion (BHI) broth (Oxoid) were embedded in agarose and lysed by incubating the plug in 4mls lysis buffer supplemented with 4 mg lysozyme (Roche, Mannheim, Germany) and 80itg.ml-1 RNAse. Plugs were then proteinase Kdigested, washed and the DNA digested overnight with Xbal restriction endonuclease (New England BioLabs Beverley, The next day the plugs were placed into the 19 wells of a 1% agarose gel prepared with PFGE-grade agarose (Bio-Rad laboratories, Hercules, California) in 0.5x TBE buffer Sambrook and Russell, 2001). Staphylococcus 00 N aureus strain NCTC 8325 digested with Smal was used as a molecular marker (Tenover et al. 1995). The pulsed-field gel electrophoresis was run on a BIO-RAD CHEF-DR® III 00 t} 5 System for 19 hours at 6.0 V.cm' at 4°C, initial switch time 2 secs, final switch time c secs. After running the gel was stained in ethidium bromide, distained in water and

O

C photographed under UV light.

(1 Data Analysis Isolates were initially separated based on PCR results. Each primer set was given a number and data entered into a spreadsheet consisting of all positive results for each strain. The spreadsheet was imported into GelCompar IV (Applied Maths, Kortrijk, Belgium) with appropriate formatting. A dendrogram of isolate PCR profiles was generated by Dice coefficient and clustering by UPGMA (Fig. 1).

Strains displaying identical PCR profiles were further analyzed by MLST for further discrimination. Sequences were initially checked in both directions using GeneBase vl.0 software (Applied maths, Kortrijk, Belgium). A minimum of seven common positive PCR regions common within each cluster were selected for MLST analysis. MLST of common positive PCR regions were analyzed by Dice coefficient and UPGMA analysis of concatenated sequences as well as by the Blast algorithm (www.mlst.net). Analyses of concatenated sequences were undertaken with GeneBase vl.0 software.

RESULTS

PCR Analysis Amplified product was obtained from at least one of the 73 S. Typhimurium isolates tested with 22 of the 25 prophage primer sets (Table The three prophage primer sets that failed to amplify with any isolate were sieA, ST64T cro and P22 c3. The most frequently amplified regions were the genes and open reading frames of bacteriophage ST64B. A range of P22 and ST64T prophage genes were routinely found in non-DT 126 isolates; these included the 5' and 3' regions of the tail gene (gene sieB, gtrC, int, and 09/08/2005 15:22 +61-8-82723255 APT PAGE 04/09 c 0 Sthe ST64T nmt gene. Amplified product from number of primer sets from P22 and ST64T such as gtrA, gtrB and P22 wnt was obtained with a small number of isolates.

Table 2. Incidence of genes/regions, and number of alleles 00 5 observed and distribution in 73 serovar Typhimurium isolates prophage gene or region positive alleles isolates for most common allele ST64B immC cl: 5' 72 3 56 immC cl: 3' 70 2 62 immC cro 71 3 62 immC put. 2 70 5 63 ORP SB6 68 3 66 ORF SB26 55 2 54 ORF SB28 63 1 63 ORF SB37 68 3 53 ORF SB46 62 5 49 ST64T gene 9: 5' 33 3 and P22 gene 9: 3' 32 3 21 gene 17 9 2 6 gtrA 4 1 4 gtrB 7 2 6 grrC 31 4 9 sieA 0 0 na sieB 33 2 cl 1 1 1 ST64T rnt 29 1 29 only c2 1 1 1 cro 0 0 na P22 only mnt 4 1 1 cro 1 1 1 int 34 4 29 c3 0 0 na housekeeping genes fhuA 73 1 73 glnA 73 2 63 manB 54 2 53 tonB 23 1 23 sucA 73 1 73 COMS ID No: SBMI-01408531 Received by IP Australia: Time 16:09 Date 2005-08-09 21 Analysis of the PCR profiles of the 72 isolates that tested positive for prophage genes is cN summarized in Figure 1. Six separate clusters containing isolates with identical profiles were identified. Four separate clusters of non-DT126 isolates with identical PCR profiles 00 were observed. Different phage types were represented in each of these four clusters.

c Eighteen non-DT 126 isolates (including the single DT 135 isolate) were all separated CI from each other through differences in their PCR profiles. One major cluster comprising S29 DT 126 isolates plus the single DT9 isolate was generated. A further cluster consisted Ci of the 4 DT 126var isolates from the 2003 N.S.W. outbreak. These 4 isolates were separated from the other DT 126 isolates from the same outbreak, as well as the other DT 126 isolates as they did not contain the SB28 region of prophage ST64B. The DT 126 isolates as well as the single DT 9 and DT 135 isolates had significantly different PCR profiles compared to the other Typhimurium phage types tested. All but three DT126 isolates (-101, -122 and -123) failed to produce a PCR product with primers derived from bacteriophages P22 and ST64T. No other trends in terms of a phage type being separated by PCR from other phage types were noted.

Three of the five housekeeping genes tested, fhuA, glnA and sucA, were positive for all 73 isolates, including isolate 01-126-114 which did not produce amplified product with any of the prophage primer sets tested. Forty nine isolates were positive for manB while 23 isolates were positive for tonB. Repeating the PCR with these negative isolates still failed to generate amplified product. No relationship between phage-type and presence or absence of PCR product for either manB or tonB was observed. The absence of manB and tonB PCR products for a number of isolates made it possible for a level of separation of isolates to be achieved with these two primer sets based on PCR profiles (data not shown).

MLST Analysis All PCR products for all relevant primer sets were sequenced with both forward and reverse primers and the number of different alleles for each primer set was determined (Table Most bacteriophage-derived primer sets produced at least two different alleles when sequenced except those primer sets where only a small number of strains 22

(N

generating a PCR product were observed. Some primer sets produced only 1 allele even when a significant number of isolates produced PCR product, for example, ST64B ORF 00 SB28 and the ST64T mnt gene. In a few cases where significant number of isolates produced product, only 1 or 2 isolates contained an allele distinct from the majority of oO t) 5 isolates. For example, a second ST64B SB26 allele was present in only one isolate, (01rc q 9-001).

SMore than one allele was detected for only 2 of the 5 housekeeping genes, glnA and C manB. Only 1 isolate, 01-126-114, had a different manB allele to the other 53 isolates that were PCR-positive for this primer set. This isolate was also the isolate where no amplification of prophage genes could be detected. A unique glnA allele was detected in the ten DT 126 and DT 126var isolates from the 2003 outbreak.

MLST analysis of the non-DT 126-clustered isolates in Figure 1 was undertaken to discriminate between these isolates (Table Separation of the 8 isolates in the first cluster produced three subgroups comprising in the first subgroup three DT 12 and two DT 108 isolates, the second subgroup containing one strain, 02-12-002 and the third subgroup containing the two DT108 outbreak isolates (03-108-022 and 03-108-023).

Separation was based on a single allele in each case, the two outbreak strains having a different ST64B SB37 allele to the other 6 isolates and 02-12-02 having a different ST64B immC cl 5' region sequence to the other 7 isolates in this cluster.

23 Table 3. Separation of non-DT 126 S. Typhimurium isolates by MLST. Based on clusters derived in Fig. 1.

Cluster Isolate Differing Allele(s)(' i

PFGE

02-108-004 03-108-014 02-12-001 02-12-005 02-12-006 02-12-002 03-108-022 03-108-023 02-108-001 03-108-015 03-108-016 02-12-004 02-170-001 02-170-002 02-108-005 01-108-008 02-12a-002 BIM1 ORF SB37 ORF SB37 immC immC, ORF SB37 unique allele(s) which separate isolate(s) from the other isolates within each cluster.

In the second PCR profile cluster which includes isolate 02-108-001, sequencing of the ST64B immC region separated this isolate from the other three isolates, all of which exhibited sequence identity with all prophage PCR products. In the third cluster all three isolates had identical sequences. The fourth cluster comprised 02-12a-002 and 01-108- 24 008. These two isolates had different sequences throughout the whole ST64B immC region as well as different ST64B SB37 genes.

The sequencing of the DT 126 isolates resulted in a much lower degree of variation between isolates (Table The thirteen isolates from the 2001 restaurant outbreak all had identical prophage sequences in their PCR products. The ten 2003 outbreak isolates (03-126-104 to 03-126-113) were identical for all prophage gene sequences, separation was only based on the glnA sequence described above. Of the remaining DT 126 isolates only 1 isolate, 01-126-123, exhibited different prophage sequences to the other DT126 isolates. These differences were observed in ST64B SB 37, SB46 and the 5' region of gene cl. It had already been noted that this isolate had already been separated from the other DT 126 isolates based on the presence of a PCR product from the P22 cro gene (Fig. 1).

Table 4: Separation of DT 126 isolates by MLST Isolate 2001 ROS 01-126-101 2003 ROS 0 2003 ROS 01-126-114 02-126-115 02-126-116 02-126-117 02-126-118 03-126-119 03-126-120 03-126-121 03-126-122 03-126-123 03-126-124 03-126-125 03-126-126 03-126-127 01-9-001 01-135-001 Housekeeping genes manB ginA manB tonB 1 1 1 1 1 2 1 2 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Variable gene(s) gtrA +ve No SB28 gene no genes detected P22 int +ve P22 cro +ve gtrB +ve (i) (ii) (iii) 2001 restaurant outbreak strains (12 of 13 isolates).

(Isolate 01-126-101 is the 13th isolate) 2003 restaurant outbreak strains 6 DT 126 isolates) 2003 restaurant outbreak strains 4 DT 126var isolates) Pulsed-field gel electrophoresis 0 The 73 S. Typhimurium isolates generated 5 different PFGE profiles (Fig. When

C

analysed, the 5 profiles formed four groups of isolates with <90% similarity between the profiles. PFGE profiles 2 and 3 displayed >90 similarity with each other, suggesting that isolates within these two groups are nearly identical to each other. All DT126 isolates r n were either of profile 4 or 5 while representatives of the remaining Typhimurium isolates were present in each of the 5 PFGE profiles. Isolates from the 2001 restaurant outbreak Swere of either group 4 or group 5. All 10 isolates from the 2003 outbreak were included in pulsed-field group 4. The remaining epidemiologically-unrelated DT 126 isolates were distributed between the two pulsed-field groups, 8 in group 4 and 6 in group While the 36 non-DT 126 isolates were found in all pulsed-field groups, most were found in PFGE profile 3 (25 isolates). The distribution of the remaining isolates within PFGE profiles were profile 1 (4 isolates), profile 2 profile 4 and profile 5 There was no relationship observed between phage-type and pulsed-field profile although the only representative non-DT126 isolate in profile 4 was the single DT 9 isolate (01-9-001).

EXAMPLE 2 Separation of Enteriditis serovars The methodology employed is substantially identical to that employed in example 1.

Primers used.

Certain modifications were made for some of the primers used as follows.

Bacteriophage ST64B immC cl BIM1F1A TGTGTCGTTTGAGTGACTGCG (SEQ ID No 61) BIM1R1A TTCTAGGCTGGCTGACTGC (SEQ ID No 62) These two new primers were designed to encompass the variable region of the cl gene of bacteriophage ST64B as described in the first MAPLT paper. These two primers "replace" the BIM1 and BIM2 primers described in Table 1.

immC cro BlM3F2 CTTCAACAGAGCTTGCTGAGC (SEQ ID No 63) Shortens the amplified fragment length of this region without compromising detection of SNPs found in the variable region of this gene. Shorten to facilitate future multiplex PCR development.

ORF SB21 SB21F1 SB21R1 CTGTATGGTTATATCGATTATCTGG (SEQ ID No 64) GATTTCCTTGCCCAGATGACG (SEQ ID No Primers designed for this region based on the findings of Figueroa-Bossi and Bossi (2004) on the potential variability in this region due to a single base deletion.

Bacteriophages ST64T and P22 gene 9 end) G9R1B TCGATAAGAAGGCCATCAACCG (SEQ ID No 66) Shortens the amplified fragment length of this region without compromising detection of SNPs found in the variable region of this gene. Shorten to facilitate future multiplex PCR development.

Salmonella Housekeeping Genes All the following primers were designed based on the regions selected by www.mlst.net for multilocus sequence typing of Salmonella enterica.

aroC aroCPF1 aroCPR1 aroCSFl aroCSRI TCGCGCTATACTACTCAGCG (SEQ ID No 67) CGACCTCTTCACCCATCCG (SEQ ID No 68) GACCGGCACCAGTATTGGC (SEQ ID No 69) TATGCGCCACAATGTGTTGCC (SEQ ID No aroCPFl and aroCPR1 are for PCR amplification of the aroC gene O 27 aroCSF1 and aroCSR1 are for the sequencing of the amplified aroC fragment 00

C

hisD hisDPF1 ATTGAAACGTTCCATTCCGCGC (SEQ ID No 71) hisDPR1 TTCCGGAAGCGTAATCACCG (SEQ ID No 72) 00 MC hisDSF1 CGTTGCCAGCAGGTTACGC (SEQ ID No 73) hisDSR1 GTAATCGCATCCACCAAATCGC (SEQ ID No 74) c hisDPF1 and hisDPR1 are for PCR amplification of the hisD gene hisDSF1 and hisDSR1 are for the sequencing of the amplified hisD fragment purE purEF1 CATGTCTTCCCGCAATAATCCG (SEQ ID No purER1 GAACGCAAACTTGCTTCATAGCG (SEQ ID No 76) thrA thrAF1 ATATCGCGGAATCGACTCGC (SEQ ID No 77) thrAR1 GATAGAACTCATCCTGCATCGC (SEQ ID No 78) Results The results of this example are set out in Table 5 wherein the isolate number is indicated in column 1 and assigned code in column 2. <T indicates the phage type ascribed to the isolate using the Anderson phage typing system. The indications across the top column refer to the gene sequence being amplified, the primers used are set out above or in Table 1 herein.

As can be seen this method was capable of distinguishing between 10 untypable isolates, as well as capable of distinguishing between different type 4 isolates.

A dendogram setting out the relationship of the isolates is shown in Figure 4. This also sets out the source of the various isolates. Sgpr indicates that the source was from Singapore.

2005203358 28 Jul 2005 Table 5. Capacity of 38 isolates of serovar Enterdifis to amplify clonalspecific sequences r ^U4 I C I YJ I I 5narIaU ~nIrnC n R2 I SR2AiI K2 SB46 1 29:5 1 v-9:j ruj r K x L I p13 2

I

U-7 I D U I D-B2 I I SB2 m I ,ii( I .Il'7 I I .sllO I n171 nlZd I nhlS D1ZL I ~IM II pr P P y~ I 05-800163 E26-001 26 05-800164 E26-002 26 05-800664 E26-003 26 05-800665 E26-004 26 05-801022 E26-005 26 05-800155 E26-006 26 05-801935 E26-007 26 03-807691 E26-008 26 03-804163 E26-009 26 02-804952 E26-010 26 05-801163 El4v-001 14var 05-801164 EI4v-002 l4var 05-801445 EI4v-003 14var 05-801446 El4v-004 L4var 05-801447 El4v-005 14var 05-801956 EI4v-006 l4var 05-801958 El4v-007 l4var 03-803156 El4v-008 I4var 03-803139 El4v-009 l4var 05-800963 Eut-001 I untype 05-800873 Eut-002 uIntype 05-800974 Eut-003 tIntype 05-800506 Eut-004 untype 05-800507 Eut-005 untype 05-800916 Eut-006 untype 05-801643 Eut-007 untype 04-807111 Eut-008 tntype 02-806703 Eut-009 uIntype 03-806683 Eut-010 uIntype 05-801934 E4-001 4 04-809694 E4-002 4 04-809786 E4-003 4 02-806354 E4-004 4 04-809445 E4-005 4 04-808005 E4-006 4 05-800513 E4-007 4 05-800522 E4-008 4 04-801413 E4-009 4 O 29

REFERENCES

Anderson et al.(1977) J. Hyg. 78:297-300.

OO

00 Baker et al., (1991) New Biologist. 3:297-308.

Bell and Kyriakides. (2002). Salmonella a practical approach to the organism and its oO 5 control in foods. Blackwell Science Ltd. London, United Kingdom.

c Boyd et al., (1994). Proc. Natl. Acad. Sci. U.S.A. 91: 1280-1284.

de Boer et al., (2000) J. Clin. Microbiol.; 38: 1940-1946.

Figueroa-Bossi and Bossi. (2004) J. Bacteriol. 186: 4038-4041.

Kotetishvili et al., (2002). J. Clin. Microbiol. 40:1626-1635.

Lindstedt et al., (2000). FEMS Microbiol Letts. 189:19-24 Maiden et al., (1998). Proc. Natl. Acad. Sci. USA. 95:3140-3145.

Maslow et al., (1993). Application of pulsed-field electrophoresis to molecular epidemiology. In: Persing, T.F. Smith, F.C. Tenover, and T.J. White, eds.

Diagnostic Molecular Microbiology Principles and Applications. Washington D.C.: American Society for Microbiology, ISBN 1-55581-056-X, pp563-572.

Mead et al., (1999) Emerg. Infect. Dis. 5:607-625.

Mmolawa et al., (2002). Int. J. Med. Microbiol. 291:633-644.

Mmolawa et al., (2003a) J. Bacteriol. 185:3473-3475.

Mmolawa et al., (2003b) J. Bacteriol. 185:6841-6845.

Nelson and Selander. (1994) Proc. Natl. Acad. Sci. U.S.A. 91: 10227-10231.

Oberto et al., (1994) Nucleic Acid Res. 22:354-356.

Popoff and Minor. (1997). Antigenic formulas of the Salmonella serovars, 7' revision.

WHO Collaborating Centre for Reference and Research on Salmonella, Institut Pasteur, Paris, France.

Ptashne et al., (1980). Cell. 19:1-11 Ridley et al., (1998) J. Clin Microbiol. 36:2314-2321.

Ross et al., (2003) Int. J. Med. Microbiol. 293: 371-375.

Sambrook and Russell. (2001). Molecular Cloning. A Laboratory Manual. 3' ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Scott et al., (2002). Int. J. Syst. Evol. Micrbiol. 52:1701-1713.

Schicklmaier and Schmieger. (1997) Gene. 195: 93-100.

Tenover et al., (1995) J. Clin. Microbiol. 33:2233-2239.

O Threlfall and Frost. (1990) J. Appl. Bacteriol. 68:5-16.

Tucker and Heuzenroeder (2004) Int. J. Med. Microbiol. 294: 59-63.

C Vander and Kropinski. (2000). J. Bacteriol. 182:6472-6481.

Vos et al., (1995). Nucl. Acids Res. 23:4407-4424.

0 5 Wang et al., (1997). J. Bact. 179: 6551-5559.

SWard et al., (1987) Epidemiol. Infect. 99:291-294.

Winokur (2003). Molecular epidemiolgical techniques for Salmonella strain discrimination. Frontiers in Bioscience. 8:14-24.

Claims (23)

1. 2. The method of testing a sample for the presence or absence of a clone as in claim 1 wherein two or more prophage test sequences are tested for the presence or absence of clonal specific sequence.
2. 3. The method of testing a sample for the presence or absence of a clone as in either claim 1 or 2 wherein 7 or more prophage test sequences are tested for the presence or absence of clonal specific sequences.
3. 4. The method of testing a sample for the presence or absence of a clone as in claim 1 wherein only seven prophage test sequences are tested for difference and 3 or more differences in the seven prophage test sequences is taken to mean that bacteria from two samples are unrelated and 2 differences is taken to mean there may be clonal identity.
4. 5. The method of testing a sample for the presence or absence of a clone as in any one of the preceding claims wherein the prophage test sequences are each distinct non- overlapping sequences.
5. 6. The method of testing a sample for the presence or absence of a clone as in any one of the preceding claims wherein each one of the prophage test sequences are longer than about 40 bases.
6. 7. The method of testing a sample for the presence or absence of a clone as in any one of the preceding claims wherein the sequences are shorter than about 600 bases.
7. 8. The method of testing a sample for the presence or absence of a clone as in any one of the preceding claims wherein the prophage is non-defective. 32 S9. The method of testing a sample for the presence or absence of a clone as in any 00oO N, one of claims 1 to 7 wherein the prophage are defective. 0 5 10. The method of testing a sample for the presence or absence of a clone as in any C one of the preceding claims wherein the bacterial target additionally carries an additional prophage and at least one respective prophage test sequence is found in each of the two prophages.
8. 11. The method of testing a sample for the presence or absence of a clone as in any one of the preceding claims wherein the one or more prophage test nucleic acid sequences includes at least one from a coding region.
9. 12. The method of testing a sample for the presence or absence of a clone as in claim 11 wherein the coding region is selected from the group consisting of c2, cl, cro and nut.
10. 13. The method of testing a sample for the presence or absence of a clone as in any one of claims 1 to 10 wherein the one or more prophage test nucleic acid sequences include at least one from a non-coding region.
11. 14. The method of testing a sample for the presence or absence of a clone as in any one of the preceding claims wherein the testing for the presence of clonal specific sequence in the prophage test site is by primer or probe binding.
12. 15. The method of testing a sample for the presence or absence of a clone as in any one of claims 1 to 13 wherein the testing for the presence of clonal specific sequence in the prophage test site is by multilocus sequence typing.
13. 16. The method of testing a sample for the presence or absence of a clone as in any one of claims 1 to 13 wherein the testing for the presence of clonal specific sequence in the prophage test site is by PCR sequencing. O 33
14. 17. The method of testing a sample for the presence or absence of a clone as in any 00 N, one of the claims 14 to 16 wherein the testing includes a nucleic acid amplification step. 00 5 18. The method of testing a sample for the presence or absence of a clone as in any c one of the preceding claims wherein the target bacterium is a salmonella. O
15. 19. The method of testing a sample for the presence or absence of a clone as in claim C 18 wherein the target bacteria is a Salmonella enterica serovar. The method of testing a sample for the presence or absence of a clone as in claim 19 wherein the serovar is selected from the group consisting of Typhimurium, Enteriditis, Virchow, Heidelberg, Bovismorbificans, Chester, Saintpaul, Anatum, Infantis, Muenchen, Ball, Mgulani Stanley Ohio, Singapore, London, Kiambu, Derby, Seftenburg, Oranienburg, Dublin, Newport, Agona, Aberdeen, Havana, Adelaide, Bredeney, Kottbus, Mbandaka, Livingstone, Orion and Sofia.
16. 21. The method of testing a sample for the presence or absence of a clone as in claim 19 wherein the serovar is either Typhimurium or Enteriditis.
17. 22. The method of testing a sample for the presence or absence of a clone as in either claim 20 or 21 wherein at least one test sequence is selected to be from the group of prophage consisting of ST46B, P22 and DT64T.
18. 23. A method of developing a test for discriminating clones of a bacterial target species or species variant, the method comprising the steps of; ascertaining the presence and distribution of one or more prophage within the bacterial target, ascertaining the nucleotide sequence of the prophage present in one of the clones, developing either primers for amplification of or probes for hybridisation with specifically a plurality of prophage nucleotide sequences of said one or more prophage, taking a plurality of distinct clones of the bacterial target and checking for polymorphism in the plurality prophage sequences, identifying sequences that exhibit the largest polymorphisms or 34 Sidentifying specific allelelic variation or both, and optionally developing further primers or probes specific for differentiating between alleles by their differential binding capacity 00 CI or to enable ascertainment of sequence differences directly or indirectly, to thereby enabling differentiation between clones of said bacterial target. 00 S24. A kit comprising a plurality of probes or primers useful for identifying clones of a bacterial target species or species variant, said bacterial target carrying a prophage, said primers or probes specific for differentiating between polymorphic alleles of a plurality of prophage nucleic acid sequences by their differential binding capacity or to enable ascertainement of sequence differences directly or indirectly, to thereby enable differentiation between alleles of said prophage nucleic acid sequences, said plurality being such that the primers and probes are collectively able to identify a unique allele or combination of alleles specific to each clone of the bacterial target.
19. 25. The kit of claim 24 comprising primers selected to amplify prophage nucleic acid sequences present in Salmonella.
20. 26. The kit of claim 25 wherein primers are selected to amplify prophage nucleic acid sequences present in serovars Typhimurium and Enteriditis.
21. 27. The kit of claim 26 wherein the primers are selected to amplify nucleic acid sequences of prophage selected from the group comprising ST46B, P22 and DT64T.
22. 28. The kit of claim 27 wherein the primers are selected to amplify nucleic acid sequences of the c2, cl, cro and nut genes.
23. 29. The kit of claim 26 comprising forward and reverse primers for amplifying nucleic acid of part thereof of sequences amplified by any two or more sequences selected from the group consisting of SEQ ID NO I to 12, 15 to 30, 41, 42, 49, 51, and 61 to 66. The kit of claims 25 comprising probes immobilised on a solid support. 00 Dated this 28th day of July 2005 00 M MEDVET SCIENCE PTY LTD c and RURAL INDUSTRIES SRESEARCH AND DEVELOPMENT t 10 CORPORATION By their Patent Attorneys Ci A.P.T. Patent and Trade Mark Attorneys